Alumni Dissertations

 

Alumni Dissertations

Filter Dissertations By:

 
 
  • THE ROLE OF Na+/H+ EXCHANGER-1 (NHE1) IN MAMMARY BRANCHING MORPHOGENESIS AND MAINTENANCE OF TISSUE ARCHITECTURE

    Author:
    Edmund Jenkins
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Jimmie Fata
    Abstract:

    Branching morphogenesis in vivo is a highly ordered process that necessitates spatially and temporally choreographed cues by growth factors and hormones, as well as mechanical and signal feedback from the extracellular matrix. Successful completion of this developmental process results in the architectural, and thereby the functional, basis for the lung, collecting ducts of the kidney, salivary, and mammary glands. The quest to understand the basic biological mechanisms underlying this developmental morphogenesis has lead to many seminal findings in the field of epithelial tube generation, as well as provided valuable insight into the pathogenesis of cancer. The primary focus of this thesis was elucidating the role of the Na+/H+ exchanger type 1 (NHE1) in branching morphogenesis of the mouse mammary gland. To accomplish this goal, we used three-dimensional (3D) primary tissue culture of mammary gland pieces (organoids) in a four day organotypic assay of growth factor induced branching morphogenesis. NHE1 is a ubiquitously expressed master regulator of intracellular pH (pHi). We found that blocking the function of this exchanger in the presence of growth factor stimulation led to altered kinase signaling, inhibition of growth factor induced alkalization, sustained proliferation after four days, ectopic expression of keratin 6 (K6), and a dramatic failure to undergo branching morphogenesis. These findings led us to question the role of NHE1 in the maintenance of branched mammary tissue architecture. We, therefore, inhibited NHE1 function on fully branched structures in our assay and found that NHE1 inhibition led to rapid loss (within 24 hours) of branched architecture by a process of branch fusion, with complete loss of the branched morphology after four days. This was not accompanied by cell death or altered proliferation, however, we did record altered intracellular pH (pHi) in the end buds of branched structures that had NHE1 inhibited. NHE1 localization, F-actin organization, and myoepthelial cell location were altered in structures that had undergone a loss of architecture, indicating a loss of tissue organization. Finally, we found that NHE1 inhibition resulted in a decrease in mammary Ecadherin. Having found that NHE1 function is vital for both branching morphogenesis and the maintenance of branched architecture, we considered the role that NHE1 could be playing in the pathology of breast cancer. Both intracellular and extracellular pH is deregulated in cancer. This could be attributed to over activity of NHE1. Additionally, NHE1 is over expressed in many cancers. We used the ER+ breast cancer cell line MCF7 to investigate the therapeutic potential of chemotherapy augmentation by NHE1 inhibition. We found that Cycylophosphamide, a DNA alkylating chemotherapeutic agent known to be more effective in an acidic environment, was roughly 5 times more effective when used along with NHE1 inhibition. These findings indicate that NHE1 is a critical regulator of branching morphogenesis and tissue stability, as well as suggests a potential therapeutic target for the treatment of breast cancer.

  • Origin and development of hematopoietic tumors in sumoylation mutants of Drosophila melanogaster

    Author:
    Marta Kalamarz
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Shubha Govind
    Abstract:

