Life Sciences Commons^™

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 …

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 …

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 …

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 …

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 …

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 …

Notch-Mediated Regulation Of Atrial Arrhythmogenesis, Catherine Lipovsky

Arts & Sciences Electronic Theses and Dissertations

Abnormalities in electrical impulse generation and/or propagation that affect the heartճ normal rhythm are extremely common. Clinically, cardiac arrhythmias are prevalent worldwide, yet the molecular mechanisms underlying their pathology remain largely unknown. Current treatments for arrhythmias primarily target symptoms rather than the underlying cause and these treatments have limited efficacy. The most common risk factor for developing an arrhythmia is a previous cardiac injury; however, the mechanisms underlying this are not well described. My thesis work has demonstrated that the Notch signaling pathway, which is crucial for cardiac patterning and development and is normally quiescent in adult cardiomyocytes (CMs), is …

Phantoms To Placentas: Mr Methods For Oxygen Quantification, Kelsey Meinerz

Arts & Sciences Electronic Theses and Dissertations

Molecular oxygen (O2) is vital for efficient energy production and improper oxygenation is a hallmark of disease or metabolic dysfunction. In many pathologies, knowledge of tissue oxygen levels (pO2) could aid in diagnosis and treatment planning. The gold standard for pO2 measures in tissue are implantable probes, which are invasive, require surgery for placement, and are inaccessible to certain regions of the body. Methods for determining pO2 both non-invasively and quantitatively are lacking. The slight paramagnetic nature of O2 provides opportunities to non-invasively characterize pO2 in tissue via magnetic resonance (MR) techniques. As such, O2 can be treated as a …

Elucidating The Effect Of Myopathy-Causing Mutations And Second-Site Suppressors On Client Processing By J-Domain Proteins, Melanie Y. Pullen

Arts & Sciences Electronic Theses and Dissertations

Defects in protein quality control may lead to protein misfolding and aggregation often associated with protein conformational disorders such as Alzheimerճ Disease and Limb Girdle Muscular Dystrophy, among others. Molecular chaperones protect against protein misfolding and aggregation. A chaperone of interest is the ubiquitously expressed type II Hsp40 co-chaperone DNAJB6, which assists in protein folding and disaggregation. Mutations within the DNAJB6 G/F domain have been associated with the dominantly inherited disease Limb-Girdle Muscular Dystrophy type 1D (LGMD1D), now referred to as LGMDD1. Our collaborators recently discovered novel LGMDD1-associated mutations in the J-domain of DNAJB6. In the enclosed body of work, …

Dendritic Cell Development And Function, Vivek Durai

Arts & Sciences Electronic Theses and Dissertations

Dendritic cells (DCs) are a group of immune cells that include both classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). cDCs are further comprised of two distinct subsets, cDC1s and cDC2s, which play critical roles in the initiation of innate and adaptive immune responses. Understanding how these lineages develop and function is therefore paramount. All DCs require the receptor tyrosine kinase Flt3 and its ligand Flt3L for their development, but the loss of Flt3L in mice leads to a more severe DC deficiency than does the loss of Flt3. This has led to speculation that Flt3L can bind to …

Mitochondrial Morphology, Oxidative Stress Resistance, And Pathogenesis In Cryptococcus Neoformans, Andrew Lee Chang

Arts & Sciences Electronic Theses and Dissertations

Cryptococcus neoformans is an important pathogen that annually kills 200,000 people worldwide. It survives in the environment as a yeast or spore and can also proliferate within host macrophages after being inhaled into the lungs. In conditions of immunocompromise, cryptococcal cells can escape from the lungs to the brain, where they cause a deadly meningoencephalitis that is both difficult and expensive to treat. Cryptococcal adaptation to the harsh lung environment is a critical first step in its pathogenesis, and consequently a compelling topic of study. This adaptation is mediated by a complex transcriptional program that integrates cellular responses to environmental …

Post-Lysosomal Cholesterol Trafficking: Novel Tools And Insights, Mckenna Rae Feltes

