River ecosystems across the US and globally face numerous stressors that impact both ecological function and water quality. In 2015-16, municipalities along the Dan River in southern Virginia experienced repeated taste and odor (TandO) issues in their drinking water that originated from the river source water. Given that the source of TandO issues during these events were unknown, this research aimed to identify patterns in the distributions of river microorganisms that could help identify potential biological causes. Monthly water, sediment, and periphyton samples were collected for a full year from the Smith and Dan Rivers to quantify actinomycete, fungi, and chlorophyll a concentrations, which have historically been linked to TandO problems, and to characterize changes in microbial community structure. Although no significant TandO event occurred during the study period, the work produced unique and valuable data that describe patterns of microbial populations and communities in a river ecosystem. Results from the study show the abundances of actinomycetes, fungi, and chlorophyll a expressing seasonal and regional variation by habitat. From a broader ecological perspective, microbial communities sampled from water, sediment, and periphyton were each unique from each other regardless of river reach and season sampled. Overall, this research adds to our understanding of river ecology by detailing the microbial abundance and diversity in three river habitats, including periphyton, that can be used to predict sources of river TandO in future events, and offers new questions regarding how microbial diversity changes over space and time. / Master of Science / In 2015-16, cities along the Dan River in Virginia experienced multiple taste and odor (T&O) events that led to earthy and musty odors in drinking water. As part of a larger project looking at a range of possible chemical and biological sources of T&O, this research aimed to identify changes in abundance of river microorganisms that might indicate potential biological causes to T&O events. Monthly samples of water, sediment, and algal growth were collected for a year from 12 sites on the Smith and Dan Rivers. Samples were analyzed for abundances of three known T&O causing groups of organisms—actinomycetes, fungi, and photoautotrophs—and to characterize changes in total microbial communities as an indicator of ecological change occurring along the rivers. Although a significant T&O event did not occur during the study period, the research produced valuable descriptions of how important microorganisms change in a freshwater ecosystem. Actinomycetes elevated in the lower Dan River during fall, fungi elevated during the spring, and chlorophyll a was highest in the upper Smith River during winter, suggesting that photoautotrophic growth was more likely to be linked to previous T&O events. The diversity and makeup of the microbial communities in the rivers was primarily dependent on where they were growing (water, sediment, or periphyton) and secondarily on the season or the river reach. Combined, these results will help to identify causes of future T&O events in the Dan River and also provide new insights into ecological patterns of microorganisms in river ecosystems.

Systems biology is an interdisciplinary field that focuses on elucidating complex biological processes (systems) by investigating the interactions among its components through an iterative cycle composed of data generation, data analysis and mathematical modeling. Our contributions to systems biology revolve around the following two axes: - Data analysis: Two data analysis projects, which were initiated when I was a co-op at GlaxoSmithKline, are discussed in this thesis. First, next generation sequencing data generated for a phase I clinical trial is analyzed to determine the altered microbial community in human gut before and after antibiotic usage (Chapter 2). To our knowledge, there have not been similar comparative studies in humans on the impacts on the gut microbiome of an antibiotic when administered by different modes. Second, publicly available gene expression data is analyzed to investigate human immune response to tuberculosis (TB) infection (Chapter 3). The novel feature of this study is systematic drug repositioning for the prevention, control and treatment of TB using the Connectivity map. - Mathematical modeling: Polynomial dynamical systems, a state- and time- discrete logical modeling framework, is used to model two biological processes. First, a denitrification pathway in Pseudomonas aeruginosa is modeled to shed light on the reason of greenhouse gas nitrous oxide accumulation (Chapter 4). It is the first mathematical model of denitrification that can predict the effect of phosphate on the denitrification performance of this bacterium. Second, an iron homeostasis pathway linked to iron utilization, oxidative stress response and oncogenic pathways is constructed to investigate how normal breast cells become cancerous (Chapter 5). To date, our intracellular model is the only expanded core iron model that can capture a breast cancer phenotype by overexpression and knockout simulations. / Ph. D.

