Filter Dissertations and Theses By:
SYSTEMATICS AND HISTORICAL BIOGEOGRAPHY OF AGKISTRODON CONTORTRIX AND AGKISTRODON PISCIVORUS
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Many studies have revealed that lineages currently inhabiting formerly glaciated areas were pushed into southern glacial refugia and have expanded into their modern range since the last glacial maximum. There have been few studies that compare the effects of glacial cycles on lineage diversification, historical demography and migration rates in closely related species with overlapping ranges. In this study I compare phylogeographic structure, historical demography, approximate lineage age, potential distributions, and migration rates in two closely related and broadly co-occurring venomous snakes in eastern North America, the cottonmouth (Agkistrodon piscivorus) and copperhead (A. contortrix) using multilocus coalescent approaches. It has recently been discovered that gene flow between closely related species with adjacent distributions may be common (Nosil 2008). However, the absence of gene flow is a primary assumption of many phylogeographic methods including species tree inference and Bayesian species delimitation. I provide a framework for examining species delimitation when gene flow between species is present and provide a taxonomic revision of A. contortrix and A. piscivorus. In addition, I explore whether hybrids between adjacent species inhabit unique environmental conditions not suitable to one or both species. Finally, I reveal that species diversification was likely a direct result of Pleistocene glacial cycles and that species with the closest proximity to formerly glaciated areas experienced population expansion following the retreat of the Laurentide Ice Sheet. A combination of population expansion out of refugia and niche expansion has resulted in hybridization between adjacent species where species distributions come into contact. It is not clear whether gene flow has persisted during speciation and subsequent interglacial periods or if it has only recently occurred following the last glacial maximum.
PHYLOGENETICS AND BIOGEOGRAPHY OF MOUSE OPOSSUMS (DIDELPHIDAE: MARMOSA)
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This research focused on the systematics and biogeography of mouse opossums of the genus Marmosa (Mammalia: Didelphimorphia: Didelphidae), with special emphasis on species found in the complicated geography of north-central South America. Via the four chapters of this dissertation, I presented information obtained through fieldwork, examination of museum specimens, DNA sequencing, phylogenetic analyses, georeferencing (including consultation of field notes and collectors), and ecological niche modeling. In Chapter 1, I conducted phylogenetic analyses of species of Marmosa based on sequence data of the mitochondrial cytochrome-b gene (CYTB), in part to test the monophyly of species previously recognized based on morphological criteria. This study revealed the existence of unrecognized species and identified novel interspecific relationships. All trans-Andean species of the subgenus Marmosa were recovered as a clade, suggesting that the uplift of the Andes might have played an important role in the diversification of the genus. In Chapter 2, I documented the presence of M. waterhousei in the Venezuelan Andes. This finding implied that the species might have crossed the dry Depresión del Táchira during a glacial period. In Chapter 3, I investigated the phylogeography of M. robinsoni, a species predominately distributed across the dry forests of northern South America. I conducted phylogenetic analyses based on sequence data of one mitochondrial and one nuclear gene. The results confirmed the monophyly of a dry-forest clade formed by M. robinsoni and M. xerophila and showed the existence of two major clades within M. robinsoni that corresponded roughly to an east/west division. Results of ancestral area reconstructions identified multiple dispersal events out of the greater Maracaibo basin. Lastly, in Chapter 4 I used ecological niche modeling to test the geographic predictions of competition between a sister species pair, M. robinsoni and M. xerophila. The results strongly suggest that M. xerophila may isolate populations of M. robinsoni in the Península de Paraguaná of northern Venezuela--representing a novel example of geographic isolation caused by competition. Together, these studies contributed to a better understanding of the taxonomy, phylogenetics, and biogeography of the genus Marmosa; provide novel information relevant to the biogeography of dry-forest species in northwestern South America; and propose a refinement of the concept of ecological vicariance to incorporate the possibility that biotic interactions could lead to geographic isolation.
