Digital Commons Network^™

Advances In The Understanding Of Auxin Signaling Through Indirect Mechanisms, Ryan Joseph Emenecker

Arts & Sciences Electronic Theses and Dissertations

The plant hormone auxin is remarkable in its capacity to seemingly regulate all plant growth and developmental processes. Because of this, plants have evolved numerous mechanisms to allow for specific responses to auxin dependent on tissue type and developmental stage. Despite the importance of this specificity, we understand very little with respect to the molecular mechanisms underlying it. In this work, we examine two mechanisms used by plants to modulate auxin response across different tissues. First, we describe our finding that auxin interactions with another plant hormone, abscisic acid, is used as a mechanism to regulate auxin responsiveness in the …

Structural Insights Into Host-Pathogen Interactions Of Alphaviruses, Katherine Basore

Arts & Sciences Electronic Theses and Dissertations

Alphaviruses are arthropod-borne, single-stranded positive-sense RNA viruses of the Togaviridae family that infect various vertebrates worldwide in tropical and temperate areas, causing emerging and reemerging diseases in humans. Mature virions are 70 nm in diameter and contain a ~11-kilobase genome encapsidated within a nucleocapsid core, a host-derived lipid bilayer, and an envelope comprised of heterodimers of the glycoproteins E1 and E2 arranged into trimeric spikes with T=4 icosahedral symmetry. Alphaviruses are categorized into two groups based on their clinical symptoms: the arthritogenic alphaviruses, such as chikungunya (CHIKV), Mayaro (MAYV), Ross River (RRV), Semliki Forest (SFV), and O’nyong-nyong (ONNV) viruses, which …

The Decision Maker: Understanding The Role Of Ifrd1 In Urothelial Plasticity And Regeneration, Bisiayo Erionmwon Fashemi

Arts & Sciences Electronic Theses and Dissertations

The bladder urothelium forms a highly specialized watertight barrier to urinary wastes. The urothelium offers an unusual example of tissue regeneration: although urothelial cells do not rapidly turn over under physiological conditions, they have an impressive capacity to regenerate tissue upon injury. Even more remarkable, depending on the modality of injury (sterile, infectious) there appear to be two distinctive modes of urothelial regeneration. We have previously shown that in response to a urinary tract infection (UTI), the urothelial stem cell niche becomes activated and induces rapid restoration of the urothelium, whereas, regeneration following sterile injury does not involve stem cell …

Charged Pore-Lining Residues Are Required For Normal Channel Kinetics In The Eukaryotic Mechanosensitive Ion Channel Msl1, Angela M. Schlegel, Elizabeth S. Haswell

Biology Faculty Publications & Presentations

Mechanosensitive (MS) ion channels are widespread mechanisms for cellular mechanosensation that can be directly activated by increasing membrane tension. The well-studied MscS family of MS ion channels is found in bacteria, archaea, and plants. MscS-Like (MSL)1 is localized to the inner mitochondrial membrane of Arabidopsis thaliana, where it is required for normal mitochondrial responses to oxidative stress. Like Escherichia coli MscS, MSL1 has a pore-lining helix that is kinked. However, in MSL1 this kink is comprised of two charged pore-lining residues, R326 and D327. Using single-channel patch-clamp electrophysiology in E. coli, we show that altering the size and …

Single‐Molecule 3d Orientation Imaging Reveals Nanoscale Compositional Heterogeneity In Lipid Membranes, Jin Lu, Hesam Mazidi, Tianben Ding, Oumeng Zhang, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

In soft matter, thermal energy causes molecules to continuously translate and rotate, even in crowded environments, thereby impacting the spatial organization and function of most molecular assemblies, such as lipid membranes. Directly measuring the orientation and spatial organization of large collections (>3000 molecules μm⁻²) of single molecules with nanoscale resolution remains elusive. In this paper, we utilize SMOLM, single‐molecule orientation localization microscopy, to directly measure the orientation spectra (3D orientation plus “wobble”) of lipophilic probes transiently bound to lipid membranes, revealing that Nile red's (NR) orientation spectra are extremely sensitive to membrane chemical composition. SMOLM images resolve …

