Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • 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.

  • A MINUS-GAMETE-SPECIFIC GENE IN FUSION-DEFECTIVE CHLAMYDOMONAS MUTANTS AND ANALYSIS OF BIOSYNTHETIC PATHWAYS UPREGULATED DURING GAMETOGENESIS

    Author:
    Dmitry Brogun
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Charlene Forest
    Abstract:

    To gain insight into the mechanism of gamete fusion during fertilization, it is crucial to identify molecular, metabolic and genetic factors required for this process. Gamete fusion in C. reinhardtii cells proceeds via four genetically defined stages: 1) flagella recognition 2) signaling 3) mating structure adhesion and 4) fusion with subsequent zygote formation. During this study we used insertional and temperature sensitive conditional mutants that do not proceed to stage 4, but can agglutinate and adhere to each other via their mating structure. A homolog of the sex-restricted HAP2/GCS1 gene has been shown to prevent gamete fusion in C. reinhardtii. A SiteFinding-PCR search was conducted on the fusion-defective Chlamydomonas insertional mutants that could not be complemented with the wild type copy of the HAP2/GCS1 gene. We confirmed that mutant clone 5 had an insert in a copia-family retrotransposon on chromosome 13. We collaborated to discover that a gene, located 3 &rsqou; to the retrotransposon is a minus-gamete specific gene (MGS). We hypothesized that MGS might be a second gene required for gamete fusion. Our main objective was to identify whether there is a defect in the DNA sequence in MGS in any of our fusion-defective mutants. We performed a chromosome walk on coding, promoter and 5 &rsqou; upstream and 3 &rsqou; downstream regulatory regions (UTR) of MGS via PCR. PCR products then where sequenced and aligned. We used qRT-PCR to determine MGS expression levels in the control and fusion defective mutants. Analysis of the sequencing and expressional results showed no defect in the MGS gene. For the systematics study we used comparative genomic and phylogenetic approaches enabling us to study metabolic pathways that are upregulated in gametes of Chlamydomonas. Congruent experimental results show that the nuclear-encoded and chloroplast localized MEP pathway leading to the biosynthesis of the isoprenoid precursor molecules is upregulated in Chlamydomonas gametes. It is expected, that the results from these studies will provide further insights into regulatory mechanisms occurring during gametogenesis, some of which might be necessary for gamete fusion in algae as well as in higher eukaryotic organisms.

  • Vascular Endothelia Growth Factor and its Receptor VEGFR2 Regulate Synaptic Protein Levels in Rat Hippocampal Neurons

    Author:
    Qin Cao
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Patricia Rockwell
    Abstract:

    Vascular endothelial growth factor (VEGF) is a well-established angiogenic factor which also elicits protective and stimulatory effects on neuronal function. Recent studies suggest that VEGF signaling plays a critical role in modulating synaptic plasticity and enhances excitatory synaptic transmission in the hippocampus. Other growth factors including brain-derived neurotrophic factor (BDNF) and insulin have been shown to regulate synaptic ¬¬¬protein levels to stimulate neural communication but it remains unclear how VEGF participates in synapse function at the molecular level. The notion that VEGF would also modulate synaptic protein levels in differentiated hippocampal neurons has not been explored. Therefore, this work addressed whether VEGF exhibits neurotropic properties in mature rat hippocampal neurons by modulating the postsynaptic protein PSD-95 and protecting against the stress induced by nutritional deprivation. The results show that VEGF signals an increase in cell viability and increases the levels of presynaptic (synaptophysin and synapsin I) and postsynaptic (PSD-95) proteins through its cognate receptor VEGFR2. VEGF signals these events via autocrine and paracrine mechanisms. Moreover, VEGF regulates PSD-95 protein levels and synapse numbers along dendrites through the PI3K/Akt/mTOR pathway. Additional studies showed that inhibition of the Rho-associated protein kinase (ROCK) increased PSD-95 protein levels which were attenuated by VEGFR2 inhibition. Furthermore, ROCK inhibition enhanced VEGF-mediated synapse formation, survival and neurite extension. Accordingly, these findings suggest that ROCK serves as a negative regulator of VEGF signaling in mature primary hippocampal neurons. Collectively, this study revealed a novel signaling mechanism for VEGF/VEGFR-2 pathway that may function in its reported capacity to stimulate synaptic transmission. These findings implicate VEGF signaling as a potential therapeutic strategy to prevent or hinder synaptic loss in neurodegenerative disorders.

