Background. Although Detrusor Hyperactivity (DH) with Impaired Contractility (IC) is a common urodynamic finding in elderly subjects, its pathogenesis remains unknown. Human detrusor biopsy studies indicate that subjects with DHIC exhibit ultrastructural evidence of both the dysfunction and degeneration patterns present in isolated DH and IC, respectively. Based on the known cellular effects of estrogen, we proposed a hypothesis that declines in ovarian hormone production could contribute to the pathogenesis of DHIC in elderly women. / Methods. In this thesis project, mature 3--14 month old female F-344 rats were studied 4 months after bilateral ovariectomy (OVx) or sham surgery. Detrusor structure was evaluated at this time point using light and electron microscopy, while classical muscle strip studies were used to measure the impact of OVx on detrusor muscle contractility. In an effort to identify estrogen-regulated proteins in the mammalian detrusor, known candidate proteins were screened using Western blotting, while identification of novel proteins was undertaken through proteomics, with two-dimensional gel protein resolution followed by microsequencing. (Abstract shortened by UMI.)

Vitamin D is very well known for its classical role in the maintenance of calcium and phosphorus homeostasis as well as in the prevention of rickets. More recent findings of its ability to inhibit cell proliferation, induce apoptosis, induce differentiation, inhibit angiogenesis, and modulate the immune system have made it a current topic of intense research, particularly in the field of cancer research. We used a murine model of breast cancer metastasis to bone to investigate the effect of vitamin D deficiency on the growth of breast cancer tumour cells within bone. We also established that these breast cancer tumour cells express the enzyme CYP27B1 (1α-hydroxylase) which is able to convert the inactive vitamin D precursor 25-hydroxyvitamin D (25(OH)D) to the active metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D). We next examined the effect of the local activation of vitamin D by tumoral CYP27B1 on the growth of these tumour cells within bone. Although we did not see a significant difference in the growth of breast cancer tumour cells in the bones of vitamin D deficient mice as compared to vitamin D sufficient mice, we have demonstrated that breast cancer tumour cells that do not express CYP27B1 grow much more aggressively within bone than breast cancer tumour cells which express CYP27B1. This suggests a very important role for the local activation of vitamin D by extra-renal CYP27B1 on the growth of breast cancer tumour cells within the bone microenvironment. These findings suggest a potential use for 25(OH)D as a treatment for breast cancer metastasis to bone either alone or in combination. / La vitamine D est bien connue pour son rôle dans le maintien des concentrations de calcium et du phosphore dans la circulation ainsi que dans la prévention du rachitisme. La découverte plus récente de sa capacité d'inhiber la prolifération cellulaire, induire leur différentiation ainsi que l'apoptose cellulaire, inhiber l'angiogenèse, et moduler le système immunitaire rend son étude un sujet de recherche très intéressant surtout dans le domaine de la recherche sur le cancer. Nous avons étudié l'effet de la carence en vitamine D sur la croissance tumorale dans un modèle murin de métastases osseuses du cancer du sein. Nous avons aussi établi que ces cellules expriment l'enzyme CYP27B1 (1α-hydroxylase) et sont donc capables d'activer la vitamine D en son métabolite actif la 1,25-dihydroxyvitamine D (1,25(OH)2D) à partir du métabolite inactif, la 25-hydroxyvitamine D (25(OH)D). Nous avons ensuite examiné l'effet de l'activation locale de la vitamine D par les cellules tumorales dérivées du sein sur la croissance de ces cellules dans le microenvironnement osseux. Nous n'avons constaté aucune différence significative entre la croissance des cellules tumorales du cancer du sein dans l'os chez les souris carencées en vitamine D en comparaison aux souris non carencées en vitamine D. Cependant, nous avons démontré que les cellules tumorales du cancer du sein qui expriment le CYP27B1 croissent beaucoup moins vite dans l'os que les cellules tumorales qui n'expriment pas le CYP27B1. Ces résultats suggèrent un rôle très important de l'activation extra-rénale de la vitamine D par les cellules tumorales du cancer du sein pour inhiber la croissance de ces cellules dans l'os. En conclusion, ces travaux indiquent que le précurseur inactif 25(OH)D pourrait être utilisé seul ou en combinaison pour le traitement des métastases osseuses du cancer du sein.

