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Browsing by Author "Abdo, Zaid, committee member"

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    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 member
    RNA-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.
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    Assessment and intervention strategies for agricultural inhalation exposures in occupational and community environments
    (Colorado State University. Libraries, 2024) Erlandson, Grant, author; Schaeffer, Joshua, advisor; Magzamen, Sheryl, committee member; Abdo, Zaid, committee member; Martenies, Sheena, committee member
    Agriculture represents an industry vital to the U.S. economy, supplying the public with nutritious food and providing millions of workers with employment. Also characterized as one of the most hazardous industries for workers, agricultural environments contain a variety of inhalation hazards capable of impacting the health of workers and adjacent community residents. Agricultural inhalation hazards include airborne organic and inorganic dusts; livestock associated gases, pesticides, bacteria, viruses, and antibiotics. This study will focus on (1) bioaerosol exposures in dairy operations and (2) inorganic dust pesticide exposures from agricultural applications. In dairy environments, workers are regularly exposed to high levels of organic dust (bioaerosols) and their inflammatory constituents (e.g., endotoxin, muramic acid, and β-glucans). Dairy bioaerosol exposure is associated with increased prevalence of respiratory disease (e.g., asthma, rhinitis, and chronic obstructive pulmonary disease) in dairy workers. While bioaerosol exposure in dairy environments has been well characterized in previous research, efforts to identify hygienic interventions that control exposure remain unsuccessful. In crop production agriculture, it is well documented that workers are exposed to high levels of pesticides associated with adverse health outcomes (e.g., respiratory and neurologic diseases). Further, in agricultural adjacent community environments, where lower chronic pesticide exposures are found, there is mounting evidence linking adverse health effects (e.g., adult and iii childhood cancers, neurologic and respiratory diseases, and birth outcomes) in residents to exposure from agricultural pesticide applications. However, weak and sometimes inconsistent associations previously reported highlight the limitations of current community exposure assessment techniques used for pesticides. For specific Aim 1, we pilot tested a novel low-cost nasal rinse intervention to modulate airway inflammation in ten bioaerosol exposed dairy workers. Dairy workers were randomly split into treatment (n = 5) and control (n = 5) groups and administered saline nasal rinses before and after their shift for five consecutive days. Treatment group participants received pre-shift hypertonic saline rinses while the control group received normotonic saline rinses. Both received normotonic rinses post-shift. Pro and anti-inflammatory cytokines were measured from recovered saline rinses. Linear mixed model results indicated treatment group participants experienced significantly higher concentrations of anti- (IL-10) and pro-inflammatory cytokines (IL-6 and IL-8) than the control group (p < 0.02, p <0.04, and p < 0.01 respectively). This study demonstrates the capacity of hypertonic saline nasal rinses to successfully upregulate anti-inflammatory cytokine production. However, conflicting upregulation of pro-inflammatory markers cloud interpretations of efficacy. For Aim 2, we further evaluated the immunomodulatory effects of hypertonic saline rinses vs. normotonic saline rinses longitudinally (2-5 shifts) in 45 bioaerosol exposed dairy workers. However, in this aim, treatment group participants received hypertonic rinses pre- and post-shift and 16S sequencing was added to analyses to capture potential washout effects on microbial diversity. No significant differences were observed between group or day for any of the measured markers or microbiome diversity metrics. Yet, non-significant increases in anti-inflammatory IL-10 concentrations across the study period were observed independent of iv treatment group suggesting the rinse itself may be more impactful than tonicity. This study provided mixed but encouraging results that justify further research on nasal rinses as an intervention in bioaerosol exposed dairy workers. For Aim 3, we evaluated the agreement between three exposure assessment techniques used to estimate residential organophosphate (OP) exposure in agricultural adjacent communities located in the Central Valley of California. OP exposure was estimated from household dust samples, California Pesticide Use Report (CPUR) pesticide use modeling, and urinary DAP metabolites across two sampling campaigns. Simple correlation tests revealed moderate correlations (ρs = 0.46) between household dust and use model exposure estimates. Estimates from urinary DAP metabolites exhibited low to no correlation with the other two estimates. Linear mixed model results also indicated no association between urinary DAP metabolites and household dust or use model estimates. This study illustrates a lack of agreement between community pesticide exposure assessment techniques regularly used in research and motivates the development of more robust assessment techniques.
