Background: Migraine headache is a chronic pain condition that affects 20% of women of reproductive age, and is often treated with triptans. Triptans are serotonin 1B, 1D, and 1F receptor agonists that act as vasoconstrictors and inhibitors of the trigeminal cervical complex as well as peripheral neurons; they cross the blood brain barrier and placenta, and as such are plausible neurodevelopmental teratogens. No studies have examined risk of neurodevelopmental problems in children with prenatal triptan exposure. This dissertation had three aims: (1) to examine risk of behavioral problems in children using in the presence of time-varying confounding by concomitant medication use; (2) to examine risk of temperamental, motor, and communication disturbances associated with prenatal triptans exposure, adjusting for unmeasured confounding by migraine type and severity; and (3) to examine changes in neurodevelopment over time associated with prenatal triptan exposure. Methods: This dissertation used data from the Norwegian Mother and Child Cohort Study, a prospective birth cohort including more than 100,000 women recruited during their first prenatal ultrasound visit. Aims 1 and 3 used marginal structural models to assess the risk of (1) neurodevelopmental problems at age 36 months (Aim 1), or (2) change in risk of neurodevelopmental problems from 18 to 36 months (Aim 3) associated with prenatal triptan exposure. Aim 2 used propensity matching and calibration to adjust for unmeasured confounding by migraine type, severity, and attitudes towards medication use in pregnancy. Neurodevelopmental outcome measures included the Child Behavior Checklist (CBCL), the Emotionality, Activity, and Temperament Scale (EAS), and the Ages and Stages Questionnaire (ASQ). Exposure to triptans was ascertained by self-report. Results: Prenatal triptan exposure was associated with greater externalizing behavior problems at 18 and 36 months, as well as greater increases in emotionality and activity from 18 to 36 months. We observed no association between triptan exposure and motor skills or communication problems; triptan use during pregnancy was associated with migraine severity but not migraine type, and adjustment for unmeasured migraine characteristics moved effect estimates towards the null. Conclusions: Prenatal triptan exposure is associated with externalizing-type behaviors and temperament in children, while migraine itself is associated with internalizing-type behaviors and temperament. The use of concomitant medications and the severity of the underlying condition both exerted substantial influence on observed effect estimates, and should be considered in any future studies of triptan medication use in pregnancy.

Chlamydia trachomatis is the most common sexually transmitted bacterial disease agent worldwide, and, though frequently asymptomatic, can cause extreme pathology including infertility. Chlamydial species exhibit a unique biphasic developmental cycle. Once attached to a cell surface, infectious elementary bodies (EB) are internalized within an inclusion, the membrane-bound structure in which EB transform to noninfectious, replicable reticulate bodies (RB). After multiple rounds of division, RB condense to form EB, which are released and can infect new host cells. In culture, exposure to stressors, such as beta-lactam antibiotics, induce chlamydiae to reversibly detour from normal development into a noninfectious, viable state termed persistence. Cell culture data suggest that persistent forms are resistant to azithromycin (AZM), a front-line antibiotic, and are able to alter the host transcriptome. Though persistence has been described in culture for over 50 years, whether or not it: i) occurs in vivo; and ii) influences chlamydial pathogenesis, transmission and therapy has remained unresolved. To address these questions, we developed an animal model of persistent chlamydial infection using amoxicillin (AMX) treatment. AMX exposure decreased shedding of infectious chlamydiae in C. muridarum-infected mice without affecting chlamydial viability, demonstrating the presence of persistent chlamydiae. Shedding of infectious EB resumed following AMX cessation. Shedding data and microarray analyses suggested that host immunity might limit chlamydia’s exit from persistence in our model. Thus, we hypothesized that cyclophosphamide (CTX) treatment would increase the magnitude of chlamydial shedding observed after AMX-treatment cessation. CTX treatment increased post-AMX shedding by more than 10-fold compared to AMX-only controls. To determine whether persistent chlamydiae are resistant to antibiotic eradication in vivo, we induced persistence by administering AMX and treated mice with various AZM dosing regimes. Persistently infected mice demonstrated increased treatment failure following AZM therapy compared to productively infected controls. These data suggest that persistent chlamydiae are refractory to treatment in vivo and provide an explanation for the observation that treatment fails in some patients. In addition to creating the first fully characterized, experimentally tractable, in vivo model of chlamydial persistence, these experiments provide evidence that persistent/stressed chlamydial forms may serve as a long-term reservoir of infectious organisms in vivo.

