<p>Regardless of their origin or pathology, many, if not all, diseases have long been regarded as complex. Yet, despite the progression in the understanding of complexity and the development of systems biology, the majority of biomedical research has been derived from qualitative principles. In comparison to the ethical, temporal and logistical limitations of human experimentation, <em>in vivo</em> animal models have served to provide a more advantageous means to elucidate the underlying disease mechanisms. However, given the additional limitations presented by such models, <em>in silico </em>models have emerged as an effective complement, and, in some cases, a replacement for <em>in vivo</em> experimentation. The <em>in silico </em>models presented in this thesis were developed using mathematical and computational methods to investigate the evolution of two complex, diverse diseases from a systems biology perspective: allergic asthma and cancer.</p> <p>We generated two novel <em>in silico</em> models of allergic asthma aimed at clarifying some dynamic aspects of allergic responses. Experimentally, we utilized an <em>in vivo</em> murine model of chronic exposure to the most pervasive aeroallergen worldwide, house dust mite (HDM), for up to 20 weeks, equivalent to at least 20 human years. Using a range of HDM concentrations, experimental data were collected to study local and systemic effects. The first model applied empirical mathematical techniques to establish equations for airway inflammation and HDM-specific immunoglobulins using an iterative approach of experimentation and validation. Using the equations generated, we showed that the model was able to accurately predict and simulate data. The model also demonstrated the non-linear relationship between HDM exposure and both airway inflammation and allergic sensitization and identified system thresholds.</p> <p>The second model used mechanistic mathematical techniques to investigate the trafficking of eosinophils as they migrated from bone marrow to the blood and, ultimately, to the lungs. Making use of a limited data set, the model determined the effect of individual processes on the system. We identified eosinophil production, survival and death as having the greatest impacts, while migration played a relatively minor role. Furthermore, the model was used to simulate knockout models and the use of antibodies <em>in silico</em>.</p> <p>In the context of cancer growth and metastasis, we developed a theoretical model demonstrating the spatio-temporal development of a tumour in two-dimensions. The model was encoded to create a computer graphic simulation program, which simulated the effects of various parameters on the size and shape of a tumour. Through simulations, we demonstrated the importance of the diffusion process in cancer growth and metastasis.</p> <p>Ultimately, we believe the greatest benefit of each <em>in silico</em> model is the ability to provide an understanding of each respective disease recognized as dynamic and formally complex, but predominantly studied in reductionist, static or un-integrated approaches.</p> / Doctor of Philosophy (Medical Science)

<p>NB: I had another committee member, Dr. Mark Larché; and would like to have his name included in the document.</p> <p>Thank you.</p> / <p><strong>Introduction: </strong>Increasing incidence of chronic immune diseases are mirrored by changing disease risk factors, which include maternal stress during pregnancy. To date, no studies have investigated the impact of prenatal stress challenge (PNS) on the fetal immune system. Fetal liver and bone marrow represent major sources of hematopoietic stem cells (HSC) at mid gestation, which differentiate and mature in the thymus. Disturbance of immune development may cause immune impairment in later life. Further, progesterone is recognized as a critical part of feto-maternal interaction. This study aimed to determine if PNS interferes with normal fetal immune development in mice and the impact of progesterone supplementation on stress effects. <strong>Methods: </strong>DBA/2J-mated BALB/c dams were sorted into three groups: control, PNS (gestation days (GDs) 12.5 and 14.5) and PNS plus progesterone supplementation (DHD). Fetal tissue was collected on GDs 16.5 and 18.5. Flow cytometric analysis examined frequency and phenotype of fetal immune cell populations: HSC in fetal liver and bone marrow, and different stages of T cell maturation and regulatory T (Treg) cells in the thymus. Fetal tails were collected to determine fetal sex by PCR analysis. <strong>Results: </strong>PNS induced a decrease in organ size on GD16.5, which was not seen on GD18.5 and was reversed