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    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 member
    Tuberculosis 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.
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    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 member
    Organ 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.
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    Development of fluidic devices to facilitate more accessible monitoring of human health
    (Colorado State University. Libraries, 2024) Cherwin, Amanda E., author; Henry, Charles S., advisor; Tobet, Stuart A., advisor; Snow, Christopher, committee member; Abdo, Zaid, committee member
    In December of 2023, the World Health Organization (WHO) Director-General Tedros Adhanom Ghebreyesus outlined the 'Five P's' of global health priorities: Promoting health, Providing health, Protecting health, Powering health, and Performing for health. Despite the mantra of 'prevention is better than cure,' many countries still prioritize treating the sick over proactive health promotion, leading to inadequate prevention of non-communicable diseases (NCDs). Access to healthcare services poses a significant barrier to early recognition and treatment of health issues, particularly in low-income communities. To address these challenges, harnessing the power of science and technology becomes imperative. Powering health involves leveraging scientific research and collaboration to understand disease mechanisms better. Physiologically relevant models, such as microfluidic systems, offer insights into disease progression. Microfluidics, especially when combined with 2D and 3D culture systems, enhances functionality by mimicking physiological conditions. These devices provide cost-effective solutions for diagnostic challenges, bridging the gap between in vitro and in vivo studies. Protecting health requires a deeper understanding of organ systems. Chapter 2 examines a microfluidic model of the gut, an organ that plays a critical role in maintaining overall health. Two devices are discussed, an organotypic device for maintaining ex vivo gut tissue explants, and an electrochemical sensor module for monitoring relevant molecules such as oxygen or hydrogen peroxide within the tissue media. Dysbiosis in the gut microbiome has been linked to various pathologies, emphasizing the need for accurate models for studying gut barrier integrity. Ex vivo models using microfluidic devices offer promising avenues for studying disease mechanisms. The devices described in Chapter 2 serve as an effective model of the intestinal barrier that can be closely monitored in real-time. Providing health involves making effective healthcare solutions universally accessible. Point-of-care (POC) diagnostics, facilitated by microfluidics, enable rapid and cost-effective disease detection. Capillary-driven flow microfluidic devices enhance accessibility by eliminating the need for bulky external pumps, making POC testing feasible even in resource-limited settings. Combining the concepts of Powering and Providing health leads to the development of innovative diagnostic devices. Capillary-driven flow microfluidics enables the development of portable devices for diagnosing conditions from viscous sample matrices like blood and saliva. These devices offer less invasive and more accessible alternatives to traditional diagnostic methods, potentially revolutionizing healthcare delivery. Chapter 3 describes a capillary flow device used to quantify levels of two salivary biomarkers (Galectin-3 and S100A7) correlated to Heart Failure (HF) outcomes. This rapid, noninvasive, accessible POC test can drastically improve the quality of life for HF patients, particularly in rural and resource-limited areas. Using an electrochemical detection method, we demonstrate successful multiplexed detection of both biomarkers in spiked buffer solutions. Chapter 4 focuses on microfluidic devices probing rheological properties of whole blood related to Sickle Cell Disease (SCD) and clotting using capillary flow. For the SCD device, our goal was to develop a low-cost Point-of-Care (POC) multiplexed device for rapid and accurate identification of SCD phenotypes using three key reagents tied to altered sickle cell blood rheology: calcium chloride, sodium metabisulfite, and adenosine diphosphate. We developed an integrated device where whole blood reacts with reagent pads, enabling rapid assessment of a patient's SCD phenotype to inform appropriate treatment. We also introduced the Paper-based Clotting Analysis Test (PCAT) for efficient, low-cost analysis of primary hemostasis. Current methods for monitoring hemostasis are expensive and slow. Our capillary flow device uses whole blood moving at high flow rates for sustained durations to induce thrombus formation. This dissertation bridges the gap between effective health monitoring and accessibility through fluidic devices using either pump-driven or capillary-driven flow. Chapters detail the development of microfluidic systems for monitoring intestinal barrier function, detecting biomarkers in saliva for Heart Failure prognosis, and processing blood samples for Sickle Cell Disease phenotyping and clotting analysis. Ultimately, these devices hold the potential to transform healthcare management, particularly in underserved communities.