Ischemic heart disease (IHD) is a leading cause of global mortality. Emerging evidence suggests a potential role of the gut microbiome in IHD development. The present study explored the association between alpha diversity, representing microbial richness and evenness, and IHD, as well as the relationship between specific gut microbial species and IHD risk. The study uses a longitudinal design with data from the two cohorts within the Swedish Infrastructure for Medical Population-based Life-course and Environmental Research (SIMPLER). Survival analysis is employed to calculate the hazard rate for all associations. Because of thecompositionality of the gut microbiome species-level data a centered log-ratio (CLR) transformation is applied before the main analysis. We find no associations between alpha diversity and risk of IHD in the age and sex-adjusted model as well as in the main model adjusted for age, sex, smoking, education, physical activity, hypertension, high cholesterol, and intakes of fish, red and processed meat, fermented dairy, fruit and vegetables and alcohol. Five species are found to be statistically significantly associated with IHD in the main model before correction for multiple testing, which is statistically non-significant after the correction.

Honeybees (Apis mellifera L.) are the major insect pollinators of many different crops. A drastic decline in the honey bee populations has been reported over the past decade. While many factors have contributed to this decline, pesticides, poor nutrition, and Varroa mites are the most common concerns noted by scientists and beekeepers. Aside from direct toxicity from pesticides, it has been observed that sublethal pesticide doses have effects on honey bee physiology and behavior such as oxidative stress, disruption of foraging and homing, and changes to honey bee neurophysiology. The primary objectives of this project were to examine honey bee gut microbiome, physiology, and pesticide exposure along an agricultural intensification gradient and to examine the interactive impacts of pesticide exposure and poor nutrition on honey bees in a controlled laboratory cage setting. Sixteen honey bee colonies were placed in four locations across Mississippi with varying degrees of natural forage availability.

Emerging clinical evidence implicates cellular senescence in the pathogenesis of various inflammatory conditions including inflammatory bowel diseases (IBDs), demonstrating that the intestinal stem cell crypts of patients with early Crohn’s disease exhibit markers positive for cell cycle inhibitor proteins. This phenomenon coupled with chronic systemic inflammation, a term coined “inflammaging," triggers many age-related pathologies and accelerates mortality. Our research evaluates the efficacy of interventions that target these death-resistant senescent cells to improve overall health and vitality. Particularly, we investigated the effects of Fisetin, a potent flavanoid with senolytic properties, in a dextran sodium sulfate (DSS) induced mouse model of colitis. Our findings reveal that Fisetin significantly inhibits senescence and inflammation in the colon while simultaneously enhancing the relative abundance of beneficial microbes, especially Akkermansia muciniphila, showcasing its potential for managing IBDs. Additionally, given the profound restoration of the microbiome and the central role of resident microbes in the production of metabolites essential for facilitating immunomodulation, we extended our investigations to further explore the effects of fecal microbiota transplant (FMT) from long-living Ames dwarf mice, characterized by low inflammatory status, into normal mice. Our results show notable shifts in microbial diversity, indicating that FMT may combat dysbiosis, a precursor to several conditions, including autoimmune, metabolic, and neurodegenerative diseases. Lastly, our exploration of potential anti-aging pharmacological interventions including Metformin (MF) and Trodusquemine (MSI-1436) during the postnatal window has demonstrated robust transcriptomic alterations of key biomarkers in the GH/Igf1 axis, such as Pi3k, Akt, and Mtor, suggesting delayed aging and improved liver function in young mice. These epigenetic changes underscore that early-life pharmacological interventions may forestall the onset of age-related metabolic disorders. All in all, there remains an urgent need for breakthroughs that can enhance healthspan to ensure that the rapidly growing population of older adults enjoys life in these extended years

