Trillions of microbes line the gastrointestinal tract to form the gut microbiome, a symbiotic organ whose supportive functions include energy production, immune homeostasis, and defense against pathogens. Disturbances to gut microbial composition, in turn, drive the pathogenesis of various metabolic, inflammatory, and carcinogenic diseases. Much effort has been dedicated to elucidating environmental triggers of gut dysbiosis, not the least of which is the consumption of medications. Antibiotics eradicate keystone commensals and enhance pathogenic behaviors of persisting pathobionts, whose resistance mechanisms can have off-target effects on human physiology and treatment response. Recent evidence indicates that the spectrum of antimicrobial compounds that disturb the gut microbiome extends far beyond traditional antibiotics, and includes commonly prescribed cardiovascular, neuropsychiatric, metabolic, and cancer medications. Although the capacity of non-antibiotic pharmaceuticals to induce gut dysbiosis is well appreciated, their impact on gut microbial function has not been studied systematically. Bacterial multi-omic profiling offers a cost-effective, high-throughput approach to understanding bacterial genetic responses to chemical perturbations, and how these functional changes might reciprocally impact relevant human phenotypes. Our laboratory, which houses a personal strain biobank of over 30,000 gut bacterial isolates spanning over 400 taxa, has established scalable pipelines for bacterial genomic and transcriptomic profiling that are readily applicable to diverse non-model gut microbes. We applied these methodologies to healthy fecal samples and bacterial isolates to elucidate strain-level responses to common pharmaceuticals with known gut microbiome associations. We first performed a gut microbiota transcriptomic screen of 19 representative fecal isolates against 20 top-prescribed orally delivered medications. Computational analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed induction of pathways associated with metabolism and multidrug resistance, including upregulation of efflux machinery by lipid-lowering drugs, antidepressants and cardiovascular medications. We discovered many bacterial responses with clinical significance, which we computationally validated using clinical metagenomic datasets. Most importantly, we showed that statin-mediated overexpression of the AcrAB-TolC efflux pump generates collateral toxicity in dietary retinol and secondary bile acids, resulting in depletion of pump-containing Bacteroidales species from patient microbiomes. We next performed the first comprehensive screen for antimicrobial activity in cancer drugs by exposing three healthy fecal samples to a panel of 41 first-line cancer therapeutics. Using 16S-genomic profiling, we identified several members of the targeted kinase inhibitor (TKI) class that induced gut dysbiosis, including first-line hepatocellular carcinoma (HCC) treatment sorafenib. We profiled natural bacterial isolates exposed to different TKI HCC treatments, and again observed transcriptional induction of conserved multidrug efflux pumps. Adaptive evolution assays identified Resistance-Nodulation-Division (RND) efflux pumps as effectors of TKI resistance. Remarkably, we demonstrated that acquired TKI resistance in evolved Bacteroidales lineages generated strain-specific cross-resistances and collateral sensitivities to several unrelated antibiotics. Collectively, our work demonstrates the importance of profiling xenobiotic impacts on the gut microbial resistome, as bacterial adaptations to pharmaceutical toxicities can feed back onto microbiome communities and the human host to affect health outcomes.