Uveal Melanoma (UM) is the most common intraocular tumor in adults. Liver metastasis is the leading cause of death in patients affected by this disease and in approximately 40% of the cases, metastasis occurs 10 years after initial diagnosis. Tumor dormancy has been considered as a leading theory for the delay of the manifestation of metastatic disease and it has been the subject of numerous studies, which includes investigating metastasis suppressor genes (MSG). Studies have shown that the MSG KISS1 plays a role in various human malignancies, including melanoma, and it seems to be involved in the dormancy phase of the metastatic cascade. Previous studies from our laboratory reported that loss of KISS1 expression is related to worse prognosis in UM. In this light, mechanisms that involve increase of KISS1 expression are of interest for UM researchers. Interestingly, pathways involved in UM progression include upregulation of c-KIT, a cell surface molecule normally found in melanoma cells. This process seems to occur at the same time that KISS1 is downregulated and overt metastasis become clinically detectable. Therefore, we verified if inhibition of c-KIT could be related to KISS1 expression in metastatic UM. In addition, considering that a newly identified class of small non coding RNAs (miRNAs) are master regulators of gene expression, we sought to investigate if miRNAs were involved in metastasis of UM. Therefore, the objective of this thesis was to identify mechanisms that increase the expression of KISS1 and to better understand UM metastasis. To address these questions, we used a c-KIT inhibitor, imatinib mesylate (IM), and miRNAs in this study. Human UM cell lines with different metastatic potential showed increased levels of KISS1, by real-time reverse transcriptase polymerase chain reaction (RT-PCR), after treatment with IM. Notably, an increase in KISS1 was observed at the protein level in a dose response manner when the most aggressive UM cell line (92.1) was treated with different concentrations of IM. Increased levels of KISS1 expression were confirmed in vivo using an experimental animal model (albino rabbits). Using a miRNA array, we were able to identify miRNAs with prometastatic and antimetastatic effects in different UM cell lines and under diverse conditions. The miRNAs 10a, 10b, 21, and let-7 were upregulated in a liver metastatic cell line compared to the primary UM cell line from the same patient. Additionally, the UM cell line with aggressive potential transfected with KISS1 showed decreased expression of the miR-221 and increased expression of miR-146 compared to the non-transfected control. Similar results were obtained when the same cell line was treated with IM. In order to translate these results to a clinical setting, in situ hybridization of miR-221 was performed in 15 human UM FFPE tissues; increased expression of miR-221 was positively correlated with metastasis in UM. In conclusion, KISS1 was upregulated following treatment with IM. In addition, down regulation of miR-221 was found in all miRNA arrays with increases in KISS1 and treatment with IM. To the best of our knowledge, this is the first study to show that treatment with a c-KIT inhibitor causes an upregulation of KISS1, and that miR-221 may promote metastasis in UM. Consequently, induction of KISS1 expression downregulates miR-221 and should be considered as potential targets for adjuvant therapy in UM metastasis. / Parmi les tumeurs intraoculaires malignes, le plus fréquent chez l'adulte est le mélanome de l'uvée (MU). Les métastases hépatiques représentent la principale cause de décès chez les patients affectés par cette maladie et dans environ 