Previously, we described prostate cancer (PCa) detection (83% sensitivity; 67% specificity) in seminal plasma by CE-MS/MS. Moreover, advanced disease was distinguished from organ-confined tumors with 80% sensitivity and 82% specificity. The discovered biomarkers were naturally occurring fragments of larger seminal proteins, predominantly semenogelin 1 and 2, representing endpoints of the ejaculate liquefaction. Here we identified proteases putatively involved in PCa specific protein cleavage, and examined gene expression and tissue protein levels, jointly with cell localization in normal prostate (nP), benign prostate hyperplasia (BPH), seminal vesicles and PCa using qPCR, Western blotting and confocal laser scanning microscopy. We found differential gene expression of chymase (CMA1), matrix metalloproteinases (MMP3, MMP7), and upregulation of MMP14 and tissue inhibitors (TIMP1 and TIMP2) in BPH. In contrast tissue protein levels of MMP14 were downregulated in PCa. MMP3/TIMP1 and MMP7/TIMP1 ratios were decreased in BPH. In seminal vesicles, we found low-level expression of most proteases and, interestingly, we also detected TIMP1 and low levels of TIMP2. We conclude that MMP3 and MMP7 activity is different in PCa compared to BPH due to fine regulation by their inhibitor TIMP1. Our findings support the concept of seminal plasma biomarkers as non-invasive tool for PCa detection and risk stratification.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

La Fibrose Kystique, causée par des mutations du canal CFTR, mène à la dysfonction du transport des fluides et des ions causant la déshydratation du liquide de surface des voies aériennes et ainsi une défaillance de la clairance mucocilliaire. Ce défaut entraine l’accumulation et l’épaississement du mucus au niveau des bronches qui devient alors un environnement idéal pour le développement d’infections chroniques et d’inflammation qui sont associées à la destruction progressive de l’épithélium chez les patients Fibrose Kystique. Même si leur rôle dans les processus lésionnels est très bien connu, l’impact de médiateurs inflammatoires sur la capacité de réparation ne l’est cependant pas. L’objectif de ma maitrise était donc d’étudier la régulation des mécanismes de réparation de l’épithélium bronchique sain et Fibrose Kystique par le facteur de nécrose tumoral (TNF)-alpha, une cytokine pro-inflammatoire cruciale dans l’initiation et la propagation de la réponse inflammatoire chez les patients FK. À l’aide d’un modèle de plaies mécaniques, nous avons montré que le TNF-alpha stimule la réparation de l’épithélium bronchique sain (NuLi-1) et Fibrose Kystique (CuFi-1). De façon surprenante, l’exposition chronique au TNF-alpha augmente cette stimulation tout comme le taux de migration cellulaire pendant la réparation. Cette augmentation de réparation semble être médiée par l’activation de la métalloprotéinase MMP-9, la relâche d’EGF par les cellules épithéliales et ainsi l’activation de la voie d’EGFR. De plus, l’activation de la réparation par le TNF-alpha semble aussi impliquer l’activation des canaux K+, dont nous avons démontré le rôle important dans la réparation. Contrairement à son effet sur la migration cellulaire et sur la réparation, le TNF-alpha diminue la prolifération cellulaire. En somme, en plus de son rôle dans les processus lésionnels, le TNF-alpha semble avoir un rôle complexe dans les processus de réparation puisqu’il stimule la migration et ralentit la prolifération cellulaire. / Cystic fibrosis (CF) pathology, caused by mutations of cftr gene, leads to ion and fluid transport dysfunction that results in mucus thickening and accumulation in the airways. This mucus accumulation promotes bacterial infection and airway inflammation associated with progressive airway epithelial damage in CF patients, unfortunately leading to respiratory failure. However, the effect of inflammatory products on the repair capacity of respiratory epithelia is unclear. Thus, the objective of my project was to study the regulation of normal and CF bronchial epithelial repair mechanisms by tumor necrosis factor-alpha (TNF)-alpha, a major component of inflammation initiation and propagation in CF. With a wound healing model, we observed that TNF-alpha stimulated the non-CF (NuLi-1) and CF (CuFi-1) bronchial wound healing rate. Surprisingly, chronic exposure to TNF-alpha enhanced this stimulation as well as the migration rate during repair. This wound healing rate stimulation by TNF-alpha seems to be due to metalloproteinase MMP-9 activation, EGF shedding by epithelial cells and subsequent EGFR transactivation. Furthermore, we recently reported a crucial relationship between the EGF response and K+ channel function, both controlling bronchial repair. We now show that TNF-alpha wound healing stimulation also implicated KvLQT1 and KATP currents activation. In contrast to its effect on cell migration, TNF-alpha downregulate cell proliferation. Thus, in addition to its recognized role in the inflammatory response leading to epithelial injury, TNF-a could exert complex actions on repair mechanisms of CF airway epithelia by upregulating cell migration while downregulating proliferation.

