UMD Theses and Dissertations
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Item SENSORY AND HORMONAL MECHANISMS OF EARLY LIFE BEHAVIOR IN A SOCIAL CICHLID FISH(2024) Westbrook, Molly; Juntti, Scott; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Studying the ontogeny of animal behavior is fundamental to ethology and allows understanding how behaviors in early life may affect later life success. The social cichlid Astatotilapia burtoni is an excellent model for examining the mechanisms of early life aggression due to the robust social hierarchy enforced by stereotyped, measurable social behaviors. We examine how hormonal signaling affects early life aggression through pharmacology and CRISPR-Cas9 mutants. We test which sensory pathways convey aggression-eliciting stimuli through sensory deprivation experiments. And we identify kinematic features that predict aggression through machine-learning video tracking algorithms. We observe that aggressive behaviors emerge around 17 days post fertilization (dpf), correlating with when the animals transition to free swimming away from the mother. We find that sex steroids subtly organize behavioral circuits for aggression and suggest that unknown additional mechanisms play a leading role. We show that thyroid hormone is not necessary or sufficient for the transition to aggressive behavior. We show that visual signals are necessary for the full expression of aggression, but in the absence of visual signal, low levels of aggression remain. We show that ciliated olfactory receptor signaling maintains low levels of aggression, as mutant animals display higher levels of aggressive behavior between 17 and 24 dpf. Finally, we demonstrate that swimming velocity has potential to predict aggressive instances of behavior. Together, we find multiple levels of control for early life aggressive bouts from sensory input to hormonal organization of brain circuits.Item THE ROLE OF GENOME ORGANIZATION AND FILAMENTOUS BACTERIOPHAGE ON GONOCOCCAL BIOLOGY AND PATHOGENICITY(2024) Kopew, Jessica; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (GC), represents a significant global health concern as it is the second most common bacterial STI and has a rising rate of antimicrobial resistance. The first study of this thesis aims to elucidate the causes and consequences of gonococcal genome reorganization. Here I found that when looking at many GC strains’ genomes, each GC strain had a unique genome organization including both inversions and translocation events. I also saw a consistent pattern of DNA high sequence similarity on both sides of the translocation or inversions, consistent with homologous recombination driven reorganization. PCR analysis of inversion events suggests that these large-scale reorganization events are both stable and rare. Growth curve analysis demonstrates a wide variability in growth rate between strains. Proteomic analysis suggests reorganization driven changes to replication termination location leads to upregulation of many kinds of proteins including energy metabolism and antimicrobial resistance associated genes. This study suggests that homologous recombination driven genome reorganization can have large impacts on gonococcal biology and pathogenicity. This study demonstrates the need for future gonococcal studies to use multiple GC strains from a diverse background to capture the wide variability in GC phenotypes. The second study of this thesis sought to uncover the role filamentous bacteriophage play in GC biology. I found that every GC strain currently in the NCBI database at the date of this study contains four filamentous bacteriophage gene regions in the GC genome. I found that FA1090Δfil (a GC strain lacking all four filamentous bacteriophage gene regions) grew poorly at 37⁰C both in broth and on agar, as compared to wild type FA1090. However, there was no difference when the strains were grown at 34⁰C or when grown without shaking, demonstrating the condition dependent nature of this growth advantage. FA1090Δfil formed larger bacterial aggregates than FA1090 WT. When these strains were analyzed for their ability to produce biofilms, no differences were seen in the overall biofilm’s biomass, yet the overall structure of the biofilms were different, with FA1090Δfil producing taller and rougher biofilms. Previous unpublished research in the Stein Lab demonstrates that filamentous phage derived proteins are capable of deteriorating the integrity of epithelial cell cultures and cervical tissue explants. The data from this chapter suggests that filamentous phage provide the gonococcus with a growth advantage, inhibit bacterial aggregation, alter the structure of the GC