Pulmonary fibrosis is a potentially deadly pathology characterized by excessive deposition of extracellular matrix (ECM), increased tissue stiffness, and loss of tissue structure and function. Recent evidence has suggested epithelial to mesenchymal transition (EMT), the transdifferentiation of an epithelial cell into a mesenchymal fibroblast, is one mechanism that results in the accumulation of myofibroblasts and excessive deposition of ECM. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin mediated interactions with ECM proteins and soluble growth factors including TGFβ. TGFβ, a potent inducer of EMT, can be activated by cell contraction mediated mechanical release of the growth factor from a macromolecular latent complex. Therefore, TGFβ activity and subsequent EMT may be influenced by both the biochemical composition and biophysical state of the surrounding ECM. Based on these knowns it was first investigated how changes in the biochemical composition of the matrix and changes in tissue rigidity together modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin specific interactions with fibronectin (Fn) variants displaying both the RGD and PHSRN binding sites facilitate cell binding through α3β1 and α5β1 integrins, and that these interactions maintain an epithelial phenotype despite engagement of increased tissue rigidities. Conversely, Fn fragments that facilitate cell binding through αv integrins drive TGFβ activation and subsequent EMT even while engaging soft underlying substrates. Adding to the complexity of studying mechanisms that contribute to pulmonary fibrosis, is exposure of the lung to injuries from environmental particulates. Therefore, we investigated how EMT is altered in response to particulate matter (PM). Here we show that PM exposure further drives TGFβ activation, EMT, and increases intracellular levels of reactive oxygen species (ROS). Additionally, cells binding the ECM through α5β1 and α3β1 integrins only partially recover an epithelial phenotype, suggesting ROS may be a secondary driver of TGFβ and EMT. Taken together these results suggest dynamic changes to the ECM microenvironment are major contributors to the control of EMT responses and provide insights into the design of biomaterial-based microenvironments for control of epithelial cell phenotype.

Central to the age-associated decrease in immune system function, characterised by the increase in the frequency and severity of infections and autoimmune diseases, is the decrease in production of naïve T cells by the thymus. This results from the targeted degeneration or involution of the thymus with age. One of the principal causes of involution is the loss of organisation and functionality of the thymic epithelium, which confers the primary function of the organ via interactive regulation of T cell development. Although the mechanisms that govern the deterioration of the thymic epithelium are poorly understood, a number of recent reports indicate that the transcription factor, Foxn1, is required to maintain this compartment in the postnatal thymus. Thus, the first aim of this study was to precisely profile Foxn1 expression levels in aging postnatal thymic epithelial cells. The second aim was to investigate the effects of upregulating Foxn1 in the aging thymus, which was achieved using a novel, regulatable Foxn1 mouse model generated during this study. In this study I show that Foxn1 is expressed at different levels in different postnatal thymic epithelial cell (TEC) sub-populations suggesting a dosage-dependent mode of action for Foxn1. Additionally, using two experimental approaches, I show that Foxn1 expression decreases with age in TECs, supporting the current data that implicate the loss of Foxn1 as a potential cause of thymus involution. Next, I generated a tissue-specific, regulatable Foxn1 mouse model that allowed me to modulate Foxn1 expression in the postnatal thymus. Firstly, using this model, I show that thymus involution can be prevented by the up-regulation and maintenance of Foxn1 expression from the onset of involution. Thymi that up-regulated Foxn1 were overtly larger and exhibited greater cellularity in both the thymocyte and epithelial compartments compared to age matched controls. Additionally, the larger TEC compartment contained a higher proportion of functional and proliferating TECs that upregulated a panel of genes involved in TEC development and function. Next, I show that Foxn1 up-regulation in aged, involuted thymi is sufficient to partially reverse involution, as shown by an increase in TEC organisation and intrathymic T cell numbers. While other strategies that promote thymic rebound or reversal have been reported, including cytokine treatment or sex steroid ablation, these approaches are complicated by side effects and toxicity. Hence, I propose a novel model for immune reconstitution through the regulation of Foxn1 expression in the postnatal thymus.