Arts & Sciences Electronic Theses and Dissertations

Cholesterol is an essential mammalian lipid. It is a major component of cellular membranes, a precursor molecule for the synthesis of hormones and bile acids, and a regulator of protein function. Although cholesterol is synthesized, de novo, in the endoplasmic reticulum, cells principally meet cholesterol requirements through uptake of lipoprotein particles. Lipoprotein-derived cholesterol is transported to the lysosome where it is transferred from the soluble lysosomal protein, NPC2, to limiting-lysosomal membrane protein NPC1. Cholesterol is then re-distributed to other cellular membranes in order to fulfill organellar cholesterol requirements; however, the cellular machineries involved in coordinating this distribution are poorly characterized. …

Conformational Basis And Small Molecule Antagonists Of E. Coli Adhesion To The Urinary Tract, Vasilios Kalas

Arts & Sciences Electronic Theses and Dissertations

Urinary tract infections (UTIs) are one of the most prevalent infections, afflicting 15 million women per year in the United States with annual healthcare costs exceeding $2-3 billion. Uropathogenic Escherichia coli (UPEC) are the main etiological agent of UTIs and employ numerous virulence factors for host colonization. The most common adhesive mechanism by which UPEC mediate host-pathogen interactions is the chaperone-usher pathway (CUP), which is responsible for the assembly of proteinaceous surface appendages termed pili. Generally, CUP pili function in adherence or invasion of host tissues and in biofilm formation on medical devices and body habitats. CUP pili are highly …

Molecular Insights Into Microbial Adhesion, Roger Davies Klein

Arts & Sciences Electronic Theses and Dissertations

Antibiotic-resistant bacterial infections are a serious and immediate threat to global public health. In the United States alone, over 2 million individuals develop antibiotic-resistant infections annually, resulting in 23,000 deaths and $20 billion in excess health care costs. Virulence factors that allow bacteria to invade and persist within the host are promising targets for novel antimicrobial agents that could be used to curb the spread of antibiotic resistance. Development of therapeutics that can selectively eliminate pathogenic bacteria while sparing the beneficial host microbiota requires a detailed molecular understanding of critical virulence factors that facilitate interactions between pathogens and their environments. …

Development Of An In Vitro Culture System For Cryptosporidium Parvum, Georgia Wilke

Arts & Sciences Electronic Theses and Dissertations

Cryptosporidium is a genus of protozoan parasites that causes diarrheal disease in humans and other animals. There are two major species that cause disease in humans: C. parvum, which infects both humans and animals, and C. hominis, which primarily infects humans. A recent study investigating the etiologies of pediatric diarrheal illness in Africa and South Asia found that Cryptosporidium is the 2nd most prevalent cause of diarrhea in infants and may be a contributing factor to chronic malnutrition. This discovery has led to renewed interest in studying this parasite and a reexamination of the barriers to studying Cryptosporidium. The main …

The Enzymatic Function Of The Tir Domain: From Axon Degeneration To Innate Immunity, Kow Essuman

Arts & Sciences Electronic Theses and Dissertations

The Toll/Interleukin-1 Receptor (TIR) domain is an evolutionarily ancient protein domain conserved from bacteria to eukaryotes, and is an essential signaling component of innate immunity pathways. In animal innate immunity, TIR domains have primarily been described for their scaffolding function in assembling protein complexes in host defense. In plant immunity, TIR domains are key components of the intracellular Nucleotide Binding Leucine rich repeat (NLR) immune receptors that confer resistance to pathogens. These NLR receptors trigger cell death and an immune response upon activation, but their mechanism has remained elusive. In bacteria, TIR domain proteins have been suggested to function as …

Structural Mechanism Of Poxvirus Sabotage Of T-Cell Costimulation, Jabari Issa Elliott

Arts & Sciences Electronic Theses and Dissertations

Poxviruses are characterized by large double stranded DNA genomes that encode numerous proteins tailored for host immune response evasion. Our lab has been investigating a sequence-diverse family of secreted poxvirus proteins that appear to share a conserved beta-sandwich fold, but differ in their immunomodulatory functions. We have termed members of this superfamily Poxvirus Immune Evasion (PIE) proteins, and there appears to be at least 20 distinct subfamilies. As it turns out, cowpox virus (CPXV) encodes 10 PIE proteins, one of which, M2, can inhibit murine T cell activation through specific interactions with co-stimulatory ligands B7. 1 (CD80) and B7. 2 …