Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Doctor of Philosophy / Department of Anatomy and Physiology / Bruce Schultz / Transforming growth factor β1 (TGF-β1) is a cytokine that reportedly affects the severity of cystic fibrosis lung disease. The goal of this project was to define the effect of TGF-β1 on vas deferens, an organ that is universally affected in male cystic fibrosis patients. In the first study, experiments were conducted using freshly isolated porcine vas deferens epithelial cells. Primary porcine vas deferens epithelial cells exposed to TGF-β1 exhibited a significantly reduced basal transepithelial electrical resistance (Rte). TGF-β1-induced reduction in Rte was prevented by SB431542, a TGF-β receptor I inhibitor, indicating that the effect of TGF-β1 requires the activation of TGF-β receptor I. Western blot and immunohistochemistry results showed the expression of TGF-β receptor I in native vas deferens epithelia, indicating that the impaired barrier function and anion secretion that were observed in cultured vas deferens cells can likely be observed in the native context. Immunohistochemical outcomes showed that TGF-β1 exposure led to loss of organization of tight junction proteins occludin and claudin-7. These outcomes suggest that TGF-β1 impairs the barrier integrity of epithelial cells lining the vas deferens. In a parallel study that employed PVD9902 cells that are derived from porcine vas deferens, TGF-β1 exposure significantly reduced anion secretion stimulated by forskolin, forskolin/IBMX, and 8-pCPT-cAMP, suggesting that TGF-β1 affects downstream targets of the cAMP signaling pathway. Real-time RT-PCR and western blot analysis showed that TGF-β1 exposure reduced both the mRNA and the protein abundance of cystic fibrosis transmembrane conductance regulator (CFTR). Pharmacological studies showed that the inhibitory effect of TGF-β1 on forskolin-stimulated anion secretion was abrogated by SB431542 and attenuated by SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. These outcomes suggest that TGF-β1, via the activation of TGF-β receptor I and p38 MAPK signaling, reduces CFTR expression, and thus impairs CFTR-mediated anion secretion. Outcomes from these studies suggest that, in epithelial cells lining the vas deferens, TGF-β1 exposure leads to an impaired physical barrier and/or reduced anion secretion, which is expected to modify the composition and the maintenance of the luminal environment and thus, is expected to reduce male fertility.

No description available.

O Mal de Alzheimer (MA) foi caracterizado pela primeira vez em 1907 pelo neuropatologista alemão Alois Alzheimer, tendo como sintomas clínicos disfunções cognitivas, fisiológicas, comportamentais, perda de memória, e eventualmente incontinência, demência, acamação e morte. É uma doença neurodegenerativa do sistema nervoso central que costuma afetar, principalmente, indivíduos em faixa etária mais avançada. Este mal é caracterizado microscopicamente pela presença de placas amilóides, que são acúmulos da proteína betaamilóide inter-neurônios, e emaranhados neurofibrilares, formados predominantemente por formas altamente fosforiladas de uma proteína associada aos microtúbulos, Tau, as quais formam massas emaranhadas que consomem o corpo celular neuronal, possivelmente levando à disfunção neuronal e finalmente à morte. MAPK p38? tem sido implicada em dois eventos associados ao MA, fosforilação da Tau e inflamação. MAPK p38? é ativada por uma via de fosforilação dupla em Thr180 e Tyr182. O planejamento de fármacos inibidores de p38? é principalmente focado em pequenas moléculas que competem pelo sítio catalítico do ATP. Aqui, nós utilizamos diferentes técnicas de modelagem molecular e planejamento racional baseado em estrutura e ligantes, tendo como base os inibidores da MAPK p38? descritos na literatura, além das estruturas depositadas no PDB. Como resultado das diferentes abordagens de triagens virtuais utilizadas neste trabalho, tais como \"docking\", farmacóforo, dinâmica molecular, campos de interação