UMD Theses and Dissertations
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Item SYSTEMS IMMUNOLOGY OF IMMUNE IMPRINTS INDUCED BY ACUTE VIRAL INFECTIONS(2023) Liu, Can; Johnson, Philip L.F.; Tsang, John S.; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Upon encountering perturbations such as viral infections, the immune system initiates a cascade of molecular and cellular responses. These alterations may persist even after recovery, resulting in enhanced or diminished response to subsequent stimuli compared to the naïve state. Such persistent changes, referred to as immune imprints or long-term non-specific memory, indicate an incomplete resolution from immunological perturbations. The primary focus of this dissertation is to systemically investigate the immune imprints resulting from acute infections and how they shape the baseline immune status to future heterologous challenges.First, we employed cutting-edge single-cell multi-omics and computational approaches to assess the immune response during the COVID-19 disease course and severity correlates at an unprecedented resolution. We identified gene expression profiles – apoptosis in plasmacytoid dendritic cells and IL-15-linked increase of fatty acid (FA) metabolism in CD56dimCD16hi NK cells – as primary correlates of disease severity. This increase of FA signature with disease severity was also concomitant with an attenuated inflammation, indicating a dysfunctional or exhaustion-like state of these NK cells. While the depressed inflammation signature in severe patients was also found in different cell types near hospitalization, it increased temporally at later time points, indicating a critical late-stage juncture in the disease course. Next, we took the opportunity of the period following the first wave of COVID-19 pandemic to study immune imprints in human cohorts who had recovered from COVID-19 before widespread vaccination and reinfection occurred. We demonstrated that individuals who recovered from mild COVID-19, exhibit distinct immune signatures through single-cell transcriptomic profiling. Male recoverees also showed heightened responses to the seasonal influenza vaccine compared to healthy individuals without a history of COVID-19 and female recoverees. These sex dimorphic imprints highlight the interplay between intrinsic factors like sex and non-intrinsic factors such as prior SARS-CoV-2 infection, in shaping an individual's immune system over time. Lastly, we also investigated the immune imprints after acute viral infection using a controlled experimental mouse model of influenza infection. After examining cellular and gene expression profiles in various organs after the infection, we found persistent changes in both adaptive and innate immune components across multiple organs. Moreover, these changes affected subsequent local IL-17 inflammatory response and secondary heterologous vaccinations in anatomically distinct organs. Together, both human and mouse studies here are important pieces toward an improved understanding of long-term immune imprints after perturbations, which can be leveraged to develop more effective and personalized vaccines and disease treatments.Item PROBING BIOPHYSICAL INTERACTIONS TO UNDERSTAND VIRAL DIFFUSION AND PARTICLE FATE IN THE AIRWAY MUCOSAL BARRIER(2023) Kaler, Logan; Duncan, Gregg A; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The mucus barrier in the airway is the first line of defense against inhaled particulates and pathogens. Within the mucus barrier, large, heavily glycosylated gel-forming mucin proteins form a network to trap particles for removal. Influenza A virus (IAV) must first cross the mucus barrier before reaching the underlying airway epithelial cells to cause infection. On the IAV envelope, hemagglutinin (HA) binds sialic acid on the surface of the cell to initiate viral entry. However, HA preferentially binds sialic acid attached to galactose by either an ⍺2,3 or ⍺2,6 linkage. In addition to the cell surface, sialic acid is found on mucins and is thought to act as a decoy receptor to entrap the IAV within the mucus layer. However, neuraminidase (NA) on the envelope of IAV cleaves the bond between HA and sialic acid, releasing the virus. While the mechanism of IAV infection has been characterized, the interplay between mucus biophysical properties and the binding of IAV within the mucus network prior to infection requires further investigation. The overall objective of this dissertation is to understand how IAV moves through the mucosal barrier to subsequently cause infection. We hypothesize the structural features of the mucus gel network are responsible for the changes in IAV movement, rather than the binding and unbinding of the virus. To investigate this, we first analyzed the movement of IAV in ex vivo mucus from human endotracheal tubes. In order to further analyze this movement, we developed a novel analysis to calculate the dissociation constant of IAV-mucus binding in a 3D gel network environment. Using this data, we established a pipeline for estimating the passage of particles, including IAV, through the airway mucosal barrier. A machine learning-based trajectory analysis was employed to classify individual trajectories in order to calculate the percentage of particles able to cross the mucus barrier within a physiologically relevant time frame. Lastly, we investigated the effect of sialic acid binding preference on diffusion of IAV through mucus collected from different