    The larval hematopoietic system of Drosophila melanogaster consists of freely circulating cells, the sessile population, and the hematopoietic organ called the lymph gland. Most hemocytes function as macrophages and play a significant role in innate immunity. Hemocytes also remodel tissues, aiding in development of the organism. Constitutive activation of immune signaling pathways, as well as various mutations in genes which are not linked to immune pathways, result in the overabundance of circulating hemocytes and tumor formation. Many aspects of tumor development, such as the identity of the affected hematopoietic population and mechanisms of tumor growth are either not characterized or not well understood. The overall goal of this project was to understand the origin and development of hematopoietic tumor formation in Ubc9 mutants. Ubc9 is an E2 enzyme, which conjugates SUMO (small ubiquitin-like modifier) to a range of target proteins. Sumoylation targets vary in function from structural components to enzymes and transcription factors. Thus, sumoylation affects multiple cellular functions via modification of protein localization, stability or activity. Loss-of-function Ubc9 mutants of Drosophila exhibit severe defects in hematopoietic and immune tissues, including hemocyte overproliferation and tumor formation during larval stages. In this dissertation, we report that the hemolymph of Ubc9 mutants contains hematopoietic cells and structures that range from aggregates (composed of only few cells) to small and large tumors. The largest tumors are less than 1 mm3 in volume. Most cells and aggregates exhibit high ration of mitotic cells, but only a few of the largest tumors in the hemolymph contain actively dividing cells. Based on staining patterns and genetic rescue experiments, we propose that the large tumors are derived specifically from overgrown posterior lobes of the hematopoietic organ. The origin of smaller tumors and aggregates is less clear. Our data suggest that these structures likely derive from circulating hemocytes and fragments of dispersed anterior lobes. Microtumors in Ubc9 mutants arise from the highly-mitotic mutant stem/progenitor cells of the lymph gland. Loss of sumoylation cascade enzymes E1 (Aos1/Uba2), E2 (Ubc9), or E3 (PIAS) leads to loss of proliferative quiescence in hematopoietic precursors localized within the lymph gland. Proliferative quiescence of these precursors is at least in part mediated by the activity of the cyclin-dependent kinase inhibitor Dacapo/p21. Expression of Dacapo homolog, human p21, in the Ubc9 progenitor cells relieves tumor formation. These studies suggest that sumoylation provides a cell-intrinsic mechanism to preserve stem/progenitor cell states for stress response, immunity and development of the fly.

  • Effects of endophyte infection, environmental stress and competition on Lolium perenne populations from the Mediterranean region

    Author:
    Kristin Kane
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Gregory Cheplick
    Abstract:

    No

  • The role of ubiquitin-mediated proteolysis in Drosophila glia development

    Author:
    Margarita Kaplow
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Tadmiri Venkatesh
    Abstract:

    Biological processes are dynamic, requiring both simple and complex mechanisms that enable cells to adapt with the ever-changing environment. Ubiquitination is one of many posttranslational modifications that result in a change in cellular activity. Mono-ubiquitination, the addition of a single ubiquitin moeity leads to endocytosis and membrane trafficking, while the addition of a multiple ubiquitin chains primarily results in protein degradation. Rap/Fzr acts as an activator of the E3 ubiquitin ligase, Anaphase Promoting Complex (APC) which has been studied for its role in the timely degradation of cell cycle regulators. My thesis work focuses on novel roles Rap/Fzr during nervous system development and specifically investigates its role during glia development. My results show that Rap/Fzr regulates glia development through its interaction with Loco, an RGS protein and Nonstop, a ubiquitin specific protease. Bioinformatic analysis revealed that both Loco and Nonstop contain Destruction box (D-box) motifs and KEN box motifs, which are amino acid sequences used by Rap/Fzr for substrate recognition. My thesis work shows that Rap/Fzr targets Loco for ubiquitination, and subsequent degradation and thus, inhibits the formation of glia from dividing neuroblasts. Furthermore, Rap/Fzr together with Nonstop, regulates the migration and the endoreplication of glia cells.