The endoplasmic reticulum (ER) is a multi-functional cellular compartment that functions in protein folding, lipid biosynthesis, and calcium homeostasis. Perturbations to ER function lead to the dysregulation of ER homeostasis, causing the accumulation of unfolded and misfolded proteins in the cell. This is a state of ER stress. ER stress elicits a cytoprotective, adaptive signaling cascade to mitigate stress, the Unfolded Protein Response (UPR). As long as the UPR can moderate stress, cells can produce the proper amount of proteins and maintain a state of homeostasis. If the UPR, however, is dysfunctional and fails to achieve this, cells will undergo apoptosis. Diabetes mellitus is a group of metabolic disorders characterized by persistent high blood glucose levels. The pathogenesis of this disease involves pancreatic β-cell dysfunction: an abnormality in the primary function of the β-cell, insulin production and secretion. Activation of the UPR is critical to pancreatic β-cell survival, where a disruption in ER stress signaling can lead to cell death and consequently diabetes. There are several models of ER stress leading to diabetes. Wolcott-Rallison syndrome, for example, occurs when there is a mutation in the gene encoding one of the master regulators of the UPR, PKR-like ER kinase (PERK). In this dissertation, we show that Wolfram Syndrome 1 (WFS1), an ER transmembrane protein, is a component of the UPR and is a downstream target of two of the master regulators of the UPR, Inositol Requiring 1 (IRE1) and PERK. WFS1 mutations lead to Wolfram syndrome, a non-autoimmune form of type 1 diabetes accompanied by optical atrophy and other neurological disorders. It has been shown that patients develop diabetes due to the selective loss of their pancreatic β-cells. Here we define the underlying molecular mechanism of β-cell loss in Wolfram syndrome, and link this cell loss to ER stress and a dysfunction in a component of the UPR, WFS1. We show that WFS1 expression is localized to the β-cell of the pancreas, it is upregulated during insulin secretion and ER stress, and its inactivation leads to chronic ER stress and apoptosis. This dissertation also reveals the previously unknown function of WFS1 in the UPR. Positive regulation of the UPR has been extensively studied, however, the precise mechanisms of negative regulation of this signaling pathway have not. Here we report that WFS1 regulates a key transcription factor of the UPR, activating transcription factor 6 (ATF6), through the ubiquitin-proteasome pathway. WFS1 expression decreases expression levels of ATF6 target genes and represses ATF6-mediated activation of the ER stress response (ERSE) promoter. WFS1 recruits and stabilizes an E3 ubiquitin ligase, HMG-CoA reductase degradation protein 1 (HRD1), on the ER membrane. The WFS1-HRD1 complex recruits ATF6 to the proteasome and enhances its ubiquitination and proteasome-mediated degradation, leading to suppression of the UPR under non-stress conditions. In response to ER stress, ATF6 is released from WFS1 and activates the UPR to mitigate ER stress. This body of work reveals a novel role for WFS1 in the UPR, and a novel mechanism for regulating ER stress signaling. These findings also indicate that hyperactivation of the UPR can lead to cellular dysfunction and death. This supports the notion that tight regulation of ER stress signaling is crucial to cell survival. This unanticipated role of WFS1 for a feedback loop of the UPR is relevant to diseases caused by chronic hyperactivation of ER stress signaling network such as pancreatic β-cell death in diabetes and neurodegeneration.

Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestine and produces the phage-encoded Shiga toxin (Stx) which is absorbed systemically and can lead to hemolytic uremic syndrome (HUS) characterized by hemolytic anemia, thrombocytopenia, and renal failure. EHEC, and two related pathogens, Enteropathogenic E. coli (EPEC), and the murine pathogen, Citrobacter rodentium, are attaching and effacing (AE) pathogens that intimately adhere to enterocytes and form actin “pedestals” beneath bound bacteria. The actin pedestal, because it is a unique characteristic of AE pathogens, has been the subject of intense study for over 20 years. Investigations into the mechanism of pedestal formation have revealed that to generate AE lesions, EHEC injects the type III effector, Tir, into mammalian cells, which functions as a receptor for the bacterial adhesin intimin. Tir-intimin binding then triggers a signaling cascade leading to pedestal formation. In spite of these mechanistic insights, the role of intimin and pedestal formation in EHEC disease remains unclear, in part because of the paucity of murine models for EHEC infection. We found that the pathogenic significance of EHEC Stx, Tir, and intimin, as well as the actin assembly triggered by the interaction of the latter two factors, could be productively assessed during murine infection by recombinant C. rodentium expressing EHEC virulence factors. Here we show that EHEC intimin was able to promote colonization of C. rodentium in conventional mice. Additionally, previous in vitro data indicates that intimin may have also function in a Tir-independent manner, and we revealed this function using streptomycin pre-treated mice. Lastly, using a toxigenic C. rodentium strain, we assessed the function of pedestal formation mediated by Tir-intimin interaction and found that Tir-mediated actin polymerization promoted mucosal colonization and a systemic Stx-mediated disease that shares several key features with human HUS.

Chronic kidney disease (CKD) progresses faster in males than females; however, the underlying mechanisms remain poorly understood. Sex differences in glomerular capillary morphology has been hypothesized to contribute, in part, to the increased susceptibility to hypertension-induced renal injury and CKD progression in males, but this has not been investigated. The goal of the present study was to assess glomerular capillary morphology in male vs. female rats with intact kidneys and after uninephrectomy (UNX). We hypothesized that glomerular capillary radii (RCAP) and length (LCAP) would be greater in male rats. Male (n=4) and female (n=4) with intact kidneys and UNX (n=4 males, n=4 females) provided a 0.4% NaCl diet and water ad libitum. Kidneys were perfusion-fixed, the left kidney was excised, and a 3 mm transverse section through the midline of the kidney was selected for further processing. Multiple 1 mm3 cubes were randomly excised from the left, middle, and right regions of the outer cortex, embedded in EPONTM, sectioned (1 μm), and stained with toluidine blue. Four glomeruli from each region were randomly selected for stereological analysis. Glomerular tuft volume (VG), RCAP, and LCAP were assessed. In rats with intact kidneys, no significant sex differences were observed in VG, RCAP, or LCAP. VG, RCAP, and LCAP were significant greater in both male and female rats with UNX vs. respective rats with intact kidneys. In rats with UNX, males exhibited a significantly greater VG and LCAP, but not RCAP, as compared to females despite no significant differences in relative kidney weight. These data indicate that males exhibit greater compensatory increases in LCAP following UNX. The greater capillary length may lead to reduced podocyte density, a well-known mechanism that increases the susceptibility to CKD progression.