Population Genomics of White-Footed Mice (Peromyscus leucopus) in New York City
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Urbanization significantly alters natural ecosystems. New York City (NYC) is one of the oldest and most urbanized cities in North America, but still maintains substantial populations of some native wildlife. The white-footed mouse, Peromyscus leucopus, is a common resident of NYC’s forest fragments, and isolated populations may adapt in response to novel urban ecosystems. Using pooled transcriptome-wide RNAseq data, individually barcoded transcriptome-wide RNAseq data, and genome-wide RADseq data, I found genetic differentiation between urban and rural P. leucopus populations and evidence suggestive of local adaptation. I compared genome and transcriptome-wide SNP data in P. leucopus from relatively large urban parks surrounded by dense urban infrastructure to large rural sites representative of native habitat. First, I built a publicly available genomic resource for P. leucopus, and then looked at patterns of genetic differentiation in protein-coding DNA sequences that showed divergence between urban and rural populations. I also looked at the unique demographic history of urban and rural populations of P. leucopus using coalescent simulations and the site frequency spectrum (SFS). Historical demographic inference supported a scenarios of post-glacial sea level rise that led to isolation of mainland and Long Island populations. I also found that several urban parks in NYC represent distinct P. leucopus populations, and the estimated divergence times for these parks are consistent with patterns of urbanization in NYC. I then looked at transcriptome sequences within urban P. leucopus to look for signatures of genetic differentiation and selective sweeps due to positive selection, and then associated outliers with environmental measures of urbanization. The majority of candidate genes were involved in metabolic functions, especially dietary specialization. Candidate loci I identified suggest that populations of P. leucopus are using novel food resources in urban habitats or metabolizing nutrients differently. Ultimately, the data indicate that cities may represent novel ecosystems with selective pressures from urbanization leading to adaptive responses in populations of Peromyscus leucopus.
Evolutionary Analyses on the Core Genome of Borrelia burgdorferi: Elucidating the Genomics of Virulence
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ABSTRACT: Adaptive Evolution in Borrelia burgdorferi by: James Haven Advisor: Dr. Weigang Qiu The availability of multiple genomes of closely related pathogen strains makes it possible to identify genome-wide variations associated with strain-specific phenotypes such as pathogenicity and virulence. One main challenge of gene-trait associative mapping in bacterial species is finding a way to minimize the effect of linkage among loci due to pervasive clonal population structures. A second concern is to distinguish selective sequence variations from random, selectively neutral differences among strains. Here we identified adaptive, strain-specific nucleotide polymorphisms (SSNPs) on the core genome of Borrelia burgdorferi, the Lyme disease pathogen. We minimized the linkage effect by comparing the genomes of seven isolates representing four genospecies (B. burgdorferi sensu stricto, B. bissettii, B. afzelii, and B. garinii) and four clonal groups of a single species (A, C, E, and K clones of B. burgdorferi sensu stricto). Identification of selective nucleotide polymorphisms was achieved by applying codon-based, tests of positive selection based on rates of synonymous (KS) and nonsynonymous (KA) substitutions. We then tested for the presence of positive selection at 824 gene loci on the main chromosome, 68 loci on the linear plasmid lp54, and 26 loci on the circular plasmid cp26. Consequently, we identified 28 genes under positive selection without regard for lineage, 12 genes associated with genospecies divergence, and 7 genes associated with the adaptive divergence of B31, a highly invasive strain. We checked results by excluding loci with high alignment uncertainties, mapping positively selected sites on protein structure models, and evaluating the possibility of false positives. Cell envelope genes are significantly over-represented among the positively selected genes. Additional categories of interest are DNA metabolism, transcription, cell division, and regulation. Focused analyses on copy number variation of established immune elicitors and a survey of intraspecific recombination support a prominent role for adaptive evolution in the maintenance of the B. burgdorferi pathogen cycle. These findings highlight immune escape as a driver of positive natural selection via surface protein variation and possibly pathogen replication dynamics.