by DHD treatment. PNS altered the percentage and absolute number of HSC within the liver and bone marrow populations, on GD16.5 and 18.5. There was a significant lag in T cell maturation as demonstrated by the altered expression of CD3 and skewed CD3-:CD3+ ratio. There was a significant decrease in Treg cells within CD3+ thymic cells in response to PNS. PNS effects in the thymus were ameliorated by DHD treatment. There was no PNS-induced sex bias. <strong>Conclusions: </strong>These results indicate that PNS compromises the developing fetal immune system, which could account for impaired immune responses in adults with chronic immune disease, and provide evidence for a therapeutic role of progesterone supplementation.</p> / Master of Science (MSc)

<p>Asthma involves a systemic element that includes the mobilization and lung-accumulation of bone marrow-derived endothelial progenitor cells (EPC). This traffic may be driven by the stromal cell derived factor-1α (SDF-1α)/CXCR4 axis, where SDF1-α is a potent progenitor cell chemoattractant.</p> <p>Interfering with EPC lung-accumulation by administering AMD3100, a CXCR4 antagonist, was previously shown to be associated with the modulation of airway angiogenesis and airway hyperresponsiveness. However, since eosinophils express CXCR4, it is unknown whether AMD3100 acted directly on EPC or indirectly through its anti-inflammatory effects on eosinophils.</p> <p>We investigated the role that eosinophilic inflammation plays in the lung-homing of EPCs and airway angiogenesis in allergic asthmatic response by utilizing eosinophil deficient (PHIL) mice.</p> <p>Wild-type BALB/c (WT) and PHIL mice underwent a chronic house dust mite (HDM) exposure protocol. Treatment groups were administered AMD3100. Outcome measurements were made 24hrs post final exposure and included: flow cytometry to enumerate lung-extracted EPCs, immunostaining for von Willebrand factor to assess bronchial vascularity, bronchoalveolar lavage for airway inflammation, haematoxylin and eosin stain to enumerate eosinophils, picrosirius red stain to assess collagen deposition, and measurement of airway resistance to increasing intranasal doses of methacholine.</p> <p>HDM exposed mice had a significant increase in EPC lung accumulation, bronchial vascularity, airway inflammation, collagen deposition and airway hyperresponsiveness (AHR) in both WT and PHIL groups, with some indices at lower levels in PHIL mice. Concurrent treatment with AMD3100 significantly attenuated EPC lung homing, bronchial vascularity, eosinophil numbers in lung tissue and AHR, but not collagen deposition in WT mice. AMD3100 treatment significantly attenuated all indices in PHIL mice.</p> <p>The findings of this study show that, EPC-driven angiogenesis and the development of AHR in allergic airway responses are independent of eosinophils, the presence of these cells, however, may have a role in worsening of the pathology of allergic airways disease.</p> / Master of Science (MSc)

<p>Obstructive lung diseases limit airflow and gas exchange and have a major impact on a patient’s long-term health. Asthma and chronic obstructive pulmonary disease (COPD) are the most prevalent obstructive lung diseases and represent a major burden on healthcare systems worldwide. It is now accepted that the pathologies associated with these diseases are heterogeneous in nature, and as the function of the lung is determined by its three-dimensional structure, methods to volumetrically evaluate the lung are important tools in furthering the study of these pathologies.</p> <p>Three-dimensional imaging methodologies, such as computed tomography (CT) and single photon emission computed tomography (SPECT), are used clinically in the diagnosis of lung disease, but results are not commonly quantified. In addition, asthma and COPD develop slowly over time and diagnosis normally takes place after the underlying pathologies are well established. Experimental models in small animals, such as rats and mice, allow for the study of disease pathogenesis in a controlled setting and development of quantitative imaging practices for these models provides translational tools for relating results back to the clinic.</p> <p>In this thesis, CT densitometry and ventilation/perfusion (V/Q) SPECT are explored as methods to investigate models of asthma and COPD. CT densitometry is shown to be capable of quantifying allergic inflammation in an asthma model but is of less use in a model of COPD, predominantly due to the relative amounts of inflammation present. However, V/Q imaging is shown to be quite sensitive to the effects of cigarette smoke in a model of COPD and has been used to better understand how pathologies associated with COPD contribute to gas exchange limitation in the lung.