Indiana University-Purdue University Indianapolis (IUPUI) / According to one theory of root resorption, occlusal trauma during orthodontic tooth movement damages the cementum covering the root dentin. The body detects the exposed dentin and seeks to remove it, and the result is root resorption. This experiment will explore an aspect of this theory by quantifying the amount and location of damage in mechanically fatigued teeth. Nine dog mandibles were sectioned at the mandibular symphysis. Each half was mounted in orthodontic resin with the incisors upright and exposed. The block was inserted into a jig and placed into a servohydraulic mechanical testing machine. The left central incisor was fatigue loaded with a 2Hz, 10-90 N sinusoidal force for 100,000 cycles (approximately 14 hours). The right central incisor served as the control. Both specimens were scanned with a micro-CT unit, stained with basic fuchsin, and then sectioned along the sagittal plane. Because the experimental and control specimens were stained before sectioning, only microdamage due to the loading process would be evident in the sections. Microdamage which occurred during the sectioning process would not be stained. Central sections through the long axes of the samples were examined for the presence of microdamage with a light microscope and a micro-CT unit. Based on preliminary findings, two types of staining patterns were measured and recorded. The first was called "diffuse stain" and consisted of large stained areas in the dentinal tubules. Diffuse stain was not associated with any visible features at the dentinocemental junction. The second type of staining pattern was called "stained defects." Stained defects were stained irregularities at the dentinocemental junction. For statistical analysis, the roots were divided into buccal-cervical, buccal-middle, buccal-apical, lingual-cervical, lingual-middle, and lingual-apical regions. Comparisons between the fatigued and non-fatigued teeth for differences in area, length, and depth were made under the generalized estimating equation (GEE) framework applied to normally-distributed data. Because the measurements were not normally distributed, a rank transformation of the measurements was performed before conducting the analyses. Comparisons between the fatigued and non-fatigued teeth for differences in presence or absence of stain or defects were made using Cochran-Mantel-Haenszel tests. Repeatability of the measurements was assessed using intraclass correlation coefficients (ICCs), paired t-tests, and Bland-Altman plots. The ICC's ranged from 0 .85 to 1.00, thus making the repeatability of the measurements generally very good. The statistical analysis showed there were no significant differences between the experimental and control teeth for stained defects or diffuse staining for length, depth, or area measurements. However, analyses comparing the distribution of stained defects and diffuse stain within the control and experimental specimens showed significant differences in the distribution of stained defects within the experimental specimens. In the experimental specimens, the stained defects were distributed in a gradient, with the most in the apical region and progressing to the least amount in the cervical region. In the control specimens, there was only a difference in the stained defects between the cervical and apical regions. This distribution is consistent with the biomechanical model which shows increasing stress moving from the cervical region towards the apex. These results show that the test and control specimens differed in how the stained defects were distributed throughout the root, even though there were no differences in the amount of staining between the control and experimental specimens. Whole tooth and histologic slides were scanned with the micro-CT unit, but the dentinocemental junction could not be delineated enough to make any measurements. No data could be collected regarding microdamage in this area using the micro-CT unit. It was recommended that future studies use a tomography unit with better resolution, use a larger samples size, employ a contrast agent when trying to visualize microdamage with the micro-CT unit, and incorporate a way to measure the intensity of the staining in addition to the location and size.

Gender has long been considered an important factor in uncovering the etiology of schizophrenia. There is now strong evidence that gender is related to the age at onset of psychotic symptoms and somewhat weaker evidence of gender differences in other aspects of schizophrenia, such as clinical and functional outcomes. Simultaneously, there is growing evidence that age at onset itself is an important prognostic factor. This dissertation seeks to investigate the complex relationship between gender, age at onset, and outcomes of schizophrenia spectrum disorders, in light of two main mechanisms, estrogen protection and psychosocial development. The estrogen protection hypothesis posits that estrogen has antipsychotic effects that result in a later age at onset and more favorable outcomes for pre-menopausal women compared with men with onset at the same age. When women lose estrogen protection around menopause, they may develop a more severe illness. The psychosocial development hypothesis posits that women who have later onset due to estrogen protection have more time to develop psychosocial resources that improve their role functioning later in life. In Chapter 2, a systematic literature review was conducted on the relationship between gender and age at onset of schizophrenia spectrum disorders in determining functional outcomes. There was some support for the psychosocial development hypothesis where age at onset mediates the relationship between gender and functional outcomes. Three studies were identified that conducted formal mediation analyses and used minimum criteria to detect mediation in the remaining studies. Together, these findings suggest that women have better functioning outcomes than men, by virtue of having later illness onset. Chapters 3 & 4 utilized a nationally representative sample of people with schizophrenia spectrum disorders in China to further examine the relationship between gender, age at onset, and other illness outcomes. Unlike many studies in the current literature, this dataset included people who had never sought formal treatment, providing a unique opportunity to explore the possibility of selection bias in other studies that primarily recruit participants from treatment settings. Furthermore, the majority of schizophrenia research has been conducted in high-income countries, and these data from China could provide some important insights from a low- to middle-income country. In Chapter 3, finite mixture techniques were used to model the entire distribution of age at onset as a function of gender and family history, taking into account any clustering patterns of observations. The analysis identified an early-onset group characterized by men and those with a family history, a mid-onset group characterized by both men and women without a family history, and a late-onset group characterized by women without a family history. Distinguishing between these groups showed that gender and family history are indeed robust predictors of age at onset. Chapter 4 investigated a) the interaction between gender and age at onset in producing clinical outcomes and b) the mediating effect of age at onset in the relationship between gender and role functioning outcomes. This study did not find evidence of interaction between gender and age at onset in producing clinical outcomes. It did find that women’s increased probability of marrying was mediated by a later age at onset. Overall, the results of this dissertation provide even further support of the well-established relationship between gender and age at onset. However, applying the estrogen protection hypothesis to schizophrenia spectrum outcomes requires further study. This dissertation also suggests that the psychosocial development hypothesis, which is not well-studied, may provide a promising new perspective on whether and why women appear to have better outcomes. This research also expands our knowledge of gender differences in schizophrenia spectrum disorders in China and highlights the importance of further research in other low- to middle-income countries. While the estrogen protection and psychosocial development hypotheses are surely important in our understanding of women with schizophrenia spectrum disorders, conducting this study using a dataset from China called attention to other social, political, and economic issues that women face that affect all aspects of their illness. These issues and their impact on women are likely different in high-income countries. Ultimately, the estrogen protection and psychosocial development hypotheses can only be understood within the complex context of women’s lived experience with schizophrenia spectrum disorders.

