Alumni Dissertations

 

Alumni Dissertations

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  • Polymerase alpha components associate with telomeres to mediate overhang processing

    Author:
    Raffaella Diotti
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Diego Loayza
    Abstract:

    Telomeres consist of TTAGGG repeats, which end with a 3' G-overhang and are bound by a six-protein complex, known as Shelterin. In humans, telomeres shorten at each cell division, unless telomerase is expressed and able to add telomeric repeats to the 3' G-overhang. However, for effective telomere maintenance, the DNA strand complementary to that made by telomerase must be synthesized. In this study, I focused on the Polα/primase complex, in particular the subunits p68 (POLA2, the regulatory subunit) and p180 (Polα, the catalytic subunit), and their potential roles at telomeres. I was able to detect p180, p68 and OBFC1, a subunit in the CST complex, at telomeres in S phase using chromatin immunoprecipitations. I could also show that OBFC1, Shelterin and Polα/primase interact, revealing contacts occurring at telomeres. Finally, depletion of p68 by shRNA and p68 and p180 by siRNA, led to increased overhang amounts at telomeres. I propose a model in which Polα-primase is important for proper telomeric overhang processing, perhaps through fill-in synthesis. These results shed light on important events necessary for efficient telomere maintenance and protection.

  • Functional Diversity of Fibroblast Growth Factor Homologous Factor Family of Proteins

    Author:
    Katarzyna Dover
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Mitchell Goldfarb
    Abstract:

    Abstract “Functional Diversity of Fibroblast Growth Factor Homologous Factor Family of Proteins” by Katarzyna Dover Thesis Advisor: Dr. Mitchell Goldfarb FHFs resemble other fibroblast growth factors on a basis of amino acid composition and crystal structure but evolved to carry on distinct, FGF unrelated functions. To date, FHFs have been implicated most clearly in modulation of voltage gated sodium channels (VGSCs). FHFs are the classical example of an increase in gene diversity through the alternative promoter usage and splicing. Hence, the multiplicity of isoforms makes this family of proteins an interesting yet, challenging research topic. Different isoforms of FHFs have distinct sub-cellular localizations and differently modulate voltage gated sodium channels. By influencing critical parameters of channel physiology, including voltage dependence of channel steady-state inactivation, recovery from inactivation and current density, the FHF family of proteins has emerged as important regulators of cellular excitability. The role of different FHF isoforms in modulation of VGSCs and their influence on cellular excitability is the main topic of this thesis. Performed experiments aimed to: (i) establish a channel-binding surface, common to all FHFs, (ii) categorize major FHF isoforms into functional groups based on the ability to modulate sodium channel Nav1.6, (iii) elucidate the mechanism involved in A-type FHF induced long-term, use-dependent channel inactivation, and (iv) determine potential differential localization of A-type FHFs in the brain and in subcellular compartments of cerebellar, hippocampal and sensory neurons.

  • Colletotrichum gloeosporioides s.l. in North America: Sex, Host, and Habitat-mediated Diversity in a Plant-associated Ascomycete

    Author:
    Vinson Doyle
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Amy Litt
    Abstract:

    Determining the factors that drive the evolution of pathogenic fungi is central to revealing the mechanisms of virulence and host preference, as well as developing effective disease control measures. Prerequisite to these pursuits is the accurate delimitation of species boundaries. Colletotrichum gloeosporioides s.l. is a species complex of plant pathogens and endophytic fungi for which reliable species recognition has only recently become possible through a multi-locus phylogenetic approach. Through intensive regional sampling that encompasses multiple hosts within and beyond agricultural zones associated with cranberry (Vaccinium macrocarpon Aiton), we have integrated North American strains of Colletotrichum gloeosporioides s.l. from these habitats into a broader phylogenetic framework and characterized some of the factors that influence species diversity. We have developed polymorphic microsatellite markers for C. fructivorum, a species determined to be responsible for cranberry fruit-rot in agricultural areas throughout North America, in order to understand the biotic and abiotic factors that shape populations within the species complex. These markers amplify across several species within the C. gloeosporioides species complex and some are variable within two species, C. rhexiae and C. kahawae, that are closely related to C. fructivorum. Broad geographical and fine-scale hierarchical sampling of C. fructivorum and C. rhexiae coupled with multilocus genotyping has allowed us to gain insight into the forces that shape populations of these species. Human-mediated dispersal is an important factor dissipating the population structure of C. fructivorum throughout its range in commercial cranberry bogs. In contrast, limited evidence suggests C. rhexiae is geographically structured within a more restricted range, implying distinct patterns of diversity between Colletotrichum species associated with wild versus agricultural hosts. We also investigate the reproductive mode of C. fructivorum using estimates of haploid disequilibrium and genotypic diversity, inferring a mixed (sexual and asexual) mode of reproduction in field populations. We discuss the importance of sexual and asexual reproduction on population dynamics and speciation within the C. gloeosporioides species complex.