  • Structure and Function in Bacteriophage Phi6

    Author:
    James Carpino
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    John Dennehy
    Abstract:

    The present study of bacteriophage Phi6 has been preceded by a great number of exploratory studies of its structure and function, and these studies have formed a basis for Phi6's development into a model organism. In this study, two aspects of the model organism have been examined. 1. There are several uncharacterized and presumed untranslated regions (UTRs) in Phi6's 13.3 kilobase-pair dsRNA genome. I examined the impact of specific modification to the 3' UTR of the small segment of bacteriophage Phi6. I determined that modification to the purported UTR of the small segment resulted in severe fitness costs, supporting a functional role for unidentified gene products, secondary RNA structure, or both. 2. Bacteriophage Phi6 packages its dsRNA genomic segments selectively and sequentially through the function of the packaging motor P4 which occupies fivefold vertices of the Phi6 procapsid, and studies support the functioning of one and only one P4 during packaging. The mechanism of this specific phenomenon is not known. I used computational reconstruction of cryoelectron microscopy and examined the occupancy of P4 on the Phi6 procapsid, and acquired insight into the mechanism of assembly and packaging.

  • A Study of the Progenitor Potential and Function of Thymic Nurse Cells Using pH91 a TNC- Specific Monoclonal Antibody

    Author:
    Rajendra Chilukuri
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Jerry Guyden
    Abstract:

    Thymic nurse cells (TNCs) are lympho-epithelial complexes that are a major component of the cortical thymic microenvironment. The functional role of TNCs in the thymus has been controversial but recent studies are beginning to elucidate the role of these cells in thymic homeostasis. In the present study, we have described the function of TNCs during the process of thymocyte selection and present results suggestive of the progenitor potential of TNCs. Using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal microscopic analyses, we show that TNCs create an intimate association with thymocytes. Thymocytes become trapped within unique extra-cytoplasmic spaces generated by the TNCs. The membrane-derived honeycomb-like fenestrae allow visualization of trapped thymocyte movement into and out of these fenestrae, a process that facilitates interactions between the two cell types. Further, we have data confirming an interaction between the abTCR expressed on trapped thymocytes and MHC class II antigen expressed on the surfaces of the TNCs. We also observe lipid-raft accumulation around the contact point between the thymocytes and TNCs. When we costained freshly isolated thymic nurse cells with TNC-specific monoclonal antibody pH91 and with K5 and K8 cytokeratin antibodies, we observed a subset of TNC that were K5+K8+ and pH91+. Previous studies have suggested that k5+k8+ thymic TECs were thymic epithelial progenitor cells. The studies presented here show that TNCs express the transcription factors Foxn1 and p63 both of which play critical role in the thymic determination as well as maintaining a proliferative subpopulation of TECs. Interestingly, when we co-stained embryonic day 11.5 (E11.5) thymic sections with pH91 and Foxn1 antibodies, their expressions were detected in this phase of early thymic organogenesis. The expression of p63 was detected a day later at E12.5. Also, we have results confirming the expression of pH91 antigen as early as E7.5 along with a stem cell marker Oct4. Finally using confocal analysis and TNC specific mAb, pH91, we show that the classical complex morphology of TNCs first appears at E17.5 stage of development. However, analyses of major histocompatibility complex (MHC) class II expression on embryonic TNCs cell surfaces show its onset from E13.5. The results show a marked increase in the expression of MHC class II, from 36.2% at E13.5 to 69.1% at E18. 5 stage of development. Taken together, these data suggest that thymic nurse cells play a significant role in the murine thymus; they create membranous spaces that facilitate MHC restriction and express markers implicating them as possessing progenitor ability.