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    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 member
    Most 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.
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    Epidemiological investigation of antimicrobial resistance in beef production using metagenomic sequencing
    (Colorado State University. Libraries, 2019) Doster, Enrique, author; Hoover, Edward A., advisor; Morley, Paul S., advisor; Belk, Keith E., committee member; Abdo, Zaid, committee member; Gow, Sheryl P., committee member
    Globally, the emergence of antimicrobial resistance (AMR) resulting in treatment failure is recognized as a growing public health threat. Antimicrobial use practices used in beef production are thought to be a direct driver of increasing antimicrobial resistance in pathogens and the environment, in part due to the higher volumes of antimicrobial drug necessary to treat cattle weighing 10 times more than an average person. This has led policy makers and public health organizations to promote "judicious use" or outright ban of antimicrobial drugs in livestock production. Use of antimicrobials is unavoidable for the treatment of disease and we must therefore learn how we can best adjust our AMD use to reduce selection of AMR pathogens. However, outside of important indicator organisms and pathogens, little is known about how different antimicrobial drug use practices affect communities of microorganisms, or microbiomes, and the AMR gene determinants, or resistome, shared between pathogen and non-pathogens alike. With advances in high-throughput sequencing (HTS), we can perform culture-independent studies and gain a better understanding of how antimicrobial drug use practices in livestock production affect AMR epidemiology. This dissertation consists of five studies that employ HTS to characterize the microbiome and resistome of samples with differing AMD exposure along the beef production line. Projects begin with a look into the short-term effects on the microbiome and resistome of feedlot cattle following treatment with a macrolide drug, tulathromycin, in the manuscript "Investigating Effects of Tulathromycin Metaphylaxis on the Fecal Resistome and Microbiome of Commercial Feedlot Cattle Early in the Feeding Period". Fecal samples collected in this project also were processed with aerobic culture, polymerase chain reaction (PCR), and lateral flow immunoassay for identification of Salmonella enterica and the comparison of these results are presented in "A Cautionary Report for Pathogen Identification Using Shotgun Metagenomics; a Comparison to Aerobic Culture and Polymerase Chain Reaction for Salmonella enterica Identification". Samples collected as part of a longitudinal study in feedlot cattle were analyzed to characterize the associations between AMD use and AMR in two bacterial species. These archived samples are leveraged for a broader understanding of AMR dynamics by adding a community-level perspective to results from aerobic culture. Results in individual cattle are presented in "Antimicrobial Drug Use in Beef Feedlots; Effects on the Microbiome and Resistome Dynamics in Individual Cattle" and results at the pen-level in "Metagenomic Investigation of the Effects of Antimicrobial Drug Use Practices on the Microbiome and Resistome of Beef Feedlot Cattle". Finally, in "Metagenomic Characterization of the Microbiome and Resistome in Retail Ground Beef" we examined the end of the beef production line by comparing the microbiome and resistome of retail ground beef products from either conventional production systems or those labeled as "raised with antibiotics" (RWA). The five studies presented in this dissertation each contribute to the collective understanding of how AMD use in livestock production system can affect the ecology of AMR in microbial communities. These projects are useful first steps in learning to manage AMR in beef production systems; encompassing a targeted look at the use of one type of AMD, characterizing the resistome dynamics in individual cattle and pens over time in a feedlot, a comparison of the resistome in ground beef products, and many other aspects of AMR epidemiology. The final study, describing limits to incorporating HTS for pathogen identification, serves as a cautionary reminder that with new technologies come new challenges and that research must keep pace.
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    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 member
    To view the abstract, please see the full text of the document.