</p> <p>The models, imaging techniques, and analysis methods described in this work provide insight into chronic obstructive lung disease and allow for future investigations into how pathologies effect gas exchange. Further, the characterization of the models described in this thesis allows for drug efficacy studies to be performed, both on established and novel treatments. Future research into asthma and COPD will benefit further from the use of threedimensional imaging methodologies because they provide volumetric information on structure and function and can act as a translational bridge between clinical disease and preclinical animal models.</p> / Doctor of Philosophy (Medical Science)

Introdução: A esclerodermia é rara doença do tecido conectivo que se manifesta através da esclerose cutânea e variável acometimento sistêmico. Duas categorias de esclerodermia são conhecidas: esclerose sistêmica, caracterizada por esclerose cutânea e acometimento visceral e a esclerodermia localizada ou morfeia que classicamente apresenta evolução benigna e autolimitada, confinada a pele e/ou tecidos subjacentes. Estudos recentes demonstram que a forma localizada possa eventualmente apresentar acometimento de órgãos internos e morbidade variável. Objetivo: Neste estudo objetivamos determinar as características demográficas, a prevalência de manifestações sistêmicas e alterações laboratoriais, bem como a associação com doenças autoimunes, em pacientes com esclerodermia da face. Métodos: Pacientes com esclerodermia localizada, incluindo os casos de esclerodermia em golpe de sabre, síndrome de Parry-Romberg e morfeia em placas com acometimento facial, foram avaliados e submetidos à investigação neurológica, incluindo exame clínico neurológico e ressonância magnética de crânio, e avaliação oftalmológica. Após 3 anos, o subgrupo de pacientes disponível para seguimento foi ressubmetido à ressonância magnética. Resultados: Foram estudados 34 pacientes com esclerodermia localizada da face. Deste total, 64,7% apresentavam uma ou mais manifestações extracutâneas, sendo cefaleia a queixa mais frequente, encontrada em 61,8% dos pacientes. Dos 23 pacientes submetidos à avaliação neurológica, 56,5% apresentaram alterações neurológicas possivelmente associadas à esclerodermia. Alterações à ressonância magnética foram observadas em 50% dos casos. O achado mais frequente foi a presença de lesões parenquimatosas com alteração de sinal em 50% dos pacientes. Dos pacientes que apresentavam alteração neurológica, 80% também apresentavam alguma alteração à ressonância magnética. Doze pacientes foram ressubmetidos a novo exame após 3 anos. Em todos os pacientes os achados de imagem se mantiveram inalterados. Durante esse intervalo de 3 anos, 25% dos pacientes apresentaram sinais de atividade da esclerodermia. Quanto à avaliação oftalmológica, 67,9% dos pacientes avaliados apresentaram alteração, sendo os achados mais frequentes a ocorrência de alterações orbiculares da esclerodermia (20,6%) e xeroftalmia (10,7%). Conclusão: Pacientes com esclerodermia localizada da face apresentam alta prevalência de alterações neurológicas e oftalmológicas. Baseado nestes achados, sugerimos que todos os casos de esclerodermia localizada da face devam ser detalhadamente examinados quanto à presença de alterações sistêmicas / Introduction: Scleroderma is a rare connective tissue disease that manifests as skin sclerosis and variable systemic involvement. Two categories of scleroderma are known: systemic sclerosis, characterized by cutaneous sclerosis and visceral involvement and localized scleroderma or morphea which classically presents benign evolution and selflimited, confined to the skin and / or underlying tissue. Recent studies show that the localized form may possibly course with involvement of internal organs and variable morbidity. Objective: This study aimed to determine the demographic characteristics, the prevalence of systemic manifestations and laboratory findings, as well as the association with autoimmune diseases, in patients with scleroderma of the face. Methods: Patients with localized scleroderma, including cases of scleroderma en coup de sabre, Parry-Romberg syndrome and morphea plaque with facial involvement were evaluated and underwent neurological examination, including neurologic examination and magnetic resonance imaging, and ophthalmology evaluation. After 3 years, the subgroup of patients available for follow-up was subjected again to MRI. Results: We studied 34 patients with localized scleroderma of the face. Of this total, 64,7% had one or more extracutaneous