Engineering Natural Competence Into The Fast-Growing Cyanobacterium Synechococcus Elongatus Utex 2973, Kristen Elizabeth Wendt

Arts & Sciences Electronic Theses and Dissertations

Synechococcus elongatus UTEX 2973 is the fastest growing cyanobacterium discovered to date. Using water, carbon dioxide, and light alone, this organism can double in 1.5 hours under optimal conditions. The accelerated doubling exhibited by Synechococcus 2973 makes it a prime candidate to serve as a model photoautotrophic system. However, Synechococcus 2973 lacks one highly desirable feature: it cannot undergo natural transformation. This thesis seeks to engineer this capacity into this fast-growing system in order to create an organism that is both fast growing and naturally competent. Synechococcus 2973 is a unique platform because it is >99% genetically identical to another …

The Role Of The Circadian Clock Protein Rev-Erba In Neuroinflammation & Synaptic Health, Percy Griffin

Arts & Sciences Electronic Theses and Dissertations

Circadian rhythms are cycles of physiological activity that are conserved across all of life’s taxa – ranging from cyanobacteria to humans - due to their importance. They are conserved to allow organisms to maximize their capacity to obtain resources in their environment. In mammals, light and dark input into the retina is the strongest synchronizer of circadian rhythms. On the molecular level, this tightly regulated transcriptional-translational feedback loop is orchestrated by proteins with cyclical expression. The loss of these proteins has functional consequences on human health and diseases.

Recently, associations have been made between circadian proteins and a host of …

A Mechanistic And Genomic Analysis Of Molluscum Contagiosum Virus Immune Evasion, Ian Benjamin Harvey

Arts & Sciences Electronic Theses and Dissertations

Molluscum contagiosum virus (MCV) is a common human-specific poxvirus with a proclivity for

infecting children and the immune-compromised. A characteristic MCV infection is restricted to

the epidermal layers of the skin and can persist for weeks to years in an otherwise healthy

individual. The high clinical burden of MCV is at odds with our limited knowledge regarding how

it successfully evades the human immune response, which is in part due to the lack of an animal

model or cell line to propagate the virus. Through this dissertation, we have uncovered and

characterized a novel mechanism by which MC80, a protein …

Pathophysiology And Proteogenomics Of Post-Infectious And Post-Hemorrhagic Hydrocephalus In Infants, Albert M. Isaacs

Arts & Sciences Electronic Theses and Dissertations

Post-infectious (PIH) and post-hemorrhagic (PHH) hydrocephalus occur as sequalae of neonatal sepsis or intraventricular hemorrhage (IVH) of prematurity, respectively. Together, PIH and PHH represent the most common form of infantile hydrocephalus, the most common indication for neurosurgery in children globally, and the leading cause of neurological morbidity and mortality worldwide. The lack of understanding of the pathophysiology of PIH and PHH, particularly with regards to the host central nervous system response to the antecedent infection and hemorrhage, perturbation of differentiating neural stems in the ventricular (VZ) and subventricular (SVZ) zones, and damage to periventricular white matter (PVWM) tracts carrying sensorimotor …

Genetic And Functional Characterization Of Novel Host Factors Regulating Virus Infection In Caenorhabditis Elegans, Luis Enrique Sandoval

Arts & Sciences Electronic Theses and Dissertations

Viruses infect the majority of eukaryotic life on the planet and remain a global threat to human health. These pathogens are constrained to intracellular life cycles, as they exploit and rely on host factors and machinery throughout their entire reproduction process. While many of these viral life cycle factors have been reported and studied, our knowledge in the identity and function of these factors remains incomplete and a challenge in fundamental virology and the development of antiviral therapeutics. Caenorhabditis elegans offers an innovative approach for discovering novel host factors required for virus infection in a multicellular and simple model organism. …