  • Novel Insights into Vascular Endothelial Growth Factor Receptor 2-Mediated Signaling to the Mammalian Target of Rapamycin/Akt Network in SK-N-SH Neuroblastoma Cells

    Author:
    Jacob Edelstein
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Patricia Rockwell
    Abstract:

    Mammalian target of rapamycin (mTOR) is a central regulator of cell growth and division that exerts many of its effects through regulating protein synthesis. The kinase Akt is a substrate and regulator of mTOR. These proteins are integral to pathological and physiological function in neuronal cells and the Akt/mTOR network is the focus of pharmaceutical interventions. Muscarinic acetylcholine receptors and vascular endothelial growth factor receptor 2 (VEGFR2) can signal protein synthesis but whether they cooperate to mediate mTOR activation has not been demonstrated. Using serum-starved SK-N-SH neuroblastoma cells, we show that the muscarinic receptor agonists carbachol and pilocarpine enhance the activation of the mTOR substrate p70 S6 Kinase (S6K) and its target ribosomal protein S6 (S6) in a VEGFR2-dependent manner. Protein kinase C (PKC) functions in an opposing fashion by positively regulating S6K and S6 phosphorylation and suppressing Akt activation. Treatments with the phosphatase inhibitors sodium orthovanadate and okadaic acid (OA) increase S6, Akt and to a lesser extent S6K phosphorylation, indicating that tyrosine and serine/threonine dephosphorylation also regulates their activity. However, OA elicited a far greater increase in phosphorylation, implicating phosphatase 2A (PP2A) as a critical determinant of their function. Furthermore, PP2A inhibition induces the appearance of novel, high molecular weight, ubiquitinated forms of Akt. The accumulation of phosphorylated Akt induced by PP2A dysfunction causes depletion of total Akt. Rapamycin potentiates Akt phosphorylation and depletion in response to OA through a mechanism regulated by a previously unknown function of VEGFR2. Although hyperactivation of Akt is a common survival mechanism in cancer cells, Akt hyperphosphorylation is associated with induction of a caspase-independent cell death mediated by oxidative stress. Taken together, these results show that the critical role of PP2A in regulating Akt activation also affects Akt ubiquitination, cleavage and removal from the cell. Furthermore, these data indicate the importance of reactive oxygen species in eliciting cell death and that PP2A promotes survival through a suppression of oxidative stress. Finally, VEGFR2 can stimulate mTOR when stimulated by ligand binding, transactivation or an unknown mechanism induced by rapamycin.

  • Systematics and biogeography of the New World scorpion genus Centruroides Marx, 1890 (Scorpiones: Buthidae)

    Author:
    Lauren Esposito
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Lorenzo Prendini
    Abstract:

    Background: The New World scorpion genus Centruroides Marx, 1890 (family Buthidae Koch, 1837) is a morphologically diverse and highly venomous taxon. Centruroides is among the most complex scorpion genera in the New World, comprising 71 described species and 5 subspecies in addition to several undescribed species. These scorpions are sexually dimorphic, the males typically exhibiting elongation of the metasoma and telson and longer, more slender pedipalp chelae. The greatest diversity of Centruroides occurs in Mexico; however the genus is distributed from the southern United States into northern South America and the Galapagos, and throughout the Caribbean. The genus includes the only scorpions of medical importance in North America, with six species that are potentially lethal to humans. Historical Taxonomic Problems: Centruroides species are problematic for systematists for several reasons. The morphological characters traditionally used (morphometrics and color) often overlap between closely related species or are vaguely defined, and many have been shown to vary within populations making the identification of species difficult. Many researchers have studied and revised small groups of species, but no comprehensive modern taxonomic treatment of the entire genus exists. The positions of Centruroides within the New World buthids, its relationship to its putative sister genus Rhopalurus Thorell, 1890, and its monophyly have never been tested. Few modern analytical methods have been applied to the genus, a problem that extends to both phylogenetic and biogeographical analysis. Aims: The broader vision of this dissertation project was to investigate and evaluate the systematics and biogeographic patterns that have resulted in one of the largest, least understood and most medically important genera of New World buthid scorpions. This was accomplished through: (1) conducting fieldwork in regions of Centruroides diversity to gather fresh material for morphological and genetic studies; (2) conducting molecular and morphological phylogenetic analyses to test the monophyly of the genus and determine its relationship to other New World buthid genera, identify the major clades and test the validity of the currently hypothesized species groups; (3) testing biogeographic hypotheses to explain the present distributions and species diversity. Conclusions: The buthid subfamily Rhopalurusinae comprising Centruroides, Rhopalurus, Physoctonus and Troglorhopalurus is supported with molecular and morphological evidence. Rhopalurus is paraphyletic with respect to Centruroides, forming two clades: one endemic to South America and the other endemic to the Greater Antilles. Centruroides is monophyletic, sister to the Greater Antilles Rhopalurus. The genus Heteroctenus Pocock, 1893 is resurrected for the Greater Antilles Rhopalurus species. Centruroides contains four, geographically delimited clades: a Caribbean clade, a North American clade, a Mesoamerican clade, and a Yucatan/Chortis block clade. A fossil-calibrated phylogeny of New World buthids dates the separation of the Greater Antilles Rhopalurus + Centruroides at {29.0, 42.9} mya. Ancestral distribution reconstruction infers this node to be South America and the Greater Antilles. The ancestral distribution of Centruroides is inferred to be North America. Both the dating and the ancestral distribution reconstruction are congruent with the GAARlandia hypothesis, which has been proposed to explain similar disjunct distributions in large mammals. The Greater Antilles distributed sister taxon of Centruroides provides evidence for a Caribbean ancestor for the genus, which subsequently colonized North and Meso-America and re-colonized the Caribbean.

  • Multifunctional Roles of APL-1 in C. elegans

    Author:
    Collin Ewald
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Christine Li
    Abstract:

    Alzheimer's disease is an age-dependent disorder and the most common type of dementia. The most prevalent mutations associated with familial Alzheimer's Disease are found in the gene encoding the amyloid precursor protein (APP) or in the presenilin genes, which encode proteases that cleave APP. In mice, knockout of the APP gene family leads to lethality and type II lissencephaly, while overexpression of APP causes a shortening in lifespan and learning defects. However, the cellular function of APP and the pathways in which APP acts are unknown. Here we investigate the role of APL-1, the Caenorhabditis elegans orthologue of APP. Specifically, we expressed APL-1 with different promoters to determine the effect of APL-1 overexpression on several parameters: lifespan, development, and learning. Overexpression of APL-1 driven by its endogenous promoter accelerated aging and thereby shortened lifespan. By contrast, overexpression of APL-1 driven by the snb-1 promoter slowed aging and thereby prolonged lifespan. This extended lifespan was dependent on signals from the gonad and activity of the transcription factor DAF-16/FOXO and the nuclear hormone receptor DAF-12/NHR. Several other APL-1 overexpression phenotypes, including slowed developmental progression, were also dependent on daf-16/FOXO and daf-12/NHR activity. Lastly, pan-neuronal expression of APL-1 caused impairments in olfactory and gustatory avoidance behaviors as well as impairments in touch habituation. These defects were rescued by decreased activity of daf-16/FOXO and daf-12/NHR. Our results suggest that signaling of neuronally expressed APL-1 perturbs learning via the insulin/IGF-1 and the TGF-β pathways. Hence, APL-1 is a multifunctional protein that signals in multiple pathways to affect lifespan, development, and learning. In light of our results, we suggest that the shortened lifespan and learning defects in mice with APP overexpression might be mediated via the insulin/IGF-1 pathway. Interestingly, AD is strongly associated with type 2 diabetes and some AD patients show brain specific diabetes.

