Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • Neuromodulatory and cytoprotective roles of zinc in the vertebrate retina

    Author:
    Ivan Anastassov
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Richard Chappell
    Abstract:

    There is increasing evidence that the role of Zn2+ in the central nervous system is more complex and widespread than originally thought. Chelatable Zn2+ is co-localized with glutamate in the terminals of mossy fiber hippocampal and first order retinal neurons. The co-release of Zn2+ with glutamate in a stimulation-dependent manner has been shown in the hippocampus and the distal retina, while the electrophysiological effects of photoreceptor-released Zn2+ suggest a neuromodulatory role at the first visual synapse. This dissertation examines the neuromodulatory and cytoprotective roles of zinc in the vertebrate retina. When endogenous Zn2+ release is chelated in a skate eyecup preparation, the photoreceptor-generated a-wave of the electroretinogram doubles. This treatment also depolarizes horizontal cells and enhances their light response, suggesting that in the absence of Zn2+ , tonic release of glutamate from photoreceptors onto postsynaptic neurons is increased. Live cell imaging demonstrates that Zn2+ is released from photoreceptor terminals following Ca2+ entry through synaptic voltage-gated calcium channels. In isolated photoreceptors from salamander, chelation of extracellular Zn2+ significantly increases Ca2+ entry at the terminal and this effect is abolished when voltage-gated calcium channels are blocked pharmacologically. In the skate, removal of retinal Zn2+ via intraocular injections of chelators severely damages the inner retina. In the absence of Zn2+ , the retina develops classic signs of glutamate excitotoxicity; cell and tissue swelling, pyknosis and spongy appearance of the inner plexiform layer. Similar tissue characteristics are observed with injections of kainate, a well-known and potent excitotoxic agent. Additionally, neurons in the ganglion cell layer become necrotic with either kainate or chelator treatments, suggesting they are particularly sensitive to overactivation of glutamate receptors. Taken together, these experiments show the importance of Zn2+ as a neuromodulatory agent at the first visual synapse, where control of glutamate release affects the transmission of the visual message and provides broad protection of the retina from excitotoxicity. Understanding the role of Zn2+ in the retina may provide novel insights into retinal diseases and contribute to our growing knowledge of zinc's important functions elsewhere in the CNS.

  • MOLECULAR PHYLOGENETICS OF OTOPHYSAN FISHES: AFRICAN ALESTIDS (CHARACIFORMES: ALESTIDAE) AND CITHARINOIDS (CHARACIFORMES: CITHARINOIDEI), AFRO-ASIAN CHEDRINS (CYPRINIFORMES: CHEDRINI), AND NEOTROPICAL LORICARIINS (SILURIFORMES: LORICARIINAE) AS CASE STUDIES

    Author:
    Jairo Arroyave GutiƩrrez
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Scott Schaefer
    Abstract:

    Otophysan fishes (Ostariophysi: Otophysi) are members of a morphologically and ecologically diverse clade of teleosts that includes most freshwater species of fish, and comprises four major lineages classified in the orders Cypriniformes, Characiformes, Siluriformes, and Gymnotiformes, respectively. Partly because of their tremendous diversity, many groups of otophysan fishes remain poorly understood phylogenetically and in a state of taxonomic disarray. This is the case--to a greater or lesser extent--of African characiforms of the suborder Citharinoidei and the family Alestidae, Afro-Asian cypriniforms of the tribe Chedrini, and Neotropical siluriforms of the subfamily Loricariinae. To address the lack of robust, comprehensive, and/or up-to-date phylogenetic hypotheses for the aforementioned groups, this doctoral dissertation investigated their systematics and evolution through phylogenetic analyses of comparative DNA sequence data, including molecular-clock analyses that resulted in the first time-calibrated phylogenies ever proposed for both alestids and citharinoids (and characiforms for that matter). The molecular phylogenies arrived at herein represent the most comprehensive hypotheses of relationships for each of the groups investigated. Although many of the relationships revealed by this study corroborated previous hypotheses based on morphological and/or molecular data, others are newly hypothesized or in conflict. Moreover, the results of this research revealed instances of para- and polyphyly in numerous nominal taxa (e.g., Brycinus [Alestidae], Nannocharax [Distichodontidae], Raiamas [Chedrini], Lamontichthys [Loricariinae]), prompting a reassessment of the taxonomies of the groups investigated. Information on the temporal context of alestid and citharinoid diversification was used to assess biogeographic hypotheses proposed to explain the Gondwanan distribution of characiforms. Likewise, the inferred chronograms shed some critical light on the historical processes that may have influenced diversification and biogeographic patterns in these and other groups of African freshwater fishes.