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    Fire-associated shifts in the soil microbiome in western conifer forests: implications for Armillaria root disease biocontrol and management
    (Colorado State University. Libraries, 2024) Fitz Axen, Ada J., author; Stewart, Jane E., advisor; Kim, Mee-Sook, committee member; Charkowski, Amy O., committee member; Abdo, Zaid, committee member
    The research presented in this thesis integrates the current understanding of environmental disturbances, plant associated microbiomes, and microbial biological control of fungal forest pathogens to contribute to improved disease management. In Chapter 2, I examined how fire disturbances affect soil microbial communities in areas where Armillaria root disease, caused by the pathogen Armillaria solidipes, is prevalent by documenting changes to bacterial and fungal community diversity and composition following three distinct levels of burn severity (high, low, and unburned) in a conifer forest in northern Idaho, United States. Expected reductions in bacterial community alpha diversity were observed when comparing burned communities with unburned communities; however, fungal communities showed a lack of significant change in alpha diversity in response to burn severity at the sampling time of 15-months post-fire. However, in both bacterial and fungal soil communities, compositional changes corresponding to burn severity levels were observed. Further examinations characterized similarities and differences between burn severity-associated communities and Armillaria species-associated communities to determine how these microbial changes might influence Armillaria root disease. At high-severity burn sites, colonization by A. solidipes and the associated microbial community was prevalent when compared with low-severity burn and unburned sites. In contrast, the presence and abundance of the weakly pathogenic species A. altimontana and its associated microbial community, including beneficial ectomycorrhizal fungi, appeared to be negatively impacted by high-severity burns. Further research is needed to determine which microbial taxa are critical for promoting or suppressing A. solidipes activity, yet the results from this study suggest that high-severity burns may create environments hospitable to this pathogen and thus monitoring for increased disease pressure following severe burns may be warranted. Chapter 3 focuses specifically on beneficial members of the native soil microbial community that exhibit antagonistic activity against A. solidipes. Because these native species are adapted to the environmental conditions and community interactions, they are more likely than foreign microbial species to successfully establish a stable population required for effective biological control. I isolated putative native biological control agents from soil samples collected under different burn severity conditions and tested their in vitro capabilities to inhibit the growth of A. solidipes with dual culture confrontation tests. Effective in vitro pathogen inhibition was observed with 10 microbial isolates, including five bacterial isolates from the genera Bacillus and Caballeronia and five fungal isolates from the genera Trichoderma and Mortierella. Further examination of the sites these microbes and communities originated from and their compositional changes documented in Chapter 2 revealed that the presence or abundance of our effective biological control organisms did not differ based on burn severity. Importantly, this suggests that fire disturbances may not directly influence the use of these species in management methods for Armillaria root disease in similar conifer forests. However, considering the increased presence of A. solidipes observed following a high-severity burn, there may be additional biotic or abiotic influences apart from biological control agents that are influencing the activity of A. solidipes after fire. These studies enhance our understanding of how abiotic and biotic influences interact to affect the presence of virulent soilborne forest pathogens and associated soil microbes. Considering the effects of these interactions is critical for the development of sustainable long-term management strategies that will help to preserve these ecosystems facing increasing environmental and pathogen-related stressors. The overall goals of this research are to build upon the growing body of research examining how the soil microbiome contributes to disease development and to provide tangible results that can be incorporated by forest managers to help reduce damage caused by Armillaria root disease in fire-prone conifer forests.
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    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 member
    Tuberculosis 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.
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    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 member
    Tuberculosis (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.
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    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 member
    The 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.