  • Effects of bisphenol-A on oxidative stress, mitochondrial dysfunction and behavior: lymphoblasts and Drosophila melanogaster studies - Potential implications in autism

    Author:
    Kulbir Kaur
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Abha Chauhan
    Abstract:

    Autism is a behaviorally defined neurodevelopmental disorder characterized by impairments in three main areas of social interaction, communication, and repetitive, restricted interests and behaviors. There has been an increase in the prevalence of autism with recent estimation of 1 in every 50 children diagnosed with autism. Though there is no single identifiable cause for autism, several studies have shown an increase in oxidative stress and decrease in antioxidants in autism. The role of environmental factors has also been implicated in autism. Bisphenol A (BPA) is a widely used chemical in the manufacturing of plastics, and its exposure has raised concerns in a variety of conditions. The present study with lymphoblastoid cells and Drosophila melanogaster identifies BPA as an environmental risk factor for the increased oxidative stress, mitochondrial dysfunction and behavioral impairments in lymphoblasts and Drosophila melanogaster. When lymphoblastoid cells were exposed to BPA, there was an increase in lipid peroxidation and free radicals (reactive oxygen species) and decrease in mitochondrial membrane potential generation suggesting BPA induced oxidative stress and mitochondrial dysfunction. The study also illustrates an increase in the mitochondrial DNA (mtDNA) copy number in the lymphoblasts in response to the BPA exposure. In neurodevelopmental disorders such as autism, behavior is an important component of the condition. We therefore attempted to detect behavioral modifications in Drosophila melanogaster following exposure to BPA. In this study, we used an open field assay to help identify disturbances in locomotion along with repetitive behavior in BPA-exposed flies. We also observed an abnormal social interaction between the BPA-exposed flies in a social setting. Along with the behavioral modifications, there was also an increase in the lipid peroxidation in the brains of the BPA-exposed flies. Furthermore there was also a delay in the development of the Drosophila embryos, although we did not detect any gross morphological changes in the peripheral nervous system of the embryos following BPA exposure. We have therefore demonstrated that Drosophila may be used as an animal model for complex neurodevelopmetal disorders, which have a poorly understood etiology.

  • Effects of The Enzyme Inhibitor Prohexadione-calcium on Hops Determined by LC-TOF-MS

    Author:
    Adam Kavalier
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Edward Kennelly
    Abstract:

    Humulus lupulus L. (hops) is an agricultural crop valued for its inflorescences, commonly known as hop cones, which produce a diverse collection of secondary metabolites. Hop cones are most valued for their terpenophenolic contents, which are essential to beer production, and the subject of biomedical research. We studied two hop cultivars, Willamette and Zeus, over five stages of development, which were characterized by detailed flower morphology, gross cone measurements, and phytochemical quantitation. By combining morphological observations with phytochemical quantitation we produced an index to inform our developmentally dependent experiments. In order to understand these developmental processes and in an attempt to induce agronomically positive effects, we perturbed hop morphological and phytochemical development using enzyme inhibition. Prohexadione-calcium (Pro-Ca) is a known inhibitor of 2-oxoglutaric acid dependent dioxygenases present in the flavonoid, gibberellic acid, and ethylene biosynthetic pathways. We treated hops with Pro-Ca at each of the five time-points over two seasons; these time-points were later characterized as five developmental stages. Pro-Ca treatment induced significant increases in terpenophenolic content by 9.1-87.3%; however some treatments also induced significant decreases. Increases in cone biomass production by 1.5-19.6% were also measured in response to treatment in both seasons. Induced changes in cone biomass production and terpenophenolic accumulation were most dependent on cultivar and the developmental stage at which plants were treated. In a second series of experiments we conducted a targeted analysis of phenolic acids, flavonoids, and terpenophenolics over 22 days following a single Pro-Ca treatment conducted during early flowering. Terpenophenolics significantly increased following treatment, and coincided with changes in the flavonoid biosynthetic pathway including accumulation of metabolic precursors upstream from flavanone-3-hydroxylase, and decreases in flavonoid products downstream from flavanone-3-hydroxylase. In addition to changes in known compounds, marker analysis revealed the presence of two markers in treated samples not previously reported from hops. One of these markers has been tentatively identified as the antimicrobial compound luteoliflavan. This research provides insight into the relationship between secondary metabolic pathways in hops and indicates targets for future research into perturbation of metabolic pathways to increase medicinal and flavor compounds in hops.