  • Population Genetics of Canine Heartworm (Dirofilaria immitis)

    Author:
    Diana Belanger
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Robert Rockwell
    Abstract:

    Dirofilaria immitis, canine heartworm, is a filarial nematode that may have genetic features that favor the development of drug resistance, including rapid rates of mutation, large population sizes, and high levels of gene flow. This parasite is currently treated with macrocyclic lactone anthelminthics, and while it has not yet shown evidence for evolving resistance to these chemotherapeutic compounds, resistance has evolved in related filarial nematodes infecting ruminants and humans. Heartworm samples from domestic dogs and coyotes were obtained via donations from veterinarians and researchers across the United States. I isolated and characterized 11 microsatellite loci for canine heartworm. Using the observed distribution of alleles, I determined the amount of genetic variability and quantified the partitioning of genetic variance. In conjunction with microsatellite data, specific mitochondrial (cox1) and Wolbachia (wsp and ftsZ) loci were used to genotype a subset of host taxa. Results indicate a lack of mitochondrial diversity and maximum likelihood trees show no discernable geographic patterning on a continental scale. This is not unexpected in a Wolbachia-infected organism like D. immitis as this bacterium has been shown to purge mitochondrial diversity in numerous model systems. After establishing baseline genetic parameters, a model of population dynamics was created to answer questions about the potential spread of drug resistance alleles. In the absence of selection, gene flow between subpopulations drives the dispersal of drug resistance alleles. Fixation time is directly proportional to selection pressure. When resistance alleles arise in a source population they spread more rapidly than if they arise in a sequestered population.

  • FUNCTIONAL CHARACTERIZATION OF THE PLANT 15-CIS-ZETA-CAROTENE ISOMERASE Z-ISO

    Author:
    Jesus Beltran Zambrano
    Year of Dissertation:
    2015
    Program:
    Biology
    Advisor:
    Eleanore Wurtzel
    Abstract:

    Vitamin A deficiency is a widespread health issue in the tropics. To solve this issue, efforts are underway to increase provitamin A carotenoids such as B-carotene in staple crops which can be achieved by breeding, metabolic engineering or a combination of both approaches. However, rational strategies to improve carotenoid content in crops require sufficient knowledge of pathway regulation. Therefore, to better understand how plants synthesize provitamin A and to guide metabolic engineering strategies in crops such as maize, the functional characterization of the new z-carotene isomerase (Z-ISO) is of significant importance. Z-ISO was recently discovered in maize and Arabidopsis (Chen et al., 2010). This new enzyme is a 15-cis-z-carotene isomerase present in all plants, diatoms and algae. Z-ISO is required in both green and non-green tissues including roots and the seed endosperm which is target for provitamin A biofortification. In this dissertation, to gain a better understanding of the role of Z-ISO in the isomerization of 15-cis-z-carotene, the Z-ISO polypeptide was biochemically characterized using extensive spectroscopy and its function was examined by developing an in vitro enzymatic assay and by an in vivo complementation system in Escherichia coli. Bioinformatic tools modeled Z-ISO as an integral membrane and heme or non-heme iron binding protein. Therefore, using the Z-ISO polypeptide sequence, we selected conserved putative residue ligands for heme and non-heme iron and mutagenized them to Alanine. These Z-ISO mutant versions were tested for enzymatic function using an E. coli complementation system. These experiments showed that from all the conserved histidines present in Z-ISO, two (H150 and H266) as well as one aspartic acid residue (D294), were essential for isomerase activity. The only cysteine (C263) residue present in Z-ISO was not required for activity. These results are in good agreement with the predicted Z-ISO model where the locations for H150 and H266 are consistent with the coordination of a common factor. Maize Z-ISO was also over-expressed and purified as a TEV protease-cleavable, maltose binding protein (MBP) fusion (MBP::Z-ISO). An in vitro assay utilizing substrate containing liposomes was developed to test Z-ISO activity. The conversion of the substrate 9,15,9'-tri-cis-z-carotene into the product 9,9'-di-cis-z-carotene by Z-ISO proceeded under reducing conditions but not under oxidizing conditions. MBP::Z-ISO purified protein was also tested for the presence of metals. Using inductively coupled plasma optical emission spectrometry (ICP-OES) iron was detected but there were no significant levels of Ca, Cu, Mg, Mn, Mo, or Zn. Therefore, we concluded that Z-ISO is a metalloprotein. E. coli culture pellets expressing MBP::Z-ISO are brown consistent with the presence of iron or heme. The presence of heme in purified MBP::Z-ISO was evaluated using heme staining and pyridine hemochrome assays. Heme staining of protein separated in SDS-PAGE gels revealed that MBP::Z-ISO and Z-ISO contain heme while MBP alone does not. Moreover, hemochrome assays showed the presence of heme b in Z-ISO. The UV-visible absorption spectrum of the intact, as isolated Z-ISO confirmed the presence of heme b in the oxidized, ferric Fe (III) state. Also, the spectrum of reduced Z-ISO is similar to that of cytochromes containing heme b with two axial histidine ligands and the reduced heme b binds carbon monoxide (CO). Z-ISO was also characterized using electron paramagnetic resonance (EPR). An X-band spectrum of the MBP::Z-ISO fusion detected high-spin ferric heme (e.g. heme b with a single histidine ligand), multiple low-spin heme species, and a non-heme iron center. EPR also indicated the presence of two low-spin heme species and that one of these hemes might have a bis-histidine coordination and the other might have a histidine-cysteine axial coordination. Reduced Z-ISO also binds NO which is consistent with the binding of CO. To gain more details on the heme cofactor, the same sample used in the EPR experiments was used to characterize the heme iron in MBP::Z-ISO using magnetic circular dichroism (MCD) which detects only heme iron. MCD characterization showed that Z-ISO has two ligand pairs (His/His and His/Cys), a result that is consistent with the EPR results. MCD experiments also showed a redox-dependent change in ligand (Cys-His) coordination of a low-spin heme b. Our data also suggested the existence of a high-spin, 5-coordinate, His-ligated heme which was detected by EPR and as a minor species by MCD. We hypothesized that substrate could bind to this intermediate or that substrate displaces an axial ligand to coordinate with the heme iron. MCD, EPR, and UV-Vis analysis showed that exogenous ligands bind the Fe(II) state but it was not known whether an exogenous ligand can displace an axial ligand when the heme is in the Fe(III) state. To test this possibility we utilized addition of cyanide (CN-), which binds preferably to the ferric rather than the ferrous heme. CN- was added to both the as isolated (oxidized) Fe(III) ferric enzyme and to the Fe(II) (dithionite-reduced) ferrous enzyme. UV-Vis absorption spectra indicated CN- binding to the ferric Fe(III) heme which suggest that Z-ISO can also bind exogenous ligands in the oxidized form. Taken together our data suggest that heme is essential for Z-ISO activity. The presence of heme and its requirement for Z-ISO activity are surprising since heme is not often described in isomerization reactions. Based on our results, we propose a mechanism for Z-ISO function.