cAMP and Polyamines Overcome Inhibition by MAG by Activating Cdk5 via Increased Expression of p35 Regulated by Activation of eIF5A
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Damaged axons in adult mammalian central nervous system (CNS) are unable to regenerate after injury although axons in the peripheral nervous system (PNS) or embryonic CNS can. The inhibitory molecules associated with myelin are one of the major obstacles to successful axon regeneration in the adult mammalian CNS. To date, three inhibitors of regeneration have been identified in myelin: NogoA, myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (OMpg) (Filbin, 2003). Interestingly, all these three ligands bind to the same receptor Nogo receptor (NgR) to mediate the inhibitory effect. p75NTR or TROY and Lingo-1( LRR and Ig domain-containing, Nogo Receptor interacting protein) are necessary components of the receptor complex as NgR is glycosyl phosphatidylinositol (GPI)-anchored and lacks a signaling domain (Wang et al., 2002a; Mi et al., 2004; Park et al., 2005; Shao et al., 2005). Activation of the receptor complex by myelin inhibitors activates the small GTPase RhoA resulting in rearrangement of the cytoskeleton and inhibition of axonal outgrowth (Hu and Strittmatter, 2004). It has been shown in our lab that elevating intracellular levels of cyclic AMP (cAMP), either via application of a cAMP analog or by prior exposure to neurotrophins (NTs) can block the inhibition of axonal regeneration by MAG and myelin (Cai et al., 1999; Cai et al., 2001; Qiu et al., 2002). Elevation of cAMP results in up-regulation of arginase I (ArgI) and subsequent synthesis of polyamines. Up-regulation of ArgI or priming with the polyamine putrescine or spermidine blocks the inhibition of axonal growth by MAG/myelin (Cai et al., 2002; Deng et al., 2009). Polyamines are known to have effects in regulating cytoskeleton organization in both the short term and the long term, but their downstream effectors have yet to be identified. Many studies have shown that Cyclin-dependent kinase 5 (Cdk5) is involved in neurite outgrowth and regulates the neuronal cytosekeleton, which prompted us to hypothesize that Cdk5 may play a role in blocking MAG/myelin-mediated inhibition. Cdk5 is a multifunctional serine/threonine kinase and its activator, p35, is expressed only in the nervous system (Tsai et al., 1994). It has been shown that activity of Cdk5 is required for neurite elongation (Nikolic et al., 1996; Paglini et al., 1998; Li et al., 2000; Harada et al., 2001). Cdk5 phosphorylates cytoskeleton proteins and regulates the organization of all three cytoskeleton elements microfilaments, microtubules and intermediate filaments (Dhavan and Tsai, 2001). Here we show that Cdk5 is required for db-cAMP and putrescine to overcome inhibition. The effect of db-cAMP and putrescine in overcoming inhibition by MAG is abolished in the presence of a specific inhibitor of Cdk5, Roscovitine. Neurons infected with dominant negative Cdk5 HSV viruses are not able to overcome inhibition by MAG in the presence of db-cAMP or putrescine. Importantly, neurons infected with HSV viruses overexpressing p35, the neuronal specific activator for Cdk5, overcome MAG's inhibition. Moreover, db-cAMP and putrescine increase the expression of p35. This in turn induces the kinase activity of Cdk5. The up-regulation of p35 by putrescine is also reflected in the increased distribution of p35 in neurites and growth cones. Furthermore, we show that putrescine up-regulates p35 protein by hypusine modification of eukaryotic Initiation Factor 5A (eIF5A), and this hypusination is necessary for putrescine to overcome inhibition by MAG. Our findings reveal a previously unknown mechanism by which polyamines encourage regeneration after CNS injury.