Microrna Gene Expression States Underlying Individual Variation In Aging And Lifespan In Isogenic C. Elegans, Holly Kinser

McKelvey School of Engineering Theses & Dissertations

Average lifespan differs greatly between species, but lifespan among same-species individuals is also highly variable. While much effort has been devoted to uncovering longevity-associated traits and lifespan-extending perturbations in humans and model organisms, how differences in lifespan arise between individuals is unknown. Studies of human identical twins demonstrate that surprisingly little of the variation in lifespan between individuals can be explained by genetics and shared environment. Furthermore, even genetically identical C. elegans reared in highly homogeneous environments display a degree of variability in lifespan similar to that of outbred human populations. Thus, longevity must be determined at least in part …

Wnt/_-Catenin Mediated Regulation Of Murine And Human Cardiac Electrophysiology And Arrhythmogenesis, Gang Li

McKelvey School of Engineering Theses & Dissertations

Cardiac arrhythmias affects millions of Americans and can lead to sudden cardiac death, accounting for more than 300,000 deaths annually. Despite the vast knowledge available for cardiac disease and associated arrhythmias, very few effective therapies exist. Current interventions include cardioverter defibrillators and antiarrhythmic drugs targeting ion channels or the _-adrenergic pathway. In most acquired and inherited arrhythmias, molecular signaling pathways are perturbed. However, little is known about the underlying mechanism of how these signaling pathways regulate cardiac electrophysiology. Therefore, a better understanding of major signaling pathways governing cardiac development or dysregulated in cardiac disease could lead to novel therapeutics for …

Delineating The Role Of Mir-124 For The Activation Of Neuronal Program, Ya-Lin Lu

Arts & Sciences Electronic Theses and Dissertations

The ectopic expression of two brain-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), can robustly and efficiently reprogram human skin fibroblasts into neurons. The miRNAs act as repressors of non-neuronal genes in fibroblasts for the induction of the neuronal program. This process is analogous to neurogenesis in vivo when the expression of miR-9/9* and miR-124 represses anti-neurogenic genes such as REST or NRSF (neuron-restrictive silencer factor/repressor element-1 silencing transcription factor). Although we have some mechanistic insights into how miR-9/9*-124 drives fate conversion by acting as negative regulators of gene expression, little remained understood of the role of miRNAs as positive regulators …

Specificity Of Ssb Binding To Its Interacting Proteins And Multiple Allosteric Effects Of Ssb C-Terminal Tail On Assembly And Dna Binding Of E. Coli Recor Proteins, Min Kyung Shinn

Arts & Sciences Electronic Theses and Dissertations

The homo-tetrameric E. coli single strand (ss) DNA binding (SSB) protein is an essential component in DNA maintenance for its role in binding and protecting single stranded DNA intermediates via its N-terminal DNA binding domain (DBD). SSB also acts as a hub to recruit at least 17 SSB interacting proteins (SIPs) involved in DNA replication, recombination, and repair via its 9 amino acid C-terminal acidic tip region. A 56 amino acid intrinsically disordered linker connects the DBD and the acidic tip and plays a role in cooperative binding to ssDNA. Using isothermal titration calorimetry, I determined that the SSB-Ct peptides …

Bumpy Road Ahead: Overcoming Dna Replication Obstacles One Barrier At A Time, Melanie Anne Sparks

Arts & Sciences Electronic Theses and Dissertations

DNA replication must occur efficiently and timely every cell cycle to protect the integrity of the genome. Stalled or slowed replication forks lead to replication stress that can cause replication fork collapse, and potentially genome instability. Scattered throughout the genome are tightly bound proteins, such as transcription factors, that are necessary for cell function and survival. These proteins have the potential to impede timely DNA replication. Furthermore, genomic DNA is packaged around histone octamers into structures called nucleosomes that both compact the DNA and provide an additional layer of information and regulation termed epigenetics. Thus, DNA replication is not only …