  • Microspherule Protein Msp58 and Ubiquitin Ligase EDD Form a Stable Complex that Regulates Cell Proliferation

    Author:
    Mario Benavides
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Hualin Zhong
    Abstract:

    A complex molecular network is put into place at specific phases of the cell cycle to prevent unscheduled cell division that could result in malignant cell growth. Emerging evidence shows that still uncharacterized proteins play crucial functions at those cell cycle transition points. Nuclear protein Msp58 and EDD E3 ubiquitin ligase have been implicated in different aspects of cell proliferation and reported to be abnormally expressed in numerous types of cancers. The molecular mechanisms underlying Msp58 and EDD functions, however, are not well understood. The work presented here shows that Msp58 and EDD form a stable protein complex that regulates cell viability and proliferation. Interestingly, knockdown of EDD by RNA interference leads to a significant accumulation of Msp58 protein, which suggests that EDD serves as a negative regulator of Msp58. In addition, our in vivo ubiquitination assays and analyses of various cell lines treated with translational and proteasomal inhibitors demonstrate that Msp58 is regulated post-translationally by the ubiquitin-proteasome pathway. These results imply that EDD ligase activity is involved in this regulatory process. Using flow cytometry analyses and biochemical characterization of Msp58 and/or EDD depleted cells, we show that the Msp58-EDD complex plays important roles in cell cycle progression via the control of cyclin gene expression. In particular, silencing Msp58 and/or EDD alters the protein levels of cyclins B, D and E. Taken together, our data suggest that a set of the biological roles attributed to Msp58 and EDD may be executed in the context of the complex that they form, thereby revealing a novel molecular mechanism for these two proteins to accomplish their functions.

  • The influence of hepatocyte growth factor during phagocytosis by retinal pigment epithelium

    Author:
    Jonathan Blaize
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    William L'Amoreaux
    Abstract:

    Processing of photoreceptor outer segments (OS) by the RPE is critical for maintaining the health of the neural retina. If any portion of OS processing is disrupted, or if the RPE suffer injury, subsequent inhibition of OS processing has deleterious effects. Therefore, it is crucial to understand OS processing in order to maintain visual health.1 Sub-retinal clearance of OS by RPE is facilitated by phagocytosis featuring both RPE-specific and Fc gamma receptor associated signaling cascades.2 Integration of these two pathways renders RPE capable of internalizing both specific and non-specific targets. To accomplish these tasks, there must be specific pathways available to present the cell with the protein machinery necessary for binding, internalizing and processing OS. The discovery that lack of c-Met signaling results in impaired phagocytosis in alveolar and hepatocyte macrophages suggests c-Met's role as modulator of phagocytosis.3 These data also suggest a role for hepatocyte growth factor (HGF), the natural ligand for c-Met activation, in preparing phagocytes for clearance of cellular debris. We propose that HGF activation of c-Met in RPE prepares these cells for phagocytosis by initiating a signaling cascade that includes activation of phosphatidylinositol-3 kinase (PI3K). Subsequent activation of Rac1 by PI3K may regulate phagosome formation.4,5

  • DNA Adducts of 10-decarbamoyl Mitomycin C Activate p53-dependent and p53-independent Cell Death

    Author:
    Ernest Boamah
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Jill Bargonetti
    Abstract:

    Mitomycin C (MC), a natural antibiotic and DNA cross-linking agent, has cytotoxic activity and is known to activate the tumor suppressor p53 protein. 10-decarbamoyl mitomycin C (DMC), a derivative of MC, has increased cytotoxicity compared to MC. Both MC and DMC induce cellular cytotoxicity in cells with wild-type p53, while only DMC shows significant cell death activity in the absence of wild-type p53. We investigated the difference in MC and DMC cytotoxicity by comparing DNA adduct composition and the cellular regulation of molecular targets in human cancer cell lines with or without wild-type p53. Compared to MC, DMC produced substantially more mitosene-1-β mono and 1-β cross-link adducts in DNA and resulted in abnormal nuclear morphology in human cancer cells with or without p53. Significantly, greater poly(ADP-ribose)polymerase (PARP) activity was observed after DMC treatment in both the presence and absence of wild-type p53. Both MC and DMC induced double strand breaks as indicated by gamma-H2AX foci formation irrespective of the p53 status, suggesting that double strand breaks cannot account for DMC's increased cytotoxicity. In cell lines expressing wild-type p53, both MC and DMC signaled for p53 stability and apoptosis induction resulting in cleavage of procaspase-3 and -8. Despite the DMC induced cellular cytotoxicity observed in cell lines lacking wild-type p53, cleavage of procaspase-3 or -8 was not observed in these cells. However, we observed an increase in caspase activity. Caspase-2 activation has been suggested as a pathway for p53-independent cell death in the absence of Chk1. Interestingly, Chk1 was depleted following DMC, but not MC treatment in cells with or without wild-type p53. This Chk1 depletion was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Additionally, gene silencing of Chk1 by siRNA increased the cytotoxicity of MC but not of DMC. DMC treatment also caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity. This suggests that DMC- mediated DNA adducts facilitate signal transduction to a pathway targeting proteins for proteolysis. In conclusion, we have found that DMC generates significantly more mitosene-1-β stereoisomeric DNA adducts than MC and causes rapid down-regulation of multiple cellular targets. These studies suggest increased mitosene-1-β stereoisomeric DNA adducts more effectively signal for a mode of cell death which does not require a functional p53 protein.

  • PREDICTING INTRODUCTIONS AND RANGE EXPANSIONS OF THE MONK PARAKEET WITH ECOLOGICAL NICHE MODELING AND LANDSCAPE GENETICS

    Author:
    Corentin Bohl
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Jason Munshi-South
    Abstract:

    The ability to predict species future geographic distributions is an important challenge in biogeography and conservation biology, with critical implications for pressing environmental issues, including the potential spread of invasive species. This research examines a two-step framework to build accurate predictions of the invasive potential of the monk parakeet (Myiopsitta monachus). This species, native to temperate South-America, has established several stable populations worldwide, and shares many of the typical traits of high-risk invaders. The proposed framework aims to 1) identify areas where the species is likely to thrive, and 2) determine which of these suitable areas the species can likely disperse to. Objective 1 requires identifying the environmental conditions suitable to the species, for which I used ecological niche modeling (Chapter 1 & 2). Objective 2 addresses the ability of the species to conquer new adjacent favorable areas via dispersal, a problem that can be addressed with landscape genetics (Chapter 3). In Chapter 1, I developed a null model approach to evaluate the performance and significance of ecological niche models. The results highlight the importance of accounting for both discrimination and overfitting and correctly estimating significance. In Chapter 2, I tested the effect of different model calibration strategies on transferability (the ability to predict independent data in different geographic regions). I used this information to make predictions about the global invasive potential of the monk parakeet. The best prediction was obtained with native calibration records and complex model settings. This prediction indicates several areas with conditions suitable for monk parakeets, including areas adjacent to existing introduced populations. In Chapter 3, I integrated ecological niche modeling and landscape genetics to make predictions about the landscape features that affect monk parakeet dispersal. I tested these predictions with genetic data from an introduced population in Florida, and assessed their significance with null models. Estimating resistance to dispersal with ecological niche modeling produced results equivalent to evaluating a range of alternative hypotheses with a stepwise regression model. The results indicate that monk parakeet may not be limited by distance and most landscape features and are likely to expand to adjacent suitable areas.

  • CANDIDA ALBICANS ALS5P AMYLOID IN HOST-MICROBE INTERACTIONS: A CEANORHABDITIS ELEGANS STUDY

    Author:
    MICHAEL BOIS
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    PETER LIPKE
    Abstract:

    Candida albicans, a dimorphic fungus and an opportunistic pathogen, possesses a myriad of adherence factors including members of the agglutinin-like sequence (Als) family of mannoproteins. The adhesin Als5p mediates adhesion to many substrates, and is upregulated during commensal interactions, but is downregulated during active C. albicans infections[1]. An amyloid forming core sequence at residues 325-331 has been shown to be important for Als5p function, because a single amino acid substitution at position 326 (V326N) greatly reduces Als5p-mediated adherence[2]. We evaluated the role of Als5p in host-microbe interactions, using Caenorhabditis elegans as a host model and feeding them Saccharomyces cerevisiae expressing Als5p on the surface. Als5p-expressing yeast had increased intestinal accumulation rates when compared to non-expressing S. cerevisiae or yeast expressing the amyloid deficient Als5pV326N, respectively. Surprisingly, this accumulation delayed S. cerevisiae-induced killing of C. elegans. Treatment with the amyloid-inhibiting dye Congo red or repression of Als5p expression abrogated the protective effect of Als5p. Being that reproductive fitness is the most important measure of a pathogen's impact on the host, we looked at oocyte quantity and quality[3]. Als5p had no effect on oocyte quantity or quality. In order to understand why nematodes exposed to Asl5p were able to harbor the cells expressing functional amyloid, we looked into the innate immune system of the nematode. Toll- Like receptors (TLRs) are important mediators of innate immune responses to Candida albicans, and several classes of scavenger receptors have been implicated in recognizing and reacting to a variety of ligands in humans[4]. The C. elegans genome encodes for a single TLR, TOL-1, and scavenger receptor CED-1[4,5]. CED-1 is the orthologue to mammalian scavenger receptor SCARF1 and is required for defense against Cryptococcus neoformans[4,6]. Our studies showed that CED-1 was necessary for prolonged survival in the presence of Als5p, and TOL-1 was required for death in C. elegans fed S. cerevisiae. We have further demonstrated the necessity of CED-1 and TIR-1 in phosphorylation of ERK-2/MPK-2. The SAM and TIR domains of TIR-1 were shown to be necessary in discriminating the presence of functional amyloid, and thus elicit specificity in downstream signaling. Remarkably, the presence of the HEAT/Armadillo domain, alone, was sufficient to increase levels of phosphorylated ERK-2/MPK-2 in nematodes fed any yeast strain in this study. This study is the first to show that expression of amyloid-forming Als5p in S. cerevisiae can: a) attenuate S. cerevisiae pathogenicity in C. elegans; b) move the yeast-host interaction towards hallmarks of commensalism; c) be discriminated against by host pathogen recognition receptors (PRRs) leading to a slower decline in viability; and d) can lead to distinct downstream MAPK responses.

  • The Effects of Thyroid Hormone Insufficiency During Development On Cortical Morphology And The Behavioural Manifestations

    Author:
    Susan Briffa-Mirabella
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Carl Dobkin
    Abstract:

    The central hypothesis of this work is that developmental thyroid insufficiency impacts the development of the rat cerebral cortex by altering cortical volume and the number of cortical neurons. In addition, as these neuroanatomical changes caused by milder forms of developmental hypothyroidism or hypothyroxinemia may have both immediate and long term consequences on certain aspects of behaviour, the investigation sought to determine if the alterations in morphology, including the change in relative cortical volume, and the change in the number of cortical neurons in the rat brain, led to behavioral manifestations. Hypothyroidism was induced by the administration of graded levels of the antithyroid agent propylthiouracil (PTU) to suppress thyroid hormone production. The number of neurons was estimated, using unbiased sampling techniques, to determine whether the cellular composition of cortex was altered following developmental TH insufficiency. To determine if these cortical alterations led to changes in behaviour, a battery of behavioural tests were performed which included maternal retrieval (PND 4), maternal separation anxiety (PND 6), Barnes maze (PND 48 and PND 86), social interaction social approach (PND 48-50), and open field (PND 46). Taken together, the results presented here support the hypothesis that developmental hypothyroidism and hypothyroxinemia induced by chemical thyroid hormone suppression (PTU) cause alterations in the morphology of the cerebral cortex by altering cortical volume and changing the number of cortical neurons in the rat brain. Furthermore these alterations ultimately lead to changes in certain aspects of behaviour. These results have important clinical relevance because several studies suggest that developmental disabilities ranging from mild dyslexia to severe mental retardation can be attributed to alterations in cortical morphology resulting from abnormal cortical development.