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Previously, the exact phylogenetic relationship between the giant panda (Ailuropoda melanoleuca) and the red panda (Ailurus fulgens) has been unclear, but with the advent of molecular sequencing technologies, these two animals are now known to be distantly related. Although taxonomically distinct, these two panda species have several things in common, primarily their almost exclusively bamboo diet, but also their similar physical adaptations to their diet and their sympatry in some locations. What was unknown was if their similarities also extended to their gastrointestinal tract (GIT) microbiota. Using next-generation 16S rRNA parallel sequencing technologies, we uncovered several factors that impact bacterial communities of the pandas and also found that the two panda species harbor distinct microbial communities. In general, the Firmicutes and the Proteobacteria dominate both pandas’ gut microbiomes, with lesser contributions by the Actinobacteria and the Bacteroidetes. However, for the red panda, sex, age, and season significantly alter GIT microbiota. For giant pandas, we found that bacterial communities differed significantly between their normal fecal samples and mucus excretions. Together, these data display the plasticity of their bacterial communities, with several factors shaping GIT microbiota. As both species are highly threatened, understanding the relationship between the panda and their gut microbiome has given insight into the overall health of these uniquely herbivorous carnivores.

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Bacteria, especially symbiotic species in the gastrointestinal tract, have lived with human for long time and are involved in many aspects of host physiology. There is growing evidence that microbiota-gut-brain axis plays an important role in modulating the response to stress in both human and animals. Alterations in the gut microbiota can change the central nervous system function through effects on the endocrine, immune and nervous systems. Recent studies suggest that probiotic treatment may help to maintain resistance against the detrimental effects of stress though the microbiota-gut-brain axis. However, how potentially beneficial bacteria interact with specific immune and neural components, to mediate beneficial effects on behavior remain unclear. Using chronic social defeat stress, a model often used in post-traumatic stress disorder research, we found that administration of Lactobacillus rhamnosus JB-1 beginning 48 hours following chronic social defeat led to persistence of fear memory and social deficits. These effects were associated with changes in gene expression related to emotion and memory in the hippocampus. This was in contrast to previous studies showing that probiotic intervention during social defeat prevents stress induced deficits in social behavior. This indicates that timing of L. rhamnosus treatment in relation to stress exposure has important implications for effects of the bacteria on behavior. In relation to the mechanism of action of L. rhamnosus on behavior, we demonstrate through depletion and adoptive transfer experiments that CD4+CD25+ T cells in mice treated with JB-1 were necessary and sufficient for JB-1 induced anxiolytic and antidepressant-like effects. Evidence also suggested that Ly6Chi monocytes may be a downstream target inhibited by Tregs involved in the behavioral effects of the bacteria. We observed that JB-1 could also reduce the number of activated microglia in the hippocampus, and attenuate hypothalamic-pituitary-adrenal axis reactivity with the integrity of vagus nerve. Crucially we demonstrated that JB-1 induced promotion of peripheral Tregs, reduction in microglia activation in the hippocampus, and attenuation of HPA axis reactivity, were all inhibited following vagotomy indicating that vagus nerve integrity is required to maintain immune and endocrine linkages from gut microbes to the brain. These studies demonstrate prerequisites for beneficial probiotic effects on stress related behaviours including a specific time window in relation to stress exposure, the activation of regulatory immune cells, and undisrupted vagal nerve signalling. These findings highlight the inter-systemic communication of the microbiota-gut-brain axis in the stress response, and might help to unveil more therapeutic opportunities in relation to stress-related mood disorders. / Thesis / Doctor of Philosophy (PhD) / Excessive exposure or dysregulated responses to stress in human and animals induces behavioral changes and the development of mood disorders. The Microbiota-gut-brain axis plays an important role in maintenance of homeostasis. However, crosstalk between the different components of microbiota-gut-brain axis, and how specific microbes can modulate these interactions, remains unclear. Thus, we sought to understand the mechanism of inter-systemic communication linking a specific gut microbe to changes in stress response and behavior. We observed immunoregulation by regulatory T cells were essential in Lactobacillus rhamnosus JB-1 induced anxiolytic and antidepressant-like effects. We also found the integrity of vagus nerve was necessary for JB-1 induced promotion of regulatory T cells and decrease in microglial activation and attenuation of hypothalamic-pituitary-adrenal axis that are associated with the anxiolytic effects of the bacteria. We also identified that the temporal relationship between exposures to stress and the bacteria is important as ingestion of JB-1 directly after chronic social defeat lead to persistence of fear memory and social deficits. This work will help us to understand mechanisms underlying the microbiota-gut-brain axis, which may allow for the development of novel microbe based therapeutic intervention against mood disorders.

