Topical application of phorbol ester tumour promoter evoked the stimulated generation of hydrogen peroxide in the mouse skin in vivo, probably resulting from its action on an associated non-target population of infiltrating inflammatory cells. This formation was demonstrated indirectly by hydrogen peroxide dependent 3-amino-1,2,4-triazole catalase complex formation and confirms the hypothesis and findings of Goldstein et al. (1983). In the assay model discussed, n-alkanes representing the range C6-C14 caused stimulated production of hydrogen peroxide but in mouse skin already containing inflammatory cells. It is proposed that the known tumour-promoting activity of dodecane and tetradecane is free radical mediated, and that this activity is expressed when these alkanes cause an inflammatory state such as occurs upon repeated dermal application. Free radical generation and possible tumour-promoting activity of lower alkanes is latent, due to lower irritancy and high volatilization, unless these alkanes are encountered in combination with an irritant. Skin explant studies demonstrated a priming potential of the phorbol ester tumour promoter to the induction of skin catalase by oxygen. This dose-dependent induction is also seen in untreated skin and may represent a possible compensatory response to oxygen toxicity. Such induction may confound the interpretation of other air-incubation studies in vitro. The generation of hydrogen peroxide was stimulated as a consequence of abrasion, a known tumour promoting agent, and this indicates that irritation, (and possibly the immune response) has a role in tumour promotion and in the variation in strain susceptibility to tumour promoters. Current techniques for the assay of enzymic activities specific for cytochrome P-450 and cytochrome P-448 failed to detect these in mouse skin. The presence of these activities in mouse hepatic tissue was demonstrated. Preliminary data is presented for a novel skin irritation screening test based on phorbol ester-stimulated oxygen consumption by inflammatory cells responding to the applied test material. The technique may offer a more precise and convenient measure of irritancy than that provided by the currently used Draize test.

The studies described in this thesis have been concerned with several aspects of follicular development in the mammalian ovary. Chapters 2, 3 6 4 deal with mathematical modelling of ovarian follicle dynamics in normal animals and comparisons with experimentally manipulated animals. Chapter 5 describes a novel aethod for estimating the clonal origin of the mouse ovarian follicle. In the final two chapters, the comparative physiology and anatomy of follicular numbers and sizes and the incidence of polyovular follicles are described for a number of species. The unifying theme of these studies is that they reveal patterns existing in follicular development and utllisation by detailed examination of one species, (CBA/ca mouse), and broadly by interspeci,fic comparisons (with relation to scaling). A detailed mathematical description of the follicular dynamics of virgin CBA/ca mice up to 98 days of age has been obtained by the application of compartmental modelling to differential follicle counts. The rates of follicle growth (migration) and death have been estimated for five ?stages of development (primordial to Graafian). The model predicts age changes in follicle growth and death rate, there being transitions in the parameters at 20 days second at 60 days. The parameters for normal animals have been compared with those of anilllals under two experimental conditions: 1) by unilateral ovariectomy at 4-2 days of age, which abruptly halves the numbers of ovarian follicles and alters the ratio of large : small follicles. 2) by blocking ovulation using progesterone implants. The dynamics of follicle growth were altered by both treatments in comparison with the controls. Follicles at all stages of development were affected by unilateral ovariectomy and differences may exist with time. The compensatory response by the remaining ovary was due to a combination of an increased preantral growth rate and a decrease in atresia at antral stages. Earlier stages of follicle development were affected this may have been incidental to the compensatory response. In progesterone treated animals follicles developed through to antral stages when they un~erwent atresia. The effects of treatment were observed at three levels of development: 1) The initiation of growth from the primordial pool, 2) Growth rate of small follicles and 3) deaths at larger stages of follicular development. Longer term observations indicated that these effects may not be constant. The modelling studies have looked at numerical changes in the follicle population with time but a greater understanding of the develomental biology of the follicle is required in order to explain the changes in growth and death rates observed. This problem has been tackled initially by studying the clonal origin of the follicular epithelium. The technique used is based on the principle that cells in females. are generally mosaic as a result of X-chromosome inactivation the use of X linked cell markers phospho-glycerate kinase-1 (PGK-1). Granulosa cells were found to be polyclonal in origin with the number of progenitor cells numbering 5 on average. Analysis of cumulus and mural granulosa cells showed that substantial cell mixing had occurred and cuaulus cells were generally founded by more than one clone. Finally, comparative studies have been conducted to look at scaling of follicle sizes and numbers and of polyovular follicles. Ovarian follicle and oocyte sizes were scaled according to body weight (ranging from .005-500Kg) using data from 22 species. Primordial and Graafian follicle sizes varied with body weight but closer correlations for the latter were obtained when the sum of the surface areas or volUiles for a preovulatory set were considered as opposed to the values for individual follicles. The numbers of nongrowtng follicles 1n reserve at young adult ages were correlated with maximum longevity of the species and related to body weight. The frequency of polyov~lar follicles varied 1n species studied and were most abundant 1n the domestic bitch. The overall incidence of polyovular folUcles 1n young bitches was 14 S, being reduced to 5~ 1n bitches at 7-11 years. The frequency of the various types of polyovular preantral folUcle varied inversely with the numbers of oocytes per follicle.