biofilm, and that phage proteins can lead to loss of the integrity of the epithelium. Taken en toto, these studies demonstrate that both alterations in bacterial genome organization and contributions from filamentous bacteriophage genomes can impact gonococcal biology and pathogenicity, which could be key to preventing and treating GC infections.Item ZIKA VIRUS RECRUITS CELLULAR PROTEINS TO SUPPORT ITS REPLICATION(2024) Chang, Peixi; Zhang, Yanjin YJ; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Zika virus (ZIKV) is a mosquito-borne pathogen with a massive impact on global public health due to its association with severe neurological complications, including microcephaly in newborns and Guillain-Barré syndrome in adults. The ZIKV epidemic in the Americas in 2015-2016 and its continuing spread in tropical regions have highlighted the urgent need to understand the molecular mechanisms of viral replication to develop effective antiviral strategies. However, many aspects of how ZIKV interacts with host cells remain unclear. This study identifies and characterizes host factors contributing to ZIKV replication. First, karyopherin alpha 6 (KPNA6) contributes to ZIKV replication by interacting with the ZIKV non-structural protein NS2B. Characterization and mutational analyses identified two essential amino acid residues within NS2B that are critical for interacting with KPNA6. The substitution of these two residues of NS2B in an infectious ZIKV cDNA clone resulted in a significant reduction in viral replication, suggesting that the NS2B-KPNA6 interaction plays a vital role in the viral life cycle. Further studies found that KPNA6 contributes to ZIKV RNA synthesis. Mass spectrometry analysis of the KPNA6 interactome showed that KPNA6 interacts with proteins involved in RNA synthesis, suggesting that ZIKV recruits these factors by promoting KPNA6-binding. Second, this study developed an effective method to isolate the ZIKV replication complex, a membranous structure where viral RNA is synthesized. Proteomic analysis of the isolated complex led to identifying numerous host proteins associated with the viral replication machinery. Among these proteins, human replication factor C subunit 2 (RFC2), an accessory factor involved in DNA replication and repair, was discovered to facilitate ZIKV replication, making it a potential target for therapeutic interventions. In conclusion, this study reveals crucial host factors essential for ZIKV infection and replication and provides insights into the ZIKV-cell interactions. These findings offer new possibilities for developing novel antiviral strategies for controlling future viral outbreaks.Item ROLE OF TRPV4 MECHANOSENSING REGULATING MACROPHAGE FUNCTIONS IN INFLAMMATORY DISEASES(2024) Dutta, Bidisha; Rahaman, Shaik O; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Macrophages are the most versatile cells of the hematopoietic system with roles in homeostasis, host-tissue development, innate immune response and tissue repair. Although the inflammatory activation and maintenance signals are tightly regulated, an imbalance in them results in unchecked inflammation resulting in cellular and tissue damage. Macrophages can affect most if not all phases of inflammation owing to their ability to adopt distinct functional states, secrete cytokines and phagocytose pathogens and debris. Recent evidence suggests that beyond biochemical cues, mechanical forces, like changing matrix stiffness in the tissue microenvironment, can shape immune cell functions involved in inflammation. These cells convert mechanical stimuli to biochemical signals in a process called mechanotransduction, regulating a multitude of cellular functions. However, knowledge about the molecular mediators of mechanotransduction and their functions in macrophage phenotypic and functional change is largely missing, highlighting the need for studying mechanosensory molecules such as ion channels. The present study focuses on the role of a specific mechanosensitive ion channel, Transient Receptor Potential Vanilloid 4 (TRPV4), in the regulation of macrophage mediated inflammatory responses. Given its emerging role in inflammatory diseases like fibrosis, arthritis, foreign body response (FBR), TRPV4’s contribution to macrophage behavior in inflammation is of growing interest. Employing cellular imaging and molecular biology techniques such as Ca2+ influx assays, immunohistochemistry, immunoblotting, and single nuclei RNA sequencing we delineate mechanisms by which biomechanical stimuli-mediated activation of TRPV4 affects macrophage function. We elucidate TRPV4’s role in macrophage mechanotransduction, providing a mechanistic understanding of