Adaptive Plasticity in the Human Saccade System
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The rapid point-to-point movements of the eyes called saccades are some of the most commonly made by humans, yet differ from nearly every other type of motor output in that they are completed too quickly to be adjusted during their execution by visual feedback. Yet, saccadic accuracy remains quite high over a lifetime despite inevitable changes to the physical structures controlling the eyes, indicating that the oculomotor system actively monitors and adjusts motor commands to achieve this consistent behavioral production. Indeed, it seems that beyond the ability to compensate for slow, age-related bodily changes, saccades can be modified following traumatic injury or pathology that affects their production, or in response to more short-term systematic alterations to post-saccadic visual feedback in a lab setting. It is, in fact, thought that all of these forms of plasticity rely on the visual detection of accuracy errors by a unified set of mechanisms that support the process known as saccade adaptation. A great deal has been learned about saccade adaptation, as it has been extensively studied as a phenomenon in its own right, as well as being used to explore the process of motor learning in general. However, many fundamental questions about saccade adaptation remain unanswered, often related to the way that saccade adaptation might operate in the natural environment with substantially more complex visual stimuli than are generally used in the lab. Here, we addressed these questions with (in some cases original) variants and more conventional examples of the frequently used intrasaccadic target step (ISS) paradigm (in which an experimenter causes saccadic error by shifting a target during the movement). By exploring the responses to whole-field-ISSs, we have inferred that saccade adaptation might be supported by a trans-saccadic integration mechanism, and may be sensitive to intrasaccadic motion signals. Challenging the oculomotor system by confronting it with multiple post-saccadic targets has revealed that saccade adaptation can occur in a target-identity specific manner, so that even if post-saccadic error varies from trial-to-trial, adaptation seems to reflect the average behavior of the target. At a more basic level, we systematically varied ISSs to determine the lower limits of the oculomotor system's sensitivity to intrasaccadic displacement during adaptation. Also at a more basic level, we looked at the effects of rendering post-saccadic feedback more intermittent during adaptation, finding it to have little effect on the magnitude or rate of adaptive dynamics, similar to other forms of motor learning but somewhat dissimilar from operant conditioning. These experiments also furnished a useful setting to develop and test a novel model of saccade adaptation which more explicitly relies on post-saccadic sensory prediction than previous models, but that is nonetheless in keeping with the ethos of modern motor learning theory. Finally, we found that the establishment and maintenance of a context for saccadic performance by adaptation could be achieved by consistently pairing a target's visual-identity with a specific ISS, extending what had been previously recognized as constituting a cue for contextual motor learning. In general, our results suggest that saccade adaptation is a highly flexible mechanism that not only supports the maintenance of accuracy, but also makes use of a wide range of brain functions to deftly tailor saccadic behavior contingent on task demands.
Systematics of Lecythidoideae (Lecythidaceae) with emphasis on Bertholletia, Corythophora, Eschweilera, and Lecythis
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Phylogenetic analyses based on morphological and DNA sequence data were generated to test the monophyly of Eschweilera and Lecythis and to investigate the relationships of these two genera and their close relatives (Bertholletia and Corythophora). The final results were applied to questions of taxonomic rearrangements and character evolution in traits related to pollination. A cladistic analysis based on morphology indicates that Bertholletia, Corythophora, Eschweilera, and Lecythis form a clade (the Bertholletia clade), but the resolution within the clade is too poor to elucidate the phylogenetic relationships among the genera. Nonetheless, the data support the monophyly of Corythophora. Eschweilera is divided into two clades: Eschweilera section Tetrapetala and Eschweilera s.s. Whether or not the two clades form a clade remains as unresolved. Lecythis is not resolved as monophyletic, but each of the Sections Corrugata, Pisonis, and Poiteaui is monophyletic. A combined analysis based on morphology and nuclear ITS and plastid ndhF, trnL-F, and trnH-psbA sequence data was conducted to improve the resolution of the phylogenetic tree. The results support the generic circumscriptions of Bertholletia and Corythophora based on morphology, but suggest the revision of Eschweilera and Lecythis. Eschweilera is paraphyletic and divided into three clades: the Eschweilera section Tetrapetala clade, the Eschweilera andina clade, and the core Eschweilera clade. Lecythis is polyphyletic and consists of five clades: the Lecythis s.s. clade, the Pachylecythis clade, the Lecythis chartacea clade, the Holopyxidium clade, and the Section Corrugata clade. Based on the results of these analyses, Section Tetrapetala clade merits recognition as an independent genus and should be segregated from Eschweilera. On the other hand, the five clades of Lecythis are distinct from each other and should be recognized as five separate genera. Androecial evolution inferred from the resulting phylogeny demonstrates that features such as floral zygomorphy, closed androecia, coiled ligules, and the production of nectar are homoplasious. Evolution of these characters most likely is because of the adaptation in response to pollinator shift and does not simply represent a process of morphological transformation from a simpler to a more complicated structure as previously suggested.