40 % des cas, les métastases apparaissent plus de 10 ans après le diagnostic initial. La latence de la tumeur est considérée comme étant la théorie principale qui expliquerait le retard de l'apparition des métastases. Cela fut le sujet de nombreuses études dont un certain nombre intéressaient par l'analyse des Gènes Suppresseurs de Métastases (GSM)Il a été démontré que le GSM KISS1 joue un rôle dans différentes maladies malignes, dont le mélanome. Ce gène parait être impliqué dans la phase de latence de la cascade métastatique. Des études antérieures conduites dans notre laboratoire ont conclu que la perte d'expression du gène KISS1 est associée à un mauvais pronostic chez les patients atteints de MU. Ainsi, les mécanismes qui impliquent l'augmentation d'expression de KISS1 présentent un intérêt pour les chercheurs travaillant sur MU.Curieusement, les voies de signalisations impliquées dans la progression de MU incluent la régulation positive de c-KIT une molécule de surface cellulaire. Ce phénomène semble coïncider avec la régulation négative de KISS1 et le moment où les métastases déclarées deviennent cliniquement détectables. Nous avons donc vérifié si l'inhibition de c-KIT pourrait-être liée à l'expression de KISS1. De plus, considérant qu'une nouvelle classe de petits ARN non codés (miARN), qui sont les principaux régulateurs de l'expression génique, vient d'être identifiée, nous avons cherché à examiner si les miARNs étaient impliqués dans les métastases de MU.Par conséquent, l'objectif de cette thèse était d'identifier les mécanismes qui augmentent l'expression de KISS1 afin de mieux comprendre l'évolution des métastases de MU. Pour aborder ces questions, nous avons utilisé un inhibiteur de c-KIT, l'Imatinib mesylate (IM), et des miARNs.Les lignées cellulaires de MU aux différents potentiels métastatiques présentent un niveau élevé de KISS1 après un traitement à l'IM. Les niveaux accrus de KISS1 ont été confirmés in vivo en utilisant le model expérimental animal (lapins albinos). En utilisant des puces à miARN, nous avons réussis à identifier les miARNs aux effets pro-métastatiques et anti-métastatiques dans différentes lignées cellulaires de MU et sous diverses conditions. Une régulation positive des miARNs 10a, 10b, 21 et let-7 fut identifiée dans une lignée cellulaire de métastase hépatique par rapport à la lignée cellulaire de la tumeur primaire du même patient. De plus, dans une lignée cellulaire de MU où une transfection de KISS1 fut réalisée, on a pu observer une diminution de l'expression de miR-221 et une augmentation de l'expression de miR-146, par rapport au control non-transfecté. Des résultats semblables ont été obtenus, quand la même lignée cellulaire a été traitée avec IM. Afin d'utiliser ces résultats dans un environnement clinique, nous avons exécuté une hybridation in situ de miR-221 sur 15 tissus humains (fixés dans le formol puis inclus dans la paraffine) de MU dans lesquels une expression accrue de miR-221 est corrélé positivement avec des métastases dans le MU.En conclusion, KISS1 est régulé positivement sous l'action d'IM. De plus, une régulation négative de miR-221 fut constatée dans toutes les puces de miARN qui présentaient une augmentation de KISS1 ainsi que pendant le traitement avec IM. À notre connaissance, ceci est la première étude à démontrer que le traitement avec un inhibiteur de c-KIT cause une régulation positive de KISS1 et que le miR-221 peut avoir un effet métastatique sur le MU. Par conséquent, l'augmentation de la expression de KISS ainsi que la suppression de miR-221 peuvent potentiellement être considérées comme des cibles potentielles dans la thérapie adjuvante de MU métastatique.