in vitro human airway epithelial cell cultures. The combined results of these studies confirmed our hypothesis that the mucus microstructure rather than the adhesive interactions of IAV to the mucins was responsible for the differences in IAV diffusion. This work provides further insight into role of the mucosal barrier in IAV infection and identifies the mucus gel network microstructure as a target for the development of therapeutics against IAV.Item Modeling Syndromic Surveillance and Outbreaks in Subpopulations(2020) Pettie, Christa; Herrmann, Jeffrey; Reliability Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This research is motivated by the need to assist resource limited communities by enhancing the use of syndromic surveillance (SyS) systems and data. Public health agencies and academic researchers have developed and implemented SyS systems as a pattern recognition tool to detect a potential disease outbreak using pre-diagnostic data. SyS systems collect data from multiple types of sources: absenteeism records, over the counter medicine sales, chief complaints, web queries, and more. It could be expensive, however, to gather data from every available source; subsequently, gathering information about only some subpopulations may be a desirable option. This raises questions about the differences between subpopulation behavior and which subpopulations’ data would give the earliest, most accurate warning of a disease outbreak. To investigate the feasibility of using subpopulation data, this research will gather and organize SyS data by subpopulation (separated by population characteristics such as age or location) and identify how well the SyS data correlates to the real world disease progression. This research will study SyS how reports of Influenza-like-illness (ILI) in subpopulations represent the disease behavior. The first step of the research process is to understand how SyS is used in environments with varying levels of resources and what gaps are present in SyS modeling techniques. Various modeling techniques and applications are assessed, specifically the Susceptible Infected Recovered “SIR” model and associated modifications of that model. Through data analysis, well correlated subpopulations will be identified and compared to actual disease behavior and SyS data sets. A model referred to as ModSySIR will be presented that uses real world community data ideal for ease of use and implementation in a resource limited community. The highest level research objective is to provide a potential data analysis method and modeling approach to inform decision making for health departments using SyS systems that rely on fewer resources.Item TRACKING ACUTE RESPIRATORY INFEECTIONS IN A COLLEGE RESIDENT COMMUNITY(2018) Adenaiye, Oluwasanmi; Milton, Donald K; Public and Community Health; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Influenza and other acute respiratory infections (ARIs) contribute significantly to human morbidity and mortality globally. Animal experiments and human challenge studies have not provided an adequate explanation about the relative importance of social, behavioral and physical environment in the transmission of ARIs and are limited due to uncertainty about the generalizability of their findings to a natural infection. Also, household transmission studies seldom characterize all potential transmission covariates e.g. environmental conditions, leaving a gap in the knowledge of transmission mechanisms. Here, we describe the design and preliminary results of an extensive college dormitory ARI transmission study that has the potential to characterize several important ARI transmission covariates; we critically appraise the design and show how the findings from such design can be applied to answer most of the vital questions that exist about the transmission of ARIs.Item Enhancing gene delivery to the mammalian nucleus for applications in viral reverse genetics and human artificial chromosome development.(2017) Brown, David; Dinman, Jonathan D; Glass, John I; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Delivery of transgenic DNA into mammalian cells is critical to realizing the potential of synthetic biology in advancing gene therapy, construction of entire chromosomes and production of new vaccines and therapeutics in cultured mammalian cells. New synthetic biology techniques such as rapid, inexpensive DNA synthesis have opened the door to engineering biology. However, now the delivery of these synthetic DNA constructs to the nucleus of a living cell is the limiting step in the development of these applications. Living cells possess numerous cellular barriers that a synthetic DNA construct needs to cross to be successfully expressed. In this dissertation, I explore two methods to enhance DNA delivery across the nuclear membrane barrier. First, a plasmid delivery system was developed involving a papillomavirus scaffolding protein that when expressed by the transfected cell line, more consistently delivered plasmids bearing a specific DNA binding site to the nucleus of mammalian cells. This technique enabled us to produce infectious influenza virus more effectively when transfecting mammalian cells with DNA copies of influenza virus genes. These improvements accelerate production of vaccine against influenza virus. Second, we improved an existing method of transferring large DNA molecules cloned in Saccharomyces cerevisiae into cultured cells through polyethylene glycol