Development And Application Of Mass Spectrometry-Based Approaches For Protein Higher Order Structure Analysis And Protein-Protein Interaction Characterization, Mengru Zhang

Arts & Sciences Electronic Theses and Dissertations

Proteins, one of the most fundamental biomolecules, adopt unique higher order structures (HOS) to enable diverse biological functions. Deciphering protein HOS is crucial to gain deeper insights of their working mechanisms and to develop biotherapeutics. Mass spectrometry (MS)-based approaches evolved rapidly in the past 30 years and are now playing critical roles in protein HOS characterization. One of those approaches is MS-based footprinting whose principle is to map the solvent accessible surface area (SASA) to deliver structural information. Protein footprinting can be achieved by reversible labeling, e.g., hydrogen-deuterium exchange (HDX), and by irreversible labeling using radical-based reagents or other targeted …

Genomic Analysis Of Diverse Bacterial Pathogens, Robert Potter

Arts & Sciences Electronic Theses and Dissertations

Bacterial pathogens have been a historical scourge for the entirety of human existence but have been significantly thwarted since the 20th century due to the development of antibiotics. However, owing to the large selection pressure of antibiotics on bacterial populations, phenotypic antibiotic resistance from the development of vertically transmitted mutations and horizontally acquired antibiotic resistance genes (ARGs) is increasing. The sum has produced multidrug resistant organisms (MDROs) which have extremely limited treatment options. Epidemiological studies have determined that carbapenem resistant Enterobacteriaceae (CRE), Acinetobacter baumannii, and vancomycin resistant Enterococcus (VRE) are some of the most problematic MDRO infections. The advent of …

Mechanisms Of Cross-Presentation By Cdc1s, Derek James Theisen

Arts & Sciences Electronic Theses and Dissertations

Classical dendritic cells (cDCs) are specialized antigen presenting cells that can be divided into distinct subsets based on the types of pathogens they respond to and the type of immune response they generate. The cDC1 subset is specialized in priming CD8 T cell responses through the process of cross-presentation. During cross-presentation, exogenous protein antigens are taken up by cDC1 and presented on MHCI molecules, allowing for the priming of CD8 T cells during conditions when DCs themselves are not directly infected. The ability to cross-present in vivo is unique to cDC1, and is essential for anti-viral responses and rejection of …

Regulation Of Yolk Microtubule Dynamics By Dachsous Cadherins, Gina Danielle Castelvecchi

Arts & Sciences Electronic Theses and Dissertations

The process of epiboly, or the thinning and spreading of a tissue, is a well-conserved morphogenetic process. As one of four conserved gastrulation cell movements, epiboly is important to help organize the overall body plan. Epiboly in zebrafish involves the thinning and spreading of the blastoderm originating from the animal pole to completely enclose the yolk. It is driven by a multitude of physical processes that involve the three cell types comprising the embryo: the yolk syncytial layer (YSL), enveloping layer (EVL), and deep cells. These physical aspects can be broadly described as involving cell-cell interactions through adhesion proteins, actomyosin …

The Good, The Brown, And The Healthy: Understanding Non-Thermogenic Brown Adipose Function In Obese Mice, Caryn Nicole Carson

Arts & Sciences Electronic Theses and Dissertations

Genetic and environmental factors heavily intertwine to affect metabolic homeostasis. To tease out the exact interactions between these two realms of influence, researchers often compare how one or multiple different inbred mouse strains react to various diets. An observation consistently seen across multiple strains on the same diet can reasonably be considered a general dietary effect, whereas an observation seen only in one strain of mice is more likely to result from a genetic cause or gene-by-environment interaction. Similarly to humans, a high fat diet causes many mouse strains to develop obesity and poor metabolic health, with varying degrees of …

Mechanisms Of Sting-Associated Vasculopathy And Immunodeficiency, Brock Gordon Bennion