Antimicrobial resistance poses a significant challenge to human health and is also a pressing One Health concern. The routine use of antibiotics as growth promoters in agricultural animals has contributed to the emergence of antibiotic resistance, which can subsequently affect human populations. Discontinuing this practice has led to a surge in infections and therapeutic antibiotic use in these animals. This increased susceptibility to infections may be linked, at least partially, to the loss of colonization resistance resulting from alterations in the microbiome. This study focuses on poultry, as the consumption of chicken meat can introduce antibiotic-resistant microbes into the human population. The overarching hypothesis for this research project is that a rationally designed consortium of microbes sourced from healthy chickens will increase colonization resistance and decrease susceptibility to infections as an alternative to growth-promoting antibiotics. The first goal was to analyze the broiler chicken’s gut microbiome and to establish a comprehensive culture collection of microorganisms from healthy chickens. Culture-enriched and culture-independent 16S sequencing was applied to assess the cultivability of the samples and to analyze their microbial profiles. Isolates were identified using MALDI-TOF and 16S rRNA gene sequencing. Frozen samples (from antibiotic-free farms) had a greater microbial diversity than fresh samples (from a university research facility). However, a greater proportion of the microbiome was recovered by culture from the fresh compared to the frozen samples. A strain collection of 1121 isolates representing 121 species was constructed. In Aim 2, I carried out a functional screen to identify isolates from the culture collection that inhibited the growth of the predominant poultry pathogens, E. coli and C. perfringens. Several isolates were identified that inhibited one or the other pathogens and a small number of isolates killed both pathogens. These microbes form the basis of therapeutic consortia to increase colonization resistance in chickens. / Thesis / Master of Science (MSc) / In the poultry industry, antibiotics have been used to promote chicken’s growth. This has contributed to the spread of antibiotic resistance to animal/human pathogens. When the use of growth-promoting antibiotics is stopped, the chickens become more susceptible to infections. These chickens have possibly lost protective bacteria that help fight pathogens. I thought that bacteria from healthy chicken’s intestine could help fight pathogens. To do this, I isolated a large collection of chicken gut’s good bacteria from healthy birds after individually separating them from the mixture using growing methods and sequencing. I separated bacteria from frozen and fresh mixtures, found that more bacteria grow from fresh mixtures. I then tested individual bacteria from this collection to see if they stop pathogenic bacteria like E. coli and C. perfringens from growing. I found that many bacteria could do this which may be used to develop a therapeutic community of good bugs to colonize chickens to make them more disease resistant.

Research on adolescent drinking shows that younger people are at greater risk of developing behavioral deficiencies that can be detrimental to their social relationships and health over time. Recent research has shown that changes within the microbiome-gut-brain axis (MGB) can affect social behavior. These changes involve microbiota populations that influence addictive behaviors after prolonged substance intake through neurochemical shifts that extend through the nervous, circulatory, and immune system. Using Massey's biosocial model, I aim to expand on the relationship between the MGB axis, social behavior, and adolescent alcohol use disorder through a meta-theoretical approach. I explore the strengths and shortcomings of Massey's biosocial model of segregation and stratification and its use of the allostatic load model, telomere length, and gene exposure to develop a stronger theoretical concept using the micro-gut-brain axis as a conceptual foundation. Can the MGB-axis model be used to identify potential pathways in which alcohol use disorder (AUD) persists from adolescence to adulthood? I find that adolescent drinking leads to changes in microbiota populations that are known to influence AUDs and increases the development of diseases such as liver disease and its effects on social behavior. The MGB axis can help us understand the effects of substance and dietary habits on disease and illness by connecting life science knowledge and sociological perspectives. With this modern application of cognitive sociology, I have shown that future research on addictive behaviors should consider the application of biomarker data to further expand on new theoretical and methodological approaches in the study of disease and addiction.

