Biology Theses and Dissertations
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Item INVESTIGATION OF A NOVEL O-GLCNAC MODIFICATION OF A VACCINIA VIRUS CORE PROTEIN(2024) Zhang, Yunliang; Scull, Margaret; Moss, Bernard; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Vaccinia virus (VACV) is a large, complex, enveloped virus that is the prototypic member of the genus Orthopoxvirus of the Poxviridae family and is well known as the live-virus vaccine that eradicated smallpox. It has a linear, double-stranded DNA genome of approximately 190 kbp that encodes about 200 proteins some of which undergo various post-translational modifications. These modifications are crucial for regulating protein function and influencing the virus behavior within the vertebrate and insect cells. Among these, O-GlcNAcylation is notable for its reversible modulation of protein function, like phosphorylation. Although over 5,000 human proteins have been documented as O-GlcNAcylated, the prevalence and function of this modification in viral proteins remain underexplored.Early studies from the Moss laboratory demonstrated the presence of a 40-kDa protein that contained N-acetylglucosamine in purified virions. The small size of the pronase-digestion product and the absence of other sugars suggested one or few glucosamines. The current study advances this understanding by pinpointing the novel O-linked β-N-acetylglucosamine (O-GlcNAc)-modified protein in VACV infectious particles. Enzymatic labeling of purified virions was performed using the mutant β-1,4-galactosyltransferase (GalT1 (Y289L)) to specifically transfer azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues. Following copper catalyzed azide-alkyne cycloaddition (CuAAC) of biotin or an infrared dye, the candidate O-GlcNAc proteins were detected by SDS-polyacrylamide gel electrophoresis and identified by mass spectrometry (MS). Then using strain-promoted cycloaddition (SPACC) chemistry to attach a polyethylene glycol mass tag of 10 kDa to the O-GlcNAc protein, a significant shift in the electrophoretic mobility of the VACV A4 protein was documented by western blotting. The presence of O-GlcNAc in A4 was confirmed by MS and by binding to specific antibodies. Multiple modification sites were pinpointed using higher-energy collisional dissociation induced electron-transfer dissociation in MS. Further evidence linking cellular protein O-GlcNAc transferase (OGT) to the modification of A4 was derived from experiments conducted with an A4-expressing cell line. Disruption of OGT activity, either through chemical inhibition or knock-down techniques, reduced A4 O-GlcNAc modification without impairing VACV infectivity. This finding suggests that the O-GlcNAc modification of A4 does not play an essential role in VACV infectivity, which is not correlated with the A4 deletion phenotype. Therefore, the specific effects of O-GlcNAc modification on the VACV lifecycle remain elusive, indicating further studies are required to determine the potentially subtle effects of O-GlcNAcylated A4 on the VACV life cycle.Item CHARACTERIZING THE ROLES AND MECHANISMS OF CYTONEMES IN ASYMMETRIC SIGNALING AND ORGANIZATIONS IN THE DROSOPHILA MUSCLE PROGENITOR NICHE.(2024) Patel, Akshay Jitendrakumar; Roy, Sougata; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tissue development and homeostasis rely on the ability of embryonic or stem cells to efficiently determine whether to multiply for self-renewal or differentiate to generate a wide range of cell types that constitute an adult body. Stem cells determine these fates in the context of a specialized microenvironment or the niche that they occupy. All stem cell niches characterized to date are known to function using two key processes - adhesive interactions and asymmetric growth factor signaling between the niche and stem cells. While adhesion to the niche maintains niche occupancy and stemness, the loss of niche adhesion and occupancy initiates stem cell differentiation. Moreover, niche cells produce secreted growth factors to support stem cell self-renewal. Despite the ability of secreted growth factors to disperse across tissues over a long range, only the niche-adhering stem cells receive the self-renewal signals. The genetically identical daughter cells that lack adhesion to the niche fail to receive self-renewal signals, even when located within one or two cell diameters away, leading to the activation of their post-mitotic fates. Therefore, understanding how asymmetric signal distribution and adhesive interactions are produced and coordinated within the niche is critical to understanding how stem cells determine their identity and prime differentiation to generate or regenerate tissues. This thesis investigated and characterized a new mechanism of asymmetric signaling and cell organization in the Drosophila Adult Muscle Progenitor (AMP) niche. By employing genetic, cell-biological, and high-resolution microscopy techniques, this work discovered that AMPs extend thin polarized actin-based filopodia, called cytonemes, by orienting toward the wing disc niche. Cytonemes play a dual role. Cytonemes help AMPs to physically adhere to the wing disc niche and also directly receive a self-renewal Fibroblast Growth Factor (FGF) through the cytoneme-niche contact sites. AMP cytonemes localize the FGF-receptor (FGFR), called Heartless (Htl), and selectively adhere to the wing disc areas that express two different Htl ligands, Pyramus and Thisbe, both mammalian FGF8 homologs. Htl on these cytonemes directly receives Pyramus and Thisbe through the cytoneme-niche contact sites. Although FGFs are long-range secreted paracrine signals and Htl is the only receptor shared by Pyramus and Thisbe, these FGFs are received and restricted only to the niche-adhering AMPs due to the contact-dependent cytoneme-mediated asymmetric delivery of the signals. Moreover, despite employing a common FGF signal transduction pathway, Thisbe- and Pyramus-signaling initiates divergence of AMP fates into two distinct muscle-specific lineages. These experiments showed that cytoneme-mediated signal communication forms the basis of asymmetric signaling and organization within the AMP niche. We next asked how AMPs determine the niche-specific polarity and affinity of cytonemes. This research discovered that FGF reception and signaling activation in AMPs are required to activate polarized cytoneme formation orienting toward the wing disc niche. Without FGF signaling, AMPs cytonemes fail to polarize and adhere to the FGF-producing niche, causing them to exit the niche and start to differentiate. Thus, while target-specific asymmetric FGF distribution relies on cytonemes, activation of FGF signaling feedback maintains the polarity and adhesion of the signaling cytonemes toward the FGF-producing niche. A consequence of this interdependent relationship between niche adhesion, polarized FGF-reception, and stimulation of FGF signaling feedback is the maintenance of the self-organized niche-specific asymmetric signaling and organization via cytonemes. We next investigated whether the niche-adhering cytonemes receive additional fate-specifying cues, particularly the mechanical cues from the niche. Recent evidence suggests a critical role of mechanical and physical cues in determining stem cell fates. This work discovered that the AMP cytonemes are enriched with a common mechano-transducer, named Talin. AMP-specific genetic manipulation of talin indicates that Talin is critical for cytoneme-mediated niche occupancy and FGF signaling. Using a Talin-based force probe expressed at the physiological levels and FLIM-FRET microscopy, we discovered that Talin experiences pN level force within the cytonemes. These findings suggest that AMPs employ cytonemes not only for receiving FGFs in a restricted polarized manner but also for a mechanosensory function. In conclusion, these results strongly suggest a critical role of cytonemes in coordinating asymmetric signaling and organization in the stem cell niche. In addition, the work provides evidence that the stem cell cytonemes are critical organelles for integrating the inputs and outputs of both growth factor signaling and mechanical cues to sculpt tissues.Item ROLES OF PLASMA MEMBRANE WOUNDING AND REPAIR IN B CELL ANTIGEN CAPTURE AND PRESENTATION(2022) van Haaren, Jurriaan Jan Hein; Song, Wenxia W; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The B cell-mediated humoral immune responses play a crucial role in neutralizing pathogens and unwanted foreign substances. B cells become activated upon antigen binding of their B cell receptor (BCR), and then internalize and process antigen for presentation on their MHCII for T cell recognition. acquiring T cell signaling through antigen presentation is essential for B cell differentiation into high-affinity antibody-producing cells and memory B cells. In vivo, Antigen encountered by B cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. When B cells engage surface-associated antigen, BCR signaling induces reorganization of the cytoskeleton, causing spreading and contraction of the B cell on the antigen-presenting surface. This allows the B cell to engage more antigen and gather the antigen into a central cluster for internalization. Internalization of surface-associated antigen has been shown to require myosin-generated forces and the exocytosis of lysosomal enzymes. However, the mechanism that initiates lysosomal exocytosis remains unknown.This research explored a possible mechanism for the triggering of lysosomal exocytosis of B cells during interaction with surface-associated antigen. We showed that BCR interaction with antigen tethered to beads, to planar lipid-bilayers (PLBs) or expressed on the surface of live cells causes permeabilization of the B cell plasma membrane (PM), an event that required strong BCR-antigen affinity, BCR signaling, and activation of non-muscle myosin IIA (NMIIA). Moreover, we showed that B cell PM permeabilization triggers a repair response that includes the exocytosis of lysosomes at the site of antigen interaction. Importantly, we showed that B cells undergoing PM permeabilization and subsequent repair internalize more antigen; and better activate T cells compared to unpermeabilized B cells. Thus, our research reveals a novel mechanism for B cells to capture surface-associated antigen: antigen affinity-dependent binding of the BCR indices localized B cell PM permeabilization and lysosome exocytosis as a repair response, which