Introduction: Irritable bowel syndrome (IBS) is characterized by gastrointestinal (GI) symptoms, and as a consequence of dysregulated communication via the gut-brain axis, is highly comorbid with mental illnesses such as anxiety and depression. With no known cure, IBS patients must manage their symptoms through lifestyle factors. Physical activity is one such lifestyle factor that reduces GI symptoms and improves mental health; however, it remains unclear whether physical activity buffers against the acute worsening of IBS symptoms following a stressor. Method: To investigate this, we evaluated the stress reactivity and recovery of 9 IBS patients and 13 healthy controls following exposure to acute stress. We exposed participants to an electronic Trier Social Stress Test (e-TSST) and measured changes in psychological stress (state anxiety), physiological stress (sympathovagal balance, where higher LF/HF ratio indicates greater stress system activation), and GI symptom severity before, during and every 20 minutes for one hour after. Physical activity was measured using the Stanford Seven-Day Physical Activity Recall questionnaire and quantified as weekly energy expenditure. Results: IBS patients had higher state anxiety (p = .05), LF/HF ratio (p = .01) and GI symptom severity (p = .01) than healthy controls. Although the e-TSST did not exacerbate these group differences, higher state anxiety at baseline (p = .03) and higher LF/HF ratio in response to an acute stressor (p < .001) were associated with more severe GI symptoms within the first 20 minutes following the e-TSST. Importantly, IBS patients who were more physically active experienced less severe GI symptoms during that same timeframe (p = .03). Conclusion: Physical activity may be a promising lifestyle factor for lessening GI symptom severity in response to an acute stressor. / Thesis / Master of Science in Kinesiology

Obesity is a growing problem that impacts both adults and children. Obesity is linked to the development of unfavorable health outcomes like excess fat accumulation in the liver, a problem known as non-alcoholic fatty liver disease (NAFLD). Brown adipose tissue (BAT), a thermogenic body fat that can be turned on by cold, produces heat by consuming circulating lipids and glucose in a futile cycle. Less active cold-stimulated BAT is linked to obesity and type 2 diabetes in adults but whether this relationship exists in children is unknown. In rodents, increases in BAT activity are associated with reductions in NAFLD, effects that may be mediated through changes in the gut microbiome and reductions in peripheral serotonin. Whether the gut microbiome and serotonin play a role in regulating BAT activity in adults and children is not known. In this thesis, we have utilized magnetic resonance imaging (MRI) proton density fat fraction (PDFF) to assess BAT in the supraclavicular (SCV) region after three hours and one hour of whole-body cold exposure in adults and children, respectively. In 60 adults (aged 18-57 years), we examined whether there is a relationship between cold-stimulated BAT activity and liver fat (assessed via MRI) and whether gut microbiota plays a role in connecting these two tissues. In children, we investigated, BAT activity after whole-body cold exposure in 26 boys (aged 8-10 years). We also explored if BAT activity was different between boys with and without overweight/obesity. Finally, in young boys, we measured the levels of serotonin in platelet-poor plasma and its metabolic end product 5-hydroxyindole acetic acid (5-HIAA) in the urine. We explored if these measures of circulating serotonin were related to cold-stimulated BAT activity and if they were different between boys with and without overweight/obesity. The findings from this research indicate that higher cold-stimulated BAT activity is associated with lower liver fat in adults, but that this relationship is unlikely mediated through changes in the gut microbiota. Additionally, boys with overweight/obesity have lower cold-stimulated BAT activity and lower 5-HIAA in their urine compared to those with normal weight. Moreover, circulatory serotonin is negatively related to total adiposity. However, circulating serotonin is not related to cold-stimulated BAT activity in this cohort. These findings are important as they indicate for the first time that increasing BAT activity in adults and children could potentially be a new avenue for the treatment of NAFLD and obesity. / Thesis / Doctor of Philosophy (PhD) / Obesity is a risk factor for the accumulation of extra liver fat, a problem known as non-alcoholic fatty liver disease (NAFLD). Brown adipose tissue (BAT) is a kind of body fat that rather than storing calories like white fat burns calories when switched on by cold. Studies in adults have shown that people with obesity and type 2 diabetes have less active BAT suggesting switching it on may be helpful to promote weight loss and lower glucose. However, whether this relationship exists in children is not known. In rodents, increased BAT activity has also been linked to reductions in NAFLD, effects that might involve a hormone called serotonin, or changes in the gut microbiome but whether this is important in children and adults is also not understood. In this thesis, we utilized magnetic resonance imaging (MRI) to examine BAT activity after whole-body cold exposure in adults (3 hours) and children (1 hour). In 60 adults (aged 18-57 years), we report that higher cold-stimulated BAT activity is linked to NAFLD, but gut microbiota does not seem to play a role in this relationship. In 26 boys (aged 8-10 years), BAT is less responsive to cold in boys with overweight/obesity compared to boys with normal weight. Additionally, serotonin is lower in boys with overweight/obesity compared to boys with normal weight. These findings suggest that increasing BAT activity in adults and children could potentially be a new avenue for the treatment of NAFLD and obesity.