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    Pathogens, pulmonary function, and the nasal microbiome of dairy workers
    (Colorado State University. Libraries, 2024) Seidel, James, author; Schaeffer, Joshua, advisor; Magzamen, Sheryl, committee member; Abdo, Zaid, committee member; Valley, Morgan, committee member
    Dairy workers are exposed to bioaerosols that are diverse in both size (0-100 µm in aerodynamic diameter) and inflammatory constituents (e.g. endotoxins, muramic acid, and β-glucans). Bioaerosol exposure at dairies is associated with a higher prevalence of chronic obstructive pulmonary disease (COPD), chronic bronchitis, asthma, respiratory pneumonitis, and asthma-like reductions in pulmonary function. More recently, opportunistic pathogens present at dairies such as the novel influenza D virus (IDV), influenza A (IAV), and livestock-associated Methicillin-resistant Staphylococcus aureus (MRSA) have also been a focus of research, as these pathogens can infect workers and pose a public health risk through community spread. Intrinsic factors such as genetics and childhood exposures likely play a major role in exposure response and respiratory disease pathology, but little research has been focused on the nasal microbiome's role in pathogen exposure and cross-shift changes of pulmonary function. From a longitudinal (2-5 working shifts) cohort of dairy workers in the High Plains Region of the US, this research analyzed pathogens found in the nares of dairy workers via pre- and post-shift nasal lavages. The same nasal lavages underwent targeted 16S rRNA gene sequencing to quantify the bacterial communities that comprise the nasal microbiome. Spirometry was also performed on dairy workers pre- and post-shift to measure cross-shift changes in pulmonary function. Overall, 32.1% (n=237) of nasal lavages tested positive for Methicillin-susceptible Staphylococcus aureus (MSSA), 11.4% tested positive for MRSA, 17.3% for IDV, 2.5% for IAV, and 1.3% for influenzas C virus (ICV). Only 1 of the original 31 participants never tested positive for a pathogen during their workweek. Differences in nasal microbiome characteristics emerged based on pathogen positivity, and differential abundance analysis revealed significant differences in genera based on the positivity of both bacterial and viral pathogens. The dairy workers in this study also experienced decreases in cross-shift pulmonary function. The average decrease in forced expiratory volume in one second (FEV1) over 108 working shift was -74.4 ml, and the average decrease of forced vital capacity (FVC) was -92.5 ml. Significant differences in microbiome characteristics did emerge based on post-shift and cross-shift spirometry performances, and taxonomic differences were noted in participants performing poorly on cross-shift FVC. The nasal microbiomes of workers also underwent community state typing, and participants in CST3 showed the most resilience to cross-shift changes in lung function. This research also investigated the efficacy of a hypertonic saline nasal lavage in improving cross-shift changes in pulmonary function. From a cohort of 44 dairy workers, 22 workers received pre- and post-shift hypertonic saline nasal lavages with an osmotic concentration of 400 milliosmole (mOsm). The 22 participants in the control group received pre- and post-shift normotonic saline (308 mOsm) nasal lavages. Based on constructed mixed linear models, the treatment improved cross-shift outcomes of the forced expiratory flow at 25-75% of the vital capacity (FEF25-75%), but had little effect on FEV1 and FVC. The use of a pre- and post-shift lavage of any osmolarity, however, appeared to reduce the burden of cross-shift pulmonary function decline often experienced by dairy workers. For the first time, this research showed that both viral and bacterial pathogens are present in the nares of US dairy workers. This work also identified the nasal microbiome characteristics that may play a role in pathogen exposure and cross-shift lung function outcomes. The use of a saline nasal lavage as an intervention was also explored, and the intervention appeared to improve cross-shift pulmonary function outcomes.
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    Read alignment using deep neural networks
    (Colorado State University. Libraries, 2019) Shrestha, Akash, author; Chitsaz, Hamidreza, advisor; Ben-Hur, Asa, committee member; Abdo, Zaid, committee member
    Read alignment is the process of mapping short DNA sequences into the reference genome. With the advent of consecutively evolving "next generation" sequencing technologies, the need for sequence alignment tools appeared. Many scientific communities and the companies marketing the sequencing technologies developed a whole spectrum of read aligners/mappers for different error profiles and read length characteristics. Among the most recent successfully marketed sequencing technologies are Oxford Nanopore and PacBio SMRT sequencing, which are considered top players because of their extremely long reads and low cost. However, the reads may contain error up to 20% that are not generally uniformly distributed. To deal with that level of error rate and read length, proximity preserving hashing techniques, such as Minhash and Minimizers, were utilized to quickly map a read to the target region of the reference sequence. Subsequently, a variant of global or local alignment dynamic programming is then used to give the final alignment. In this research work, we train a Deep Neural Network (DNN) to yield a hashing scheme for the highly erroneous long reads, which is deemed superior to Minhash for mapping the reads. We implemented that idea to build a read alignment tool: DNNAligner. We evaluated the performance of our aligner against the popular read aligners in the bioinformatics community currently — minimap2, bwa-mem and graphmap. Our results show that the performance of DNNAligner is comparable to other tools without any code optimization or integration of other advanced features. Moreover, DNN exhibits superior performance in comparison with Minhashon neighborhood classification.