  • The investigation of the antidiabetic Dominican traditional medicinal plants Costus spicatus Sw. and Momordica charantia L.

    Author:
    Amy Keller
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Edward Kennelly
    Abstract:

    Diabetes is a serious disease affecting many people throughout the world, and is expected to increase in the coming decades. Traditional medicine is used in many places around the globe, including the Dominican Republic, for the treatment of diabetes along with modern medicines. Fieldwork conducted in the Dominican community by the Institute of Economic Botany identified Costus species and Momordica charantia L. as being widely used for diabetes treatment, thus pointing to these plants for further investigation. In an in vivo study, Costus spicatus Sw. tea or water were fed ad libitum to a C57BLKS/J mice (KS) db/db mouse model of obesity and type 2 diabetes mellitus (T2DM). The C. spicatus tea did not improve glucose or insulin tolerance, or moderate hyperglycemia or insulin sensitivity. To analyze the hypoglycemic effect of Momordica charantia fruit, initial studies were conducted in vitro. Both an ethanol extract and saponin-rich fraction of fruit, along with the five isolated triterpene saponin compounds 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al, momordicine I, momordicine II, 3-hydroxycucurbita-5,24-dien-19-al-7,23-di-O-β-glucopyranoside, and kuguaglycoside G, were tested to assess their potential stimulation of insulin secretion. The saponin-rich fraction, along with mormordicin II and kuguaglycoside G, were active in the assay, suggesting saponins as the active hypoglycemic compounds in M. charantia. To further characterize the antidiabetic activity of Momordica charantia, a saponin-rich fraction and ethanol extract of the fruit was gavaged daily to C57BL/6 mice fed a high-fat diet. Both treatments lowered fasting glucose and improved glucose tolerance after three weeks. Also, the ethanol-extract treated group had significantly less β-cell mass at the end of the study, pointing to improved β-cell function. The results of this study again suggest saponins in M. charantia as the therapeutic constituents. In conclusion, the studies described aimed to investigate the efficacy of traditional medicine in a rigorous scientific setting and found that although Costus spicatus was not active, Momordica charantia displayed significant antidiabetic activity. Information about safety and efficacy of herbal medicine will continue to be important as these traditional treatments increase in use around the world for health conditions, including diabetes.

  • Postnatal refinement of interareal circuits in ferret visual cortex

    Author:
    Reem Khalil
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Jonathan Levitt
    Abstract:

    Visual cortical areas are presumed to subserve different perceptual functions as a result of their rich network of interareal anatomical circuits. Interareal circuits have been shown to undergo extensive remodeling in the postnatal period. Revealing the timing of when brain circuits mature may help us assign particular neural substrates to particular visual functions. To illuminate perceptual development, we assessed the postnatal anatomical refinement of interareal feedforward and feedback projections in ferret visual cortex. We also described the developmental trajectory of zinc levels in ferret visual cortex, reflecting a subset of glutamatergic interareal feedforward and feedback processes. We find that the period of major reorganization in feedback circuits, feedforward circuits, as well as the dramatic decline in zinc levels in ferret visual cortex occurs in the month following eye opening. In chapter 1, we demonstrated that zinc histochemistry can be reliably used to distinguish visual cortical areas in juvenile visual cortex and further reveals circuit refinement. We show that the postnatal decline in levels of synaptic zinc follows a broadly similar timecourse in multiple areas of ferret visual cortex. In chapter 2, we assessed the developmental refinement of feedback projections between primary visual cortex and extrastriate areas in the juvenile ferret brain. We reveal substantial refinement in the spatial organization of feedback projections arising from multiple visual areas to primary visual cortex of the ferret during the period after eye opening. We find that while certain aspects of feedback circuitry refine with a similar timecourse in all areas, other aspects refine asynchronously. In chapter 3, we investigated the postnatal development of feedforward projections from V1 to different target areas. Before eye opening, at 4 weeks postnatal, synaptic bouton density is very high, and interbouton interval along individual axons is quite short. In all areas examined, both bouton density decreased, and interbouton interval increased substantially from 6 weeks to 8 weeks postnatal. Therefore, feedback and feedforward cortical circuits appear to share a broadly similar developmental trajectory. Our findings are consistent with the notion that visual experience is necessary and crucial in the refinement of these cortical circuits. Furthermore, our findings suggest that at least some aspects of cortical maturation occur largely synchronously in multiple visual areas.