facilitates antigen internalization and presentation through the extracellular release of lysosomal hydrolases. In addition, we explored the molecular mechanism required for B cell PM permeabilization in response to surface-associated antigen. We showed that B cells that undergo PM permeabilization in response to PLB-associated antigen spread over the PLB at a faster rate and to a larger area in comparison to cells that remain intact. Furthermore, we showed that B cells that undergo PM permeabilization recruit more NMIIA at a faster rate, and display a higher level of NMIIA organization at the immune synapse. We additionally discovered a 2o B cell spreading and NMIIA recruitment event, approximately 25-30 minutes after antigen engagement, that facilitates B cell PM permeabilization. Thus, B cell PM permeabilization requires the engagement of a large amount of antigen through B cell spreading on the presenting surface, as well as strong NMIIA recruitment and organization at the immune synapse. This research suggests that B cell PM permeabilization in response to surface-associated antigen plays an important role in distinguishing B cells with various levels of BCR activation, providing novel insights into the mechanisms responsible for affinity differentiation during B cell activation.Item SEX DIFFERENCES IN THE FOREBRAIN DOPAMINERGIC CIRCUIT(2022) Manion, Matthew Timothy Coon; Glasper, Erica R; Wang, Kuan Hong; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Several psychiatric disorders exhibit different incidence rates in men and women and areassociated with dysfunctions in forebrain dopaminergic circuits. Although anatomical and functional sex differences in the brain have been studied, little is known about sex differences in the forebrain dopaminergic circuits associated with behavioral dysfunction. We hypothesized that known sex differences in forebrain dopamine circuit-associated behaviors would be the result of sex differences in forebrain dopamine circuit anatomy. As a first step to address this hypothesis, we combined a mouse transgenic driver line (tyrosine hydroxylase promoter-driven Cre recombinase) with virally encoded fluorescent reporters (FLEX-tdTomato and SynaptophysinGFP) to compare the density of midbrain dopaminergic axon projections and terminal boutons in dopamine projection target regions. Using this technique, we analyzed projections from the ventral tegmental area (VTA) to prefrontal cortex and basolateral amygdala (BLA) in male and female adult mice. Multiple analyses at 10x and 25x magnification revealed higher bouton density in BLA in males compared to females. To determine if this anatomical difference is mediated by gonadal steroid hormones, subjects were treated with a drug used to reduce gonadal steroid hormone production in clinical populations, leuprolide acetate (Lupron), before anatomical measures. Leuprolide administration resulted in a reduction in circulating testosterone, but did not show an effect on dopamine circuit anatomy. The finding of an anatomical sex difference in the forebrain dopamine circuit provides a structural foundation for further investigation of how sex differences in brain circuits may underlie behavioral dysfunction that play roles in psychiatric illnesses.Item DEVELOPMENT OF A BIOINFORMATICS PLASMID-SEARCH ENGINE FOR CRONOBACTER SPECIES.(2021) Negrete, Flavia; El-Sayeed, Najib NE; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cronobacter species. are foodborne pathogens that cause serious disease in neonates, infants, and adults. Plasmid classification lays the groundwork for understanding the stable coexistence of various extrachromosomal replicons in a single bacterium, and thus the organization of its genome. This study developed a bioinformatics plasmid-search engine to identify genomic attributes contained on Cronobacter plasmids. A database containing 32 Cronobacter plasmid sequences from all seven Cronobacter species was developed. Another database containing 683 draft and closed plasmids and genomes was also developed. Each strain’s plasmid content was sorted into six different categories based on their genetic attributes: virulence, Type-IV, heavy-metal, cryptic, multi-drug resistant, or mobilization. An in-house BLAST+-python script was used to perform a Linux-BLAST analysis to create a formatted %ID output matrix of plasmid genes. This thesis represents the first bioinformatics plasmid-search engine developed for Cronobacter. Understanding the role of plasmids in virulence and persistence underpins future mitigation strategies to be developed for controlling this pathogen.Item UNRAVELING QUALITY CONTROL PATHWAYS: THE ROLE AND FUNCTION OF PARKIN-INDEPENDENT MITOPHAGY(2021) Shah, Hetal Vinod; Carr, Catherine; Youle, Richard; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mitochondrial dysfunction is attributed to several human diseases, including neurological, immunological, and cardiological. Given the underlying mitochondrial dysfunction in these disorders and lack of therapeutic options, it is important to understand the mechanisms by which mitochondrial homeostasis is maintained. Characterization of mitochondrial quality control pathways, such as mitophagy, have greatly aided in understanding the