manifestation, headache being the most frequent complaint found in 61,8% of patients. Of the 23 patients undergoing neurological evaluation, 56,5% had neurological changes possibly associated with scleroderma. MRI changes were observed in 50% of cases. The most frequent was the presence of parenchymal lesions with signal alteration in 50% of patients. Of the patients who had neurological deficits, 80% also had a change to MRI. Twelve patients were subjected again to another MRI scan after 3 years. In all patients, imaging findings were unchanged. During this interval of 3 years, 25% of patients showed signs of activity of scleroderma. As for ophthalmologic evaluation, 67,9% of patients showed abnormalities, with the most frequent findings being the occurrence of orbicular changes of scleroderma (20.6%) and xerophthalmia (10.7%). Conclusion: Patients with localized scleroderma face have a high prevalence of neurological and ophthalmological changes. Based on these findings, we suggest that all cases of localized scleroderma of the face should be thoroughly examined for the presence of systemic changes

Allogeneic hematopoietic chimerism leading to central tolerance has significant therapeutic potential. Establishment of hematopoietic chimerism created by stem cell transplantation has been shown to prevent and cure a number of autoimmune diseases and induce the most robust and long-lasting form of transplantation tolerance known. However, the realization of the vast clinical potential of hematopoietic chimerism for induction of transplantation tolerance has been impeded by the toxicity of the host conditioning regimen and the development of graft-versus-host disease (GVHD). This thesis describes the development of stem cell transplantation protocols that 1) reduce the host conditioning regimen; and 2) abrogate the development of GVHD. When applied to the treatment of autoimmune diabetic NOD mice, a model of type 1 diabetes, stem cell transplantation was able to 3) prevent autoimmune recurrence; and 4) permit curative pancreatic islet transplantation. I first describe a tolerance-based stem cell transplantation protocol that combines sub-lethal irradiation with transient blockade of the CD40-CD154 costimulatory pathway using an anti-CD154 antibody. With this protocol, I established hematopoietic chimerism in BALB/c mice transplanted with fully allogeneic C57BL/6 bone marrow. All chimeric mice treated with anti-CD154 antibody remained free of graft vs.host disease (GVHD) and accepted donor-origin but not third party skin allografts. It was similarly possible to create allogeneic hematopoietic chimerism in NOD/Lt mice with spontaneous autoimmune diabetes. Pancreatic islet allografts transplanted into chimeric NOD/Lt mice were resistant not only to allorejection but also to recurrence of autoimmunity. I conclude that it is possible to establish robust allogeneic hematopoietic chimerism in sub-lethally irradiated mice without subsequent GVHD by blocking the CD40-CD154 costimulatory pathway using as few as two injections of anti-CD154 antibody. I also conclude that chimerism created in this way generates donor-specific allograft tolerance and reverses the predisposition to recurrent autoimmune diabetes in NOD/Lt mice, enabling them to accept curative islet allografts. In order to further reduce the impediments associated with the implementation of allogeneic hematopoietic chimerism as a therapeutic modality, I adapted a costimulation blockade-based protocol developed for solid organ transplantation for use in stem cell transplantation. The protocol combines a donor-specific transfusion (DST) with anti-CD154 antibody to induce peripheral transplantation tolerance. When applied to stem cell transplantation, administration of DST, anti-CD154 antibody, and allogeneic bone marrow led to hematopoietic chimerism and central tolerance with no myeloablation (i.e. no radiation) and no GVHD in 3 different strains of mice. The development of donor-specific tolerance in this system was shown to involve deletion of both peripheral host alloreactive CD8+ T cells and nascent intrathymic alloreactive CD8+ T cells. In the absence of large numbers of host alloreactive CD8+ T cells, the cell transfusion that precedes transplantation need not be of donor-origin, suggesting that both allo-specific and non-allo-specific mechanisms regulate engraftment. Agents that interfere with peripheral transplantation tolerance partially impair establishment of chimerism. I conclude that robust allogeneic hematopoietic chimerism and central tolerance can be established in the absence of host myeloablative conditioning using a peripheral transplantation tolerance protocol.