The Sustainable Management and Conservation Santalum yasi (Sandalwood) in Fiji and Tonga: A Combined Ecological and Genetic Approach
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Valued internationally for the aromatic oil found within its heartwood, Sandalwood (Santalum, Santalaceae) is one of the most heavily exploited groups of plants across its range. While historically, most oil has been harvested from Santalum album in Southeast Asia and India, the decrease of S. album sources has caused a widening gap between supply and demand, which creates profitable market opportunities and increasing harvest pressure for alternative sandalwood oil sources. Santalum yasi, a quality alternative, has been harvested extensively in Fiji and Tonga, yet is vastly under-studied. The absence of basic data on population dynamics and genetic variation for remnant populations remains a major constraint to the sustainable management of this culturally and economically valued resource. This dissertation focuses on the ecological and genetic data and analyses that can aid in developing sustainable management strategies. Population size-class structure data was collected using transects in the three densest natural populations of Santalum yasi. Population dynamics, current species distribution, and ecological threats were investigated to find that the few remaining wild stands display discontinuous size class structures, are under regenerative stress and that the natural distribution has diminished significantly, even to local extinction in some areas. Using a nuclear microsatellite analysis, genetic variability within and between populations was investigated. Results suggest that there is no significant genetic variation between populations, but that most of the genetic variation lies within populations. This genetic distribution suggests that there is a significant level of gene flow between and among populations, most likely through human induced dispersal, showing a more panmictic trend than previously supposed. This may provide molecular evidence confirming the Western documentation and traditional oral history of extensive interaction between Fiji and Tonga and their trade of plants and culture. Based on these dwindling levels of yasi population size and unstructured genetic variation in Fiji and Tonga, further enumerations and resource surveys are not practical to conduct at this time. Rather, forestry and governmental efforts should be focused on promotion of local involvement in assisted natural regeneration of wild stands and preservation of genetic variation through in situ, community-mediated conservation.
THE ROLE OF Na+/H+ EXCHANGER-1 (NHE1) IN MAMMARY BRANCHING MORPHOGENESIS AND MAINTENANCE OF TISSUE ARCHITECTURE
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Branching morphogenesis in vivo is a highly ordered process that necessitates spatially and temporally choreographed cues by growth factors and hormones, as well as mechanical and signal feedback from the extracellular matrix. Successful completion of this developmental process results in the architectural, and thereby the functional, basis for the lung, collecting ducts of the kidney, salivary, and mammary glands. The quest to understand the basic biological mechanisms underlying this developmental morphogenesis has lead to many seminal findings in the field of epithelial tube generation, as well as provided valuable insight into the pathogenesis of cancer. The primary focus of this thesis was elucidating the role of the Na+/H+ exchanger type 1 (NHE1) in branching morphogenesis of the mouse mammary gland. To accomplish this goal, we used three-dimensional (3D) primary tissue culture of mammary gland pieces (organoids) in a four day organotypic assay of growth factor induced branching morphogenesis. NHE1 is a ubiquitously expressed master regulator of intracellular pH (pHi). We found that blocking the function of this exchanger in the presence of growth factor stimulation led to altered kinase signaling, inhibition of growth factor induced alkalization, sustained proliferation after four days, ectopic expression of keratin 6 (K6), and a dramatic failure to undergo branching morphogenesis. These findings led us to question the role of NHE1 in the maintenance of branched mammary tissue architecture. We, therefore, inhibited NHE1 function on fully branched structures in our assay and found that NHE1 inhibition led to