Current state of knowledge concerning the effects of alcohol on biological membranes was reviewed. The influence of chronic alcohol administration on the structure and function of rat liver plasma membranes was studied. Functionally, a diminished response to both glucagon and epinephrine was detected in the isolated perfused rat liver preparation after chronic alcohol feeding. Receptor binding studies identified a decrease in glucagon and alpha(,1)-adrenergic receptor numbers. The binding affinities were not altered. This condition remained stable through 72 hours of alcohol withdrawal. / Further characterization with isopycnic centrifugation showed that the alcoholic plasma membrane spanned a different equilibrium density range with a significantly raised peak density value. This anomaly persisted after 48 hours withdrawal from alcohol. Analyses of lipid composition revealed significant alterations in the phospholipids as well as the fatty acid content in some phospholipid classes of the experimental plasma membranes. However, these modifications were reversed during 48 hours of alcohol withdrawal. Additional changes were noted in some fatty acids of certain phospholipids, reflecting readaptation to the new condition without alcohol. / The discrepancy in the time course of reversal of various alcohol-induced effects suggests that adaptation of the plasma membrane to alcohol includes both the lipids and proteins. A combination of alteration in both these components would contribute to the overall modification of structure and function of rat liver plasma membrane subsequent to chronic alcohol administration.

The prevalence of allergic diseases is increasing worldwide for unclear reasons. This sudden widespread increase, particularly in children, suggests causes that are not due to genetic makeup of individuals, but rather a change in environment and lifestyle factors. Growing epidemiological and immunological evidence supports the hygiene hypothesis, which states that decreased exposure to immune stimulatinginfections in early childhood is the cause of the rise in allergic diseases. Studies have shown that even bacterial components such as lipopolysaccharide (LPS) can have a protective effect. The receptor for LPS is Toll-like receptor 4 (TLR4), an innate immunological receptor, which may play an essential role in regulation of allergic disease. We investigated the expression of TLR4 in the nasal mucosa of allergic and non-allergic children and adults, the effects of LPS on allergic inflammation and the regulation of TLR4 expression. We hypothesized that LPS, through TLR4, could regulate allergic inflammation and that long-term allergic inflammation would limit these responses. / Children and adults undergoing nasal surgery were recruited from the local hospitals. Nasal biopsies from the patients were explanted and cultured ex vivo with allergen and LPS. LPS could prevent allergen-induced Th2 cytokine production and inflammatory cell increases in explants from atopic children; this was through induction of Th1 cytokines and IL-10. TLR4 was also detected on CD4+CD25+ T cells. LPS did not provide the same protection in adults. Adults, especially atopic subjects, had less TLR4 immunopositive cells; hence, their reduced responsiveness to LPS. This suggests that factors could downregulate TLR4 in allergic adults. The U-937 monocytic cell line was used as an in vitro model to study the regulation of TLR4 by interleukin-4 (IL-4), a T-helper type 2 (Th2) cytokine involved in allergic inflammation. U-937 cells cultured with IL-4 had decreased LPS responsiveness, TLR4 protein surface expression, TLR4 mRNA expression and transcriptional activity of the upstream region of TLR4. This effect was tyrosine kinase and STAT6 dependent. A STAT6 binding site was determined in an area of the TLR4 gene necessary to mediate IL-4's inhibitory effects on TLR4 transcription. IL-4 was also determined to reduce TLR4 expression in PBMCs, especially in CD4+ T cells purified from the blood of children. / As predicted in epidemiological studies and animal studies, LPS was shown to provide anti-inflammatory effects against allergen in human nasal tissue. LPS may directly stimulate regulatory T cells (CD25+CD4+) to produce anti-inflammatory cytokine production. The effects of LPS exposure may be lost due to reduced expression of TLR4 by inflammatory cells, and this may be caused by the allergic inflammation itself. Therefore, development of allergic inflammation can down-regulate anti-inflammatory mechanisms and promote long term chronic inflammation.

Idiopathic pulmonary fibrosis (IPF) belongs to the group of the interstitial lung diseases and is characterized by inflammation, proliferation of fibroblasts and type II pneumocytes, and increased collagen deposition. Inflammatory cells, by releasing mediators and cytokines, participate in the pathogenesis of IPF. Endothelin-1 (ET-1), a vasoconstrictor and mitogenic peptide, is one of the mediators that has been shown to be involved in the fibrotic process of IPF in humans. There are, however, no studies examining the role of ET-1 in animal models of IPF. We used the rat model of pulmonary fibrosis, induced by bleomycin, to study the role of ET-1 and endothelin converting enzyme-1 (ECE-1) in IPF using immunohistochemistry (IHC). We also studied by morphometry the effect of bosentan, the mixed ET-A/B receptor antagonist, on the severity of the fibrosis. We found increased ET-1 and ECE-1 immunoreactivities in the lungs of the fibrosis group compared with the control group (P $<$ 0.05), principally in epithelial cells. By morphometry, we found a decrease in the volume fraction (Vv) of air and an increase in the Vv of connective tissue in the fibrosis group compared with control. The fibrosis was significantly reduced by bosentan (P $<$ 0.05). These results are consistent with the notion that ET-1 is an important mediator of bleomycin-induced pulmonary fibrosis.

Patients with systemic amyloidosis frequently show gastrointestinal (GI) tract and adrenal gland involvement with amyloid. However, the evolution of amyloid-related pathological changes in humans is unknown. Conversely, although GI amyloidosis has been described in casein-stimulated mice, the chemical nature of the GI amyloid have not been carried out. Here we report studies on these and related aspects using alveolar hydatid cyst (AHC)-infected mouse model of reactive amyloidosis. Paraffin sections from the adrenal gland, stomach, small and large intestine were obtained from AHC-mice at different time period post-infection (p.i.) and stained with Congo red or immunohistochemically with antibodies against mouse AA amyloid (RAA) or bovine ubiquitin (RABU). The GI-amyloid was purified by column chromatography and analyzed. In the adrenal gland, amyloid deposits were first detected at 4 weeks p.i. in the cortical-medullary junction which then extended into the adrenal parenchyma. In the GI tract, submucosal blood vessels, the first site of amyloid deposition, became amyloidotic at 1 week p.i. With time, the amyloid deposits extended into the lamina propria of the mucosa. Ileum was the most severely affected region in the GI tract. Both RAA and RABU reacted specifically with the GI amyloid. Amyloid was purified by exclusion chromatography in 4M guanidine. The purified amyloid on Western blotting reacted with RAA and on N-terminal amino acid sequence analysis revealed homology with murine serum amyloid A$ sb2$ (SAA$ sb2$) indicating its derivation from SAA$ sb2.$ Both the similarities and the differences between the GI amyloid-related pathological changes in humans and mice are discussed.