molecular, predição de atividade e toxicidade, cálculo de propriedades farmacocinéticas e físico-químicas, foi selecionado um total de 14 compostos que atendem aos critérios adotados de baixa ou nenhuma toxicidade potencial, bom perfil farmacoterapêutico predito, atividades calculadas em valores comparáveis aos obtidos para os compostos de referência, além da manutenção das principais interações observadas para os inibidores mais potentes. Estes compostos podem ser adquiridos para estudos de inibição in vitro frente à enzima MAPK p38?, contribuindo assim na busca de um potencial candidato à fármaco no tratamento do Mal de Alzheimer. / Alzheimer\'s disease (AD) was first characterized in 1907 by the German neuropathologist Alois Alzheimer, whose clinical symptoms includes cognitive, physiological and behavioral dysfunctions, memory loss, eventually incontinence, dementia, and death. It is a neurodegenerative disease of the central nervous system that usually affects individuals group in older age. This is characterized microscopically by the presence of amyloid plaques, which are accumulations of beta-amyloid protein inter-neurons, and neurofibrillary tangles formed predominantly by highly phosphorylated forms of the microtubule-associated protein, tau, which form tangled masses that consume neuronal cell body, possibly leading to neuronal dysfunction and ultimately death. p38? MAPK has been implicated in both events associated with AD, tau phosphorylation and inflammation. p38? MAPK pathway is activated by a dual phosphorylation at Thr180 and Tyr182 residues. The drug design of p38? MAPK inhibitors is mainly focused on small molecules that compete for ATP in the catalytic site. Here, we used different techniques of molecular modeling based on p38? MAPK structure deposited in the PDB and its inhibitors described in the literature. As a result of different virtual screening approaches used in this work, such as \"docking\", pharmacophore, molecular dynamics, molecular interaction fields, activity and toxicity predictions assays, pharmacokinetic properties and physicochemical, was selected a total of 14 compounds that meet these criteria of low or no toxicity potential, good pharmacotherapeutic profile, predicted activities calculated values comparable to those obtained for the reference compounds, while maintaining the main interactions observed for the most potent inhibitors. These compounds should be acquired for in vitro inhibition studies against the enzyme p38? MAPK, thereby helping in the search of a potential drug candidate for the treatment of Alzheimer\'s disease.

La myélofibrose primitive (MFP) est un néoplasme myéloprolifératif (NMP) chronique BCR-ABL1-négatif associant une dérégulation de l’hématopoïèse (myéloprolifération, dysmégacaryopoïèse et migration des cellules souches et progéniteurs hématopoïétiques (CSH/PH)) à une altération du stroma médullaire et splénique (fibrose ostéomyélosclérose, néoangiogenèse). Le mégacaryocyte (MK) est un acteur majeur de sa pathogenèse, via la production de cytokines et facteurs fibrosants, dans un contexte inflammatoire. Plusieurs arguments suggèrent que les mutations JAK2V617F et MPL515L/K qui caractérisent les NMP ne sont pas les événements initiaux de la MFP car elles ne sont retrouvées que chez la moitié des patients. L’objectif de mon travail a été de rechercher si d’autres anomalies, géniques ou non, pouvaient expliquer la pathogenèse de la MFP. Pour cela, parallèlement à une démarche génomique (transcriptome et CGH array), nous avons développé une approche de biologie cellulaire ciblée sur le rôle du stroma hématopoïétique. Bien que n’ayant pas identifié d’autres anomalies génomiques que celles décrites dans la littérature et en particulier, la délétion 13q, les approches génomiques que nous avons développées nous ont permis de préciser les bornes de cette délétion dans les PH CD34+ et les polynucléaires des patients. Cette délétion (région chromosomique minimale 13q14-13q21) est située à 2 mégabases (télomérique) du cluster FLT où est localisé le gène