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At present, Drug-Induced Liver Injury (DILI) is the most frequent cause of acute liver injury, with an incidence ranging from 0.023-1.5‰ in developed countries. DILI can be separated into two different types. Whereas intrinsic DILI concerns predictable dose-dependent hepatoxicity, idiosyncratic DILI refers to unpredictable adverse drug reactions for which there is no existing pharmacological explanation. Many prescription and over-the-counter drugs causing DILI do so in an undiscovered and idiosyncratic pattern. It is the latter that causes DILI to be the most frequent cause of safety-related drug withdrawals. This causes delays in the availability of treatments and is responsible for 20% to 40% of liver transplants and hepatic failures. While several mechanisms might be responsible for idiosyncratic DILI, a broad industry-wide consensus was reached in 2016 on the importance of the liver bile salt export pump (BSEP) and the need to test for BSEP inhibition at an early stage of drug development. An assay for BSEP-mediated bile acid transport was established to study the biochemical mechanisms of inhibition of BSEP activity by organic compounds. The four selected compounds showed competitive inhibition of taurocholate transport in Lineweaver-Burk plots but also other types of inhibition in Eadie-Hofstee plots. These results indicate that these inhibitors might compete with taurocholate for binding in the substrate-binding pocket and possibly, that the inhibitors are transported by BSEP. Interestingly, this work shows a strong inhibitory effect of excess Mg2+ on the ability of BSEP to transport glycocholic acid; the transport of taurocholic acid was not inhibited by Mg2+. To get more insight in structure-function relationships in BSEP, a condense and accurate database of all known BSEP point mutations from published literature was compiled. This database shows a total of 264 known mutations associated with several pathogenic and non-pathogenic phenotypes. To date, there is still little known about the effects of many of these mutations on BSEP activity. Only 82 of the 264 mutations have currently been investigated for their impact on function and expression level of BSEP. 2D and 3D primary cultured hepatocytes were used to establish the effect of DILI-inducing drugs on the synthesis of bile acids and their distribution across the cell and the media. Multivariate analyses of the data demonstrate that drugs can change the bile acid composition in hepatocytes and their environment. This might occur by regulating bile acid synthesis pathways and/or by modulating the selectivity of BSEP itself. Interestingly, some of the detected changes in bile acid concentrations might be early indicators of cell stress and cell death. The experiments also suggest that drugs can cause a change in BSEP expression in the plasma membrane, through changes in the trafficking of protein to and from the plasma membrane. These changes could explain the existence of non-pathogenic BSEP inhibitors. These findings increase our understanding of the influence of compounds on bile acid production and BSEP distribution and their potential roles in idiosyncratic DILI.