The prevalence of diabetes is approaching epidemic proportions worldwide. There is currently no cure for type 1 diabetes, and successful treatment requires constant monitoring of blood sugars and use of exogenous insulin to prevent hyperglycemia. Diabetes will be curable when pancreatic β-islet cells can be transplanted into diabetes patients without requiring long-term immunosuppression. This will require learning more about the induction of functional tolerance, a state that maintains the competence of the immune system to most antigens but protects graft-specific antigens from immune rejection, permitting transplantation. One known mechanism of peripheral tolerance is T cell anergy, a phenotype of hypo-reponsiveness in CD4+ T cells. The focus of this thesis is a description of factors shown to be specific to the induction and maintenance of T cell anergy, whose loss reverses the anergic phenotype, restoring the ability of the cells to proliferate in response to antigen. The first of these is Egr-2, a zinc-finger transcription factor, whose presence is required for the induction of anergy induced in T cell clones by TCR stimulation in the absence of costimulation. Egr-2 is shown to be important to anergy induction but not anergy maintenance. In contrast, a negative costimulation receptor, PD-1, is shown to be necessary for the maintenance of anergy. It is possible that learning more about the genetic factors that orchestrate T cell anergy will prove useful in the development of tolerance-based protocols for organ and tissue transplantation without the use of long-term immunosuppression.

Introdução: A esclerodermia é rara doença do tecido conectivo que se manifesta através da esclerose cutânea e variável acometimento sistêmico. Duas categorias de esclerodermia são conhecidas: esclerose sistêmica, caracterizada por esclerose cutânea e acometimento visceral e a esclerodermia localizada ou morfeia que classicamente apresenta evolução benigna e autolimitada, confinada a pele e/ou tecidos subjacentes. Estudos recentes demonstram que a forma localizada possa eventualmente apresentar acometimento de órgãos internos e morbidade variável. Objetivo: Neste estudo objetivamos determinar as características demográficas, a prevalência de manifestações sistêmicas e alterações laboratoriais, bem como a associação com doenças autoimunes, em pacientes com esclerodermia da face. Métodos: Pacientes com esclerodermia localizada, incluindo os casos de esclerodermia em golpe de sabre, síndrome de Parry-Romberg e morfeia em placas com acometimento facial, foram avaliados e submetidos à investigação neurológica, incluindo exame clínico neurológico e ressonância magnética de crânio, e avaliação oftalmológica. Após 3 anos, o subgrupo de pacientes disponível para seguimento foi ressubmetido à ressonância magnética. Resultados: Foram estudados 34 pacientes com esclerodermia localizada da face. Deste total, 64,7% apresentavam uma ou mais manifestações extracutâneas, sendo cefaleia a queixa mais frequente, encontrada em 61,8% dos pacientes. Dos 23 pacientes submetidos à avaliação neurológica, 56,5% apresentaram alterações neurológicas possivelmente associadas à esclerodermia. Alterações à ressonância magnética foram observadas em 50% dos casos. O achado mais frequente foi a presença de lesões parenquimatosas com alteração de sinal em 50% dos pacientes. Dos pacientes que apresentavam alteração neurológica, 80% também apresentavam alguma alteração à ressonância magnética. Doze pacientes foram ressubmetidos a novo exame após 3 anos. Em todos os pacientes os achados de imagem se mantiveram inalterados. Durante esse intervalo de 3 anos, 25% dos pacientes apresentaram sinais de atividade da esclerodermia. Quanto à avaliação oftalmológica, 67,9% dos pacientes avaliados apresentaram alteração, sendo os achados mais frequentes a ocorrência de alterações orbiculares da esclerodermia (20,6%) e xeroftalmia (10,7%). Conclusão: Pacientes com esclerodermia localizada da face apresentam alta prevalência de alterações neurológicas e oftalmológicas. Baseado nestes achados, sugerimos que todos os casos de esclerodermia localizada da face devam ser detalhadamente examinados quanto à presença de alterações sistêmicas / Introduction: Scleroderma is a rare connective tissue disease that manifests as skin sclerosis and variable systemic involvement. Two categories