2020-
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Browsing 2020- by Author "Abdo, Zaid, committee member"
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Item Open Access A GPU accelerated RNA-RNA interaction program(Colorado State University. Libraries, 2021) Gildemaster, Brandon, author; Rajopadhye, Sanjay, advisor; Chitsaz, Hamidreza, committee member; Abdo, Zaid, committee memberRNA-RNA interaction (RRI) is important in processes like gene regulation, and is known to play roles in diseases including cancer and Alzheimer's. Large RRI computations run for days, weeks or even months, because the algorithms have time and space complexity of, respectively, O(N3M3) and O(N2M2), for sequences length N and M, and there is a need for high-throughput RRI tools. GPU parallelization of such algorithms is a challenge. We first show that the most computationally expensive part of base pair maximization (BPM) algorithms comprises O(N3) instances of upper banded tropical matrix products. We develop the first GPU library for this attaining close to theoretical machine peak (TMP). We next optimize other (fifth degree polynomial) terms in the computation and develop the first GPU implementation of the complete BPMax algorithm. We attain 12% of GPU TMP, a significant speedup over the original parallel CPU implementation, which attains less than 1% of CPU TMP. We also perform a large scale study of three small viral RNAs, hypothesized to be relevant to COVID-19.Item Open Access Computational tools to identify correlates of vaccine-induced protection against tuberculosis(Colorado State University. Libraries, 2021) Fox, Amy, author; Henao-Tamayo, Marcela, advisor; Anderson, Brooke, advisor; Abdo, Zaid, committee member; Fosdick, Bailey, committee memberTuberculosis is a significant threat to human health. While the BCG vaccine exists to protect children from disseminated forms of tuberculosis, it fails to protect against pulmonary tuberculosis. Thus, a better vaccine is needed. However, the immune system in response to tuberculosis and the BCG vaccine is incompletely understood. We sought to develop novel analysis methods to help understand the immune system. This dissertation describes an analysis tool, cyto-feature engineering, that rapidly identifies flow cytometry immune cell populations utilizing experimental controls. The tool was corroborated through testing the pipeline on different types of flow cytometry datasets. Cyto-feature engineering was then utilized to understand the immune response to two immunomodulatory drugs—losartan and propranolol—when used in conjunction with the BCG vaccine. This study identified an increase in T cell responses due to drug administration, but ultimately failed to decrease bacterial burden in the lung and spleen. Other studies employed a new method for identifying immune cells correlated with various metabolites in the context of tuberculosis. The method can be utilized to generate hypotheses from secondary data sources and gain new biological insight. Using this method, we identified a potential correlation between CD45RA and arachidonic acid metabolism which could serve as a potential target for future vaccination studies. The research outlined in this dissertation will hopefully lead to better immunological analyses of data and the development of a better tuberculosis vaccine.Item Open Access Design and evaluation of an instrumented microfluidic organotypic device and sensor module for organ-on-a-chip applications(Colorado State University. Libraries, 2020) Richardson, Alec Evan, author; Henry, Charles, advisor; Tobet, Stuart, advisor; Bark, David, committee member; Abdo, Zaid, committee memberOrgan and tissue-on-a-chip technologies are powerful tools for drug discovery and disease modeling, yet many of these systems rely heavily on in vitro cell culture to create reductionist models of tissues and organs. Therefore, Organ-on-chip devices recapitulate some tissue functions and are useful for high-throughput screening but fail to capture the richness of cellular interactions of tissues in vivo because they lack the cellular diversity and complex architecture of native tissue. This thesis describes the design and testing of 1) a microfluidic organotypic device (MOD) for culture of murine intestinal tissue and 2) a microfluidic sensor module to be implemented inline with the MOD for real-time sensing of analytes and metabolites. The MOD houses full-thickness murine intestinal tissue, including muscular, neural, immune, and epithelial components. We used the MOD system to maintain murine intestinal explants for 72 h ex vivo. Explants cultured in the MOD formed a barrier between independent fluidic channels perfused with media, which is critical to recapitulating intestinal barrier function in vivo. We also established differential oxygen concentrations in the fluidic channels and showed that more bacteria were present on the tissue's mucosal surface when exposed to near-anoxic media. The sensor module is a reversibly sealed microfluidic device with magnetic connections that can withstand high backpressures. Further, electrodes housed in commercial finger-tight fittings were integrated into the sensor module in a plug-and-play format. Future work will include developing electrochemical/optical sensors for various biological compounds relevant to intestinal physiology. Ultimately, the MOD and sensor module will be implemented in