Alterations in glucocorticoid (GC) biosynthesis and metabolism are associated with a variety of pathophysiological disorders including cholestasis, diabetes and other metabolic disorders. Bile acids (BA) are also important modulators of metabolic functions and regulate cholesterol, triglyceride and glucose homeostasis as well as being critical for dietary fat digestion, enterohepatic function, and postprandial thermogenesis. In intact cells and in vivo, the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme converts inactive GC precursors (cortisone in humans, and 11-dehydrocorticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively) thereby amplifying local intracellular GC levels. Interconversion by 11β-HSD1 of other sterols has also been described. These include conversions of 7keto-cholesterol to 7β-hydroxycholesterol, 7-oxodehydroepiandrosterone (7-oxo-DHEA) to 7α-hydroxy- and 7β-hydroxy DHEA, 7- oxo-lithocholic acid (LCA, a bile acid; BA) to chenodeoxycholic acid (CDCA, a 7α- hydroxylated BA) and ursodeoxycholic acid (UDCA, a 7β-hydroxylated BA) in human liver microsomes. In the liver, BA inhibit 11β-HSD1 but whether 11β-HSD1 regulates BA homeostasis is unclear. Evidence of molecular regulation of the enterohepatic recycling of bile acids by liver glucocorticoid receptor (GR) in mice does suggest a role for 11β-HSD1. It was therefore hypothesised that disruption of 11β-HSD1 expression in mice would impair BA recycling and might affect the relative concentrations of BA within the enterohepatic circuit. The primary objective of the current work was to investigate the impact of altered 11β-HSD1 on BA homeostasis. This was achieved using genetically modified mouse models with altered 11β-HSD1 expression, either globally or restricted to hepatocytes. BA are stored in the gall bladder and are released postprandially, to aid digestion. It was hypothesised that 11β-HSD1 deficiency might the affect the process of postprandial gall bladder emptying/refilling. Mice with global 11β-HSD1 knockout (Hsd11b1-/-) and age-matched control mice (C57Bl/6) were either fasted for 4h and culled or fasted for 4h and re-fed for another 4h before culling. Their response to fasting and re-feeding was assessed with specific focus on organs associated with BA recycling in the enterohepatic circuit (liver, gall bladder, serum and small intestine). Gall bladders of fasted Hsd11b1-/- and C57Bl/6 mice had similar volumes of bile but in fasted Hsd11b1-/- mice, BA concentrations were higher in serum and liver. As expected, re-feeding caused gall bladder emptying in C57Bl/6 mice with consequent increased serum and liver bile acid concentrations. In Hsd11b1-/- mice, the gall bladder did not empty and serum and liver BA concentrations were similar to the fasted state. To explore possible reasons for this, levels of mRNA encoding proteins known to be involved in hepatic BA transport were quantified using real-time q-PCR. Levels of mRNA encoding NTCP/ SCL10A1/ SCL10A1, the transporter responsible for most hepatocyte BA uptake, were increased in livers of fasted Hsd11b1-/- mice whereas levels of Slc51b mRNA, encoding the OST- transporter that facilitates BA removal from liver to the systemic circulation, and levels of Mrp2 and Atp8b1/FIC1 mRNAs (both encoding proteins which transport BA from liver into gall bladder) were decreased. This suggests that in fasted Hsd11b1-/- mice, BA transporter expression is altered to increase BA influx into hepatocytes and decrease efflux, to compensate for reduced levels of liver BA. These data together imply that bile acid recycling is controlled by 11β-HSD1 activity which regulates gall bladder emptying, hepatic BA concentration and BA transporter activity to ensure continuity of BA recycling within the enterohepatic circuit compartments. These changes may also affect digestion of lipids and fat-soluble micronutrients. Because 11β-HSD1 can directly metabolise secondary BA, it was predicted that 11β-HSD1 deficiency would lead to changes in the BA profile. Profiling of BA in the gall bladder was performed using mass spectrophotometry. In Hsd11b1-/- mice, 7α-hydroxylated BA predominated (cholic acid [CA]>α-muricholic acid [α- MCA]>CDCA>others), in contrast to C57Bl/6 mice in which 7β-hydroxylated BA predominated (ω-MCA>β-MCA>UDCA>others). The ratio of 7α:7β acids was therefore >100-fold greater in Hsd11b1-/- mice. This suggests that 11β-HSD1 either directly or indirectly controls the epimerisation of 7α- to 7β- hydroxylated BAs. Measurement of mRNAs encoding proteins important for hepatic BA biosynthesis in livers of fasted Hsd11b1-/- mice showed decreased expression of Scarb1/SR-B1, Cyp39a1 and Cyp27a1 (though with no change in levels of CDCA, the product of CYP27A1, in liver or bile fluid), compared to fasted control mice. Hepatic levels of Gpbar1/TGR5/GPBAR1 and Cyp3a11 mRNAs, encoding proteins important in BA detoxification, were increased and decreased, respectively. This suggests that Gpbar1/TGR5/GPBAR1, encoding G-protein coupled bile acid receptor (also called TGR5/GPBAR1) and an FXR target, could be induced to detoxify 7α-hydroxylated BA whereas expression of Cyp3a11, which catalyses the conversion of LCA to hyodeoxycholic acid (HDCA) is decreased; bile fluid of Hsd11b1-/- mice contained lower levels of LCA and little to no HDCA, though LCA and HDCA levels in liver were unaltered. Currently, the functional differences between 7α- and 7β- hydroxylated BA are not clear. However, these findings could have significant implications for bile acid-mediated transcription which, in turn, might affect lipid and sterol metabolism. Also, alterations in BA composition may have other physiological consequences via other pathways. Because cholesterol is the precursor of BA synthesis, it was hypothesised that western diet (WD) (containing cholesterol) would exacerbate and/or alter the phenotype of Hsd11b1-/- mice. Gall bladder weights of fasted Hsd11b1-/- and control C57Bl/6 mice did not change with western diet compared to chow diet. In control C57Bl/6 mice, the total BA concentration in the gall bladder increased in response to WD in comparison to chow diet. In contrast, Hsd11b1-/- mice showed no change in total BA concentration when fed on WD in comparison to chow. These data indicate that 11β-HSD1 is required by mice for the normal increase in total BA concentration in bile in response to dietary cholesterol. BA profiling of bile from control mice fed on WD showed no difference in the relative amounts of 7β-hydroxylated BA and 7α-hydroxylated BA to littermates fed on chow diet with the exception of β–MCA which increased, and α–MCA which decreased. Like chow-fed Hsd11b1-/- mice, BA profiling of bile from WD-fed Hsd11b1-/- mice showed a significant decrease in relative levels of 7β-hydroxylated BA (UDCA < β-MCA < others) and an increase in percentage of 7α-hydroxylated BAs (CA>α-MCA>CDCA>others) compared to C57Bl/6 controls. These data show that Hsd11b1-/- mice fail to show the normal increase in 7β-hydroxylated BA and decrease in 7α-hydroxylated BA observed in control mice in response to a cholesterol containing diet, suggesting 11β-HSD1 deficiency blunts the influence of cholesterol on BA composition. Measurement of hepatic mRNAs encoding BA transporters suggest that hepatocyte uptake of BA is decreased in C57Bl/6 on WD compared to those mice on chow diet, whereas this was not the case in Hsd11b1-/- mice where hepatic expression did not change with diet. Thus, Hsd11b1-/- mice failed to increase expression of Ntcp/ Scl10a1/ Scl10a1 appropriately, suggesting impaired hepatic BA uptake, while Slc51b (encoding OST-β) expression was increased, compared to control mice, possibly to reduce hepatic BA concentration by transporting BA out of hepatocytes into the systemic circulation., Therefore, Hsd11b1-/- mice may adapt to a cholesterol-induced increase in hepatic BA by blunting hepatic BA uptake via NTCP/ SCL10A1/ SCL10A1 and increasing hepatic efflux via OST-β. The effects of 11β-HSD1 deficiency upon BA recycling and BA profile and concentration within the enterohepatic circuit, could reflect 11β-HSD1 action within the liver or could be due to actions in other tissues. To investigate the role of hepatic 11β-HSD1 specifically, 11β-HSD1 liver-specific knockout (Hsd11b1LKO), 11β- HSD1 liver-specific over-expressors (Hsd11b1LOE) and control mice with exon 3 of the Hsd11b1 gene “floxed” (Hsd11b1F) were studied. Findings from this study indicate a role for 11β-HSD1 in adaption to dietary cholesterol and suggest that hepatic 11β-HSD1 (as opposed to 11β-HSD1 in extra-hepatic tissues) is the main factor regulating BA metabolism. Also, work from this thesis demonstrates 11β-HSD1 is an important regulator of gall bladder emptying and filling, an important component of enterohepatic bile acid recycling. Based on these findings it is anticipated that therapeutic use of 11β-HSD1 inhibitors will result in BA imbalances within the enterohepatic circuit and therefore BA homeostasis. Care must therefore be observed when implementing therapeutic use of 11β-HSD1 inhibitors, with particular focus on patients with cholestasis, Addison’s disease and critically ill patients who already have known BA imbalances in their enterohepatic system.