Glioblastoma Multiforme (GBM) is an aggressive and fatal cancer of the brain. It is characterized with augmented morbidity and elusion to therapies due in part to the incessant infiltration and spread of tumor cells in normal brain. We investigated the function of Matrix metalloproteinase-1, an important enzyme noted to be responsible for invasion in other cancers, in GBM and its regulation by epidermal growth factor receptor (EGFR) signaling. Previous studies from our laboratory demonstrated elevated levels of MMP-1 in GBM. Further studies indicated the involvement of MMP-1 in GBM invasion. The GBM cell lines T98G, U251MG and U87MG were used for this study. In T98G cell lines, inhibition of MMP-1 by siRNA significantly suppressed basal in vitro invasion without impacting cell viability. The over-expression of MMP-1 was accomplished in U251MG and U87MG using the mammalian expression vector, pIRES, encoding full length MMP-1 cDNA. The MMP-1 over-expressing U251MG and U87MG cells exhibited significantly enhanced invasion in vitro with no modification in the cell proliferation rates. A majority of GBM patients present defective EGFR signaling due to over-expression, amplification or mutation in the receptor. MMP-1 is known to be up-regulated by various stimulatory agents including growth factors. We examined the regulation of MMP-1 by EGFR activation and observed the induction of MMP-1 after EGF treatment. Inhibition of the receptor by pharmaceutic inhibitor treatment and genetic approaches led to reduction in MMP-1 levels. We also observed that this regulation is primarily mediated by the downstream MAPK pathway. Inhibition of MAPK and not PI3K pathway resulted in diminished MMP-1 protein levels even in the presence of EGF. These studies demonstrate the importance of the EGFR-MAPK signaling pathway in the induction of MMP-1 in glioma cell lines. In addition, MMP-1 plays a role in glioma cell invasion in vitro. These results along with the reports of MMP-1 over-expression in GBM warrant future studies examining the function of MMP-1 in vivo.

During cardiac development and in cardiac disease changes in hemodynamic load initiate events leading to remodeling of the ECM. This study addresses the hypothesis that interactions between Integrins and Metalloprotienases function to modulate cell adhesion in the cultured cardiac fibroblast. The fibroblast is positioned to detect and respond to changes in the mechanical load on the heart. Functionally the cardiac fibroblast is the primary cell type responsible for the production, maintenance, and remodeling of the cardiac interstitium. Matrix Metalloproteinases, specifically the Gelatinases, are expressed in concert during development and in disease with changes in the hemodynamic loading of the heart. Our studies have identified by a complex on the surface of the cardiac fibroblast composed of the a3b1 integrin, MMP-2, and TIMP-2. Putatively, this complex is involved in the maturation of adhesions. Inhibition of MMP-2 was associated with a decrease in the strength of adhesion of cell plated on collagen and fibronectin. Confocal imaging and analysis indicate that a predominate interaction occurs between MMP-2 and the a3 integrin chain. Taken together biochemical, functional, and microscopic data have identified a complex on the surface of the cardiac fibroblast that represents elements of mechanotransduction and matrix metabolism in a single site that functions in the maturation of adhesion

A memory aid, that used brief (33ms) presentations of previously learned information (target words), was assessed on its ability to reinforce memory for target words while the subject was performing an interference task. The interference task required subjects to learn new words and thus interfered with their memory of the target words. The brief presentation (momentary memory priming) was hypothesized to refresh the subjects' memory of the target words. 143 subjects, in a within subject design, were given a 33ms presentation of the target memory words during the interference task in a treatment condition and a blank 33ms presentation in the control condition. The primary dependent measure, memory loss over the interference trial, was not significantly different between the two conditions. The memory prime did not appear to hinder the subjects' performance on the interference task. This paper describes the experiment and the results along with suggestions for future research.