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    The adventures of Lactobacillus acidophilus: evaluating a recombinant probiotic rotavirus vaccine from host and microbial perspectives
    (Colorado State University. Libraries, 2024) Gilfillan, Darby L., author; Vilander, Allison, advisor; Dean, Gregg, advisor; Abdo, Zaid, committee member; Wilkins, Mike, committee member
    Rotavirus is an enteric infection of global importance causing diarrheal-associated illness that can be fatal in young children and the elderly. There is a gap in vaccine efficacy between high- and lower-middle-income countries (LMIC) with LMIC often experiencing diminished vaccine-conferred protection. Rotaviruses, whether attenuated vaccine strains or primary pathogens, do not exist in isolation within the host's gastrointestinal tract. Other actors present within the microbiome can inhibit or augment vaccine efficacy by influencing the vaccine itself or the mucosal immune response. Understanding and exploiting interactions between host and microbe is a promising frontier for mucosal vaccinology. This dissertation will explore the probiotic Lactobacillus acidophilus (LA) as a vaccine platform for a microbiome-minded, next-generation approach to rotavirus immunization. We developed and confirmed a novel recombinant LA (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results demonstrate the potential of rLA platforms to generate protective mucosal immunity. Additionally, metagenomic and metatranscriptomic analyses of exogenous probiotic administration within the murine small intestine revealed differences between LA genome expression and the whole metatranscriptome in recombinant- versus wild-type LA-vaccinated mice. LA genome expression in rLA-vaccinated mice had decreased carbohydrate metabolism and increased stress responses. We also detected antigen and adjuvant transcript expression only in mice exposed to the rLA platform. There was relative enrichment of probiotic species in the wild-type group with overall increased α- and β-diversity in the buffer compared to probiotic groups. These results highlight the interactions between an exogenous probiotic and the host microbiome at an immune inductive site. Finally, we used an in vitro model to evaluate modulation of polyunsaturated fatty acid (PUFA) metabolism on host cell and (r)LA interactions. Both (r)LA and PUFA treatments significantly changed pathogen recognition receptor expression. (r)LA treatment mainly altered inflammatory cytokine expression while PUFA supplementation primarily influenced mucin expression. rLA strains adhered more to host cells than wild-type LA while the rLA strain expressing both antigens and adjuvants may better prevent E. coli adhesion. These results and methodologies provide a starting point for further investigation into PUFA metabolism as a mechanism for improving rLA immunogenicity and competition against other enteric pathogens.
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    The epidemiology and ecology of Escherichia coli O157 on U.S. dairies
    (Colorado State University. Libraries, 2018) Stenkamp-Strahm, Chloe Marie, author; Reynolds, Stephen, advisor; McConnel, Craig, advisor; Magzamen, Sheryl, committee member; Lombard, Jason, committee member; Abdo, Zaid, committee member
    Escherichia coli O157 (O157) is a bacterium that causes human foodborne disease outbreaks worldwide. Beef and dairy cattle are reservoirs for O157, as they harbor the bacteria in their lower gastrointestinal (GI) tracts and shed it in feces without clinical illness. Humans become infected with O157 after contacting cows or manure, or ingesting the bacteria on dairy, meat or produce products. Dairy cattle are a central part of the U.S food supply, providing milk for a multitude of dairy products, and 15-20% of the beef produced. Transmission of O157 from dairy cattle to humans is reduced by techniques that limit bacterial survival after food is harvested (i.e. post-harvest). However, O157 outbreaks occur after post-harvest dairy pasteurization and slaughter laws are applied across the U.S food chain. Due to these outbreaks, an emphasis has been placed on developing methods that reduce O157 presence prior to harvest (i.e pre-harvest) at the dairy farm. An understanding of dairy cow O157 prevalence and magnitude of shedding, and animal-level correlates for shedding, may aid in the development of pre-harvest O157 strategies. We hypothesized that life history features (parity, history of disease, others) would be associated with O157 shedding by adult cows on Colorado dairies, and that shedding in early lactation would be correlated with shedding detected during the pre-weaning period of these dams' calves. Although overall prevalence was low (3.0%) and only one individual shed O157 at a high magnitude (>103 CFU/g feces), a higher number of adult cows shed O157 between June and October. Dams were at increased risk of shedding if they were a lower parity, earlier days in milk, or had a history of antibiotic