Bile acids are an essential part of the human digestion system and play an important role in lipid absorption, bacteria movement in the small intestine, and regulation of the farnesoid X receptor (FXR) and G protein-coupled bile acid receptors (GPBAR) responsible for homeostasis. They are synthesized in the liver, stored in the gallbladder and the excess is carried on to the small intestine. Many liver and intestinal diseases are diagnosed based on bile acid metabolism pathways and the structures of those bile acids. Therefore, the bile acid structure determines the function of the molecule. Many bile acids are similar in structure and only differ by an orientation of a hydroxyl or other group. This difference can be the key to diagnosing a patient with a lifechanging disease. In analytical chemistry, ion mobility mass spectrometry (IM-MS) is used to measure the difference in size and shape of the molecules by calculating the collision cross-section (CCS). In this study, we aim to analyze different bile acids using Structures for Lossless Ion Manipulations (SLIM) ion mobility and comparing nitrogen and helium buffer gas to determine the best method of separation. The calculated CCS values were analyzed to determine trends that would accurately identify these bile acids. Additionally, this method will be applied to test a clinical assay for its sensitivity. A concentration study will be performed on specific bile acids that are used in the total bile acid test to be applied in clinical settings. This test is important to improve patients’ diagnosis and have quicker results than the typical laboratory test. The goal is to increase scientific knowledge of ion mobility in the clinical field, not only to further biological analysis, but also to enhance clinical care around the world.

The human gut microbiome significantly affects host biology. In particular, gut bacteria modify important pools of molecules that impact host physiology. Therefore, further studies of gut bacterial metabolism will provide insights into the significance of gut bacteria and their impact on host health and disease. Here, I describe my projects that aimed to contribute to our understanding on the gut bacterial modifications of bile acids. Chapter 2 describes our use of a chemical tool to modulate gut bacterial metabolism in vivo. AAA-10 inhibits bile salt hydrolase (BSHs), enzymes that catalyze the gateway step for all gut bacterial metabolism of bile acids. We showed successful modulation of bile acid pools using AAA-10 in both monocolonized and conventional mice, which led to significant changes in metabolic phenotypes and host gene expression, respectively. AAA-10 treatment improved glucose tolerance and reduced liver steatosis in monocolonized mice, while AAA-10 treatment led to preliminary gene expression changes in genes related to the circadian rhythm and immune response in conventional mice. These studies will serve as a guide for future in vivo experiments modulating gut bacterial metabolism of bile acids. However, AAA-10’s effectiveness as a BSH inhibitor in vivo has room for improvement, and my efforts to understand mechanisms of insensitivity to AAA-10 are presented in Chapter 3. Here, we describe our discovery of a bacteria strain, Lactobacillus intestinalis ASF 360, that has a strong BSH activity that is insensitive to AAA-10 inhibition. Using heterologous expression and bacterial assays, we showed evidence for a transport mechanism that may be involved in removing the inhibitor from the cell during normal growing conditions. We also discovered a new lead inhibitor, DCA-FMK, that inhibited BSH activity in L. intestinalis. Our new lead inhibitor together with our understanding of the resistance mechanism will guide us towards developing a next-generation BSH inhibitor. Finally, Chapter 4 describes our efforts to elucidate the biosynthesis of the bile acid metabolite isoallolithocholic acid (isoalloLCA), a compound that enhances the differentiation of naïve T cells into Treg cells both in vitro and in vivo. Using a combination of microbial genetics, protein expression, analytical chemistry, and synthetic organic chemistry, we identified a gene cluster that is responsible for isoalloLCA production in the abundant gut bacterial phylum Bacteroidetes. We showed that the gene cluster is necessary and sufficient for both in vitro and in vivo production of isoalloLCA, and both isoalloLCA and the isoalloLCA-producing genes are associated with inflammatory bowel disease (IBD) in human patients. These results highlight a new mechanism by which gut bacterial metabolism mediate host immune tolerance. Overall, my projects cover a range of topics about gut bacterial metabolism of bile acids. Together, these studies contribute to our understanding of how gut bacteria impact on host health and disease by chemically modifying compounds found in the gut.

Cholesterol equilibrium is meticulously orchestrated, and its proper regulation is pivotal inmitigating the risk of cardiovascular disorders. To preserve this delicate balance,cholesterol must activate sensing mechanisms at organelle membranes within the cell’sinterior. The recently discovered Aster protein family (Aster-A, -B, and -C) partakes innonvesicular cholesterol import to organelles from the plasma membrane (PM). Despitetheir high coding sequence homology, each Aster protein exhibits distinct tissue expressionpatterns, conferring unique functions. This work elucidates their nuanced roles inregulating whole-body cholesterol flux. It scrutinizes how existing therapeutics modulatecholesterol flux, emphasizing their effects on key proteins that are known regulators ofcholesterol homeostasis. The introductory chapter reviews the intended and off-targeteffects of these existing therapeutics. Aster proteins are then introduced as novelmodulatory nodes in cholesterol regulation. Aster-C, prominently expressed in sterol-regulatory tissues like the liver and testis, presents an intriguing target, especially inpotential therapeutic applications for dyslipidemia-associated disease pathology. The bodychapter delves into Aster-C’s role in whole-body homeostasis, unveiling its contribution tocholesterol balance and revealing modest effects on bile acid metabolism under low-cholesterol dietary conditions. Aster-C knockout prompts upregulation in the messengerribonucleic acid (mRNA) levels of paralogs Aster-A and -B in murine liver tissue,suggesting nuanced roles and potential redundancy with other Aster proteins. Comparative nalysis with existing literature indicates that Aster-C’s hepatic transcription is inducibleby FXR stimulation. These findings, intersected with our own research on the Aster-Cknockout-induced disruption of bile acid homeostasis, suggest an integral role for Aster-Cin mediating the rate of reverse cholesterol transport within hepatic tissue. Overall, wedeconstruct cholesterol metabolism, identify existing therapeutic interventions, and thenpropose potential benefits of targeting Aster proteins as novel nodes by which the rate ofreverse cholesterol transport can be stimulated. Ultimately, we aim to explore noveltherapeutic applications in Aster modulation by potentiating clinical relevance in our futuredirections section. Herein, we hope to broaden perspectives on inter-tissue cholesterolhomeostasis dynamics by emphasizing the need for further investigations into the role ofAster proteins in cholesterol trafficking.