Adhesion G-protein coupled receptors (GPCRs) are the second largest class of GPCRs, yet their functions and ligands remain predominantly unidentified. Polymorphisms in the gene encoding the adhesion GPCR latrophilin 3 (ADGRL3) have been associated with an increased risk for attention deficit hyperactivity disorder (ADHD) and substance use disorder (SUD) in various linkage and association studies. Disrupting the function of ADGRL3 homologs across mammalian and invertebrate model systems leads to changes in various dopaminergic phenotypes such as hyperactivity, sleep impairment, and changes in sensitivity to psychostimulants, suggesting that ADGRL3 contributes to behavior by modulating dopamine signaling. Here, I use behavioral and imaging studies to delineate an important role for Cirl, the Drosophila homolog of ADGRL3, in a recently characterized dopaminergic sleep circuit. Sleep impairment is a common symptom in both SUD and ADHD, and sleep studies are well established in Drosophila. Our work shows that fruit flies that carry a null mutation for Cirl are hyperactive and display a deficit in sleep that is enhanced by adult thermogenetic activation of dopamine neurons. Though Cirl displays high expression within dopamine neurons, conditional knockout of Cirl in dopamine neurons does not recapitulate sleep deficits seen in Cirl null flies, and specific rescue of Cirl in a knockout background does not ameliorate them. Intriguingly, activating dopamine neurons in Cirl null flies throughout development rescued the sleep deficits, indicating that this dopaminergic intervention induces lasting changes that can ameliorate lack of Cirl function. Imaging studies reveal that Cirl shows high expression in the central complex, which is involved in sleep and receives dense dopaminergic input. I demonstrate that Cirl functions within different populations of the central complex downstream of dopaminergic innervation to differentially affect night and daytime sleep through both dopaminergic and non-dopaminergic mechanisms. This work delineates a novel role for an adhesion GPCR in modulating sleep behavior, and further characterizes ADGRL3 as a potential therapeutic target for disorders characterized by dysregulation of dopaminergic neurotransmission.

Recent studies have revealed that crosstalk between glioma cells and the brain microenvironment is a crucial regulator of cancer initiation and progression. A vast majority of glioma patients suffer from seizures, and this pathological neuronal activity has been proposed to contribute to increased glioma cell proliferation. Glioma patients also suffer from additional neurological symptoms, including deficits in attention, concentration, memory, and language. These neurological effects of gliomas along with the limited therapeutic options underscore the need for novel therapies. This thesis investigates the neuronal alterations at the margins of glioma which contribute to the neurological symptoms (Chapter 2), and on the effect of sensory stimulation on the glioma cells and microglia in the glioma microenvironment (Chapter 3). The work describes the development of new mouse models in which glioma cells are infiltrating the somatosensory cortex in mice that express GCaMP in neurons or microglia. Methodologies include a combination of in vivo two-photon calcium imaging and tissue-based analysis to determine the glioma-induced alteration on whisker stimulation-evoked responses of these different cell types. This work also tests the effects of pharmacologically inhibiting mTOR signaling on neuronal responses (Chapter 2) or purinergic signaling on microglial responses (Chapter 3). Together these studies demonstrate that glioma infiltration induces local effects in functionally-responsive cortex, and that many of these glioma-induced effects on neurons and microglia are ameliorated by pharmacological inhibition of mTOR or purinergic signaling. This reveals a highly dynamic and plastic nature of the glioma-induced alterations, and points towards new strategies to treat glioma-associated neurological symptoms while potentially slowing tumor progression.