inflammatory disease pathophysiology which could lead to the development of potential therapeutics for disease intervention.Item EXAMINING INTERACTIONS AMONG FUNGAL INFECTION, SLEEP, AND HOST DEFENSE IN POPULATIONS OF WORLDWIDE, SLEEP INBRED PANEL, AND MUTANT DROSOPHILA MELANOGASTER(2024) Nan, Mintong; St. Leger, Raymond; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Previous studies with mutant Drosophila melanogaster lines and the Drosophila Genetic Reference Panel (DGRP) reveal substantial variation in susceptibility to infection with Metarhizium anisopliae strain Ma549. These differences likely arise from variations in immunity, physiology, and behavior, yet the role of selection pressures in maintaining these disparities remains understudied. Using global Drosophila populations, the Sleep Inbred Panel (SIP), and sleep-deprived mutants, we investigated the interactions among fungal infection, sleep, and host defense. Testing 14,957 male and 15,287 female flies from 43 lines across 28 locations, we found that resistance to Ma549 correlates with latitudinal gradients in sleep duration, temperature, and humidity. Tropical populations may exhibit stronger defenses due to fungal diversity; however, the most disease-resistant males were also more susceptible to desiccation, indicating trade-offs between abiotic stress and disease resistance. Longer-sleeping males and virgin flies survived infections longer, and increased daytime sleep post-infection was particularly protective in resistant flies. These findings suggest that sleep and disease resistance are interrelated traits possibly shaped by clinal evolution. Using 10,917 males and 11,166 females selected for extreme long or short nighttime sleep duration, we found that short-sleepers, despite having fragmented sleep when healthy, outlived long-sleepers after Ma549 infection. Resistance differences were sex-specific: males were more resistant among short-sleepers, while females showed higher resistance among long-sleepers. Daytime sleep bout numbers correlated with dimorphic disease resistance. Additionally, Ma549 infection increased daytime sleep in both short- and long-sleepers, with short-sleeping males nearly matching long-sleepers in sleep duration post-infection. Virgins, regardless of sleep status, slept more and survived infection longer, indicating that sleep traits, sex, and mating status are closely linked to disease resistance. The study highlights that circadian rhythms influence sleep and immunity, with the sleep-deprived Shmns mutant failing to undergo sickness sleep and succumbing quickly to infection. Mutants with disrupted circadian rhythms (PER and CLK) also showed impaired sickness sleep; however, only the per gene offered protection against disease, while the Clk mutant had increased survival. Independent of mutant status, males slept more than females, and virgins slept more than mated flies, emphasizing the significance of circadian rhythms in sleep and disease resistance.Item Roles of Female Sex Hormones in Regulating Neisseria gonorrhoeae Colonization of the Human Cervix(2024) Di Benigno, Sofia; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) is a human-exclusive pathogen that infects the genital tract. Gonococcal infection may present with or without symptoms and can lead to a variety of serious sequelae if left untreated, especially in female patients. Despite this, there are few models that can effectively mimic GC infection in the female reproductive tract (FRT); of these, even fewer consider the impact of the menstrual cycle, an important feature of the FRT, on GC infection. I used the human cervical tissue explant model previously developed in our lab, which can recapitulate GC infection in vivo. Tissue explants were treated with the sex hormones estradiol and progesterone to mimic various stages of the menstrual cycle and examine its impact on GC infectivity. Estradiol was used to mimic the late proliferative phase, and a combination of estradiol and progesterone was used to mimic the middle of the secretory phase. The effects of hormones on GC infectivity were examined after 72 total hours of hormone treatment and 24 hours of inoculation with GC of strain MS11. My results show that treatment with estradiol and with a combination of estradiol and progesterone both increase the level of GC colonization on the endocervix, but not on the ectocervix, compared to controls that were not treated with hormones. However, the hormone treatment did not affect GC penetration of the cervical epithelium. Both hormone treatments increased the number of GC colonies on the endocervical epithelium, and a combination of estradiol and progesterone produced an additional