Over the last few years, there has been increasing recognition that the healthy gut microbiome is dynamic. It is also well known that the microbiome plays a crucial role in gastrointestinal health. Disruptions to the gut microbiome can cause or exacerbate various metabolic diseases, including obesity, type 2 diabetes, liver disease, gastrointestinal cancer, and inflammatory bowel disease. Additionally, a loss in the cycling of bacterial populations has been linked to some of these metabolic diseases. Microbes significantly influence host metabolism through the breakdown and production of metabolites. For example, short-chain fatty acids (SCFAs) and bile acids (BAs) are microbially produced metabolites that modulate metabolism and reduce inflammation. However, until recently, most microbial studies have focused on investigating changes in microbial populations rather than microbial functions. In this thesis, I explore the impact of time on microbial functions and how these functions modulate the development of metabolic diseases.In the first part of the thesis, I explore how the blood metabolite pool changes before and after Transjugular Intrahepatic Portosystemic Shunt (TIPS) placement which showed there is compensatory shunting in the liver even before TIPS that affects the susceptibility to hepatic encephalopathy (HE). Additionally, we identified some microbial metabolites affected by TIPS that may be protective against HE. In the second part of the thesis, I describe how the dietary intervention time-restricted feeding (TRF) does not prevent the development of Metabolic dysfunction-associated steatohepatitis (MASH) in a streptozotocin/high fat diet (STAM/HFD)-induced MASH model. Moreover, we found a Lachnospiraceae and Oscillospiraceae microbe that may be protective against MASH through the modulation of fatty acids. Finally, the last chapter demonstrates that metatranscriptomics is a useful method for understanding time-dependent changes in microbial activity induced by TRF. This chapter also introduces a new bacterial therapeutic we engineered based on our metatranscriptomics results. These three chapters demonstrate how accounting for time and microbial functions provide new insights into microbe-host relationships and their role in mediating or protecting against diseases.

Recent advancements in microbiome research have unveiled promising avenues for therapeutic interventions while underscoring the need for mechanistic studies of microbiome function and temporal dynamics. This dissertation explores the interplay between the gut microbiome, luminal bile acid modifications, and circadian dynamics in the context of women's health and neurodegenerative disease.Chapter 1 reviews the current research on engineered microbial strains as novel therapeutics for gynecological health, including polycystic ovary syndrome (PCOS). Engineered probiotics, designed to express specific therapeutic functions such as antiviral peptides and anti-biofilm agents, offer targeted approaches for treating conditions like HIV and Candida albicans infections. The chapter underscores the integration of synthetic biology with clinical medicine, highlighting the therapeutic potential of targeting the microbiome.Chapter 2 delves into one of these specific microbiome functions that impacts metabolism and the hypothalamic-gonadal-pituitary axis in the context of PCOS. Through murine models, this chapter demonstrates how bacterial bile acid modifications by bile salt hydrolase (BSH) can ameliorate metabolic dysfunction and some reproductive parameters associated with PCOS. This mechanistic insight illustrates the therapeutic promise of microbiome-targeted therapies, and highlights the role of bile acid receptors FXR and TGR5 in glucose homeostasis and hormonal regulation.Chapter 3 explores the impact of time-restricted feeding (TRF) on the gut microbiome in an Alzheimer's disease (AD) mouse model. The study reveals that TRF modulates gut microbial composition, function, and rhythmicity, influencing metabolites crucial for regulating metabolism, neuroinflammation, and amyloid pathology. The microbiome emerges as a key player contributing to the mechanism of TRF’s mitigation of AD-related cognitive decline and neuropathology.Together, these chapters contribute to a deeper understanding of how characterization and manipulation of the microbiome can elucidate mechanisms of disease and shape therapeutic strategies across diverse health conditions, from reproductive health challenges like PCOS to neurodegenerative diseases such as Alzheimer's. Continued research using synthetic biology and multi-omic analyses to elucidate mechanistic functions of the microbiome will further contribute to improved scientific tools and healthcare outcomes.

Background and aims: Liver regeneration is essential for the preservation of homeostasis and survival. Bile acids (BAs)-mediated signaling is necessary for liver regeneration, but BAs levels need to be carefully controlled to avoid hepatotoxicity. We studied the early response of the BAs-fibroblast growth factor 19 (FGF19) axis in healthy individuals undergoing hepatectomy for living donor liver transplant. We also evaluated BAs synthesis in mice upon partial hepatectomy (PH) and acute inflammation, focusing on the regulation of cytochrome-7A1 (CYP7A1), a key enzyme in BAs synthesis from cholesterol. Methods: Serum was obtained from twelve human liver donors. Mice underwent 2/3-PH or sham-operation. Acute inflammation was induced with bacterial lipopolysaccharide (LPS) in mice fed control or antoxidant-supplemented diets. BAs and 7α-hydroxy-4-cholesten-3-one (C4) levels were measured by HPLC-MS/MS; serum FGF19 by ELISA. Gene expression and protein levels were analyzed by RT-qPCR and western-blot. Results: Serum BAs levels increased after PH. In patients with more pronounced hypercholanemia, FGF19 concentrations transiently rose, while C4 levels (a readout of CYP7A1 activity) dropped 2 h post-resection in all cases. Serum BAs and C4 followed the same pattern in mice 1 h after PH, but C4 levels also dropped in shamoperated and LPS-treated animals, without marked changes in CYP7A1 protein levels. LPS-induced serum C4 decline was attenuated in mice fed an antioxidant-supplemented diet. Conclusions: In human liver regeneration FGF19 upregulation may constitute a protective response from BAs excess during liver regeneration. Our findings suggest the existence of post-translational mechanisms regulating CYP7A1 activity, and therefore BAs synthesis, independent from CYP7A1/Cyp7a1 gene transcription.