rapid loss (within 24 hours) of branched architecture by a process of branch fusion, with complete loss of the branched morphology after four days. This was not accompanied by cell death or altered proliferation, however, we did record altered intracellular pH (pHi) in the end buds of branched structures that had NHE1 inhibited. NHE1 localization, F-actin organization, and myoepthelial cell location were altered in structures that had undergone a loss of architecture, indicating a loss of tissue organization. Finally, we found that NHE1 inhibition resulted in a decrease in mammary Ecadherin. Having found that NHE1 function is vital for both branching morphogenesis and the maintenance of branched architecture, we considered the role that NHE1 could be playing in the pathology of breast cancer. Both intracellular and extracellular pH is deregulated in cancer. This could be attributed to over activity of NHE1. Additionally, NHE1 is over expressed in many cancers. We used the ER+ breast cancer cell line MCF7 to investigate the therapeutic potential of chemotherapy augmentation by NHE1 inhibition. We found that Cycylophosphamide, a DNA alkylating chemotherapeutic agent known to be more effective in an acidic environment, was roughly 5 times more effective when used along with NHE1 inhibition. These findings indicate that NHE1 is a critical regulator of branching morphogenesis and tissue stability, as well as suggests a potential therapeutic target for the treatment of breast cancer.
Origin and development of hematopoietic tumors in sumoylation mutants of Drosophila melanogaster
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The larval hematopoietic system of Drosophila melanogaster consists of freely circulating cells, the sessile population, and the hematopoietic organ called the lymph gland. Most hemocytes function as macrophages and play a significant role in innate immunity. Hemocytes also remodel tissues, aiding in development of the organism. Constitutive activation of immune signaling pathways, as well as various mutations in genes which are not linked to immune pathways, result in the overabundance of circulating hemocytes and tumor formation. Many aspects of tumor development, such as the identity of the affected hematopoietic population and mechanisms of tumor growth are either not characterized or not well understood. The overall goal of this project was to understand the origin and development of hematopoietic tumor formation in Ubc9 mutants. Ubc9 is an E2 enzyme, which conjugates SUMO (small ubiquitin-like modifier) to a range of target proteins. Sumoylation targets vary in function from structural components to enzymes and transcription factors. Thus, sumoylation affects multiple cellular functions via modification of protein localization, stability or activity. Loss-of-function Ubc9 mutants of Drosophila exhibit severe defects in hematopoietic and immune tissues, including hemocyte overproliferation and tumor formation during larval stages. In this dissertation, we report that the hemolymph of Ubc9 mutants contains hematopoietic cells and structures that range from aggregates (composed of only few cells) to small and large tumors. The largest tumors are less than 1 mm3 in volume. Most cells and aggregates exhibit high ration of mitotic cells, but only a few of the largest tumors in the hemolymph contain actively dividing cells. Based on staining patterns and genetic rescue experiments, we propose that the large tumors are derived specifically from overgrown posterior lobes of the hematopoietic organ. The origin of smaller tumors and aggregates is less clear. Our data suggest that these structures likely derive from circulating hemocytes and fragments of dispersed anterior lobes. Microtumors in Ubc9 mutants arise from the highly-mitotic mutant stem/progenitor cells of the lymph gland. Loss of sumoylation cascade enzymes E1 (Aos1/Uba2), E2 (Ubc9), or E3 (PIAS) leads to loss of proliferative quiescence in hematopoietic precursors localized within the lymph gland. Proliferative quiescence of these precursors is at least in part mediated by the activity of the cyclin-dependent kinase inhibitor Dacapo/p21. Expression of Dacapo homolog, human p21, in the Ubc9 progenitor cells relieves tumor formation. These studies suggest that sumoylation provides a cell-intrinsic mechanism to preserve stem/progenitor cell states for stress response, immunity and development of the fly.