  • Down Regulation of Neuronatin by MicroRNA-151 Overcomes Inhibition of Axonal Growth by Myelin-based Inhibitors

    Author:
    Dawn Kochanek
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Marie Filbin
    Abstract:

    Down Regulation of Neuronatin by microRNA-151 Overcomes Inhibition of Axonal Growth by Myelin-based Inhibitors by Dawn Marie Kochanek Thesis Advisor: Dr. Marie T. Filbin After injury, the axons of the adult central nervous system (CNS) fail to regenerate. This failure is due to the cellular environment and the neuronal response to that environment. One factor for environmentally-mediated axonal inhibition are the proteins that are present in myelin, such as myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein (OMgp). Previously, our lab has shown that elevating the ubiquitous second messenger cyclic-adenosine monophosphate (cAMP) overcomes MAG/myelin inhibition. MicroRNAs (miRNA or miR), are small fragments of RNA that have been shown to bind to target mRNAs and regulate their translation. We hypothesized that miRNAs might be playing a role in the ability of cAMP to overcome MAG/myelin-based axonal growth inhibition. To investigate if miRNAs have a role in the cAMP effect we performed a miRNA microarray with cAMP treated vs. control dorsal root ganglion (DRG) neurons. One miRNA that increased more than two fold with cAMP treatment was miRNA-151. As previously stated, the axons of the mammalian CNS do not regenerate after injury. However, there is one situation in which they have been shown to spontaneously regenerate. DRG axons are bifurcated with one branch extending into the CNS and the other into the peripheral nervous system (PNS). Studies have shown that if a lesion is made to the PNS branch and then subsequently to the CNS branch, the CNS branch will regenerate. This conditioning lesion-induced regeneration has been found to be dependent upon increased levels of cAMP. We next investigated whether like after treatment with cAMP, miR-151 was also increased after a peripheral conditioning lesion. We found similar significant increases in miR-151 levels in DRG neurons following a peripheral conditioning lesion. To determine a functional role for miR-151 in overcoming MAG/myelin-mediated neurite outgrowth inhibition we next performed overexpression and knockdown analyses of miR-151 and then subsequently subjected the neurons to a neurite outgrowth assay. Overexpression of pre-miR-151 in DRG neurons overcame MAG/myelin-mediated neurite outgrowth inhibition and conversely, knockdown of miR-151 with anti-miR-151 in DRG neurons attenuated the ability of db-cAMP to overcome MAG/myelin-mediated inhibition. To investigate the mechanism by which overexpression of miR-151 overcomes MAG/myelin-mediated inhibition we sought to identify miR-151 target mRNAs, using target prediction algorithms. One putative target was Neuronatin, a 9 kD transmembrane proteolipid protein with unknown neuronal function. We next wanted to assess if miR-151 could bind to the 3'UTR of Neuronatin and inhibit its translation. To test this, 293-T cells were co-transfected with miR-151 and a luciferase reporter gene fused to a wildtype or mutated Neuronatin 3'UTR. MiR-151 overexpression decreased the luciferase activity of the wildtype, but not the activity of the mutated Neuronatin 3'UTR, thus validating that Neuronatin is a miR-151 target. Likewise, we found that both treatment of DRG neurons with db-cAMP or overexpressing miR-151 led to a significant decrease in Neuronatin protein levels, while Neuronatin mRNA levels were unaffected. Finally, using siRNA we knocked-down Neuronatin in DRG neurons and then subjected the neurons to a neurite outgrowth assay. Knockdown of Neuronatin led to a significant increase in total neurite length on both MAG-expressing CHO cells and purified myelin. Our findings suggest that the cAMP-induced miR-151 plays an important role in overcoming MAG/myelin-mediated axonal growth inhibition.