Campylobacter jejuni (C. jejuni) and Clostridium perfringens (C. perfringens) are two leading pathogenic bacteria responsible for both human and animal diseases. In chapters Ⅱ to Ⅳ, I presented an overview of current knowledge on the role of immune response, microbiota, bile acids and their microbial metabolites on campylobacteriosis, and C. perfringens induced necrotic diseases.In chapter Ⅴ, I investigated effects of deoxycholic acid-modulated anaerobes (DCA-Anaero) on attenuating chicken transmission-exacerbated campylobacteriosis. SPF Il10-/- mice were gavaged with clindamycin for 7 days followed by infected with three C. jejuni strains, Cj-P0 (C. jejuni AR101), Cj-P1 (from chicken pre-infected with C. jejuni AR101), and Cj-P1-DCA-Anaero (from C. jejuni AR101 infected chicken pre-colonized with DCA-Anaero) at day 0 and euthanized at day 8. As a result, Cj-P1 induced more severe histopathology compared to Cj-P0, suggesting that chicken transmission increased C. jejuni virulence. Importantly, mice infected with Cj-P1-DCA-Anaero showed attenuation of intestinal inflammation compared to Cj-P1, which suggest that DCA-Anaero attenuate chicken-transmitted campylobacteriosis in mice and it is important to control the elevation of C. jejuni virulence during chicken transmission process. In our previous research, we also found that DCA could control chicken necrotic enteritis (NE) by reducing C. perfringens invasion and sporulation. In chapter Ⅵ, I developed vaccines using C. perfringens sporulation proteins (CP-spor-super1&2) and examined their effects on reducing NE in chickens. Broiler chickens were vaccinated at day 0, and had a booster vaccination at day 10, followed by infected with Eimeria maxima at day 16, and C. perfringens at day 20. Chickens were euthanized, and samples were collected at day 21. Vaccinated birds were resistant to severe clinical NE on histopathology and body weight gain loss. CP-spor-super1 vaccine reduced NE-induced proinflammatory Ifnγ gene expression as well as C. perfringens luminal colonization and tissue invasion in the small intestine (SI). Together, this study showed that CP-spor-super vaccines reduced NE manifestations such as histopathology and productivity loss. Inspired by the colonization susceptibility of C. jejuni on different conventional animals with various gut bile acids compositions, in chapter Ⅶ, I investigated the role of specific bile acid microbial metabolites on C. jejuni infection. Chickens gavaged with CA developed mild resistance to C. jejuni colonization and had increased CA levels in SI. C. jejuni-resistant conventional Il10-/- mice became highly susceptible to campylobacteriosis when fed CDCA diet, while no pathogen and colitis were found in basal diet group. Mechanism studies showed that mouse microbiota or bile acid alone reduced C. jejuni in vitro growth moderately, while CDCA microbial metabolites promoted C. jejuni in vitro growth, and CA microbial metabolites inhibited C. jejuni in vitro growth. The results suggest animal susceptibility to C. jejuni is mediated by microbiota-metabolized host-specific bile acids. Taken together, this dissertation showed that DCA-Anaero attenuated C. jejuni chicken transmission-worsened enteritis in clindamycin treated mice; vaccines using C. perfringens sporulation proteins reduced NE in chickens; host-specific CDCA and CA microbial metabolites differentially mediated C. jejuni infectiousness. All of these findings showed that specific bile acid microbial metabolites played important roles in C. jejuni or C. perfringens infection.

The mammalian gut is home to a vibrant community of microbes. The ecological interactions that shape this environment are distinct across gut locations. As host and microbial community co-evolved, they formed a complex yet stable relationship that prevents invading microorganisms, such as the spore-forming bacterium, Clostridioides difficile, from establishing within the gut. Great strides have been made over the past several years in characterizing C. difficile infection physiology, particularly in how gut microbes and their host work together to provide colonization resistance. I designed the work in this thesis to characterize how the mammalian small and large intestines shape C. difficile germination and outgrowth. Using observational and experimental approaches, I used human, murine and bioreactor models of the gut microbiota to study varying levels of ecological complexity. I show that the small intestinal microbiota is predominated by Firmicutes and that fluctuations in the microbiota are associated with changes in pH and bile acids. The bile acid population from the duodenum to the mid jejunum consists mainly of conjugated primary and secondary bile acids, with little microbial metabolism of these compounds occurring across the proximal small intestine. Since conjugated bile acids tend to promote C. difficile germination, this environment supports C. difficile’s transition from spore to vegetative cell at high efficiencies, suggesting that colonization resistance is tied to preventing the establishment of vegetative cells later in the gut. I also present work characterizing the effect of dietary xanthan gum on C. difficile colonization in a murine model of infection. Xanthan gum administration modified the microbiota and led to increased production of short chain fatty acids. However, it also interfered with the activity of the orally administered antibiotics used to render mice susceptible to C. difficile colonization. As a result, C. difficile colonization resistance was maintained in mice fed xanthan gum. Finally, I use a bioreactor model to characterize how founder effects shape microbial community establishment and influence C. difficile colonization resistance. Dilution increases the variability of the microbiota and abrogates its ability to resist invasion by a non-indigenous microbe. Additionally, we provided some reactor communities with additional concentrations of the dietary polysaccharide inulin. While some communities responded to inulin by producing additional butyrate, more dilute communities concurrently lost their ability to produce additional butyrate and resist C. difficile colonization. These data demonstrate that a particular level of microbiota cohesiveness is required to produce both functions and suggests that metabolic activity of butyrate-producing microbes is tied to colonization resistance. Together, my work demonstrates the importance of understanding the environmental interactions that shape microbial physiology, particularly that of C. difficile, in both the small and large intestines. In the small intestine, a variable microbiota with low biomass is shaped by host-driven processes, such as bile acid secretion. However, in the large intestine, a high biomass microbiota shapes the environment by metabolizing complex nutrients and preventing invasive taxa, such as C. difficile, from becoming established. Future work can leverage these findings to develop treatment methods that incorporate the dynamics of the intestinal environment to improve efficacy.