mechanisms that control proper mitochondrial function. While there is a robust understanding of the molecular machinery and mechanisms of PINK1/Parkin-mediated mitophagy, Parkin-independent mitophagy mechanisms are less understood. The aim of this thesis is to investigate the requirements for iron chelation-induced mitophagy and to investigate the requirements for the mitochondrial-anchored E3 ubiquitin ligases, MUL1, MARCH5, and MARCH9 in mitophagy. Iron chelation is a potent activator of mitophagy and iron chelation-induced mitophagy is thought to be independent of both Parkin and PINK1. We show that iron homeostatic pathways may also govern mitophagy caused by iron loss. We also found that iron chelation-induced mitophagy utilized canonical autophagy machinery for autophagosome formation and fusion. However, our data indicates that mitophagy stimulated by iron chelation does not use the canonical ATG16L1 protein, but rather ATG16L2, a protein that has not been implicated in autophagy. Finally, we show that the autophagy receptor protein, NDP52, may mediate mitophagy in a ubiquitin-dependent manner. The second aim of this dissertation is to understand the role of the mitochondrial-anchored E3 ubiquitin ligases, MUL1, MARCH5, and the unreported MARCH9, in mitochondrial quality control systems. Mitochondrial E3 ligases are thought to help maintain mitochondrial integrity by mediating basal turnover of integral mitochondrial membrane proteins as well as by participating in quality control pathways. This dissertation shows that knockout of any of the mitochondrial-anchored E3 ubiquitin ligases alone does not affect either PINK1/Parkin-dependent mitophagy nor Parkin-independent mitophagy. However, we found that combinatorial knockout of the mitochondrial-anchored E3 ubiquitin ligases did not affect PINK1/Parkin-dependent mitophagy but did cause a defect in Parkin-independent mitophagy. This indicates a role for the mitochondrial-anchored E3 ubiquitin ligases in Parkin-independent mitophagy. In sum, this dissertation begins unraveling the role and function of Parkin-independent mitophagy mechanisms.Item The Nanoarchitecture of the Outer Hair Cell Lateral Wall: Structural Correlates of Electromotility(2021) Sun, Willy Weiyih; Carr, Catherine; Kachar, Bechara; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Proper mammalian hearing depends on an outer hair cell-based mechanism that amplifies the sound-induced travelling waves in the cochlea. Outer hair cells (OHCs) contribute to this cochlear amplification through their electromotile property—voltage-dependent somatic length changes that can operate at acoustic frequencies. This unique form of motility is driven by prestin, a member of the solute carrier 26 family of anion transporters that is highly expressed along the OHC lateral plasma membrane. The lateral plasma membrane is supported by a cortical actin-spectrin lattice and a smooth ER system known as lateral cisternae to form a regular layered structure along the entire OHC lateral wall. The detailed structural organization of each layer and how they interact to transduce prestin conformational changes into whole-cell motility are not well understood. In this dissertation, I combine cryogenic sample preparation methods and electron tomography to elucidate the functional architecture of the OHC lateral wall complex. In chapter 1, I review the biology of the mammalian auditory system. In chapter 2, I detail how the combined methodological approach used can preserve and reveal the three-dimensional nano-architectures in cells at near-native state. In chapter 3, I describe the successful use of this methodology to elucidate the structure-function relationships in a comparable model structure, the glycocalyx on the surface of enterocytes. In Chapter 4, I provide the details on the organization of each layer of the OHC lateral wall complex and how they are structurally integrated. I show that the lateral plasma membrane contains closely tiled microdomains of orthogonally packed putative prestin protein complexes. The cortical lattice connects the plasma membrane to the adjacent lateral cisternae through two independent cross-bridging components. The lateral cisternae are in turn integrated through inter and intra-cisternal cross-bridging systems. Finally, mitochondria are attached to the lateral cisternae through another set of linker elements. By quantifying the dimensions of each of these components and mapping their distribution I provide a detailed blueprint of the nano-architecture of the OHC electromotile apparatus and discuss how its cohesive structure allows effective transmission of forces generated by prestin to the rest of the cell to drive cochlear amplification.Item ARABIDOPSIS THALIANA GLUTAMATE RECEPTOR-LIKE 3.7 UNDERLIES ROOT MORPHOLOGY AND SIGNALING VIA MEMBRANE POTENTIAL HOMEOSTASIS(2021) Barbosa-Caro, Juan Camilo; Feijó, José A; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plants perceive highly variable environments and biotic interactions through membrane receptors like the GLutamate Receptor-like (GLR) family, related to the ionotropic Glutamate Receptors that underlie information transmission in neurons. GLRs underpin information transduction and morphological