long-term microbiome studies to elucidate the relationship among microbial, epithelial, neuro and immune components of the gut wall in health and disease.Item Open Access Development of Lactobacillus acidophilus as an oral vaccine vector and effects of rice bran ingestion on the mucosal health of Malian infants(Colorado State University. Libraries, 2020) Vilander, Allison C., author; Dean, Gregg, advisor; Abdo, Zaid, committee member; Dow, Steven, committee member; MacNeill, Amy, committee member; Ryan, Elizabeth, committee memberMost pathogens enter the body at the mucosa and induce innate and adaptive immune responses at these surfaces essential for protection against infection and disease. Induction of mucosal immune responses is best achieved locally but mucosal vaccines have been difficult to develop with few currently approved for use. Almost all are attenuated live vaccines which limits their use and efficacy in some populations. Strategies to enhance the mucosal immune response to vaccination and move away from attenuated live vaccines are needed. Prebiotics (nondigestible food ingredients that promotes growth of beneficial microorganisms) and probiotics (live microorganisms that are beneficial when ingested) are an active area of interest for improving mucosal health and increasing oral vaccine performance. Here we present the development of the probiotic Gram-positive lactic acid bacteria Lactobacillus acidophilus (LA) as a novel oral subunit vaccine. LA has many advantages as an oral vaccine vector including endogenous acid and bile resistance, heat tolerance, and numerous proteins that interact with the mucosal immune system. We show that LA can induce immune responses to weakly immunogenic neutralizing peptides from HIV-1 and rotavirus. To enhance the immune response, we developed the E. coli type I pilus protein, FimH, as a LA vaccine adjuvant. FimH increased the immune response to vaccination and increased LA trafficking by antigen presenting cells to the mesenteric lymph node, an important site of mucosal immune induction. We also evaluate the effects of ingestion of the nutrient dense prebiotic rice bran on mucosal health in a cohort of healthy Malian infants at risk for malnutrition and the subclinical condition environmental enteric dysfunction. Rice bran ingestion was found to decrease episodes of diarrhea, decrease the age to elevated fecal microbiome α-diversity, and stabilize total fecal secretory IgA concentrations over time. These results indicate that rice bran protects from diarrhea and improves the mucosal environment.Item Open Access Exploration of novel antimicrobials and comparative genomic analysis for the control of foodborne pathogens(Colorado State University. Libraries, 2020) Jia, Mo, author; Yang, Hua, advisor; Belk, Keith, committee member; Martin, Jennifer, committee member; Abdo, Zaid, committee memberTo view the abstract, please see the full text of the document.Item Open Access From BCG vaccination routes to lung and gut microbiota: avenues to tackle Mycobacterium tuberculosis infection(Colorado State University. Libraries, 2021) Silva-Angulo, Fabiola, author; Henao-Tamayo, Marcela, advisor; Weir, Tiffany, committee member; Abdo, Zaid, committee member; Izzo, Angelo, committee memberTuberculosis is an infectious lung disease responsible for approximately 1.4 million human deaths, world-wide every year. The causal agent of tuberculosis, Mycobacterium tuberculosis (M. tuberculosis), has been estimated to latently infect one-third of the human population. Currently, the BCG vaccine, a live attenuated strain of Mycobacterium bovis, is the only vaccine available to control the disease. Although the BCG vaccine has been the most widely administered worldwide and has been used for more than 100 years, tuberculosis dissemination remains uncontrolled and highly prevalent, especially in developing countries. Several questions about the effect that local microbiota and the administration route of BCG vaccination make on tuberculosis immunopathogenesis remain unanswered. These questions are critical for developing new approaches to control the disease. BCG vaccination is administered intradermally, however, some studies have suggested that BCG vaccination efficacy may be dependent on the administration route. Vaccination through the natural route of M. tuberculosis infection and a combination of other routes have been studied in animal models with varying results. Currently, the analysis of vaccination through the natural infection site is an attractive approach to priming innate immunity. The first study of this thesis examined the immune response induced after BCG vaccination using different routes (aerosol, subcutaneous, intravenous, and intranasal) in C57BL/6 mic and their response to pulmonary M. tuberculosis infection. The study was focused on specific markers of both CD4+ and CD8+ T cells. Our data suggested differences in the adaptive immune response based on the route of BCG vaccination and mainly elicited by CD4+ T cell immune response, with the intranasal delivery the most effective in decreasing the growth of M. tuberculosis in lungs. Another crucial question is the effect of M. tuberculosis infection and BCG vaccination on the structure, diversity, and potential function of the host lung and gut microbiota. Thus, the objective for the second study of this thesis was to characterize the effect of BCG vaccination and