use. Calf shedding was not detected on the Colorado dairies studied; no correlation between dam and calf O157 shedding was present. We hypothesized that the lack of calf shedding was due to the sampling time-frame, calf management, and the geographic region of study. Using fecal samples collected by the National Animal Health Monitoring System (NAHMS) from dairy calves across the U.S, we estimated the prevalence of O157 shedding and managerial, environmental, and calf-level variables associated with pathogen presence. U.S calf shedding of O157 was low (2.5 %) and not influenced by geographic region. Calves were at increased risk to shed if they received colostrum from their own dam, which suggests that increased time spent with the dam is associated with shedding. Results indicated that the passive transfer status of calves also influenced shedding, but was affected by the temperature and humidity index (THI) calves were exposed to during pre-weaning. Calves experiencing thermoneutral or heat-stress THIs were more likely to shed O157 if they had poor or moderate passive transfer. Calves were unlikely to shed if they had excellent passive transfer, regardless of THI. Herds of cattle likely have uniform levels of O157 exposure, but only some individuals shed the bacteria. We hypothesized that the GI microbial community influenced which cows become colonized with O157 post-ingestion. After measuring microbial communities in naturally infected cows on Colorado dairies, lower microbial richness (i.e. total number of unique species) was associated with intermittent or multi-day shedding of O157. The species Bacillus coagulans was lower in abundance in fecal samples that contained O157, while the genus Moryella spp was higher in abundance. The results of this dissertation highlight factors associated with O157 shedding by dairy cows and calves. This information may be used when developing techniques that reduce transmission between dairy cows, or dissemination of O157 beyond the dairy. Because O157 does not adversely affect cows, the future adoption of O157 mitigation strategies relies on whether or not these approaches benefit the dairy operation. Based on our results, we hypothesize that ill health and cow stress is associated with shedding, but is difficult to measure and monetarily quantify. At the current time, development of reduction strategies should focus on methods that reduce O157 while simultaneously improving cow health and production (e. g reducing stress during cow transition periods, improving passive transfer and limiting dam exposure of calves, feeding probiotics that improve GI health, creating multi-pathogen vaccines). We propose that future studies should also focus on determining whether O157 augments milk production and cow fertility.
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    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.
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    Theory of graph traversal edit distance, extensions, and applications
    (Colorado State University. Libraries, 2019) Ebrahimpour Boroojeny, Ali, author; Chitsaz, Hamidreza, advisor; Ben-Hur, Asa, committee member; Abdo, Zaid, committee member
    Many problems in applied machine learning deal with graphs (also called networks), including social networks, security, web data mining, protein function prediction, and genome informatics. The kernel paradigm beautifully decouples the learning algorithm from the underlying geometric space, which renders graph kernels important for the aforementioned applications. In this paper, we give a new graph kernel which we call graph traversal edit distance (GTED). We introduce the GTED problem and give the first polynomial time algorithm for it. Informally, the graph traversal edit distance is the minimum edit distance between two strings formed by the edge labels of respective Eulerian traversals of the two graphs. Also, GTED is motivated by and provides the first mathematical formalism for sequence co-assembly and de novo variation detection in bioinformatics. We demonstrate that GTED admits a polynomial time algorithm using a linear program in the graph product space that is guaranteed to yield an integer solution. To the best of our knowledge, this is the first approach to this problem. We also give a linear programming relaxation algorithm for a lower bound on GTED. We use GTED as a graph kernel and evaluate it by computing the accuracy of an SVM classifier on a few datasets in the literature. Our results suggest that our kernel outperforms many of the common graph kernels in the tested datasets. As a second set of experiments, we successfully cluster viral genomes using GTED on their assembly graphs obtained from de novo assembly of next-generation sequencing reads. In this project, we also show how to solve the problems of local and semi-global alignment between two graphs. Finally, we suggest an approach for speeding up the computations using pre-assumption on a subset of nodes that have to be paired.