mediated fusion of the yeast and cultured cells. Creating a reporter yeast strain allowed us to track the percentage of fused cells and the percentage that achieved YCp delivery allowing us to easily optimize the process. By synchronizing recipient cells in mitosis when the nuclear envelope is broken down we increased the delivery efficiency of large YCps ten-fold. This was accomplished by fusing yeast spheroplasts harboring large YCps (up to 1.1 Mb) with cultured cell lines. A statistical design of experiments approach was employed to further boost the vector delivery rate 300-fold to achieve a YCp delivery rate of 1/840 cells. This method was adapted to deliver a 152-kb herpes simplex virus genome cloned in yeast into mammalian cells to produce infectious virus. Finally, we discuss future applications for this technology including the development of human artificial chromosomes and applications in viral reverse genetics for vaccine development.Item THE VIRAL GENOMICS REVOLUTION: BIG DATA APPROACHES TO BASIC VIRAL RESEARCH, SURVEILLANCE, AND VACCINE DEVELOPMENT(2015) Schobel, Seth A.; Cummings, Michael P; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Since the decoding of the first RNA virus in 1976, the field of viral genomics has exploded, first through the use of Sanger sequencing technologies and later with the use next-generation sequencing approaches. With the development of these sequencing technologies, viral genomics has entered an era of big data. New challenges for analyzing these data are now apparent. Here, we describe novel methods to extend the current capabilities of viral comparative genomics. Through the use of antigenic distancing techniques, we have examined the relationship between the antigenic phenotype and the genetic content of influenza virus to establish a more systematic approach to viral surveillance and vaccine selection. Distancing of Antigenicity by Sequence-based Hierarchical Clustering (DASH) was developed and used to perform a retrospective analysis of 22 influenza seasons. Our methods produced vaccine candidates identical to or with a high concordance of antigenic similarity with those selected by the WHO. In a second effort, we have developed VirComp and OrionPlot: two independent yet related tools. These tools first generate gene-based genome constellations, or genotypes, of viral genomes, and second create visualizations of the resultant genome constellations. VirComp utilizes sequence-clustering techniques to infer genome constellations and prepares genome constellation data matrices for visualization with OrionPlot. OrionPlot is a java application for tailoring genome constellation figures for publication. OrionPlot allows for color selection of gene cluster assignments, customized box sizes to enable the visualization of gene comparisons based on sequence length, and label coloring. We have provided five analyses designed as vignettes to illustrate the utility of our tools for performing viral comparative genomic analyses. Study three focused on the analysis of respiratory syncytial virus (RSV) genomes circulating during the 2012- 2013 RSV season. We discovered a correlation between a recent tandem duplication within the G gene of RSV-A and a decrease in severity of infection. Our data suggests that this duplication is associated with a higher infection rate in female infants than is generally observed. Through these studies, we have extended the state of the art of genotype analysis, phenotype/genotype studies and established correlations between clinical metadata and RSV sequence data.Item Going Viral: Internet and Social Media Based Surveillance Systems for Detecting Influenza Activity in Maryland(2015) Bowen, Lisa; Gold, Robert S; Epidemiology and Biostatistics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Influenza surveillance is essential for detecting and managing outbreaks. The Maryland Department of Health and Mental Hygiene (DHMH) currently includes the number of emergency room and physician visits for influenza-like-illness (ILI) to track flu activity. Recently, internet and social media based surveillance methods have emerged as useful in detecting outbreaks. This study aims to determine if internet and social media based surveillance methods are useful in monitoring ILI in Maryland through assessing how Google Flu Trends (GFT) and tweets compare to portions of DHMH’s formal reporting system. Innovations of this study include using symptom based keywords and incorporating a variety of sources of surveillance data. Results show tweets had a strong positive correlation with all other surveillance sources, Pearson’s correlation coefficients ranged from 0.62-0.68. GFT were more highly correlated with DHMH data. Further research should investigate automating collection of tweets, application to other diseases, and standardized methods for location determination.Item Zoonotic Transmission of Influenza H9 subtype through Reassortment(2013) Kimble, James Brian; Perez, Daniel R; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Influenza A virus causes disease across a broad host range including avian and mammalian species. Most influenza viruses are found in wild aquatic birds, are of low consequence and refrain from zoonotic transmission. However, some strains occasionally cross the species barrier, into domestic birds and a plethora of mammalian species, most notably swine and humans. Many of these infections are dead ends and quickly disappear from the