Schneiderian papillomas (SP) are tumors arising from the surface epithelium (Schneiderian epithelium) of the nasal cavity and paranasal sinuses. Evidence points towards a viral etiology, specifically Human papillomavirus (HPV). Although substantial data indicates HPV as a likely etiology, little is known about the role of HPV in benign nasal pathologies or in normal nasal mucosa. Objective. To characterize the role of HPV in SP, chronic sinusitis (CS) and its prevalence in normal nasal mucosa. A case controlled study was undertaken, matching patients with SP to patients with chronic sinusitis (CS). Patients with normal nasal mucosa served as a control group. All patients had their tissues analyzed for the presence of various HPV types using polymerase chain reaction (PCR) coupled with a line blot assay. Results. A total of 168 patients were identified (74 SP, 74 CS, 20 control). Of these, 70 (41.7%) had detectable DNA, and 9/70 (12.9%) had detectable HPV of types 6, 11, and 16. None had detectable HPV type 18. Significant differences were detected in the presence of HPV in CS, SP and control groups, as well as in the presence of low risk versus high-risk types amongst investigation and control groups. Conclusions. Significant differences exist in the distribution of HPV between SP, benign nasal pathologies such as CS and normal nasal mucosa. HPV may play an important role, at least as cofactor, in the development of SP, with types 6, 11 and 16 more pivotal than other types. Line blot assay may provide a useful technique in identifying HPV in SP.

Studies on normal and regenerated rat aortic endothelium at 1, 2, 3 and 4 weeks following segmental balloon denudation injury were carried out to evaluate: (1) the permeability to intravenously injected Evans blue (EB) and horseradish peroxidase (HRP) using en face aortic preparations and (2) the volume density of stress fibers by morphometry using thin section electron microscopy. The results of these studies indicated that: (1) the permeability of the regenerated endothelium to both EB and HRP was identical to normal endothelium at all time points studied and (2) stress fiber volume density significantly increased in regenerated endothelium at 1 week as compared to control, however, returned to and remained at normal value at and after 2 weeks following segmental balloon injury. These results are consistent with the view that structural and functional changes in regenerated vascular endothelium, if present, are transient in nature and the integrity of endothelial monolayer is eventually reestablished during the repair process that follows a single injury.

We assessed clonal development and extent of progression of hemopoietic malignancies (dysmyelopoietic syndrome (DMPS) and acute myelogenous leukemia) by examining in vitro growth patterns of their normal and leukemic progenitors. Additional phenotypic and cytogenetic analysis of an in vitro human myeloid leukemia model (HL-60) and its variant sublines were performed. These were aimed at determining cytogenetic abnormalities associated with phenotypic changes which accompany the derivation of these variant sublines. Our findings indicate that in vitro bone marrow cultures can be used clinically to rule out preleukemia, and that quantitations of bone marrow culture (CFU-C) can determine the potential for the development of acute leukemia in the DMPS patients. Acute leukemia developed in 48% of DMPS patients with a median transformation of 10 months. / In acute leukemia, there was a preferential growth of normal karyotype in the in vitro cultures even among the phenotypically specified "blast" colonies. / Analysis of HL-60 variant sublines demonstrated the development of specific chromosomal abnormalities (1q+, iso8q, iso17q) in two cell lines (clones resistant to chemical induction) in association with loss of differentiation. These specific chromosomal abnormalities are known to be associated with tumor progression. The development of 1q+ abnormality was associated with loss of myeloperoxidase reaction and persistence of primary granules in that specific variant. A group of variant subclones was also associated with loss of differentiation, cytogenetically however, they demonstrated a revert to near diploid near normal karyotypes.