FLT3. Plusieurs arguments nous ont ensuite conduits à rechercher si le couple Flt3-ligand/Flt3 était impliqué dans la dérégulation de l’hématopoïèse et plus particulièrement dans la dysmégacaryopoïèse observée chez les patients. Parmi ceux-ci, citons : 1) l’existence d’une modulation d’expression de gènes inclus dans la zone de délétion 13q et dans le cluster FLT, dont le gène FLT3 et 2) le fait que Flt3, un récepteur clé de la régulation de l’hématopoïèse primitive, soit souvent impliqué dans la pathogenèse d’hémopathies malignes et que son ligand, Flt3-ligand, soit majoritairement produit par le stroma hématopoïétique. Notre étude montre une dérégulation de Flt3 et des MAPKs p38 dans les PH CD34+ et les MK des patients atteints de MFP et ceci, quelque soit leur statut mutationnel Jak2. Elle démontre également que la persistance de la stimulation de l’axe Flt3/p38 en réponse à une production accrue de Flt3 ligand, participe à la dysmégacaryopoïèse qui caractérise la maladie. En effet, nous avons mis en évidence : 1) une augmentation du taux sérique de Flt3 ligand et de son expression par les cellules du stroma médullaire et splénique ainsi que par les PH des patients atteints de MFP, 2) une surexpression spécifique de son récepteur Flt3 et de sa phosphorylation dans les CSH/PH CD34+ et les progéniteurs mégacaryocytaires (MK), qui persistent au cours de la différenciation MK, quelque soit le statut mutationnel de Jak2 des patients, 3) une activation de Flt3 dans les progéniteurs MK en réponse au Flt3 ligand conduisant à la phosphorylation en cascade de la voie de signalisation des MAPKs p38 et à l’expression de ses gènes cibles tels que AP-1, p53, NFATc4, ATF2, IL-8, 4) une restauration de la mégacaryopoïèse et une inhibition de la migration (Flt3-ligand)-dépendante des progéniteurs MK des patients après inhibition de Flt3 ou de p38.Nos résultats confirment l’importance d’une altération des MAPKs dans une dérégulation de l’hématopoïèse et soulignent le rôle d’une activation persistante de la voie p38, via le couple Flt3-ligand/Flt3, dans la dysmégacaryopoïèse qui caractérise la myélofibrose primitive. Ils suggèrent également que cette dérégulation participe au processus inflammatoire à l’origine de la réaction stromale et « lit » d’une transformation leucémique potentielle. Ce dialogue altéré entre les cellules hématopoïétiques pathologiques (Bad seeds), en particulier mégacaryocytaires et les cellules stromales (Bad soil), conforte notre concept « Bad seeds in Bad soil ». / The primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (NMP) BCR-ABL1-negative associating a dysregulation of hematopoiesis (myeloproliferation, dysmegacaryopoiesis and egress of hematopoietic stem and progenitor cells (HSC / PH)) from an altered bone marrow stroma (osteosclerosis, fibrosis, angiogenesis) to the spleen. The megakaryocyte (MK) is a major player in its pathogenesis through the production of cytokines and fibrotic factors in an inflammatory context. Several arguments suggest that mutations JAK2V617F and MPL515L / K which characterize the NMP are not the initial events of the PMF since they are found only in half of patients. The aim of my work was to investigate whether other abnormalities, genetic or otherwise, could explain the pathogenesis of the PMF. For this, a process parallel to genomics (transcriptome and CGH array), we developed a cell biology approach focused on the role of hematopoietic stroma.Although we have not identified other genomic abnormalities as those described in the literature and in particular, deletion 13q, by genomic approaches we have clarified the limits of this deletion in the PH CD34+ and polymorphonuclear patients. This deletion (chromosomal region 13q14-13q21 minimum) is located 2 megabases (telomeric) of the cluster where is located the FLT gene FLT3. Several arguments have then led to inquire whether the couple was involved in Flt3-ligand/Flt3 deregulation of hematopoiesis, especially in the dysmegakaryopoiesis observed in patients. Among these are: 1) the existence of an expression