The genotoxicity of energetic compounds (i.e., explosives) that are known to be present in contaminated soils at military training sites has not been extensively investigated. Thus, the Salmonella mutagenicity and Muta(TM)Mouse assays were employed as in vitro assays to examine the mutagenic activity of twelve explosive compounds, as well as three soil samples from Canadian Forces Base Petawawa. Salmonella analyses employed strains TA98 (frameshift mutations) and TA100 (base-pair substitution mutations), as well as the metabolically-enhanced YG1041 (TA98 background) and YG1042 (TA100 background), with and without exogenous metabolic activation (S9). For Salmonella analyses, the results indicate that ten of the explosive compounds were mutagenic, and consistently elicited direct-acting, base-pair substitution activity. All three soil samples were also observed to be mutagenic, eliciting direct-acting, frameshift activity. Mutagenic potencies were significantly higher on the metabolically-enhanced strains for all compounds and soil samples. For Muta(TM)Mouse analyses on FE1 cells, the results indicate that the majority of explosive compounds did not exhibit mutagenic activity. All three soil samples elicited significant positive responses (PET 1 and PET 3 without S9, and PET 2 with S9), and although there is some evidence of a concentration-related trend, the responses were weak. Correspondence of the mutagenic activity observed with the two assay systems, for both the explosive compounds and soil samples, was negligible. The differential response is likely due to differences in metabolic capacity between the two assay systems. Furthermore, it is likely that there are unidentified compounds present in these soil samples that are, at least in part, responsible for the observed mutagenic activity. Additional testing of other explosive compounds, as well as soil samples from other military training sites, using a variety of in vitro and in vivo assays, is warranted in order to reliably estimate mutagenic hazard and subsequently assess risk to human health.

The initiating events of ovarian cancer remain unknown, but an established risk factor is use of estrogen therapy by post-menopausal women where there is a positive correlation between duration of use and risk for disease. Mouse models of ovarian cancer have shown that exposure to exogenous 17β-estradiol (E2) accelerates tumour onset so this study aims to investigate the E2 signalling mechanisms responsible for sensitizing ovarian epithelial cells to transformation. By developing model systems that are responsive to E2 manipulation, we showed that E2 induces the formation of epithelial dysplasias both in vitro and in vivo. microRNA microarray was used to discover that E2 up-regulates microRNA-378 via the ESR1 pathway, resulting in the down-regulation of a tumour suppressor gene called Disabled-2 (Dab2). E2 suppression of Dab2 was found to result in increased proliferation, loss of contact inhibition, epithelial dysplasia, and increased sensitivity to transformation. This mechanism was also found to be active in mouse fallopian tube epithelium and human ovarian cancer cells. Single-cell RNA sequencing and trajectory analysis was subsequently used to explore additional signalling mechanisms that might contribute to the emergence of dysplastic lesions induced by E2. Multiple molecular signalling pathways dysregulated by E2 were identified and this revealed several possible biomarkers to be investigated for early detection of ovarian cancer. In the context of a current lack of strategies for ovarian cancer prevention or early detection, this work represents a significant advance in our understanding of how E2 promotes ovarian cancer initiation.