Increased exposure to antibiotics has led to the dissemination of genes conferring resistance to antimicrobial metabolites throughout human microbiomes globally via horizontal gene transfer (HGT). This has resulted in the emergence of new resistant strains leading to a rising epidemic of deaths from previously treatable infections. Evidence suggests that before the age of anthropogenic antibiotic use, microbes living within a community produced antibiotic metabolites and, subsequently, maintained such genes for several useful functions and a balance of diversity in nature. The question of the origin of these resistant genes is difficult to answer, but with continued advancements in ancient genomic analysis, researchers have developed methods of acquiring a more accurate representation of the microbiome associated with our human ancestors by extracting fossilized microbial specimens from dental calculus and directly sequencing the metagenomes. This thesis outlines the production of taxonomic and functional profiles of 20 different human and non-human oral microbiome samples using metagenomics tools originally developed for living individuals, altered for use with ancient microbial specimens. Putative antimicrobial resistant (AMR) genes derived from these profiles were reconstructed and conserved functional regions were identified. From the data that is available regarding the human microbiome from a range of time points throughout history dating back to Neanderthal specimens, it is possible to elucidate relationships between these AMR genes and to better understand the evolutionary trajectory of antibiotic resistance.

There is increasing evidence indicating that exposure to air pollutants may be associated with the onset of several respiratory diseases such as allergic airway disease and chronic obstructive pulmonary disorder (COPD). Many lung diseases demonstrate an outgrowth of pathogenic bacteria belonging to the Proteobacteria phylum, and the incidence of occurrence of these diseases is higher in heavily polluted regions. Within the human body, the lungs are among the first to be exposed to the harmful effects of inhaled pollutants and microbes. Research in the past few decades have expounded on the air-pollution-induced local and systemic inflammatory responses, but the involvement of the lung microbial communities has not yet been well-characterized. Lungs were historically considered to be sterile, but recent advances have demonstrated that the lower respiratory tract is replete with a wide variety of microorganisms - both in health and disease. Recent studies show that these lung microbes may play a significant role in modulating the immune environment by inducing IgA and mucus production. Air pollutants have previously been shown to alter intestinal bacterial populations that increase susceptibility to inflammatory diseases; however, to date, the effects of traffic-generated air pollutants on the resident microbial communities on the lungs have not been explored. The microbiome is influenced by several factors, including diet and environmental exposures. A large percentage of the Western world population consumes a high-fat (HF) diet which has resulted in the epidemic of obesity. Consumption of an HF diet has been shown to alter the intestinal microflora and increase baseline inflammation. We aimed to understand whether diet might also contribute to the alteration of the commensal lung microbiome, either alone or related to exposure. Thus, we investigated the hypothesis that exposure to air pollutants can alter the commensal lung microbiota, thereby promoting alterations in the lung's immune and inflammatory responses; in addition to determining whether these outcomes are exacerbated by a high fat-diet. We performed two studies with exposures to different components of air pollutant mixtures on C57Bl/6 mice placed on either a control (LF) diet or a high-fat (HF) diet. Our first exposure study was performed on C57Bl/6 mice with a mixture of gasoline and diesel engine emissions (ME: 30 µg PM/m3 gasoline engine emissions + 70 µg PM/m3 diesel engine emissions) or filtered air (FA) for 6h/d, 7 d/wk for 30 days. The ME study investigated the alterations in immunoglobulin A (IgA), IgG and IgM, and lung microbiota abundance and diversity. Our results revealed ME exposures alongside the HF diet causes a decrease in IgA and IgG when compared to FA controls, thereby decreasing airway barrier protection. This was accompanied by the expansion of bacteria within the Proteobacteria phylum and a decrease in the overall bacterial diversity and richness in the exposed vs. control groups. In our second study, we exposed C57Bl/6 mice to only the diesel exhaust particle component (35µg DEP, suspended in 35µl 0.9% sterile saline) or sterile saline only (control) twice a week for 30 days. We investigated immunoglobulin profiles by ELISA that revealed a significant increase in IgA and IgG in response to DEP. We also observed an increase in inflammatory tumor necrosis factor (TNF) - α, Interleukin (IL) -10, Toll-like receptors (TLR) - 2,4, nuclear factor kappa B (NF-κB) histologically and by RT-qPCR. Mucus production and collagen deposition within the lungs were also significantly elevated with DEP exposures. Microbial abundance determined quantitatively from the bronchoalveolar lavage fluid (BALF) by qPCR revealed an expansion of bacteria belonging to the Proteobacteria phylum in the DEP exposed groups on the HF diet. We also observed an increase in reactive oxygen and nitrogen species (ROS-RNS) products (nitrates), within the groups that revealed an expansion of Proteobacteria. These observations are most likely due to the unique metabolic capabilities of Proteobacteria to proliferate in inflammatory environments with excess nitrates. We assessed if treatments with probiotics could attenuate the DEP-induced inflammation by supplementing a separate group of study animals on the HF diet with 0.3 g/day of Winclove Ecologic® Barrier probiotics in their drinking water throughout the study. With probiotic treatments, we observed a significant decrease in ROS-RNS that was accompanied by complete elimination of Proteobacteria suggesting that in the absence of nitrates, the expansion of Proteobacteria is curbed effectively. We also observed a decrease in proinflammatory TNF-α and collagen deposition with probiotic treatments, and an increase in IgA levels within the BALF, suggesting that probiotics aid in balancing proinflammatory responses and enhance beneficial immune responses to efficiently mediate the DEP-induced inflammation. Both studies showed that air pollutants alter the immune defenses and contribute to lung microbial alterations with an expansion of Proteobacteria. The immunoglobulin profiles discordant between the two studies can be explained by the route and/or duration and composition of air pollutant exposure. Collectively these studies suggest that exposure to air pollutants alter immune responses and/or increase the availability of inflammatory by-products within the lungs that can enable the selective outgrowth of pathogenic bacteria. The observed detrimental outcomes are further exacerbated when coupled with the consumption of an HF diet. Importantly, these results may shed light on the missing link between air pollution-induced inflammation and bacterial expansion and also point to therapeutic alternatives to curb bacterial outgrowth in lung disease exacerbations observed in patient populations living and/or working in heavily polluted regions.