Molecular profiling of blood for several purposes, 1) To identify prostate cancer biomarkers, 2) to identify commonalities between asthma and mood disorders, and 3) to identify mRNAs that may be involved in psychobiotic changes to behaviour. / Every cell of the body has the opportunity to secrete molecules into the blood. These molecules: proteins, RNAs, and DNAs, can be secreted freely, or within extracellular vesicles (EV). The complement of specific molecules secreted by cells can vary in accordance with changes to their immediate environment, such as disease in a particular organ. Cells of the immune system which circulate in the blood may also change the rates at which they produce these molecules in response to a disease or unusual event occurring somewhere within the body. The full complement of proteins, RNAs, or DNAs from all sources within the blood can therefore be measured to garner information about disease states and communication between every tissue of the body. In this body of work, we leveraged this to address three separate challenges within medical science. First, we utilized blood as a source of biomarkers for disease and disease severity; isolating EVs from the blood of prostate cancer patients and healthy subjects and characterized their proteins with mass spectrometry to identify potential biomarkers for prostate cancer and its stages. Next, we explored the ability of blood to identify commonalities between distinct but often comorbid diseases; here we utilized publicly available datasets to identify transcripts or gene sets potentially facilitating the relationship between PTSD, MDD, and asthma. Finally, we utilized differential gene and gene sets expression to gain mechanistic insight into microbiota-gut-brain axis; investigating the hippocampus and blood of mice fed one of two psychobiotic bacteria: Lactobacillus rhamnosus JB1, Lactobacillus reuteri 6475. The analysis identified several mRNA expression differences potentially responsible for the mood-altering characteristics of these psychobiotic bacteria. This body of work illustrates the utility of blood omics data for addressing many problems within medical science, and highlights the large scale of information stored within the blood. / Thesis / Doctor of Philosophy (Medical Science) / Every cell of the body has the opportunity to secrete molecules into the blood. These molecules: proteins, ribonucleic acids (RNAs), and deoxyribonucleic acids (DNAs), can be secreted freely, or within small membrane compartments called extracellular vesicles (EV). Specific molecules are secreted more or less by cells depending on changes to their immediate environment, such as disease in a particular organ. We leveraged this to the benefit of medical science in three separate scenarios: 1) using the molecular contents of EVs to determine when someone has prostate cancer, and at what stage; 2) examining RNAs of the blood to determine why so many with asthma also have depression or PTSD; 3) measuring RNAs in the blood and hippocampus of mice to better understand how certain bacteria in the gut can alleviate depression. This work illustrates the utility of blood in tackling many challenging problems within medical science.