of scleroderma are known: systemic sclerosis, characterized by cutaneous sclerosis and visceral involvement and localized scleroderma or morphea which classically presents benign evolution and selflimited, confined to the skin and / or underlying tissue. Recent studies show that the localized form may possibly course with involvement of internal organs and variable morbidity. Objective: This study aimed to determine the demographic characteristics, the prevalence of systemic manifestations and laboratory findings, as well as the association with autoimmune diseases, in patients with scleroderma of the face. Methods: Patients with localized scleroderma, including cases of scleroderma en coup de sabre, Parry-Romberg syndrome and morphea plaque with facial involvement were evaluated and underwent neurological examination, including neurologic examination and magnetic resonance imaging, and ophthalmology evaluation. After 3 years, the subgroup of patients available for follow-up was subjected again to MRI. Results: We studied 34 patients with localized scleroderma of the face. Of this total, 64,7% had one or more extracutaneous manifestation, headache being the most frequent complaint found in 61,8% of patients. Of the 23 patients undergoing neurological evaluation, 56,5% had neurological changes possibly associated with scleroderma. MRI changes were observed in 50% of cases. The most frequent was the presence of parenchymal lesions with signal alteration in 50% of patients. Of the patients who had neurological deficits, 80% also had a change to MRI. Twelve patients were subjected again to another MRI scan after 3 years. In all patients, imaging findings were unchanged. During this interval of 3 years, 25% of patients showed signs of activity of scleroderma. As for ophthalmologic evaluation, 67,9% of patients showed abnormalities, with the most frequent findings being the occurrence of orbicular changes of scleroderma (20.6%) and xerophthalmia (10.7%). Conclusion: Patients with localized scleroderma face have a high prevalence of neurological and ophthalmological changes. Based on these findings, we suggest that all cases of localized scleroderma of the face should be thoroughly examined for the presence of systemic changes

<p>Food allergies are immune system-driven diseases that lead to reproducible adverse reactions which can be fatal. These severe systemic reactions are primary mediated by immunoglobulin E (IgE) that is derived from B cells which have been activated by T helper type 2 (Th2) cells. While much work has advanced the clinical and pharmacological management of patients with allergic diseases, much remains to be elucidated about how individuals initially acquire allergy. This Thesis details a mechanism linking initial gastrointestinal exposure to peanut (PN) allergen, to the generation of Th2 cells: PN allergen activates epithelial cell secretion of interleukin (IL)-33 and eosinophil degranulation of eosinophil peroxidase, which causes CD103+ dendritic cell (DC) activation and migration to mesenteric lymph nodes where DC OX40L engages naïve T cells to secrete IL-4 in an autocrine/paracrine manner to promote and consolidate Th2 cell differentiation. These events are followed by B cell activation and PN-specific IgE production, which sensitizes mast cells to be hypersensitive to PN re-exposure by causing immediate allergic reactions including anaphylaxis. This is later followed by eosinophilic inflammation that is partially mediated by innate lymphoid cells. As food allergy also serves as a unique model to better understand mechanisms of adaptive immunity, especially Th2 immunobiology, both basic science and clinical implications are discussed in this Thesis. Major themes include Th2 and disease heterogeneity, identification of ‘the original source of IL-4’, an unprecedented <em>in vivo </em>requirement for eosinophils in priming adaptive immune responses, and the need to weigh basic science findings against the human disease <em>in natura </em>litmus test. Looking forward, many questions remain to be answered in the field of food allergy research, but the findings of this Thesis may be one step towards the prevention, management or cure of a disease with growing public concern, potentially fatal consequences, and an unmet need in understanding its pathogenesis.</p> / Doctor of Philosophy (Medical Science)