L’invasion de la matrice extracellulaire est le premier obstacle que les tumeurs solides rencontrent lors de leur dissémination métastatique. Il existe deux mécanismes principaux: 1) la transition épithélio-mésenchymateuse au cours de laquelle les cellules épithéliales, à l’échelle individuelle, perdent leur cohésion intercellulaire et acquièrent des capacités de motilité pour quitter leur site d’origine ; 2) l’invasion collective dans laquelle des cellules en groupe coopèrent pour migrer collectivement en se frayant un passage à travers le microenvironnement. Dans les modèles murins, on sait maintenant que des groupes ou agrégats de cellules tumorales circulantes issus du détachement de tumeurs primaires présentent une capacité accrue à coloniser des organes à distance. Cependant, les facteurs et mécanismes sous-jacents conduisant au détachement des cellules tumorales en groupe sont peu connus. Pour étudier ces mécanismes, nous avons utilisé des sphéroïdes tumoraux, modèle expérimental tri-dimensionnel (3D), qui mime le microenvironnement ainsi que les caractéristiques morphologiques et fonctionnelles de la tumeur primaire. Nos résultats indiquent que l'inhibiteur de l’activateur du plasminogène (PAI-1), une protéine matricellulaire trouvée en forte concentration dans les cancers invasifs, favorise le détachement en groupe et l’invasion collective de cellules du cancer du sein (lignée cellulaire MCF7) et du colon (lignée cellulaire HCT116) organisées en sphéroïdes tumoraux. PAI-1 a des propriétés dé-adhésives sur des cellules étalées en monocouche sur des matrices pro-adhésives et génère l’agrégation des cellules entre-elles. Ces cellules conservent leurs caractéristiques épithéliales malgré une modification importante de la dynamique du réseau d'actine.Nos résultats suggèrent que les récepteurs cellulaires de la famille des lipoprotéines de basse densité (LDL-R) sont impliqués dans les propriétés dé-adhésives de PAI-1. Dans la cellule, la transduction du signal passe par les kinases ROCK (kinase associée à la GTPase RhoA) et Janus (JAK), ce qui conduit à une phosphorylation de la chaîne légère de la myosine II (MLC2), nécessaire pour l’activité contractile de la myosine et des facteurs de transcription STAT3. En outre, ROCK, un régulateur connu de la contraction du cytoskelette d’actomyosine, est également impliqué dans l'activation de STAT3. L'inhibition de ROCK ou JAK rétablit l’adhésivité des cellules en 2D et réduit leur migration en 3D. Nos données suggèrent que PAI-1 génère des zones de forte activité contractile membranaire dans les cellules en périphérie de la tumeur via ROCK-MLC2 et JAK-STAT, ce qui promeut le détachement de groupe de cellules hautement invasives. Ce nouvel axe de signalisation fonctionnelle de PAI-1 représente une cible anti-métastatique potentielle. / Invasion of the extracellular matrix is the first obstacle that solid tumours encounter during their metastatic dissemination. There are two main mechanisms: 1) epithelial to mesenchymal transition wherein epithelial cells lose their intercellular cohesion and convert to individual migratory behaviours to escape their point of origin; 2) invasion in a collective manner, in which a group or cluster of cohesive cancer cells detaches from the tumour mass and progressively, pushes its way through the microenvironment. It is now known that circulating tumour cell clusters may result from the evasion of cohesive small groups of cells from tumours and such tumour cell clusters display an increased propensity to colonize distant organs in mouse models. However, the extracellular factor/s and the underlying mechanism that enable cell detachment, as clusters are largely unknown. To study the process of tumour cell detachment and invasion, we used a three-dimensional (3D) multicellular tumour spheroid (MCTS) model, which mimics the microenvironment as well as morphological, functional and symmetric geometry features of the primary tumour. Our results strongly indicate that the plasminogen activator inhibitor-1 (PAI-1), a matricellular protein found in high concentration at the invasive front of most cancers, promotes cancer cell cluster detachment and a collective invasion phenotype within MCTS of breast cancer MCF7 and colon cancer HCT116 cell lines. We found that PAI-1 has a de-adhesive effect which induced a multilayered cell clustering of cells spread out on a pro-adhesive matrices in 2D monolayer cultures. Cells retained their epithelial characteristics and membrane-localized E-cadherin despite significant modification of actin dynamics. PAI-1 functions as a de-adhesive molecule most likely involved low-density lipoprotein receptors at the cell surface. We report that the downstream intracellular events may be mediated through the Rho-associated kinase (ROCK) and Janus kinases (JAK) signalling pathway, resulting in phosphorylation of either myosin light chain 2 (MLC2), which is required for myosin II-mediated contractility or STAT3 transcription factors. In addition, ROCK, a known contributor to actomyosin contractility is involved in STAT3 phosphorylation and activation. Inhibition of ROCK and JAK restored adhesion of cells on 2D substratum and reduced their migration/invasion within 3D MCTS model. Our data support a model in which PAI-1 generates actomyosin contractility and high membrane activity at the tumour periphery in a JAK-STAT/ROCK-MLC2 dependent manner promoting the detachment of highly invasive cell clusters. This novel axis of functional signalling of PAI-1 is a potential anti-metastasis target.

The genotoxicity of energetic compounds (i.e., explosives) that are known to be present in contaminated soils at military training sites has not been extensively investigated. Thus, the Salmonella mutagenicity and Muta(TM)Mouse assays were employed as in vitro assays to examine the mutagenic activity of twelve explosive compounds, as well as three soil samples from Canadian Forces Base Petawawa. Salmonella analyses employed strains TA98 (frameshift mutations) and TA100 (base-pair substitution mutations), as well as the metabolically-enhanced YG1041 (TA98 background) and YG1042 (TA100 background), with and without exogenous metabolic activation (S9). For Salmonella analyses, the results indicate that ten of the explosive compounds were mutagenic, and consistently elicited direct-acting, base-pair substitution activity. All three soil samples were also observed to be mutagenic, eliciting direct-acting, frameshift activity. Mutagenic potencies were significantly higher on the metabolically-enhanced strains for all compounds and soil samples. For Muta(TM)Mouse analyses on FE1 cells, the results indicate that the majority of explosive compounds did not exhibit mutagenic activity. All three soil samples elicited significant positive responses (PET 1 and PET 3 without S9, and PET 2 with S9), and although there is some evidence of a concentration-related trend, the responses were weak. Correspondence of the mutagenic activity observed with the two assay systems, for both the explosive compounds and soil samples, was negligible. The differential response is likely due to differences in metabolic capacity between the two assay systems. Furthermore, it is likely that there are unidentified compounds present in these soil samples that are, at least in part, responsible for the observed mutagenic activity. Additional testing of other explosive compounds, as well as soil samples from other military training sites, using a variety of in vitro and in vivo assays, is warranted in order to reliably estimate mutagenic hazard and subsequently assess risk to human health.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death in the United States. Aldo-keto reductase 1 B10 (AKR1B10) is highly expressed in colon and small intestine of normal humans, but its expression is lost or markedly down-regulated in tissues of patients with ulcerative colitis (UC) and CRC. AKR1B10 is a monomeric cytosolic enzyme with strong enzymatic activity to α, β-unsaturated carbonyl compounds, protecting cells from carbonyl lesions; AKR1B10 also mediates de novo synthesis of long chain fatty acids and membrane lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2). To study the etiopathogenic role of AKR1B10 in UC and CRC, our lab generated AKR1B8 deficient (AKR1B8 -/-) mice. AKR1 B8 is the orthologue in mice of human AKR1B10,