Type 2 Diabetes (T2D) is a metabolic disease characterized by chronic elevation in plasma glucose levels.T2D affects millions of people worldwide. One risk factor for developing T2D is accelerated postnatal growth, or “catch-up growth” (CUG), in early life. Consequences of interventions to prevent CUG, including early-postnatal undernutrition (UN), on adaptation to environmental stress have not been investigated. I hypothesize that mice exposed to postnatal UN are protected from weight gain and glucose intolerance, but have decreased pancreatic plasticity and gut-intrinsic factors. An ICR/CD1 mouse model for CUG prevention using early postnatal caloric restriction was generated and challenged with high fat diet (HFD). Postnatal UN mice are unable to adapt to HFD resulting in decreased insulin secretion and worsened glucose tolerance. Pancreatic islet area and number were quantified using H&E staining. Mice with postnatal UN had decreased islet area and number on chow but not on HFD. Islet composition was not different in postnatal UN mice. Insulin secretion is restored during an oGTT. Therefore, regulatory genes for incretin and bile acid synthesis, lipogenesis, and gluconeogenesis were investigated. This showed that lipogenesis and intestinal bile acid regulatory genes were reduced on HFD when CUG is prevented. This study shows that prevention of CUG using postnatal UN results in reduced glucose-stimulated insulin secretion and islet area on chow as well as reduced expression of lipogenesis and bile acid regulatory genes and worsened glucose metabolism when challenged with HFD.

Obesity constitutes a major global public health threat. It can lead to a cluster of metabolic disorders and a higher risk of developing various types of pathological conditions and diseases. Among multiple environmental factors, high fat diets (HFD) play an important role in the development of obesity. Flavonoids are one of the most abundant categories of bioactive compounds present in the human diet. My dissertation work identified mechanisms by which the flavan-3-ol (-)-epicatechin (EC), could mitigate high fat diet-induced obesity-associated intestinal permeabilization and metabolic endotoxemia. Chapter one looked at the protective effects of EC from HFD-induced intestinal barrier permeabilization and endotoxemia and its implications for steatosis and insulin resistance in vivo. We subsequently found that the luminal content of total bile acid and select secondary bile acids, particularly deoxycholic acid, were increased by HFD consumption. The second chapter focused on investigating if EC could prevent DCA-induced intestinal permeabilization in vitro. Such effect could in turn, help prevent the paracellular transport of lipopolysaccharides (LPS) from the lumen into the bloodstream. The last chapter provided additional mechanistic insight into how flavan-3-ols and procyanidins (PCAs) can mitigate HFD-induced endotoxemia through their capacity to prevent chylomicron- dependent transcellular transport of LPS in Caco-2 monolayers and high fat-induced postprandial endotoxemia in mice. In summary, this thesis works provide evidence that EC can decrease HFD-associated endotoxemia, characterizing the underlying mechanisms. This action would be critical to the capacity of EC to mitigate HFD- and obesity-associated comorbidities.

Necrotic enteritis (NE) has re-emerged following restriction of antimicrobial usage and costs $6 billion every year worldwide. The primary objective of the studies was to evaluate prevention and treatment of NE using a microbiota metabolic product, secondary bile acid deoxycholic acid (DCA), in drinking water. Day-old birds were tagged and placed in floor pens. In experiments 1 and 2, the birds were infected with Eimeria maxima (Em) at d 18 and C. perfringens at d 23 and 24 and the birds were euthanized at d 26. In experiment 3, birds were infected with Eimeria at d 20 and C. perfringens at d 25 and 26 and euthanized at d 27. DCA was administered in drinking water at 1.91, 0.95, or 0.48 mM, respectively. Small intestinal content and tissue were collected for histopathology and molecular study. Data were analyzed using one-way ANOVA followed by t-test. NE birds in experiment 1 developed subclinical signs of NE, while NE birds in experiment 3 suffered acute NE. DCA at 1.91 mM attenuated the BWG loss in experiments 1 and 3. Consistently, it reduced NE-induced intestinal histopathology in experiments 1 and 3. However, in experiment 2 because of minimal body weight alteration in Em and NE birds compared to non-infected birds, no tissue was collected. DCA reduced NE-induced expression of inflammatory gene Infγ in experiment 1. Similarly, DCA reduced elevation of NE-associated C. perfringens luminal colonization in experiment 1. Using targeted metabolomics (LC-MS/MS) of bile acids, we found that NE was associated with reduction of total bile acids in ileal content. The second objective of the studies was to examine improvement of broiler chickens growth through modulating gut microbiota. Mouse specific pathogen free (SPF) stool was cultured on Brain Heart Infusion (BHI) agar under anaerobic or aerobic condition and collected as SPF anaerobic (SPF-Anaero) and aerobic (SPF-Aero) microbiota. Day-old birds were tagged, weighed and randomly assigned to 9 pens. The birds were then orally gavaged with PBS, 108 CFU/bird SPF-Anaero, or 108 CFU/bird SPF-Aero. Body weight was measured at d 0, 14, and d 28, and euthanized at d 28. Data was analyzed using one-way ANOVA followed by t-test. Birds colonized with SPF-Anaero grew 15% heavier during d 0-14 (28 vs. 24 g/day/bird, P= 0.0009) compared to control birds, while birds colonized with SPF-Aero gained 8% more body weight compared to control birds (26 vs. 24 g/day /bird, P= 0.02). Consistently, SPF-Anaero birds grew faster compared to control birds during d14-28 (67 vs. 60 g/day/bird, P= 0.007). SPF-Anaero and SPF-Aero birds grew 15 and 4%, respectively, heavier on accumulative body weight gain compared to control birds (47 vs. 42 g/day/bird, P= 0.0009; 44 vs. 42 g/day/bird, P= 0.03, respectively). In conclusion, microbial metabolic product DCA in drinking water prevents NE-induced BWG reduction and histopathology, perhaps through reducing C. perfringens colonization and/or bile acid restoration. Modulating microbiota could be used as an antimicrobial free alternative to improve bird body weight gain. These findings may offer an alternative strategy to use of DCA and microbiota for NE prevention and growth promotion.

