Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • Systematics and Phylogeny of Arcoid Bivalves (Arcoida: Pteriomorphia: Bivalvia)

    Author:
    Louise Crowley
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Ward Wheeler
    Abstract:

    The Arcoida is a large group of mostly marine bivalves, with a global distribution. Familiar taxa in this group include the arks, bittersweets and dog cockles. Relationships among the higher-level taxa of the Arcoida are not well understood and the classification of this group has been the subject of debate and rearrangement. While many views exist as to the evolution of this group, none of them are based explicitly on a phylogenetic analysis. In this study, the phylogenetic relationship of the Arcoida is inferred from a systematic analysis based on both morphological and molecular data. This is the first analysis in which representatives of all seven nominal families are included. 141 morphological characters from the external shell and internal anatomy were coded for 131 taxa. The phylogenetic signal of both these character types was explored. Few non-homoplastic synapomorphies for the group were recovered; shell tubules are confirmed as the sole non-homoplastic synapomorphy for the order. Shell characters failed to recover the majority of the higher taxonomic ranks that they were initially used to describe. Little coherent signal was received from the analysis of anatomy alone. Four molecular markers, the nuclear 18S rRNA, 28S rRNA, protein coding histone H3 and mitochondrial cytochrome c oxidase I, were also investigated using direct optimization as implemented in POY (Varón et al., 2008). These data were analyzed individually as well as simultaneously with the morphological data. A Sensitivity Analysis (Wheeler, 1995) of the molecular data was also performed--this explores the effects of parameter costs (i.e. indels and transition/transversion ratios) on the phylogenetic results. The results of these phylogenetic analyses do not reflect the current classification of the group. In this study, the majority of the higher taxonomic groups of Newell (1969) were not recovered, including the two superfamilies Arcoidea and Limopsoidea, as well as five of the families; only the monophyly of the Glycymerididae and Noetiidae is supported. A major taxonomic review of the order is necessary. This analysis is the largest and most comprehensive phylogenetic analysis of the Arcoida to date.

  • Molecular genetic studies of sulfur nutrient response in Arabidopsis thaliana

    Author:
    Hanbin Dan
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Zhi-Liang Zheng
    Abstract:

    The aim of this study was to isolate the components that are critical for the S nutrient response in plants. We used Arabidopsis thaliana as a model system. We first developed a S deficiency-responsive promoter:GUS reporter system. We confirmed that At2g44460 encoding a putative thioglucosidase exhibits the strongest induction by S deficiency. Interestingly, At2g44460 induction by S deficiency was suppressed by the application of auxin, a plant hormone. Together with other physiological and genetic evidence, we showed that auxin plays a negative regulatory role in S deficiency response. Furthermore, we found that S deficiency-induced expression of At2g44460 and a sulfate transporter gene (SULTR4;2) is dependent on the availability of C and N, which exhibit a synergistic interaction. Therefore, we designed a genetic screen by using the At2g44460 promoter:GUS reporter line (designated GHF1) with an aim of isolating the mutants that alter the expression of At2g44460 in response to C,N and S status. Screen of the mutants resulted in the isolation of two allelic mutations on the SEL1 gene, which encodes a high-affinity transporter called SULTR1;2. SULTR1;2 is mainly responsible for transporting sulfate from the soil into the root. The two alleles, designated sel1-15 and sel1-16, have distinct missense mutations on the putative transmembrane domains, but they did not seem to cause mislocalization of the protein. As expected, these two mutations, like a SULTR1;2 null allele (sel1-10), abolish the sulfate uptake in both yeast and plant systems. They also reduced the accumulation of internal sulfate. However, a dose response study indicates that expression of the S deficiency-upregulated genes, At2g44460, SULTR4;2, LSU1 and SDI1, is higher in the mutants than that in WT under either the high sulfate treatment or under different sulfate treatments that result in similar levels of internal sulfate. Furthermore, these mutants reduced the sensitivity to external application of the high concentration of sulfate metabolites, suflite, Cys and GSH. Taken together, these results indicate that besides the sulfate transport function, SULTR1;2 likely acts as a sensor for S nutrient, adding this transporter to the growing list of nutrient transceptors.