Arts & Sciences Electronic Theses and Dissertations

STING N153S in mice and STING N154S in humans cause spontaneous autoimmunity. Specifically, these mutations reduce the numbers of T cells and NK cells, and cause lung disease. However, mice develop perivascular lung inflammation that is distinct from the pulmonary fibrosis observed in human patients. Viral infections are known to exacerbate autoimmunity and foment pulmonary fibrosis. Therefore, we hypothesized that exposure to a virus may influence STING N153S disease in mice. To test this, we infected STING N153S animals with the gammaherpesvirus family member, gHV68, and found that 14 days post infection pulmonary fibrosis was readily observed by histological staining. …

The Role Of Escrt-Iii-Like Subunit Ist1 In Membrane Trafficking Pathways, Amy Kate Clippinger

Arts & Sciences Electronic Theses and Dissertations

ESCRTs (Endosomal Sorting Complex Required for Transport) are a modular set of proteins with membrane remodeling activities that include the formation and release of intralumenal vesicles (ILVs) to generate multivesicular endosomes. ESCRT-III filaments have an established role in membrane fission for ILV formation and the topologically related processes of viral budding and cytokinesis. Among the 12 ESCRT-III proteins most have established roles in ILV formation, but the cellular roles of IST1 remain elusive. We found that IST1 and another ESCRT-III subunit CHMP1B form filaments that spiral around the outside or cytoplasmic surface of membrane tubules. Consistent with a role in …

The Role Of Cdx4 During Patterning Of Definitive Hemogenic Mesoderm, John Philip Creamer

Arts & Sciences Electronic Theses and Dissertations

The current standard of treatment for a variety of hematopoietic malignancies and genetic disorders is allogenic bone marrow transplantation, where donor hematopoietic stem cells (HSCs) engraft within the host and give rise to all of them hematopoietic lineages necessary for homeostasis. In many cases, finding a compatible human leukocyte antigen (HLA) matching donor is not possible, due to the large amount of genetic variation at those loci, but with the advent of induced pluripotent stem cells (iPSCs), unlimited sources of patient matched cells can be derived. Hematopoietic differentiations of human pluripotent stem cells (hPSCs) have been shown to recapitulate the …

Modeling Her2 Mutations In Colorectal Cancer Using A Her2 Transgenic Mouse Model And Gastrointestinal Organoids, Elisa Murray

Arts & Sciences Electronic Theses and Dissertations

Amplification or mutations in members of the epidermal growth factor receptor family, such as HER2, have been identified in several human diseases. In particular, mutations in the intracellular kinase domain have been identified in breast, colon, and lung cancers. The Cancer Genome Atlas has identified HER2 mutations or gene amplification in seven percent of colon cancer patients. These mutations are well known to promote enhanced cell growth and transformation of colon cancer cell lines. Previous studies have found HER2 mutations to confer anchorage independent growth and activation of downstream signaling pathways such as MAPK. Although HER2 mutations have been extensively …

Convex Relaxations For Particle-Gradient Flow With Applications In Super-Resolution Single-Molecule Localization Microscopy, Hesam Mazidisharfabadi

McKelvey School of Engineering Theses & Dissertations

Single-molecule localization microscopy (SMLM) techniques have become advanced bioanalytical tools by quantifying the positions and orientations of molecules in space and time at the nanoscale. With the noisy and heterogeneous nature of SMLM datasets in mind, we discuss leveraging particle-gradient flow 1) for quantifying the accuracy of localization algorithms with and without ground truth and 2) as a basis for novel, model-driven localization algorithms with empirically robust performance. Using experimental data, we demonstrate that overlapping images of molecules, a typical consequence of densely packed biological structures, cause biases in position estimates and reconstruction artifacts. To minimize such biases, we develop …

Structural Mechanism For Gating Of A Eukaryoticmechanosensitive Channel Of Small Conductance, Zengqin Deng, Grigory Maksaev, Angela M. Schlegel, Jingying Zhang, Michael Rau, James A.J. Fitzpatrick, Elizabeth S. Haswell, Peng Yuan