Obesity is rising around the world as a result of many factors, including an imbalance in food intake, an increased consumption of high-calorie diets, and a lack of physical exercise. Obesity not only leads to an increased risk for type 2 diabetes, cardiovascular disease and cancer, but also negatively affects longevity and quality of life. Anti-obesogenic approaches which are safe, widely available, and inexpensive are required to combat metabolic diseases. Mushrooms contain antioxidants, fibre and other phytochemicals, and have been shown to have anti-obesogenic and anti-diabetic effects through the modulation of diverse cellular and physiological pathways. In this thesis, three edible mushroom species (white button mushroom, shiitake mushroom and oyster mushroom) were selected as sustainable and functional food ingredients. Wheat-flour noodles were enriched with mushroom powder sourced from different parts (cap, stem, whole) at different levels (5%, 10%, 15%) to investigate the effects of mushroom addition on the nutritional composition, physical and textural properties. Noodles containing mushrooms had a nutritional advantage with higher protein and dietary fibre contents compared with wheat-flour noodles. The addition of 15% mushroom powder in the noodles resulted in a significant (p < 0.05) decrease in glycaemic glucose equivalents after in vitro digestion. The antioxidant levels of the samples increased following the in vitro digestion, and were positively correlated with the fibre content (p

The dawn of a new era of medicine has begun as clinicians and researchers shift their focus to more individual-centric diagnostic, treatment, and disease management strategies. Precision medicine is a multidisciplinary approach to human health care that takes into account a person’s genetic makeup, behaviors, and environmental factors when evaluating pathophysiology, tailoring treatments, and designing novel therapeutic moieties. In this dissertation I break down the critical subfields of this discipline to explain and apply the emerging tools and techniques we now have at our disposal to better understand the underlying biology of complex human psychiatric disorders.We begin with pharmacokinetics and pharmacodynamics, two branches of precision medicine that are involved directly with the temporal dynamics of pharmaceutical therapies and aid in disentanglement of how the body processes drugs versus how the drugs affect our bodies. I discuss detailed research across three separate drugs; risperidone, methamphetamine, and nicotine, integrating quantitative metabolic studies, genetic assessment, neuroimaging, and receptor analysis to clearly define inter-patient variability in risk and response. Following this is work I accomplished in the realm of gene and environment interactions in young children experiencing anxiety disorders, which over time, led to what I hold as my largest contribution to the personalized medicine field; microbiome and host interactions. I am attempting to unlock a more direct, biological mechanism to something known as antipsychotic-induced weight gain (AIWG) through the examination of bile acids and the gut microbiome and their crosstalk and interplay with host physiology. Results are abundant throughout this document and each study presented here within brings a unique piece of the precision medicine puzzle to the table.Pharmacokinetics, pharmacodynamics, genomic technology, gene-environment interactions, and the host-microbiome axis are the salient concepts in my toolbox of personalized medicine techniques that I believe can be leveraged in a variety of combinations to accomplish large goals in the medical and biotechnology fields. Whether it be through careful patient assessment with companion diagnostics, proper medication selection based on risk vs. reward value in harmony with an individual’s personal makeup, perseverance of high level disease progression and treatment monitoring, or even one day tailoring drug discovery to the highly specific receptors and biological pathways involved in these grievous diseases, it is clear precision medicine will pave the way for a better life for many people in the future.

The human gastrointestinal tract is populated by a diverse and mutualistic microbial flora that has profound effect on human health. Identification and characterization of microbial-metabolites produced in response to human dietary intakes may provide insights into host-microbiota interactions to better understand the role of the microbiota in human health and diseases. However, much of this interaction is poorly understood as many metabolites remain unidentified or understudied at the present time. Starch is ubiquitously present in human diet and is a major energy source to all living cells. Resistant starch (RS) is the starch portion that is not absorbed in the small intestine of healthy individuals but is fermented in the gut by the gut microbes. Recent advances in the fields of metabolomics and microbiome analyses have resulted in the newly identified functional roles of short chain fatty acids in the context of health benefits of resistant starches. This thesis encompasses a retrospective examination of the effects of resistant starch type 4 on medium and long chain fatty acids and bile acids on individuals with metabolic syndrome. Study samples were obtained and used from a completed/published 2-arm crossover, placebo controlled, double blind, human dietary intervention trial that reported several immunometabolic health benefits and gut-microbial alterations after resistant starch consumption. Eighteen fecal and plasma samples were subjected to methylation and analyzed with gas-chromatography, for the medium and long chain fatty acids, and liquid chromatography for the bile acids followed by mass-spectrometry measurements. Differential changes in the test and control groups post intervention in 46 fatty acids and 23 bile acid species were compared and presented in this thesis. Further research is needed to determine how these changes correlated with the previously reported microbiome changes in the participants from this study to meaningfully interpret the implications of the changes reported here. To conclude, this work presents novel changes in the fatty acid and bile acid profiles after resistant starch consumption that were previously unknown in the literature and opens up new direction for future investigations within the rapidly growing discipline of microbial metabolomics.