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Advances in sequencing technologies have sparked the discovery of new genetic etiologies for neurological and neurodevelopmental disorders. As new disease-causing mutations are unveiled, questions into the specific mechanisms of pathogenicity and potential therapeutic approaches arise. To address these questions, in vivo and in vitro models have been generated and analyzed; but how best to utilize these models, and how well they recapitulate the human brain, is still not fully understood. Within the work discussed in this thesis, we address this problem through the transcriptomic and functional interrogation of these models in the context of neurodevelopment and disease. In Chapter 2 of this thesis, we describe the use of single-cell RNA-sequencing to examine the longitudinal transcriptomic profiles of neuronal network establishment and maturation in ex vivo mouse cortex- and hippocampus-derived cultures. Our data highlights unique developmental transcriptomic profiles for individual genes, disease gene subclasses, and biological processes, and discusses cell population-specific divergent transcriptomic profiles between genes associated with neurological diseases, focusing on epilepsy and autism spectrum disorder. We also compared the data from our ex vivo system to transcriptomic data collected from in vivo neonatal and adult mouse brains and human cortical organoids, highlighting the importance of the generation and consideration of system-specific transcriptomic datasets when looking into a gene, disease, or biological process of interest, and serves as a vital resource for researchers. In Chapter 3, we propose a high-throughput drug discovery paradigm utilizing the application of transcriptome reversal for neurodevelopmental disorder-associated genes that affect the transcriptome. This approach describes the idea that if gene dysregulation is causal for the pathogenicity of a disease, then correcting the transcriptional signature should have a therapeutic effect. We demonstrated that small-molecule induced gene expression changes vary between both cell lines and neural cell populations, and highlight both the importance of selecting the appropriate model of disease and creating cell population-specific signatures for compounds and disease. In Chapter 4, we focus on the utilization of multi-electrode arrays for the electrophysiological characterization of primary cortical networks derived from mouse models of epileptic encephalopathy. This technique allows for the analysis of numerous neuronal and network synchronization metrics for spontaneous longitudinal activity and responses to external stimuli in the form of electrical stimulation and compound addition. In particular, mouse models with mutations in the genes Grin2a, Gnb1, and Scn1a were analyzed. We discovered significant hyperexcitability, bursting, and synchrony phenotypes, and discuss how acute and chronic compound addition can be used to interrogate biological pathways and reverse disease activity signatures.

This thesis explored the mechanism of action of MPTP and its toxic metabolite, MPP⁺, and compared it to the mechanism of action of haloperidol metabolites, some of which are found in schizophrenic patients. Experiments assessed the effects of these compounds on several aspects of amine uptake in mouse brain synaptosomes. Both MPTP and MPP⁺ were inhibitors of labeled neurotransmitter (serotonin and dopamine) uptake consistent with previous studies. MPP⁺ had a higher inhibitory potency in the dopaminergic system, while MPTP had a higher inhibitory potency in the serotonergic system. Haloperidol metabolites (HPP⁺, R-HPP⁺, and HPTP) also inhibited both amine transport systems with a greater affinity for the serotonergic system. Additional studies demonstrated that all of the above compounds showed reversible inhibition of serotonin uptake following drug removal by centrifugation and resuspension. In the dopaminergic system, both MPTP and MPP⁺ were reversible; however, HPP⁺ was not. This finding suggests that HPP⁺ treatment may result in irreversible poisoning of the nerve terminal or it may demonstrate a slow off-rate for its interaction with the dopamine transporter. Furthermore, HPP+ showed non-competitive inhibition of both serotonin and dopamine uptake. Amine uptake in the presence and absence of HPP* had a decreased maximal inhibitory effect and no potency change. The reversible inhibition of serotonin uptake by HPP⁺ might suggest competitive inhibition, but apparently, the comparative rates of binding and unbinding of HPP⁺ and serotonin resulted in a non-competitive interaction. These experiments support the use of MPTP as a model system for analyzing the neurotoxic potential of toxins, drug metabolites, and pesticides. The similar in vitro potencies suggest that the haloperidol derivatives could have effects similar to those of MPP⁺ in vivo. / Master of Science