M. tuberculosis infection on the lung and gut micro- and mycobiota of C57BL/6 mice. Results indicated that BCG vaccination and M. tuberculosis infection in mice altered the relative lung abundance of Firmicutes and Bacteroidetes phyla compared to the control non-vaccinated, non-infected group. Lung diversity was most affected after M. tuberculosis infection. A multivariate regression approach was used to compare the profile evolution of gut and lung microbiota. More genera had modified relative abundances associated with BCG vaccination status at the gut level compared with lung. Conversely, genera with modified relative abundances associated with M. tuberculosis infection were numerous at lung level, and indicated that the local host response against infection impacted the whole microbial flora while the immune response after vaccination modified mainly the gut microbiota. This study demonstrated that parenteral vaccination with a live attenuated microorganism induced both lung and gut dysbiosis, which may play a crucial role in the immune response to M. tuberculosis infection.Item Open Access Investigation of RelBE1 toxin-antitoxin function in the carbon-dependent metabolic adaptation of Mycobacterium tuberculosis(Colorado State University. Libraries, 2022) Starkey, Julie M., author; Slayden, Richard, advisor; Dobos, Karen, committee member; Abdo, Zaid, committee member; Tjalkens, Ron, committee memberTuberculosis (TB) is a devastating disease with suboptimal treatment regimens and a single vaccine with variable efficacy. Reducing the global burden of TB requires a refined arsenal of methods to prevent and treat the disease, which necessitates a better understanding of M. tuberculosis (Mtb) pathogenesis during infection. Mtb undergoes continuous metabolic reprogramming throughout acute and chronic stages of infection in order to survive and persist harsh host conditions, and the regulatory network responsible for mediating metabolic adaptation has not been fully defined. Mtb harbors at least 88 Toxin-antitoxin (TA) loci that have been proposed to function as regulatory modules in response to stress. TA systems are uniquely abundant in Mtb, making them viable targets for the treatment of both active and latent infection. Several RelBE TA systems are present in Mtb, and the RelE toxins function as ribonucleases to inhibit translation when not bound to RelB antitoxins. The genes encoding relBE1 are adjacent to a gene that encodes an enzyme involved in central carbon metabolism, which could suggest a regulatory role for RelBE1 in carbon metabolism. We aimed to explore the relationship between the RelBE1 TA system and carbon-mediated metabolic adaptation. This work incorporated in vitro transcriptional and genetic studies under defined carbon sources to investigate the activity of RelBE1 and the requirement of RelE1 in Mtb metabolism, growth, and viability in the presence of different carbon sources. We observed transcriptional and physiological trends consistent with the hypothesis that RelBE1 contributes to iii adaptation of Mtb metabolism in the presence of cholesterol and oleate. Additionally, we found evidence that supports the necessity of RelE1 in Mtb metabolism under conditions depleted of nutrients. To investigate if multiple RelBE systems work redundantly or cooperatively in Mtb metabolic adaptation, we applied CRISPRi to simultaneously silence three RelBE TA loci. CRISPRi construction of knockdown mutants resulted in variable success but did not fully resolve the question regarding the cooperative or redundant functions of RelBE systems in Mtb metabolism. Nonetheless, the study provided the building blocks for efficient genetic manipulation of multiple TA systems in Mtb that are essential for exploring the coordination of TA systems in their contribution to Mtb pathogenesis. This thesis work contributes to the debate regarding TA system function in Mtb stress response and adaptation during infection. Given the limitations of the presented studies, further work is warranted to elucidate the relationship between TA systems and Mtb pathogenesis. Expanding our understanding of TA systems in TB disease would provide novel avenues in research to improve treatments against TB.Item Open Access Lophodermella needle cast pathosystem: the phylogenetic relationships, host-mycobiota interactions, and molecular diagnosis of Lophodermella pathogens on Pinus(Colorado State University. Libraries, 2022) Ata, Jessa Pude, author; Stewart, Jane E., advisor; Abdo, Zaid, committee member; Kim, Mee-Sook, committee member; Mondo, Stephen J., committee member; Norton, Andrew P., committee memberThe impact of needle diseases in conifer stands has increased worldwide due to regional variations of warmer and wetter climates that spur the activity of needle pathogens. Heavy needle cast infection results in loss of growth among pine stands which can lead to losses in biomass production and decline in ecosystem goods and services. Despite this threat, a well-informed disease management strategy is lacking due to limited research on many needle pathogens that remain to have unclear taxonomy, uncharacterized fungal biology, and unknown trophic lifestyles and interactions. Thus, this research applied molecular tools to understand conifer needle pathosystems, particularly Lophodermella needle casts that have caused epidemics on Pinus contorta stands in Colorado, USA. Specifically, this research