species, but occasionally, a stable lineage is established and becomes endemic in an animal population. Avian Influenza virus (AIV) H9N2 was predominantly found in wild ducks and shore birds across the globe with occasional infections in turkeys until the late 1980's, at which point the virus became established in Eurasian poultry populations. In the late 1990's the virus again jumped hosts, first into swine, and then into humans. Across many regions, these viruses appear to be gaining human-like virus characteristics. Here, the influenza receptor distribution in a range of poultry species has been characterized and shown that many of the birds were able to bind human-like binding viruses. While no large-scale H9N2 human infections have occurred, the threat is there. The most likely route for this to occur is through reassortment with human viruses. The 2009 human pandemic H1N1 (pH1N1) is a likely candidate as it is found in multiple species and seems to readily reassort. The two viruses were shown to be compatible for reassortment and H9:pH1N1 viruses would readily infect and transmit in both ferrets (a human model animal) and swine. Finally, a novel method of modeling reassortment in vivo was developed, which simultaneously tests the breadth of possible reassortant and utilizes natural host selective pressure to select the most-fit progeny. Furthermore, the characterization of these viruses in ferrets showed they readily infect, efficiently transmit, and exhibit mild to moderate pathological consequences. Taken together, these findings broaden our understanding of natural observations, characterize the potential for zoonosis, highlight the dangers H9 viruses may pose to humans, and give scientists a new tool to deepen our understanding of reassortment.Item A Difference in Heterosubtypic Immunity Induced by a Modified Live Attenuated Avian Influenza Backbone in Mice and Ferrets(2011) Hickman, Danielle; Perez, Daniel R; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The unprecedented emergence of multiple avian influenza virus (AIV) subtypes with a broad host range poses a major challenge in the design of vaccination strategies that are effective against multiple subtypes of influenza. The present study focused on the protective effects of a modified AIV as a backbone for epidemic and pandemic influenza. In addition, the ability of this backbone to induce heterosubtypic immunity (Het-I) was also analyzed. Het-I is the ability of one influenza subtype to protect against a different influenza subtype. Previously, a live attenuated AIV with the internal backbone of A/guinea fowl/Hong Kong/WF10/99 (H9N2) (WF10), called WF10att, protected chickens against a lethal influenza challenge. To characterize the WF10att backbone as a master donor strain and determine its ability to induce Het-I, we evaluated its protective efficacy in mice and ferrets. Vaccinated mice were protected against homologous challenge with A/WSN/1933 (H1N1) (WSN), mouse-adapted A/California/04/2009 (pH1N1) and A/Vietnam/1203/2004 (H5N1) (HPAI H5N1) viruses, and ferrets survived homologous challenge with HPAI H5N1. H7N2att vaccinated mice were protected against both H1N1 and HPAI H5N1 challenge; however, Het-I was observed in H9N2att vaccinated ferrets challenged with HPAI H5N1. We found that both B and T cells are involved in the Het-I induced by our WF10att backbone. Cross-reactive non-neutralizing antibodies to viral proteins were detected. JhD-/- mice, which lack mature B-lymphocytes, were vaccinated with the recombinant vaccines and challenged with HPAI H5N1. None of the vaccinated mice survived challenge further suggesting a role for Het-I. In addition, cells isolated from the lungs of H7N2att vaccinated mice had cross-reactive antibody-secreting cells targeted to HPAI H5N1. Together, these results suggest a role for B cells in Het-I. Although B cells are important, T cells may also play a role in Het-I. Both IFN-γ and Granzyme B secreting cells were detected in lung and spleen cells isolated from H7N2att vaccinated mice and stimulated with HPAI H5N1 suggesting a role for T cells in Het-I. The ability of our WF10att backbone to induce Het-I depends on the surface glycoproteins expressed and the challenge virus subtype. In addition, WF10att uses both B and T cells to induce Het-I.Item Phylogenetic analysis of swine influenza viruses isolated from humans in Alma-Ata, Kazakhstan(2009) Padmanabhan, Rangarajan; Perez, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Continuous surveillance of influenza becomes important considering the economic, epidemic and pandemic implications of influenza infections. This study details phylogenetic & molecular analysis of the genes of four swine influenza viruses isolated from humans in Alma-Ata, Kazakhstan. Phylogenetic analysis placed the eight segments of the four viruses in the classical H1N1 swine clade, along with the isolate A/sw/Jamesburg/1942, except for the HA of A/Alma-Ata/32/98, which was placed in the human H1N1 lineage, along with the isolate A/WS/1933. On amino acid analysis, the viruses displayed mutations on HA and ribonucleoproteins which putatively disrupt antigenic recognition of the virus by the host immune system. The presence of these viruses relatively unchanged for 6 decades after their initial isolation could be speculated to be a combination of laboratory leaks in southern USSR in 1980s, low divergence of classical H1N1 viruses in pigs, and the low population density of Kazakhstan.