  • DIVERSITY, RESOURCE PARTITIONING, AND SPECIES TURNOVER IN NEOTROPICAL SAPROXYLIC BEETLES (COLEOPTERA: CERAMBYCIDAE, CURCULIONIDAE) ASSOCIATED WITH TREES IN THE BRAZIL NUT FAMILY (LECYTHIDACEAE)

    Author:
    Joyce Fassbender
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Amy Berkov
    Abstract:

    Deforestation and global changes in temperature and moisture associated with rising levels of greenhouse gases are expected to have strong, direct effects on abundance of wood-boring beetles through loss of larval feeding substrates, and indirect effects through climate and microclimate change. This dissertation examines Neotropical saproxylic beetle (Coleoptera: Curculionidae) diversity, niche breadth, and resource partitioning, and predicts possible impacts of climate change. Data from beetle rearing experiments conducted in French Guiana and Peru were analyzed to assess species richness, abundance, host specificity, seasonality and stratification of wood-boring beetles associated with the Brazil nut family (Lecythidaceae). Niche stability was assessed over time (French Guiana 1995-96, 2007-08) and space (Peru 2003-05). In French Guiana, resource partitioning was analyzed among the most abundant subfamilies of Curculionidae (Conoderinae, Scolytinae, Platypodinae) and Cerambycidae (Cerambycinae, Lamiinae). Species richness was higher in Peru than French Guiana, with high beta-diversity between sites; largely due to the prevalence of rare species in Peru. In both localities, most beetle species were disproportionately associated with the host Eschweilera coriacea (DC) S.A. Mori. In French Guiana, comparatively large cerambycids were more abundant during the dry season and seemed relatively drought tolerant. Small-bodied curculionids were most abundant during the rainy season, with weevils and platypodines best represented at ground stratum. In Peru, weevils were more abundant during the dry season. Cerambycinae, which are preferentially associated with the dry season canopy stratum, are expected to thrive should regional climates become warmer and drier. Lamiinae may respond by seasonally alternating stratum. Many Neotropical weevils and bark/ambrosia beetles seem strongly moisture-dependent, and populations are expected to be negatively impacted by increased drought. The Brazil nut family is threatened by both habitat fragmentation and climate change. The favored host species, E. coriacea, has a wide geographic distribution that extends into western Amazonia, which is not expected to experience severe precipitation changes, and could provide refuge for saproxylic beetles currently associated with Lecythidaceae. Saproxylic beetles, especially curculionids, may be less impacted by direct effects of host loss than indirect effects of climate change, especially northeast Amazonia which is expected to experience declining precipitation and longer dry seasons.

  • Evolution of Song Culture in the Zebra Finch

    Author:
    Olga Feher
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Ofer Tchernichovski
    Abstract:

    Cumulative cultural evolution is when behavior in subsequent generations of learners builds on the accumulated information of previous generations to such an extent that no individual learner can produce the behavior on its own. Many examples exist in humans, but in nonhuman animals there are only a handful of suspected cases. Here, we provide the first demonstration of cumulative cultural evolution in the laboratory in nonhuman animals. We raised zebra finches in complete acoustic and social isolation to create "uncultured" animals. Isolate zebra finches sing unstructured songs that are different from wild-type songs in many aspects such as spectral details of syllables and syntactic organization. We developed an automated procedure to quantify the differences between isolate and wild-type song at different timescales of song structure: spectral features, duration of sounds, and song rhythm. We then used the isolate birds to teach their songs to juveniles who became the tutors for the next generations of learners and so on recursively. We followed the evolution of isolate song over multiple generations. We found that isolate song was gradually transforming into wild-type song over 3-4 learning generations. In addition to this experiment where we trained young birds in individual tutor-pupil pairs, we established a semi-natural colony with an isolate founder and tracked song changes over multiple generations of learners. In the colony, the song also progressed towards wild-type song in a few generations, but some of the details of the changes differed between the two conditions. The rapid evolution indicates that wild-type song culture is encoded in every bird, but it takes multiple generations to surface. The young birds used imitation biases to change isolate song features into wild-type features.

  • C. elegans ADAMTS ADT-2 regulates body size and cuticle collagen organization

    Author:
    Thilini Fernando
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Cathy Savage-Dunn
    Abstract:

    The regulation of body size is a fundamental feature of animals critical to their survival and fitness, yet its underlying mechanisms remain poorly understood. In C. elegans, the DBL-1 signaling pathway plays a major role in growth control. The mechanisms by which other pathways regulate body size function, however, are less well understood. To identify additional genes involved in body size regulation, a genetic screen for small mutants was previously performed. One of the genes identified in that screen was sma 21. I now demonstrate that sma 21 encodes ADT-2, a member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases. ADAMTS proteins are believed to remodel the extracellular matrix (ECM) and may modulate the activity of extracellular signals. Genetic interactions suggest that ADT-2 acts in parallel with known size regulatory pathways. I further demonstrate that ADT-2 activity is required for normal cuticle collagen fibril organization and adt-2 regulatory sequences drive expression in glial-like cells. ADT-2::GFP fusion protein is localized in the alae and the annuli of the cuticle. We therefore show that ADT-2 is secreted into the cuticle where it may act to proteolytically process secreted collagen or other ECM molecules required for normal cuticle structure and body size.

  • The effects of chronic habitat degradation on the physiology and metal accumulation of eastern oysters (Crassostrea virginica) in the Hudson Raritan Estuary

    Author:
    Allison Fitzgerald
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    William Wallace
    Abstract:

    The Hudson Raritan Estuary (HRE) was once home an abundant population of the eastern oyster, Crassostrea virginica. Years of habitat degradation, via removal of habitat and shell substrate, overfishing of the population, and inputs of organic and inorganic contaminants, all led to the decline of this previous keystone species. The HRE today is a highly eutrophic environment, with increased sediment inputs, periods of low dissolved oxygen, algal blooms, and hotspots of contaminants throughout. The current study was designed to understand how a chronically degraded habitat, as is present in the HRE now, affects both juvenile and adult oyster physiology. There are three parts to this study: in the first, a large scale field transplant study was deployed to determine how juvenile oyster health and subcellular physiology are altered over a continuum of sites across the HRE, and if subcellular metal accumulation related to alterations in physiology. Using eight sites across the HRE, it was apparent that there are many site-specific factors that affect oyster physiology, and the synergistic effects of these abiotic and biotic factors together influence oyster physiology the most. There was no one factor that could be isolated as a key parameter to determine future oyster restoration. The second part used a field transplant study to examine the role of a degraded habitat on adult oysters and reproduction. Using Vitellogenin protein and energy expenditures to estimate oyster reproduction, it was seen that adult oysters respond much slower than juveniles and no differences were seen between highly degraded habitats and less degraded habitats. Thirdly, both juveniles and adults were observed to accumulate non-essential metals (Cd and Hg) in the field. In order to determine if metal accumulation is the sole cause of physiological alterations, a laboratory exposure was designed to determine if changes in subcellular physiology could be correlated specifically to subcellular accumulation of Cd or Hg, when no other abiotic factors are able to influence oyster health. It was observed that Cd accumulation lead to physiological changes, but Hg accumulation did not. Using this information about the site-specific nature of oyster physiology and how metal accumulation can alter physiology will allow researchers to choose future restoration sites and set up projects that will allow for maximum growth and survival.