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    Time series analysis of limber pine (Pinus flexilis) health in the U.S. Rocky Mountains in response to white pine blister rust (Cronartium ribicola) and bark beetles
    (Colorado State University. Libraries, 2018) Leddy, K. A., author; Stewart, Jane E., advisor; Abdo, Zaid, committee member; Sloan, Dan, committee member; Schoettle, Anna, committee member; Liber, Howard, committee member
    From 2004-2007, 106 permanent limber pine monitoring plots were established and measured throughout the U.S. Rocky Mountains (MT, WY, CO) to characterize health trends in response to white pine blister rust (WPBR) and bark beetles (including mountain pine beetle, "MPB", and Ips spp., "Ips") over time. These plots were subsequently measured in 2011-2013 and again in 2016-17 to form a time series analysis of limber pine health. Data were gathered on 8,206 monumented trees (4,176 limber pine) and included measurements on various stand, ground cover, and landscape characteristics over the three time intervals. The overall percentage of live trees infected with WPBR was 29.4% in 2004-07 and 25.7% in 2016-17, with incidence decreasing in parts of Wyoming (Pole Mountain, Laramie Peak), increasing in southern Colorado (Sangre de Cristo Mountains), and stable in other subregions. However, of limber pines that were healthy during the first measurement, 22.2% were declining/dying and 21.1% had died by the end of the study period due to WPBR and/or bark beetle damages. Due to this, it is likely that new WPBR infections are occurring as the large number of live, infected trees dying during the survey may have masked newly infected trees in incidence calculations. In heavily WPBR-infected areas such as Pole Mountain, Wyoming, 65% of live trees were infected (in 2004-07), and of trees that began the study as healthy, 23% were declining or dying and 38% had died by the end of the study period (2016-17). Additionally, WPBR severity increased significantly from the beginning of the study with 4 previously uninfected sites gaining WPBR infections, 29 sites advancing to 'moderately infected' and 5 sites becoming 'heavily infected'. The overall average number of cankers per tree (3.5) was stable, but the number of infected limber pine with a canker in the lower 1/3 of the stem (18%) increased significantly (+4.2%, P = 0.001). When examining all limber pine in the study, 8%, 3% and 3% were killed by MPB/Ips., WPBR, and combined effects of these agents, respectively. Of the 887 live, but declining or dying limber pine, 52% had WPBR infections and 38% had damage from twig beetles (Pityophthorus spp., Pityogenes spp.) in 2016-17. Though all sites had ≥ 20% limber pine composition, 34% of sites had no limber pine regeneration and 7% had no regeneration of any tree species over the entirety of the study period. The results of this time series indicate that limber pine populations in the U.S. Rocky Mountains are declining due to effects from WPBR and MPB/Ips. Long-term surveys capture the effects of these damage agents on native tree populations and provide critical guidance for future management and restoration of these ecologically valuable species. Limber pine is at risk due to the various biotic and abiotic agents threatening their health. Thus, future directions involve restorative management practices for highly impacted areas where limber pine is a climax species and proactive management for healthy limber stands to promote resilience to likely damage agents. In highly impacted areas (WPBR incidence, mortality, or bark beetle damage on >50% of trees and low limber pine density and regeneration), where limber pine co-exists with other tree species, it may be favorable to allow the natural succession of other tree species to become dominant. However in xeric, harsh sites where limber pine is a climax species, these highly impacted areas are at-risk for losing all tree cover and should be considered for protective and restorative planting strategies. As natural resistance to WPBR occurs on the landscape, genetic screening and protection of mature limber pine carrying either complete or partial resistance to the pathogen should be pursued to preserve this genetic diversity. A priority should be to protect resistant against bark beetles and fire using established management practices. Additionally, seed-sourcing from resistant trees can allow for resistant progeny to be out-planted into high priority areas, thus buffering stands at risk for high WPBR mortality. Moreover management plans that promote diversification of age and diameter classes within stands can provide resilience against pest and pathogen attacks, as bark beetles vary in diameter preference and WPBR infections tend to cause higher mortality in smaller diameter trees. Lastly in healthy limber pine stands, proactive management of pest impacts to promote stand resilience is recommended as in Schoettle & Sniezko (2007) in order to preserve these healthy populations.