abstract: An estimated 267 million women worldwide are HSV-2 seropositive, including roughly 20% of reproductive-aged American women. HSV-2 is a neurotropic virus that establishes a persistent, life-long infection that increases risk for STI acquisition in individuals. The vaginal epithelium represents a critical first line of defense against infection, and during acute infection, underlying immune mechanisms in the epithelium may be critical to protect against disease pathogenesis. The recently identified pro-inflammatory cytokine IL-36gamma has been shown to be expressed at mucosal epithelia, including the female reproductive tract (FRT) and may be an important factor in host defense. Although IL-36gamma has been shown to be induced in the FRT after exposure to microbial products, the contributions of IL-36gamma to host defense mechanisms in response to this clinically relevant STI pathogen are not well understood. This dissertation describes the regulation of IL-36gamma in the FRT and explores its contribution to the host response against genital HSV-2 infection. To test the hypothesis that IL-36gamma is a key regulator of mucosal inflammation and immunity in the FRT, hormonal regulation of IL-36gamma in the FRT was investigated using estrogen- and progesterone-conditioned mice. From this preliminary study, it was shown that progesterone dampens IL36G expression relative to estrogen and may potentially increase susceptibility to infection. Next, the impact of IL-36gamma treatment on HSV-2 infection and replication in human 3-D vaginal epithelial cells was explored. In parallel, the impact of intravaginal IL-36gamma delivery on HSV-2 disease pathogenesis was evaluated using a lethal murine challenge model. IL-36gamma pre-treatment significantly limited HSV-2 replication in vitro and in vivo and was associated with transient neutrophil infiltration that corresponded with decreased disease severity and increased survival in mice. Last, the requirement for IL-36gamma in host defense was investigated utilizing IL-36gamma-/- mice in a lethal HSV-2 murine challenge model. Following infection, IL-36gamma-/- mice exhibited significantly impaired neutrophil recruitment, decreased overall survival time, and significantly increased viral neuroinvasion relative to wild type mice. Collectively, these data indicate that IL-36gamma is a crucial regulator of HSV-2-induced neutrophil infiltration and appears to function in a previously uncharacterized manner to limit viral neuroinvasion in genital HSV-2 disease pathogenesis. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2019

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of many studies, the effects that environmental changes have on this form of movement are poorly understood. In certain pathologies where the cornea exists in a chronic hypoxic state, wound healing is delayed. The goal of this work is to examine the changes in corneal structure in hypoxic corneas that may affect migration and to determine the effects that changes in basement membrane stiffness and focal adhesion turnover have on epithelial cell migration. We analyzed migration after injury in organ cultures and determined that hypoxic corneas exhibited alterations in leading edge morphology. Under hypoxia, fibronectin localization to the apical epithelium and anterior stroma was reduced. Investigators have suggested that alterations in basal lamina composition may increase the stiffness of the membrane. To examine the effect that increased stiffness has on collective migration we performed traction force microscopy. Using multi-layered corneal epithelial sheets, we developed a novel method to analyze the generation of cellular traction forces and the directionality of sheet movement on polyacrylamide gels. We determined that the leading edges of corneal epithelial sheets undergo contraction prior to migration. Alterations in stiffness affected the amount of force exerted by cells at the leading edge. On stiffer surfaces, individual cells within sheets exhibited greater movement compared to cells on softer substrates. To further assess sheet dynamics, we examined the activation of focal adhesion proteins in hypoxic corneas and in human corneal limbal epithelial (HCLE) cells seeded onto soft and rigid substrates. Wounded hypoxic corneas displayed alterations in the localization of the focal adhesion proteins paxillin and vinculin. In HCLE cells cultured on stiff substrates, there was an elevation in vinculin pY1065 phosphorylation after injury, a reduction in vinculin-positive focal adhesions, and decreased actin bundle thickness. Our results demonstrate that changes in membrane stiffness may affect cellular tractions and vinculin dynamics, possibly contributing to the delayed healing response associated with certain corneal pathologies.

No / To investigate whether there are differences between members of the Burkholderia cepacia complex in their ability to invade human respiratory epithelial cells, 11 strains belonging to genomovars I-V were studied in an antibiotic protection assay using the A549 cell line. Strains belonging to genomovars II and III were more invasive than those of genomovars I, IV and V. There was also intra-genomovar variation in invasiveness. No correlation between invasiveness and other putative virulence factors of importance in B. cepacia infection in individuals with cystic fibrosis, cable pilus and B. cepacia epidemic strain marker was identified.