Indiana University-Purdue University Indianapolis (IUPUI) / Enteric viruses are infectious human pathogens that initiate infection in the gastrointestinal tract. They follow a fecal-oral route of transmission and are spread by contamination of food, water, or contact between individuals. Furthermore, enteric viruses also cause significant morbidity, mortality, and economic burdens yearly. Coxsackievirus (CV) is commonly isolated among enteric viruses and is an etiological agent of hand, foot, and mouth disease, hemorrhagic conjunctivitis, and myocarditis. The virus predominantly infects infants and young children and accounts for 11% of the fatality rate in neonates. Despite CV’s impact on human health, there are no treatments or vaccines for CV infections. Using a mouse model to study a key CV, Coxsackievirus B3 (CVB3), our laboratory has found two critical factors that impact CVB3 replication and pathogenesis. First, we have demonstrated that intestinal bacteria enhance intestinal CVB3 replication. We found that certain specific bacteria (Salmonella enterica) and its cell wall components, like lipopolysaccharides (LPS), enhanced CVB3 stability and infectivity in vitro. Additionally, we found that particular constituents of LPS are required for stability to occur. These data suggest that specific bacteria may be integral in maintaining CVB3 infectivity in the intestine. Besides virus-microbiome interaction, CVB3 is also impacted by sex hormones. Using castrated mice models, we observed a sex bias to CVB3 infection, with male mice succumbing to CVB3-induced disease at an increased rate compared to female mice. Our data suggest that testosterone, a predominant male sex hormone, enhanced CVB3 intestinal replication and viral dissemination to organs in male and female mice, but lethality only in male mice. Moreover, testosterone also affected the immune response by reducing the activation of the CD8+ T cells. CD8+ T cells are required to clear the viral infection and are integral in vaccine development. In contrast, we found an enhanced CD8+ T cell response in female mice to CVB3 infection, suggesting a sex-dependent T cell response that may underlie the sex bias in disease. Overall, these data represent an essential advancement in the CV field and will help develop future therapeutics and aid in vaccine design to limit CV infections.

The human gut microbiota plays a crucial role in human health and therefore imbalances in gut microbiota functioning can lead to the development of metabolic disorders. Short-chain fatty acids (SCFA), tricarboxylic acid (TCA) cycle metabolites, bile acids (BA) and other metabolites are the essential compounds for the metabolome. Per- and Polyfluoroalkyl substances (PFAS) are a group of chemicals that can be absorbed onto the lumen by food. Since many PFAS has shown to possess a long list of adverse effects on human health, the short term impact of PFAS on gut microbiota functioning using an in vitro model that mimics fermentation in the human colon was explored. Samples obtained from in vitro fecal fermentation were then analysed through high-performance liquid chromatography coupled to time-of-flight mass spectrometry (LC-qToF-MS) using targeted and non-targeted approaches. Fecal samples were donated by four donors which were put through a fermentation over 24 hours and treated with no PFAS mixture (control), low concentration mixture of PFAS (PFAS-L) and high concentration mixture of PFAS (PFAS-H). The PFAS mixture contained perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorohexanesulfonic acid (PFHxS), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA). For the fermentation, samples were collected after 0, 2, 4, 6, 8 and 24 hours. The samples were spun down and the supernatant was collected. Two approaches were applied to explore the metabolism in the fermentation extracts: The first one was a “targeted SCFA and TCA cycle metabolites analysis’’ and the second one was “untargeted analysis of polar and nonpolar metabolites’’. Results show that all TCA cycle metabolites displayed different trends with the compound and little to no variation between the treatments. However, although there was no significant difference, 4 core compounds of the TCA metabolism were lower after treatment with PFAS-L or PFAS-H. For the untargeted method, 78 of 2855 compounds were identified. When comparing control samples, PFAS-H and PFAS-L, 200 features showed statistically significant differences. Most of these had higher concentrations for PFAS-treated samples. When PFAS-H was compared only with PFAS-L, 30 features were found to be statistically significant, indicating that distinct concentrations of PFAS differentially affect gut microbiota metabolism. For future identification, further analysis must be done, preferably with MS/MS, to obtain more structural information for these significant metabolites, since the masses of the fraction ions are needed to narrow down the search in databases used to obtain the identity of an ion. PFAS and the concentration seem to have an impact on the gut microbiota, the study should be done with additional donors to archive trends applicable to a whole population.

No description available.