  • Phylogeny, taxonomy and morphological evolution in Conostegia (Melastomataceae: Miconieae)

    Author:
    Ricardo Kriebel
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Fabian Michelangeli
    Abstract:

    The genus Conostegia comprises 77 species of shrubs and trees ranging from Central America to northern South America and the Caribbean. They are ecologically important as they provide pollen for native bees and fruits for birds. One of the main questions of this study is if the genus Conostegia is actually monophyletic. I address this question for the first time by gathering genetic data from four chloroplast regions and two nuclear ribosomal regions of DNA. Phylogenetic analyses of these data revealed that Conostegia is not monophyletic and that a group of species in the genera Clidemia and Miconia fall within it. It is noteworthy that species of these genera that fall within Conostegia are for the most part restricted to southern Central America. Morphological studies that include the use of anatomy, morphometrics, as well as different types of microscopy were conducted to attempt and identify potential characters that support a more broadly circumscribed Conostegia. These studies revealed several characters such as structured variation in herkogamy, a stele within the style and presence of mucilage inside the ovary in many species as potential synapomorphies for clades within the Conostegia clade. Not only are some of these characters useful but also novel in the systematics of the family Melastomataceae. Having identified a broader Conostegia clade, a taxonomic revision was conducted, including ample documentation of the morphology of all the species in the clade, descriptions and maps for 77 species. Lastly, taking advantage of the results of the molecular phylogeny as guidance, three main hypotheses were tested using morphometric approaches. The first hypothesis stated that diversification in floral morphology had indeed occurred within the Conostegia clade, all species of which are buzz pollinated. It has been said in the past that groups in which buzz pollination has evolved, tend to conserve their floral morphology and pollinators. That being said, no study has addressed how conserved flowers in these lineages actually are. This question was addressed by quantifying floral morphology over a broad sample of species within the Conostegia clade and found four major floral types. Transitions between floral types and their possible biological significance are discussed. The second hypothesis that was tested was that leaf venation in Conostegia is in fact quantifiable using geometric morphometrics and that such variation has a phylogenetic component. Variation in leaf venation, in particular the position of origin of the main parallel veins that characterize the family, has been historically used to distinguish groups of species. These different types of leaf veins have also been proposed as potential synapomorphies for major clades within the family. Possibly because variation in leaf venation is a continuous character, its use in Melastomataceae systematics has been difficult. A general framework is here proposed and it is shown that geometric morphometrics is an efficient tool for grasping leaf venation. In addition, it is shown that one of the three major clades in Conostegia is in fact different in its leaf venation from the other two clades and that the other two clades are similar to each other. The third and last hypothesis tested was that seeds in Conostegia can be quantified using elliptic Fourier analysis (EFA). The latter is a powerful geometric morphometric technique based on outlines. Traditionally, seeds in the Melastomataceae have been used in the systematics of many groups in a qualitative framework by coding binary or multistate discrete variables. For the first time a continuous framework using EFA was attempted. The results show that EFA can efficiently quantify many seeds in a small amount of time and summarize their variation using multivariate statistics in few axes. Furthermore, the EFA analyses revealed that one of the clades within Conostegia is significantly different than the other two but that the latter two are not different between each other. These results corroborate, in a different structure, the results obtained in the leaf venation analyses. In conclusion, identifying a natural or monophyletic group allowed for a revision of the taxonomy of a noteworthy component of Neotropical forests providing a tool to the general public for their identification. Subsequently, having clarity with respect to the relationships and identity of the species within the clade, biological questions were addressed experimenting with new available tools. This process yielded several discoveries.