Clostridium difficile infection is the most common nosocomial infection in the US and other developed countries, yet standard antibiotic therapy for this infection fails to cure nearly a quarter of patients. These individuals experience a recurrence of the infection, and a large subset of patients with a single recurrence go on to develop recurrent C. difficile infection syndrome (R-CDI), characterized by vicious cycles of repeated antibiotic use and recurrence of disease. Although fecal microbiota transplantation (FMT) is now widely recognized as an extremely effective therapy for R-CDI, its mechanisms were heretofore unknown. This substantial gap in our knowledge limited both our ability to design novel therapies for R-CDI and our understanding of the role our native intestinal microbes play in our health. The work herein summarizes our efforts to identify and test the mechanisms behind this important treatment. Our findings indicate that fecal bile acid composition, which is significantly altered following FMT, also dramatically impacts C. difficile physiology. Bile acids present after FMT, compared to those present prior to the procedure, prevent germination of C. difficile spores and limit the growth of vegetative cells. With this knowledge, we demonstrated that a bile acid already clinically available, ursodeoxycholic acid (UDCA), can also prevent C. difficile germination and growth. Although unfortunately UDCA is a poor treatment option for many patients due to its absorption in the small intestine, we showed both that a non-absorbed derivative of UDCA, C7-sulfated UDCA, may be a promising novel therapy for R-CDI, and that UDCA may still have clinical utility in patients with altered gastrointestinal anatomy. These findings have paved the way for novel treatments for this widespread infection.

The application of natural products to newly identified phenotypic and target-based screens is central to the drug discovery process. Currently the research and development (R&D) process for a drug can span 10 – 15 years which includes the discovery, development, and final application of the therapeutic. This dissertation aims to describe the progression of 3 compounds along our internal R&D process. Chapter 1 presents a perspective on how natural products have impacted our fundamental understanding of chemicals structure and reactivity. It finishes by highlighting lead compounds in the area of HIV latency and bacterial biofilm inhibition. Chapter 2 describes the discovery, structural assignment, and biological annotation of abyssomicin 2 as a reactivating agent of latent HIV. Subsequent studies showed the mechanism of action (MOA) was not PKC activation or HDAC inhibition, which suggests a novel MOA. Chapter 3 addresses the development of quinoline amino alcohols as disruptors and dispersal agents of V. cholerae biofilms. This has led to the discovery of a lead compound, which future studies aim to integrate as the bioactive component for medical implant devices. Chapter 4 highlights the application of the TBADT/NFSI fluorination reaction to the recently reported bile acids, which have been identified as V. cholerae biofilm inhibitors. These studies identified N-Me taurine analogs with improved activity and fluorinated analogs with reduced activity highlighting key structure activity relationships for these compounds.

Bile acids are physiologically important metabolites, which are synthesized in liver as the end products of cholesterol metabolism and then secreted into the intestines. They play a critical role in the digestion and absorption of fats and fat-soluble vitamins through emulsifications. The amphipathic and chiral nature of bile acids makes their unique building blocks for assembling supramolecular structures including vesicles, fibers, ribbons and hollow tubes. Lithocholic acid (LCA) is a secondary bile acid. Our studies show LCA can selfassemble into helical tubes in aqueous solution by the linear aggregation and fusion of vesicles. The objective of this dissertation is to tune the structure of helical tubes and functionalize them by the co-assembly of ionic LCA and cationic cetyltrimethylammonium bromide (CTAB) and ionic LCA and cationic cyanine dye (CD), respectively. The first part of this dissertation focuses on the ionic-assembly of LCA and CTAB to synthesize the helical tubes with varied diameters and pitches. Our studies show that LCA and CTAB can self-assemble into helical tubes in NH4OH aqueous solution. The diameter of the helical tubes can be changed by adjusting the molar ratio of LCA and CTAB. The pitch of the helical tubes can be tuned by varying NH4OH concentrations. Differential scanning calorimetry studies indicate that there is a homogeneous composition distribution in the LCA/CTAB helical tubes. X-ray diffraction analysis studies show that the helical tubes have multibilayer walls with an average d-spacing of 4.11nm. We demonstrate that the helical tubes with varied diameters and pitches can be transformed into helical silica through the sol-gel transcription of tetraethoxysilane (TEOS). The second part of this dissertation is to use the ionic self-assembly of LCA and CD to design light-harvesting tubes iv by mimicking green sulfur bacteria that are known to be a highly efficient photosynthesizer. Xray diffraction and optical spectra show that LCA and CD can co-assemble into J- or Haggregate tubes, depending the condition under which the self-assembly occurs. We demonstrate the feasibility of using the J-aggregate nanotubes in the sensitive and selective detection of mercury (II) ions by the photoinduced electron transfer under sunlight. The presence of mercury (II) ions in aqueous solution could be detected for concentrations as low as 10 pM.

The self-assembly of chiral amphiphilic molecules in aqueous solutions is of particular interest because the chirality of individual molecules is often expressed in their supermolecular structures. Self-assembled tubes made of chiral amphiphilic molecules represent useful supramolecular architectures which hold promise as controlled release vehicles for drug delivery, encapsulates for functional molecules, and nanoreactors for chemical reactions. Lithocholic acid (LCA) is a secondary bile acid with the concentration being identical to that of cholesterol in the hepatic bile and gallbladder. It has a rigid, nearly planar hydrophobic steroid nucleus, with four hydrogen atoms and one hydroxyl group directed toward the concave side, and the convex side with three methyl groups. The ionic head with a carboxyl group is linked to the steroid nucleus through a short alkyl chain. In this thesis work, I study the self-assembly behavior of LCA at the liquid-solid interface, in confined spaces, and bulk solution. We find that the initially formed LCA vesicles further assemble into fractal tubes on glass slides by diffusion-limited aggregation and pronglike tubes by the capillary flow generated in an evaporating vesicle solution confined by two parallel glass slides. While in bulk solution, the LCA vesicles linearly aggregate and fuse into spiral tubes at pH 12.0. The spiral tubes can transition into a straight shape as the pH of solution is reduced to 7.4. The shape transition of the tubes is reversible as the pH of solution is adjusted back to 12.0. The pH-switchable shape transition suggests that the self-assembled LCA tubes can act as a supramolecular chemical spring. Finally, the LCA tubes are endowed with optical functionality by embedding cadmium sulfide nanopaticles (CdS) in the tube walls by the co-assembling synthesis of cadmium sulfide iv (CdS) nanoparticles with lithocholic acid (LCA) molecules. The fluorescent composite tubes can undergo pH switchable spiral/straight, which are a promising system for a variety of materials and biological applications.