Viruses, and particularly bacteriophages, are key players in many microbial ecosystems and can profoundly influence the human microbiome and its impact on human health. While the bacterial and archaeal fraction of the human microbiome can now be profiled at an unprecedented resolution via cultivation-free metagenomics, viral metagenomics is still extremely challenging. The lack of universal viral genetic markers limits the de-novo discovery of viral entities, and the low number of available viral reference genomes from cultivation studies does not cover well the phage diversity in human microbiome samples. Viral-like particle (VLP) purification has been proposed as a set of experimental tools to concentrate viruses in samples prior to sequencing, but it remains unclear how efficient and reproducible such tools are in practice. In this thesis we aim to address some of these challenges and better exploit the potential of viral metagenomics in the context of the human microbiome. First, we performed and studied the performance of VLP procedures on freshwater and sediment samples. We found that bacteria can still be abundant at the end of the filtration process, thus lowering the efficiency of the enrichment. Analyzing samples with a low enrichment may lead to inconsistent conclusions, as the residual bacterial contamination might misdirect the computational analysis. To better quantify the extent of non-viral contamination in VLP sequencing, we designed ViromeQC, a novel open-source tool able to assess and rank viromes by their viral purity directly from the raw reads. In ViromeQC, rRNA genes and bacterial single-copy proteins are used as a proxy to estimate non-viral contamination. With the ViromeQC, we conducted the largest meta-analysis on the degree of enrichment of thousands of viral metagenomes, and concluded that the vast majority of them are three-fold less enriched than a standard metagenome. ViromeQC was then used to select the human gut viromes that had the highest enrichment as a starting point for a novel reference-free pipeline for the discovery of previously uncharacterized viral entities. The approach included metagenomic assembly of the enriched viromes as well as extensive mining of many thousands of assembled metagenomes, and led to a catalog of 162,876 sequences of highly-trusted viral origin. Most of these predicted viral sequences had no match against any known virus in RefSeq even though some of them showed a prevalence in gut metagenomes of up to 70%. Our analyses and publicly available tools and resources are helping to uncover the still hidden virome diversity and improve the support for current and future investigations of the human virome.