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This investigation studied the occurrence, distribution and morphology; and the transplantation, ultrastructural and in vitro characteristics of histiocytic lymphoma in the BALB/c and C57BL/6 strains of mice. The disease was more common in aged females of both strains. The liver was the major organ involved in the males and the liver and uterus were commonly involved in the females. Subcutaneous transplants readily metastasized to the lungs. Ultrastructurally the neoplastic cells resembled histiocytes and did not contain microfilaments or a basement membrane. The results suggest that this neoplasm is histiocytic in origin.

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The adapter protein Shc is a ubiquitously expressed Src homology 2 (SH2) domain protein implicated in the transmission of activation signals to Ras. She proteins become phosphorylated on tyrosine in cells stimulated with a variety of growth factors and in v-𝘴𝘳𝘤 transformed cells and are able to transform fibroblasts and differentiate PC12 cells in a Ras-dependent fashion. To assess the transforming ability of Shc in the mouse mammary gland, I generated transgenic mice harbouring the p52ˢʰᶜ cDNA under the transcriptional control of the mouse mammary tumor virus long terminal repeat (MMTV LTR). While p52ˢʰᶜ expression was correlated with multiple enlarged terminal end buds in virgin mouse mammary glands, multiparous mice developed mammary hyperplasias and mammary carcinomas. The frequency, latency and focal nature with which these tumors arose suggests that additional events are necessary to induce malignant conversion of primary mammary epithelial cells. To directly test the role of Shc in established mammary tumor models, I have generated two strains of bigenic mice. When Shc was overexpressed with NDL 1-2, a constitutively activated form of the Neu receptor tyrosine kinase, latency of tumor onset was decreased over that of parental MMTV/NDL 1-2 mice. Polyomavirus middle T antigen (PyV MT) mutants with a functionally inactive Shc binding site (MT Y250F) are debilitated in mammary tumor formation compared to wild-type PyV MT transgenic animals. Concurrent overexpression of Shc with MT Y250F accelerated tumor kinetics and increased the propensity for metastasis to the lungs of bigenic animals. / Thesis / Master of Science (MSc)

Asthma is .a respiratory.,disease.that affects over 300 million people world-wide and is involved in over 250000 deaths annually. Asthma is classically thought of as an allergic disease with variable airflow obstruction and airway hyperresponsiveness (AHR) associated with airway remodeling, although phenotype variations are observed in the population. Due to multiple factors including genes, gender, exposure to pathogens, environmental pollutants, diets, and obesity, a clear picture of the complex interactions between an individual's genes, the environment which they live in, and the development of an asthmatic phenotype remains elusive. Despite the availability of treatment strategies for asthma, varying degrees of airway inflammation, remodeling, and AHR remain present in an asthmatic patient. Our general hypothesis that was the basis for all studies was the following: "Airway remodeling in response to allergen exposure is a major contributing factor to AHR observed in asthmatics. Understanding the mechanisms behind the different components of airway remodeling will provide new avenues for therapeutic development aimed at improving lung function above and beyond current treatment strategies." The currently available therapeutics and management strategies for asthma are unable to prevent or reverse components of airway remodeling. It is possible that with greater understanding of the processes involved in the various indices of airway remodeling new classes of therapeutics could be developed to selectively target this broad, ill-managed aspect of asthma. Collectively the studies contained in this thesis have been linked by the general themes of greater characterization of in vivo mouse models of allergen exposure, the application of these models to mechanistically explore the biological pathways involved in the different components of airway remodeling, and the testing of novel therapeutic strategies targeting these pathways of interest. / Thesis / Doctor of Philosophy (PhD)