population of large GC colonies, leading to an increase in the average colony size. These increases in colony number and size were not associated with an increase in the expression of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which are the host receptors for GC Opa proteins. In contrast, treatment with estradiol induced a redistribution of CEACAMs from the luminal surface to the inside of epithelial cells. Additionally, estradiol altered the morphology of endocervical epithelial cells from columnar to cuboidal, but the integrity of cell-cell junctions was unchanged. The increase in colonization under high estradiol conditions was correlated with a decrease in levels of certain pro-inflammatory cytokines and chemokines, but this decrease was not sufficient to fully explain the increase in colonization. Next, I investigated the impact of cervical mucus on GC infectivity and interactions, as gel-forming mucin MUC5B but not MUC5AC increases with estradiol at the proliferation phase. Under both hormone treatment conditions, GC were able to establish close interaction with the luminal surface of the endocervical epithelial cells, displacing membrane-spanning mucin MUC1 in the membrane. Furthermore, GC were able to diffuse through an artificial mucin hydrogel and diffused more efficiently through a MUC5AC-dominant than a MUC5B-dominant hydrogel. Gel-forming mucins collected from cervical tissue explants enhanced GC aggregation in vitro, even at very low concentrations. However, mucins collected from estradiol-treated tissues showed less impact on GC aggregation than those collected from untreated tissues or tissues treated with both estradiol and progesterone. MUC5B and MUC5AC purified from cows and pigs also increase GC aggregation in vitro with GC aggregating more in a MUC5AC- than a MUC5B-dominant mucin mixture. Taken together, my research reveals for the first time that female sex hormones regulate GC colonization at the human cervix by changing the composition of the cervical mucus, providing a mechanism of hormonal regulation underlying the varying susceptibility of female patients to mucosal GC.Item Cell Population Shifts and Clinical Heterogeneity in Sjögren's Disease(2024) Pranzatelli, Thomas J; Johnson, Philip L.F.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sjögren's disease (SjD) is a systemic autoimmune disease that causes loss of function of the salivary and lacrimal glands. Those with the disease, overwhelmingly female with an onset of disease in the fourth or fifth decade of life, commonly suffer from dry mouth, cavities and damage to the eyes. Patients present with a wide variety of clinical phenotypes, with variation in degree of immune infiltration and glandular damage as well as positivity for autoantibodies. This thesis uncovers the changes in cell population and gene expression in the gland that underpin diversity in disease severity. SjD patients lose the majority of a specific epithelial population in their labial salivary glands and, as the number of immune infiltrates grows the surviving members of this population can be found colocalizing with invading GZMK+ T cells and expressing markers of increased proliferation. Standard differential gene expression analysis highlighted gene markers of cell types changing in proportion with disease; an unenlightening result when the cell population changes are well-characterized. To avoid this pitfall an ensemble of random forests was trained to find genes predictive of patient subtypes without being correlated with diagnosis. Genes with high importance for autoantibody positivity were enriched for GO terms related to antigen processing and presentation. A master regulator of salivary gland identity, ZBTB7B, was identified from chromatin accessibility data. Mice with this transcription factor knocked out lose salivary flow and develop pockets of tissue in their glands that resemble other glands, eg., labial gland epithelium inside of parotid glands. This work supports a clinical presentation-specific approach to therapy and paves the path for reengineering the glands to correct the effects of disease.Item TRANSLATION, REPLICATION AND TRANSCRIPTOMICS OF THE SIMPLEST PLUS-STRAND RNA PLANT VIRUSES(2024) Johnson, Philip Zhao; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plus (+)-strand RNA viruses are among the most common pathogens of plants and animals. Furthermore, they present model systems for the study of basic biological processes, including protein translation and RNA replication, and shed light on the versatile roles that RNA structures play in these processes. After cell entry, the next step in the (+)-strand RNA viral life cycle is translation of