modulation of genes included in the area of deletion 13q and FLT in the cluster, as gene FLT3 and 2) the fact that Flt3, a key receptor the regulation of primitive hematopoiesis, is often implicated in the pathogenesis of hematologic malignancies and its ligand, Flt3-ligand, was predominantly produced by the hematopoietic stroma.Our study shows dysregulation of Flt3 and p38 MAPKs in CD34+ and PH MK from patients with PMF and this, whatever their Jak2 mutation status. It also shows that persistent stimulation of the axis Flt3/p38 in response to increased production of Flt3 ligand, participates in the dysmegacaryopoiesis that characterizes the disease. Indeed, we have highlighted: 1) an increase in serum Flt3 ligand and its expression by stromal cells and bone marrow and spleen by PH patients with PMF, 2) a specific overexpression of its receptor Flt3 and its phosphorylation in HSC / PH CD34+ and megakaryocytic progenitors (MK), which persist during the MK differentiation, regardless of the mutational status of Jak2 patients, 3) activation of Flt3 in MK progenitors by the Flt3 ligand leads to phosphorylation cascade signaling pathway, p38 MAPK and expression of its target genes such as AP-1, p53, NFATc4, ATF2, IL-8, 4) a restoration of megakaryopoiesis and inhibition of migration (Flt3-ligand)-dependent patients after of MK progenitors by Flt3 or p38 inhibitors.Our results confirm the importance of an alteration of MAPKs in a deregulation of hematopoiesis and highlight the role of a persistent activation of the p38 pathway, via the couple Flt3-ligand/Flt3 in the dysmegakaryopoiesis that characterizes idiopathic myelofibrosis. They also suggest that this dysregulation contributes to the inflammatory process at the origin of the stromal reaction and "bed" of a leukemic transformation potential. The dialogue among impaired hematopoietic cell disease (Bad Seeds), especially the stromal cells and megakaryocyte (Bad Soil), reinforces our concept of "Bad Seeds in Bad Soil". This work could help improve the dialogue with therapeutic approaches targeting the axis Flt3-ligand/Flt3 mediated by activation of p38 which, by reducing the inflammatory process, re-establish a link between the "seed" and the "Soil".

Orientador: Miriane de Oliveira / Resumo: Introdução: O hormônio triiodotironina (T3) influencia o metabolismo e desenvolvimento do tecido adiposo (TA), modulando a proliferação e diferenciação de adipócitos, podendo agir sobre os reguladores do processo de adipogênese, como o receptor ativado por proliferador de peroxissomo (PPARy). O TA está envolvido na regulação da energia corporal, sintetizando e secretando substâncias denominadas adipocinas, dentre elas a adiponectina e leptina. A adiponectina está relacionada ao aumento da sensibilidade à insulina, enquanto a leptina está envolvida com o gasto energético. O T3 pode desencadear ações por ativação de vias extranucleares, dentre elas a via MAPK/ERK e integrina αVβ3. Objetivo: Verificar a ação do T3, com participação das vias extranucleares MAPK/ERK e integrina αVβ3, na modulação de adiponectina e leptina, além de avaliar os parâmetros relacionados ao perfil adipogênico e dano de DNA. Métodos: Adipócitos, 3T3-L1, foram tratados com T3 (10nM) por uma hora, na ausência ou presença dos inibidores de MAPK/ERK – PD98059 (PD, 50uM) e da integrina αvβ3 – ácido tetraiodotiroácetico (Tetrac, 10-4M). A ausência de qualquer tratamento foi considerada grupo controle (C). Após o período de tratamento foi realizado PCRq-RT para analisar a expressão de mRNA de adiponectina e leptina, e Western Blot para expressão proteica de adiponectina, leptina, PPARy, pAKT e pERK; a viabilidade celular foi realizada pelo ensaio de MTT; a quantificação do acúmulo lipídico pelos ens... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: The hormone triiodothyronine (T3) influences the metabolism and development of adipose tissue (TA), modulating the proliferation and differentiation of adipocytes, and can act on regulators of the adipogenic differentiation process, such as the peroxisome proliferator activated receptor). TA is involved in the regulation of body energy, synthesizing and secreting substances called adipokines, among them adiponectin and leptin. Adiponectin is related to increased insulin synaptic, since leptin is involved in energy expenditure. T3 can trigger actions by activation of extranuclear pathways, including MAPK / ERK and integrin α Vβ3. Objective: Given the role of T3 in TA and the importance of adipokines, the objective of this study is to verify the action of T3 with the participation of extranuclear pathways in the modulation of adiponectin and leptin and the parameters related to the adipogenic profile. Methods: Adipocytes, 3T3-L1, were treated with a physiological dose of T3 (10nM) for one hour, in the absence or presence of MAPK / ERK-PD98059 (PD) and integrin αvβ3 - tetraiodothyrocetic (Tetrac) integrin inhibitors. The absence of any treatment was considered as a control group (C). After the treatment period PCRqRT was performed to analyze the expression of leptin and adiponectin mRNA, and Western Blot for protein expression of adiponectin, leptin, PPARγ, pAKT and pERK; cell viability was performed by the MTT assay; the quantification of lipid accumulation by the... (Complete abstract click electronic access below) / Mestre

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Colorectal cancer is the second most common cause of cancer death in Western Europe and North America. Current therapies are largely ineffective and are associated with considerable morbidity. Activating mutations in KRAS and BRAF genes are frequent in colorectal cancer, especially at later stages of the disease, and result in constitutive activity of the MAPK pathway, leading to increased proliferation and tumour survival. The MEK1/2 inhibitor AZD6244, that targets the MAPK pathway downstream of these mutations, has been tested as novel therapy for colorectal cancer. However, clinical trials have been disappointing due to an apparent intrinsic and/or acquired resistance to treatment. Mechanisms underlying this resistance have been studied using cell lines and tumour xenografts. However, the relevance of these data to advanced human colorectal cancer is unclear. One of the difficulties in testing and developing novel therapies for colorectal cancer is the lack of representative models of human disease. Thus, the initial aim of my PhD was to develop a method to culture human colorectal cancers ex vivo in order to use this as a platform for investigating response to AZD6244 and other therapies. These studies indicated that regardless of growth conditions, colonic tumour explants suffered extensive apoptosis in the first 24h in culture, which limited their application in drug response assays. Therefore, as an alternative to long term culture of human colorectal explants, I tested the effects of AZD6244 using acute treatments. Twenty three fresh colonic tumours were obtained from patients and treated for 1h with AZD6244 ex vivo in dose response studies. In all samples, MEK1/2 inhibition occurred within 1h of treatment. In one group of particularly sensitive tumours, the drug also had a distinct phenotypic effect. In these tumours, I found that the agent induced a dose-dependent decrease in proliferation and increase in apoptosis within 1h of treatment. Analysis of markers for this sensitivity indicated it was not clearly dependent of the presence of KRAS or BRAF mutations, which have previously been shown to confer sensitivity. Other markers of sensitivity / resistance were also examined. In addition to studies with AZD6244 alone, I examined the combined effects of this agent and aspirin in colon cancer cells lines and in tumour explants, with promising results. Whilst the use of fresh patient tumour tissue has some technical and logistical challenges, these data suggest that such methodologies are worthy of further investigation as a means to examine determinants of sensitivity and resistance to novel therapies, or their likely activity in combination.

No description available.