Estrogenic endocrine disrupting chemicals (EEDCs) are defined as chemicals that bind to estrogen receptors (ERs) and augment estrogenic functions, either through promoting or blocking estrogen receptor signaling. Recent reports highlight the growing concern surrounding environmental exposure to EEDCs and immune system modulation. A commonly prescribed EEDC, 17α-ethinyl estradiol, is a synthetic analog of 17β-estradiol (E2), and is also found in many environmental reservoirs of human and animal exposure. Little is known regarding the immunomodulatory effects of this EEDC. Autoimmune diseases, such as systemic lupus erythematosus (SLE), are characterized by a dysregulated immune system that has lost tolerance to self-antigens. The pathogenesis of SLE is still poorly understood. However, it is likely that genetics, epigenetics, hormones, and environmental factors, such as EEDC exposure, contribute to the pathogenesis and severity of SLE. The work presented in this dissertation focused on investigating the immunomodulatory effects of exogenous estrogens in mouse models of SLE. Chapter 1 describes an overview of environmental endocrine disruptors and autoimmune disease, with a particular emphasis on estrogens. Chapter 2 represents a review of the current and pertinent literature surrounding the contributions of sex differences, hormones, and EDCs to the induction of autoantibodies and development of autoimmunity, as well as the contributions of anti- microbial responses to SLE. We explored the contribution of dietary components to SLE disease severity. Mice fed a diet devoid of exogenous phytoestrogens developed significantly reduced glomerulonephritis and glomerular immune complex deposition compared to mice fed a diet containing soy isoflavones. Diet also influenced cytokine production and epigenetics of LPS-stimulated splenic leukocytes. We identified similar effects of E2 and EE implantation with regards to innate immunity, and distinct cellular subset, cytokine production profiles, gene expression, and epigenetic responses between E2 and EE treated NZB/WF1 mice. Oral exposure to a very low human relevant dose of EE promoted glomerulonephritis and augmented responses to viral and bacterial mimics in MRL/lpr mice. Overall, our findings suggest that chronic exposure to environmental EEDCs exacerbates lupus nephritis and alter an already dysregulated immune system in genetically susceptible individuals and have greatly expanded the current body of knowledge surrounding 17α-ethinyl estradiol. / Doctor of Philosophy / Chemicals that can disrupt the normal effects of hormones are termed endocrine disrupting chemicals (EDCs). Estrogenic EDCs promote or suppress the ability of estrogen receptors to carry out normal functions within the body. Normal immune system functions require a fine balance of inflammatory and anti-inflammatory cellular responses. This delicate balance is a prime target for dysregulation by EDC exposure. Autoimmune diseases, such as systemic lupus erythematosus, are characterized by a loss of immune tolerance to ones’ own cells and tissues. There is a lack of knowledge surrounding the immunomodulatory effects of a commonly prescribed EDC, 17α-ethinyl estradiol, especially as it pertains to autoimmune disease patients. The aim of this dissertation work is to investigate the immunomodulatory effects of exogenous EDC exposure in mouse models of SLE. We found that MRL/lpr mice fed a diet devoid of phytoestrogens had reduced kidney disease and immune-complex deposition and had augmented cytokine response and epigenetics in LPS-stimulated splenic leukocytes compared to mice fed a diet high in isoflavones. We next compared the immunomodulatory effects of chronic pharmacologic dose exposure to 17β-estradiol or EE, and found that while both estrogens have similar effects on innate immune cellular responses, EE has distinct effects on T cell population subsets, cytokine production, gene response and epigenetic alterations in female NZB/WF1 mice. Finally, chronic low-dose oral exposure to EE exacerbated clinical signs of kidney disease and suppressed the normal response of toll-like receptor 9 in MRL/lpr mice. Overall, we have found that chronic exposure to environmental estrogenic EDCs exacerbates lupus nephritis and alter an already dysregulated immune system in genetically susceptible individuals.