adaptations in plants. However, mechanistic understanding is scarce. In Arabidopsis thaliana roots, we investigated how GLRs underlie amino acid-induced electric and Ca2+ excitability. We also assessed the contribution of GLR3.7 in root hair elongation. We present GLRs as mediators of a local, glutamate-induced electric and Ca2+ response in roots, with the same initiation kinetics of wound-induced Slow Wave Potentials (SWP). We identify GLR3.7 as mediator of root hair elongation through maintenance of membrane depolarization at the growing cell apex. These results propose a parallel between glutamate-triggered signals and SWP initial phase as local and chemically induced, and posit GLR3.7 as a possible contributor to Ca2+ homeostasis in root hair apical growth.Item CONSERVED ROLE OF EMX2 IN ESTABLISHING POLARITY OF SENSORY HAIR CELLS(2016) Jiang, Tao; Carr, Catherine E; Wu, Doris K; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sensory hair cells in the inner ear are responsible for relaying information such as sounds and head positions to the brain. Stereocilia, which are specialized microvilli, are arranged in a staircase-pattern with the longest row sitting adjacent to the kinocilium. These two structures together form the stereociliary bundle (hair bundle), which are polarized asymmetrically at the apical surface of the hair cell. Deflection of the stereocilia towards the kinocilium opens the mechanotransduction channels at the tip of the stereocilia, which enables ion influx to depolarize the hair cell and activates action potentials in the postsynaptic neurons. Deflection towards the opposite direction results in hyperpolarization. Thus, the stereociliary bundle polarity defines the directional sensitivity of a given hair cell. Each sensory hair cell organ displays a specific pattern of stereocilia polarity. In the maculae, which detect linear acceleration in all directions, HCs can be divided into two regions with opposite polarity by a line of polarity reversal (LPR). Similar LPR is also present in the neuromast of the zebrafish lateral line system that detect pressure change of surrounding water. My results show that the homeodomain transcription factor Emx2 is essential for establishing the LPR. Expression of Emx2 in the maculae and neuromasts determines the stereocilia polarity pattern in a cell-autonomous fashion. Gain- and loss-of-function in the sensory hair cell organs of mouse and zebrafish indicate that the role of Emx2 in polarity reversal is both necessary and sufficient. In addition, my results demonstrated that Emx2 mediates this polarity reversal via one of the heterotrimer G-proteins, Gαi. In summary, my results show that Emx2 has a conserved role in dictating stereociliary bundle polarity.Item Physiological Effects of Alcohol on Crayfish Escape Circuitry(2016) Swierzbinski, Matthew Edward; Herberholz, Jens; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Alcohol is one of the oldest and most widely used drugs on the planet, but the cellular mechanisms by which it affects neural function are still poorly understood. Unlike other drugs of abuse, alcohol has no specific receptor in the nervous system, but is believed to operate through GABAergic and serotonergic neurotransmitter systems. Invertebrate models offer circuits of reduced numerical complexity and involve the same cell types and neurotransmitter systems as vertebrate circuits. The well-understood neural circuits controlling crayfish escape behavior offer neurons that are modulated by GABAergic inhibition, thus making tail-flip circuitry an effective circuit model to study the cellular mechanisms of acute alcohol exposure. Crayfish are capable of two stereotyped, reflexive escape behaviors known as tail-flips that are controlled by two different pairs of giant interneurons, the lateral giants (LG) and the medial giants (MG). The LG circuit has been an established model in the neuroscience field for more than 60 years and is almost completely mapped out. In contrast, the MG is still poorly understood, but has important behavioral implications in social behavior and value-based decision making. In this dissertation, I show that both crayfish tail-flip circuitry are physiologically sensitive to relevant alcohol concentrations and that this sensitivity is observable on the single cell level. I also show that this ethyl alcohol (EtOH) sensitivity in the LG can be changed by altering the crayfish’s recent social experience and by removing descending inputs to the LG. While the MG exhibits similar physiological sensitivity, its inhibitory properties have never been studied before this research. Through the use of electrophysiological and pharmacological techniques, I show that the MG exhibits many similar inhibitory properties as the LG that appear to be the result of GABA-mediated chloride currents. Finally, I present evidence that the EtOH-induced changes in the MG are blocked through pre-treatment of the potent GABAA receptor agonist, muscimol, which underlines the role of GABA in EtOH’s effects on crayfish tail-flip circuitry. The work presented here opens the way for crayfish tail-flip circuitry to be used as an effective model for EtOH’s acute effects on aggression and value-based decision making.