  • Q'eqchi' Maya Reproductive Ethnomedicine, Estrogenic Plant Use, and Women's Healing Traditions in Belize

    Author:
    Jillian De Gezelle
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Michael Balick
    Abstract:

    The Q'eqchi' Maya of Belize have an extensive ethnopharmacopoeia of medicinal plants used traditionally for reproductive health and fertility. Ethnobotanical research was carried out in the Q'eqchi' communities of the Toledo District of Southern Belize from 2007-2011 on medicinal plant species used for reproductive health. Data was gathered primarily through semi-structured interviews and plant collecting trips with 6 traditional healers, 3 midwives, and 12 female herbalists. The Belizean Q'eqchi' are utilizing more than 60 plant species for reproductive health treatments, with the most species from the family Piperaceae. Ten species were selected for investigation of their estrogenic activity using a reporter gene assay: Clidemia crenulata Gleason, Drymonia serrulata Jacq. (Mart.), Gouania lupuloides (L.) Urb., Miconia oinochrophylla Donn. Sm., Mimosa pudica L., Piper jacquemontianum Kunth, Piper peltatum L., Psychotria acuminata Benth., Psychotria poeppigiana Müll. Arg., and Tococa guianensis Aubl. These plants are used to treat female infertility, male infertility, menopausal symptoms, heavy menstruation, uterine fibroids, k'uub'sa' (a Q'eqchi' womb disorder), for miscarriage prevention, for use as female contraception, and for male contraception. Methanol extracts of the leaves of all species were assayed, as well as the stems of G. lupuloides, roots of M. pudica, and the roots of P. peltatum. All the extracts displayed estrogenic activity, except for M. pudica roots and P. jacquemontianum leaves, which were both cytotoxic to the MCF-7 breast cancer cell line. Nine of the species assayed were estrogenic, four of the species were also antiestrogenic, and two of the extracts were cytotoxic to the MCF-7 cell line. Women's healing traditions are being lost in the Q'eqchi' communities of Belize at an accelerated rate, due to a combination of factors including: migration from Guatemala disrupting traditional familial lines of knowledge transmission; perceived disapproval by local biomedical authorities; women's limited mobility due to domestic obligations; and lack of confidence stemming from the devaluation of women's traditional knowledge. Medicinal plant knowledge is highly gendered with women and men commonly using different species in reproductive health treatments. Revitalizing women's healing practices is vital for maintaining the traditional knowledge needed to provide comprehensive healthcare for Belize's most remote indigenous communities.

  • COMPARATIVE PHYLOGEOGRAPHY, PHYLOGENETICS, AND POPULATION GENOMICS OF EAST AFRICAN MONTANE SMALL MAMMALS

    Author:
    Terrence Demos
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Michael Hickerson
    Abstract:

    The Eastern Afromontane region of Africa is characterized by striking levels of endemism and species richness which rank it as a global biodiversity hotspot for diverse plants and animals including mammals, but has been poorly sampled and little studied to date. Using mtDNA and multi-locus nDNA sequence data, genome-wide RAD-Seq SNP data, and morphological data, I identify major cryptic biogeographic patterns within and between 11 co-distributed small mammal species/species groups across the Eastern Afromontane region. I focus on two endemic montane small mammal species complexes, Hylomyscus mice and Sylvisorex shrews, co-distributed across the Albertine Rift (AR) and Kenya Highlands (KH) of the Eastern Afromontane Biodiversity Hotspot. I characterize patterns of phylogeographic structure, demographic history, phylogenetic relationships and undescribed biodiversity across these taxa. Putative independently evolving lineages are inferred using a combination of distribution data, coalescent species delimitation and historical demographic inference. Hypotheses put forward to account for the high diversity of the region include both retention of older palaeo-endemic lineages across major regions in climatically stable refugia, as well as the accumulation of lineages associated with more recent differentiation between allopatric populations separated by unsuitable habitat at the LGM. Populations have persisted since the Pliocene to mid-Pleistocene across a climatic gradient from the AR in the west to the KH in the east for both focal taxa. Deeply divergent and sympatric cryptic lineages, previously unidentified, are strongly supported in both mice and shrews, highlighting the broad temporal scale at which cyclical climatic changes over the last 5 Ma may have contributed to high species diversity and endemism in the Eastern Afromontane Hotspot. Complete genome-wide SNP matrices for Hylomyscus and Sylvisorex are used in population genetic analyses that support lineages not uncovered by the 3-6 locus dataset. Graphs of population splits and admixture support substantial gene flow from AR into KH shrew populations subsequent to isolation that occurred 2.5-3.5 million years earlier, possibly by intermittent colonization. A new species, Hylomyscus kerbispeterhansi, is described from Kenya using combined morphological and multi-locus data sets.

  • 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.