aims to analyze the phylogeny of Lophodermella species using molecular data and identify shared derived characters for taxa delimitation; investigate the interaction of the mycobiota and the P. contorta host in healthy versus diseased states; and develop molecular tools for the rapid diagnosis of Lophodermella needle cast. To achieve these objectives, this research is divided into five chapters. The first chapter gives an overview of the emerging needle diseases worldwide and the needle cast epidemics on P. contorta in Colorado caused by Lophodermella concolor and L. montivaga. It discusses current knowledge on the Lophodermella pathogens and management strategies for needle diseases. The second chapter highlights the relationship of Lophodermella species from North America (L. arcuata, L. concolor and L. montivaga) and Europe (L. sulcigena and L. conjuncta), and their potential synapomorphic characters. It also revealed a newly identified, genetically unique rhytismataceous species on Pinus flexilis that is morphologically similar to L. arcuata. The third chapter discusses the adverse impact of the diseases to needle mycobiota and the defense strategies of the P. contorta host. It further shows, for the first time, the endophytic lifestyle of Lophodermella pathogens on P. contorta. The fourth chapter details the efficiency of the PCR- based markers developed from multi-copy and single-copy gene regions to identify and detect L. concolor and L. montivaga on P. contorta, and L. arcuata and Bifusella linearis on P. flexilis. And lastly, the fifth chapter summarizes the important results of this research and discusses their potential implications on the management of emerging needle diseases. My dissertation closes with recommendations on future research that will address further questions of needle diseases caused by Lophodermella species and other pathogens.Item Embargo The epitranscriptome in heat-loving Archaea enhances thermophily(Colorado State University. Libraries, 2023) Fluke, Kristin Alison, author; Santangelo, Thomas J., advisor; Wilusz, Carol, committee member; Sloan, Daniel, committee member; Abdo, Zaid, committee member>170 RNA modifications are known to decorate the transcriptome across all three Domains of life. The totality of RNA modifications in a cell is called the epitranscriptome. Modifications expand the form and function of RNA, often invoking new structures, activities, and interactions. The molecular consequences, fitness impacts, transcriptome-wide distribution, and genesis of the vast majority of modifications are largely unknown, but more > 100 human diseases are linked to mutations in the genes that encode RNA modifying enzymes. It is therefore critical to elucidate the generation and impact of RNA modifications on fitness and function. 5-methylcytidine (m5C) is one of the most abundant and conserved modifications across Domains and is generated through the post-transcriptional activities of several RNA m5C methyltransferases (R5CMTs). RNA modifications, especially m5C, have largely been studied in the context of abundant rRNA and tRNAs while research into the impact of mRNA modifications is lacking due to their low abundance in the cell. Archaeal model organisms have been shown to incorporate a higher abundance of select modifications compared to Eukarya, proving a new avenue to resolve fundamental questions regarding the phenotypic consequences of epitranscriptomic changes. In the model hyperthermophilic archaeon, Thermococcus kodakarensis, I comprehensively mapped m5C to the transcriptome. I identified at least five R5CMTs that site-specifically generate m5C and showed an unprecedented level of m5C incorporation that includes 10% of unique transcripts, mainly in mRNA. I demonstrated that R5CMTs target mRNAs for modification with both sequence and structural specificity. Cells lacking m5C exhibit a severe temperature dependent growth defect, indicating the m5C epitranscriptome is critical for cellular fitness under heat stress. The extensive m5C epitranscriptome coupled with the large collection of R5CMTs indicate that T. kodakarensis is the ideal model system to pursue fundamental questions regarding the epitranscriptome. Efforts to identify RNA methyltransferases that install m5C led to the discovery of a novel modification, N4,N4-dimethylcytidine (m42C) and the enzyme responsible for its in vivo and in vitro installation. I showed that m42C is robustly resistant to bisulfite-driven deamination, potentially indicating that all bisulfite-sequencing datasets may be falsely reporting m5C sites that are instead occupied by m42C. I mapped a single m42C residue to the ribosomal decoding center in the 16S rRNA and showed that cells lacking m42C exhibit a severe growth defect at higher temperatures. Structural studies of the enzyme that generates m42C, tentatively named m42C synthase, demonstrate it adopts a canonical class I Rossman fold at the C-terminal lobe and a unique N-terminal lobe. I showed that m42C synthase methylates assembled ribosomes and defined the catalytic amino acid residue. Taken together, I report a novel writer enzyme and show that both m5C and m42C promote hyperthermophilic growth. The dense and chemically diverse epitranscriptome argues that Thermococcus provides an excellent model system for further epitranscriptomic studies that probe the impact of both ubiquitous and rare modifications on core biological processes.