the viral genome to produce the viral RNA-dependent RNA polymerase (RdRp) and associated replication proteins necessary for viral replication to occur. For many (+)-strand RNA viruses lacking a 5´cap and 3´ poly(A) tail, translation depends upon RNA structural elements within their genomes capable of hijacking the host translation machinery, which for plant viruses are commonly located in their 3´ proximal regions and are termed 3´ cap-independent translation enhancer (CITE) elements. In Chapter 2, I report upon my work characterizing a new subclass of panicum mosaic virus-like translation enhancer (PTE) elements, which bind and co-opt for viral use the host translation initiation factor 4E (eIF4E) – the translation initiation factor normally responsible for binding and recognition of mRNA 5´caps during canonical eukaryotic translation initiation. Thus, PTE 3´CITEs present a novel mechanism for co-opting the critical host factor eIF4E. My work characterizing a new subclass of PTE 3´CITEs further revealed characteristics common among all PTE 3´CITEs pertaining to their mechanism of binding eIF4E.After translation of the necessary viral replication proteins, replication of the viral RNA occurs, which again is in large part mediated by RNA structural elements within the viral genome that can bind to the viral RdRp and/or host factors involved in viral replication. Indeed, RNA structural elements often serve dual roles in viral translation and replication and/or are located proximal to RNA structural elements involved in the alternate function. In Chapter 3, I discuss my work characterizing novel replication elements in the 3´ terminal regions of umbraviruses (family Tombusviridae). The uncovered replication elements appear to be specific to umbraviruses and are located immediately upstream of replication/translation elements that are common throughout the Tombusviridae, lending greater complexity to the already complex 3´ proximal structures of umbraviruses. While the study of (+)-strand RNA viruses has historically focused on their protein-coding transcripts, (+)-strand RNA viruses also commonly produce additional non-coding transcripts, including recombinant defective RNAs, typically containing 5´ and 3´ co-terminal viral genome segments, and (+/-)-foldback RNAs, composed of complementary (+)- and (-)-strand viral sequences joined together. Long non-coding RNAs that accumulate to high levels have also been reported for plant and animal (+)-strand RNA viruses in recent years, and truncations of viral transcripts also commonly arise due to host nuclease activity and/or premature termination of replication elongation by the viral RdRp. The rise of long-read high-throughput sequencing technologies such as nanopore sequencing presents an opportunity to fully map the complexity of (+)-strand RNA viral transcriptomes. In Chapter 4, I present my work performing this analysis, employing direct RNA nanopore sequencing, in which the transcripts present in an RNA sample of interest are directly sequenced. This analysis revealed for the umbra-like virus citrus yellow vein-associated virus (CY1): (i) three novel 5´ co-terminal long non-coding RNAs; (ii) D-RNA population dynamics; (iii) a common 3´ terminal truncation of 61 nt among (+)-strand viral transcripts; (iv) missing 3´ terminal CCC-OH motif in virtually all (-)-strand reads; (v) major timepoint- and tissue-specific differences; and (vi) an abundance of (+/-)-foldback RNAs at later infection timepoints in leaf tissues. This work also sheds light on the current shortcomings of direct RNA nanopore sequencing as a technique. Finally, the importance of RNA structural biology in the study of (+)-strand RNA viruses presents the need for specialized RNA structure drawing software with functionality to easily control the layout of nucleobases, edit base-pairs, and annotate/color the nucleobases and bonds in a drawing. It is through the visual exploration of RNA structures that RNA biologists routinely improve upon the outputs of RNA structure prediction programs and perform crucial phylogenetic analyses among related RNA structures. Large RNA structures, such as whole viral genomes thousands of nucleotides long, can only be studied in their entirety with the aid of RNA structure visualization tools. To this end, I have developed over the course of my doctoral education the 2D RNA structure drawing application RNAcanvas, which is available as a web app and has grown popular among the RNA biology community. RNAcanvas emphasizes graphical mouse-based interaction with RNA structure drawings and has special functionality well suited for the drawing and exploration of large RNA structures, such as automatic