Osteoarthritis (OA) is a debilitating, degenerative joint disease that affects over 32.5 million adults in the United States and nearly 595 million people globally. OA is a major cause of pain and disability and is among the most expensive conditions to treat, carrying an annual healthcare cost of over $16.5 billion. The disease has classically been characterized by the degradation of articular cartilage and subchondral bone; however, changes to the synovium have recently garnered appreciation as synovitis has been linked to OA symptoms and progression. While the importance of the synovium in diarthrodial joint health and pathology is now widely accepted, quantitative structure-function data remains sparse. There is a need to investigate synovium structure-function relationships to better understand the synovium’s role in joint homeostasis and disease. The role of sex-based differences in OA has gained attention as epidemiological studies reveal that the incidence and prevalence of OA is higher in women than in men. Sex as a variable has rarely been considered in preclinical animal studies and in vitro laboratory experiments that explore the mechanisms of OA development and progression. Furthermore, therapeutic approaches for the treatment of OA have not adequately considered sex-based differences. As the population of those at risk for OA grows, the influence of sex-based differences in OA warrants more attention, particularly in the regenerative strategies for cartilage repair. This dissertation seeks to address persistent questions regarding OA etiology and the mechanisms underlying disease progression, as well as strategies to enhance cartilage tissue engineering therapies. The objectives of this dissertations are three-fold: (1) to further the understanding of synovium tribology (2) to develop a tissue-engineered (TE) human synovium to facilitate the study of synovium structure-function relationships and (3) to elucidate sex-based differences in cartilage regenerative medicine strategies. In Chapter 2, we assess the hypothesis that tissue glycosaminoglycan (GAG) content contributes to the low friction properties of the synovium. Bovine and human synovium tribological properties were evaluated using a custom friction testing device. Following proteoglycan depletion, synovium friction coefficients increased while GAG content decreased. In a second study, synovium samples were treated with interleukin-1 (IL) to observe inflammatory-induced structural changes. IL treatment elevated GAG concentration and decreased friction coefficients. No changes to collagen content were observed following IL treatment. For the first time, a relationship between synovium friction coefficient and GAG concentration was demonstrated. The study of synovium tribology is necessary to fully understand the mechanical environment of the healthy and diseased joint. Chapter 3 documents the development of a human TE synovium and its ability to recapitulate native tissue properties and responses to chemical stimuli. A mixed donor population of primary human fibroblast-like synoviocytes was combined with a commercially available extracellular protein mixture to fabricate TE synovium constructs. At baseline, mature TE synovium exhibited characteristics of native synovium such as the formation of an intimal lining and the expression of critical proteins like lubricin, cadherin-11, and collagen type IV. In response to IL and dexamethasone treatment, TE synovium underwent biochemical changes that mimicked the changes observed in human explants. In addition, solute transport measurements were performed to highlight the relationship between synovium extracellular matrix (ECM) composition and its functional properties, resulting in a proposed link between tissue GAG content and diffusion coefficient. A human TE synovium enables the investigation of synovium structure-function relationships in a controlled manner and can serve as a platform for disease modeling and drug screening, which may accelerate the development of new treatments for maintaining joint health that specifically target the synovium. In Chapter 4, sex-based differences in the ECM properties of canine engineered cartilage and in its degradative response to IL insult are evaluated. Isolated chondrocytes from male or female cartilage donors were encapsulated in agarose to create cylindrical cartilage constructs. Mechanical and biochemical measurements demonstrated that the sex of the donor chondrocytes did not influence intrinsic, de novo tissue formation after 42 days of tissue maturation. Following IL treatment, the mechanical, biochemical, and media analyses revealed that the sex of the donor cells did not influence the engineered cartilage’s response to IL insult. By understanding how sexual dimorphism impacts cartilage growth and susceptibility to proinflammatory cytokine insult, we may better direct cell-based strategies for cartilage repair that are personalized to account for patient sex.