Biology Faculty Publications & Presentations

Mechanosensitive ion channels transduce physical force into electrochemical signaling that underlies an array of fundamental physiological processes, including hearing, touch, proprioception, osmoregulation, and morphogenesis. The mechanosensitive channels of small conductance (MscS) constitute a remarkably diverse superfamily of channels critical for management of osmotic pressure. Here, we present cryo-electron microscopy structures of a MscS homolog from Arabidopsis thaliana, MSL1, presumably in both the closed and open states. The heptameric MSL1 channel contains an unusual bowl-shaped transmembrane region, which is reminiscent of the evolutionarily and architecturally unrelated mechanosensitive Piezo channels. Upon channel opening, the curved transmembrane domain of MSL1 flattens and expands. …

The Mechanosensitive Ion Channel Msl10 Potentiates Responses To Cell Swelling In Arabidopsis Seedlings, Debarati Basu, Elizabeth S. Haswell

Biology Faculty Publications & Presentations

The ability to respond to unanticipated increases in volume is a fundamental property of cells, essential for cellular integrity in the face of osmotic challenges. Plants must manage cell swelling during flooding, rehydration, and pathogen invasion-but little is known about the mechanisms by which this occurs. It has been proposed that plant cells could sense and respond to cell swelling through the action of mechanosensitive ion channels. Here, we characterize a new assay to study the effects of cell swelling on Arabidopsis thaliana seedlings and to test the contributions of the mechanosensitive ion channel MscS-like10 (MSL10). The assay incorporates both …

Developing Tools For Identifying Tissue-Specific Epigenetic Marks And Predicting Dna Hydroxy/Methylation, Yu He

Arts & Sciences Electronic Theses and Dissertations

A single genome can derive phenotypically unique cell types through various epigenetic modifications that instruct specific gene expression patterns. Histone modifications, DNA methylation, and DNA hydroxymetylation are the most common epigenetic modifications. To understand the mechanisms how these epigenetic modifications regulate gene expression, one often needs to map these marks genome-wide through profiling methods. Firstly, for histone modifications, Roadmap Epigenomics Consortium generated The Human Reference Epigenome Map, containing thousands of genome-wide histone modification datasets that describe epigenomes of a variety of different human tissue and cell types. This map has allowed investigators to obtain a much deeper and more comprehensive …

Effects Of Agonistic Anti-Cd137 Antibody On Chikungunya Virus Infection And B Cell Responses, Jun Hong

Arts & Sciences Electronic Theses and Dissertations

CD137, a member of the tumor necrosis factor receptor superfamily of cell surface proteins, acts as a costimulatory receptor on T cells, natural killer cells, B cell subsets, and some dendritic cells. Agonistic anti-CD137 monoclonal antibody (MAb) therapy has been combined with other chemotherapeutic agents in human cancer trials. Based on its ability to promote tumor clearance, we hypothesized that anti-CD137 MAb might activate immune responses and resolve chronic viral infections. We evaluated anti-CD137 MAb therapy in a mouse infection model of chikungunya virus (CHIKV), an alphavirus that causes chronic polyarthritis in humans and is associated with reservoirs of CHIKV …

Pathophysiology And Treatment Of Murine Globoid Cell Leukodystrophy, Yedda Li

Arts & Sciences Electronic Theses and Dissertations

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a rapidly progressing, invariably fatal pediatric disorder first described in 1916. Krabbe disease is caused by a deficiency in the lysosomal enzyme, galactosylceramidase (GALC), and is characterized clinically by failure to thrive, limb stiffness, seizures, developmental regression, and death by 2-4 years of age. Galactosylceramidase degrades the cytotoxic glycolipid, galactosylsphingosine (psychosine). In the absence of GALC activity, psychosine accumulates primarily in oligodendrocytes and Schwann cells, resulting in profound demyelination. In 1972, psychosine was hypothesized to be responsible for the clinical signs associated with Krabbe disease. However, the ‘Psychosine Hypothesis’ has never been …