layout adjustment and maintenance, complementary sequence highlighting, motif finding, and performance optimizations. Large viral structures such as that of the 2.7 kb CY1 genomic RNA could not have been characterized without the aid of RNAcanvas. In Chapter 5, I present my work developing RNAcanvas.Item Becoming a Father: Disentangling the Causes and Consequences of Caregiving Behavior in California Mouse Males(2024) Colt, Maria; Carleton, Karen L; Fisher, Heidi S; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In mammals, parental care is critical to offspring survival, however, it can also lead to measurable changes in parents. In the biparental California mouse, Peromyscus californicus, fathers actively care for their offspring, including grooming, huddling, retrieving their pups, and building nests. When fathers engage in caregiving behavior, they also experience increased memory, decreased anxiety-like behavior, and structural neuroplasticity of the hippocampus. However, the trigger, reproduction or caregiving experience, and the molecular pathways that regulate these behavioral and neurological changes, remain unclear. In the first chapter, I compared caregiving behaviors in fathers who have cared for their pups and pup-sensitized non-fathers who have cared for unrelated pups, and I found that pup-sensitized non-fathers were slower to approach pups but eventually spent more time grooming pups, whereas fathers spent more time nest-building. I then compared recognition learning, anxiety-like behaviors, and reproductive investment in fathers, pup-sensitized non-fathers, non-fathers with no caregiving experience, and virgins that were socially housed but had no caregiving experience. I found that experienced fathers exhibited increased recognition memory and decreased anxiety-like behavior compared to virgins and non-fathers, and that virgins had smaller testes and fewer sperm compared to non-fathers yet that first-time fathers had larger testes compared to non-fathers. In the second chapter, I first show that hippocampal dendritic spine density is positively associated with males’ caregiving experience. Then, I compared hippocampal gene expression in fathers, non-fathers, and pup-sensitized non-fathers and performed gene ontology, network, and pathway analyses to identify suites of RNA expression patterns associated with caregiving experience. I found that fathers exhibit an upregulation of genes associated with neurogenesis, glutamatergic synapses, neuronal signaling, cellular components of dendritic spines, and some biological pathways previously linked to maternal care, such as regulation of actin cytoskeleton. Together, my results suggest that caregiving behavior induces important behavioral, structural, and transcriptional changes in the brains of males, even if they are caring for offspring that are not their own.Item COORDINATED TRAFFICKING OF HEME TRANSPORTERS BY CARGO SORTING COMPLEXES IS ESSENTIAL FOR ORGANISMAL HEME HOMEOSTASIS(2025) Dutt, Sohini; Hamza, Iqbal IH; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biologicalfunctions and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. Unlike other metazoans, Caenorhabditis elegans cannot synthesize heme but requires heme for sustenance. Thus, C. elegans is an ideal animal model to identify heme trafficking pathways as it permits organismal heme homeostasis to be directly manipulated by controlling environmental heme. Heme is imported apically into the intestine by HRG-1-related permeases and exported basolaterally by MRP-5/ABCC5 to extra- intestinal tissues. Loss of mrp-5 causes embryonic lethality that can be suppressed by dietary heme supplementation raising the possibility that MRP-5-independent heme export pathways must exist. Here we show, by performing a forward genetic screen in mrp-5 null mutants, that loss of the vesicular cargo sorting Adaptor Protein complexes (AP-3) fully rescues mrp-5 lethality and restores heme homeostasis. Remarkably, intestinal heme accumulation due to mrp-5-deficiency causes a concomitant deficit in the lysosomal heme importer HRG-1 abundance and localization. Loss of both MRP-5 and AP-3 subunits resurrects HRG-1 levels and localization, thus underscoring the crucial role of HRG-1 in dictating mrp-5 mutant phenotypes. In the absence of MRP-5, heme is exported by SLC49A3 homolog, a previously uncharacterized transporter. Live- cell imaging reveals vesicular coalescence that facilitates heme transfer between the importers and exporters at the interface of lysosomal-related organelle. These results define a mechanistic model for metazoan heme trafficking and identifies SLC49A3 as a promising candidate for heme export in mammals.