Ovarian carcinoma afflicts over 22,000 women each year with a 5 year survival rate of only 18% for stage IV patients [23]. Current treatment options are limited due to high rates of drug resistance and recurrence. Further, the identity of "precursor lesions" which give rise to various subclasses of epithelial ovarian cancer has been evasive. This is due to discovery of the cancer at already an advanced stage. Interestingly, endometriosis a benign but invasive gynecological disease has been described as a "precursor lesion" in the development of specific subtypes of ovarian cancer. Endometriotic cyst development involves the accumulation of "old blood" components including iron-rich heme. Published evidence implicates excess iron that is involved in the transformation of normal surface epithelial cells inducing morphological characteristics of clear cell ovarian cancer cells [13, 34]. Due to excess iron in endometriotic cysts, this essential element may play a transformative role in the development of clear cell ovarian cancer and possibly other subtypes [13, 35-38]. Further, studies show increased risk of developing ovarian cancer, particularly clear cell and endometrioid ovarian subtypes, in patients diagnosed with endometriosis [36, 37, 39, 40] . This thesis aims to initiate an investigation regarding the contribution of iron and endometriotic lesions in the development and progression of specific subtypes of epithelial ovarian cancers. Since there is a lack of well-validated and characterized endometriotic cell lines that could be used for endometriosis studies, we sought to develop an immortalized cell line for future endometriotic in vitro and in vivo studies. Thus, in Chapter 3 we present our efforts in developing a novel life-span extended epithelial endometriotic cell line. The cells were derived from the endometriotic tissue of a patient with endometriosis. We describe our attempts at immortalization and the characterization of this endometriotic cell line in relation to previously reported/available endometrial/endometriotic cell lines. In Chapter 4 we investigated the role of iron in modulating functional aspects of various gynecological cell lines. Although our expectation was that iron could transform normal ovarian surface epithelial cells (OSE) to a carcinoma-like phenotype, we instead discovered that ovarian cell lines containing Ras mutations (or with H-Ras overexpression) responded to iron (presented as ferric ammonium citrate (FAC)) with a reduced growth response. Further treatment with iron induced an apoptotic/necrotic death response in the Ras mutated HEY ovarian carcinoma cell line. Interestingly, we identified that iron induced autophagic activation in all ovarian cell lines investigate, although autophagy contributed only modestly to the cell death event. Furthermore, we noted that iron activated the MAPK pathway and its inhibition (via U0126, a MAPK inhibitor) allowed survival of cells. In Chapter 5, we briefly explore the role of iron in ovarian cell types growing under anchorage-independent conditions. We found that the cell lines displayed increased cleaved PARP and apoptosis when placed under these conditions. Treatment with iron led to a reduction in cleaved PARP suggesting that iron promotes cell survival in anchorage-independent conditions. Further, inhibition of autophagy via chloroquine led to increased cleaved PARP suggesting that autophagy may mediate a protective role against anchorage-independent apoptotic response In Chapter 6, we attempted to elucidate the downstream mechanism following Ras/MAPK activation in response to iron. This study identified several signaling pathways including that involved in translational control, iron metabolism, as well as mitochondrial function. The inhibition of the iron regulatory and translation control pathway did not significantly lead to rescue of iron-induced cell death of Ras mutated/overexpressed cells. However, we noted mitochondrial stress and damage including altered expression of mitochondrial markers (TOM20/TOM70, outer membrane transporters) which occurred concurrently with iron-induced cell death. The inhibition of iron import into mitochondria using a calcium uniporter channel inhibitor (Ru360) led to a marked reversal of the cell death response. Collectively, these studies suggest that increased mitochondrial permeabilization may be responsible for the observed iron-induced cell death response. Overall, the studies presented in this thesis have revealed novel responses to iron in the gynecological cell types investigated. We initially sought to understand the role of iron in precursor lesions which included the development of a novel life-span extended epithelial endometriotic cell type. Remarkably, our findings revealed a Ras driven sensitivity to excess iron. Treatment with iron caused decreased cell growth and increased cell death in cell types containing Ras mutation/overexpression. Further, we found that the mechanism leading to the iron-induced cell death events was mediated via the MAPK pathway. We then determined that the cell death response was associated with mitochondrial permeabilization. Loss of mitochondrial integrity occurred in Ras sensitive cell lines and inhibition of iron import into the mitochondria (via the calcium uniporter channel inhibitor, Ru360) led to reversal of this response. We show herein the cellular response of excess iron and its potential implication in ovarian cancer research.