Alumni Dissertations

 

Alumni Dissertations

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  • Glutamate Receptor Signaling is a Mediator of Neurite Outgrowth Inhibition

    Author:
    Sarit Derey
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Marie Filbin
    Abstract:

    Unlike the peripheral nervous system (PNS) or embryonic neurons, the adult mammalian central nervous system (CNS) does not spontaneously regenerate after injury. This is due, in part, to the presence of myelin-associated inhibitors, such as myelin-associated glycoprotein (MAG). Our lab has shown that elevation of intracellular cAMP blocks these inhibitors in vitro and in vivo in a transcription-dependent manner. Subsequent microarray analysis revealed that elevation of cAMP results in upregulation of Arginase I (Arg1), a key enzyme in the synthesis of polyamines. Our lab has demonstrated that administration of polyamines is sufficient to block MAG and myelin-induced inhibition of axonal outgrowth in vitro as well as enhance CNS axon regeneration in vivo. Others have shown that binding of polyamines to ionotropic glutamate receptors (iGluRs), which include N-methyl-D-aspartate receptors (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors (KAR), blocks their activity. In addition, it is well established that iGluR activity is disrupted following CNS injury. Thus, since polyamines overcome MAG/myelin-mediated inhibition and given that polyamines block iGluRs activity, we set out to examine the role iGluR-mediated signaling plays in the MAG and myelin inhibitory pathways. We found that blocking NMDAR or AMPA/KA receptor activity with pharmacological antagonists was sufficient to block MAG-induced inhibition of neurite outgrowth in dorsal root ganglia (DRG) and hippocampal neurons (HNs). Likewise, exposure to iGluR agonists increased MAG-mediated inhibition of neurite outgrowth. To determine whether prior exposure of iGluR inhibitors or priming is sufficient to overcome MAG inhibition as in the case of polyamines, HNs were treated with iGluR antagonists 18 hours before exposing neurons to MAG. We found that priming with iGluR antagonists was not sufficient to overcome MAG-mediated inhibition, suggesting that GluR antagonists exert their effect by intercepting MAG inhibition signals, while not changing the ability of neurons to respond to MAG. Because polyamines require priming whereas iGluR antagonists have no effect when used to prime neurons, we therefore conclude that in our model system, polyamines do not overcome inhibition by blocking iGluR activity. It is well established that upon activation, NMDARs produce influx of Ca2+ inside the cell, which activates Ca2+-dependent kinases such as extracellular signal-regulated kinase (ERK) and conventional protein kinase C (PKC), both of which are important for synaptic plasticity, long term potentiation (LTP), and long term depression (LTD). Importantly, conventional PKC and epidermal growth factor receptor (EGFR)-regulated ERKs are also known to be activated in response to myelin inhibitors in a Ca2+-dependent manner. Blocking PKCs or EGFRs is sufficient to overcome inhibition by myelin inhibitors in vitro and promote axonal regeneration in vivo. To establish which downstream target activation is affected in response to iGluR activity block as well as myelin, PKC and ERK signaling pathways were examined. We found that myelin induces robust ERK activation, and importantly, inhibiting iGluR activity was sufficient to block ERK activation. Next, to determine whether myelin-induced ERK activation is inhibitory for neurite outgrowth, HNs were treated with the ERK inhibitor U0126 and then subjected neurite outgrowth assay in the presence of MAG. We found that blocking ERK was sufficient to overcome MAG-mediated inhibition in a dose dependent manner. Our findings demonstrate that exposure to myelin induces ERK activation and that ERK activation is regulated by iGluR activity. Since ERK is one of the main downstream targets activated by NMDAR-induced Ca2+ influx, our findings support the idea that exposure to myelin results in iGluR activation. In synapses, repeated activation of NMDAR leads to long-term potentiation (LTP) through mechanisms involving the activation of PKC and ERK as well as rapid forward trafficking of NMDARs to the membrane. This is consistent with reports that PKC induces phosphorylation in the C-terminal of the NMDA NR1 subunit, leading to increased surface insertion of NMDAR channels. To determine whether myelin-associated inhibitors affect NMDAR trafficking, cortical neurons were treated with myelin and then the amounts of surface NMDARs was assessed. We found that myelin induced a rapid increase in surface NMDAR number. In addition, myelin treatment induced phosphorylation of ser890 and ser896 C-terminal NMDA NR1 subunits, which have been shown to promote forward NMDAR trafficking to the cell surface. Thus, our findings suggest that myelin-induced PKC activation promotes surface incorporation of NMDARs, which can potentially enhance NMDAR-mediated currents. Taken together, our results implicate iGluR activity as well as ERK signaling as new mediators of MAG/myelin-induced inhibition, offering new insights into the molecular mechanism of myelin-induced block of neurite outgrowth.

  • 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.