Neutrophils infiltrate myometrium and decidual tissue prior to parturition. Activated neutrophils release reactive oxygen species (ROS) and tumor necrosis factor α (TNFα), which might increase expression of pro-labor genes such as matrix metalloproteinase-1 (MMP-1), progesterone receptor (PR) A/B ratio, and cause demethylation of DNA. These changes might cause labor. Decidual tissue was obtained from consented, healthy women at term (37+ weeks of gestation) not in labor (no contractions, without cervical effacement), term labor and preterm labor (under 37 weeks of pregnancy). Decidual and myometrial cells in culture were treated with (1) ROS, (2) TNFα, or (3) 5-aza-2’-deoxycytidine. Total RNA was extracted, converted to cDNA and evaluated by qRT-PCR for MMP-1, PR-A+B and PR-B. TNFα increased MMP-1 by 17 fold in decidual cells and more than 12 fold in myometrial cells. PR-A/B was increased by 5.6 fold in decidua. ROS up-regulated MMP-1 by 6 fold and elevated the PR-A/B ratio by 4.5 fold in decidual tissue. DNA demethylation increased MMP-1 by about 4 and 11 fold in decidual and myometrium, respectively. The PR-A/B ratio was increased by 4 fold in decidua and the PR-B was decreased by 40% in the myometrium due to DNA demethylation. Decidual tissue in preterm labor showed a 7-fold increase in MMP-1 over term laboring and over a 15-fold increase over term not in labor tissue. In conclusion, MMP-1 expression and PR-A/B ratio was increased by neutrophil products possibly through a mechanism of DNA methylation in decidua and myometrium. Preterm decidua showed a dramatic increase in MMP-1 over normal labor tissue. TNFα and ROS increased expression of MMP-1 to possibly initiate parturition. These data might help explain mechanisms responsible for preterm labor unrelated to infection or premature rupture of membranes.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Le cancer du sein triple négatif (TNBC) représente entre 15-20% des cancers du sein et est l'un des types les plus agressifs. De plus, un sous-groupe de ces patientes est résistant à la radiothérapie (RT) et développe fréquemment une récidive hâtive de la maladie. Des études précédentes ont démontré que l’inflammation induite par la RT accélère la progression du cancer et le développement des métastases. Cette hypothèse a donc été validée dans un modèle pré-clinique de TNBC en implantant les cellules de carcinome de souris triple négatives D2A1 dans les glandes mammaires de la souris Balb/c. Premièrement, la tumeur primaire à été irradiée à une dose sous-curative une semaine post-implantation des cellules. En deuxième lieu, le tissu mammaire de la souris a été pré-irradié avant d'implanter les cellules cancéreuses afin de bien discerner l'effet du microenvironnement irradié sur celles-ci. Ces deux modèles ont mené à une augmentation significative des cellules tumorales circulantes ainsi que du nombre de métastases pulmonaires. Plusieurs molécules inflammatoires dont l'interleukine-1 bêta (IL-1β), l'interleukine-6 (IL-6) ou encore la cyclooxygénase 2 (COX-2) ont été identifiées comme facteurs clés impliqués dans la migration radio-induite des cellules cancéreuses du sein. Conséquemment, un inhibiteur large-spectre comme la chloroquine (CQ), entre autres utilisé comme traitement anti-malarien et anti-inflammatoire, a su prévenir ces effets secondaires associés à la RT. Étant donné que l'action de la CQ est peu sélective, une répression de l'expression de l'ARNm de la métalloprotéinase (MMP) de membrane de type 1 (MT1-MMP), une MMP de surface impliquée notamment dans la migration cellulaire, l'invasion tumorale et l'angiogenèse, a été réalisée afin d'éclaircir le mécanisme d'inhibition des métastases radio-induites. Cette répression de la MT1-MMP prévient la formation des métastases pulmonaires radio-induites, démontrant ainsi un des mécanisme important de l'invasion radio-induite. Ce résultat confirme donc l'importance de la MT1-MMP dans ce phénomène et son potentiel comme biomarqueur de prédiction de l'efficacité des traitements de RT, particulièrement chez les patientes atteintes de TNBC.