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Chez la levure Saccharomyces cerevisiae, plusieurs des protéines impliquées dans l'assemblage et le remodelage de la paroi cellulaire sont produites sous forme inactive ou nécessitent une régulation négative une fois leur travail effectué. Des enzymes protéolytiques doivent donc intervenir à des moments précis pour réguler leur activité. Parmi ces enzymes on retrouve Kex2p, une protéase à sérine impliquée dans la maturation protéolytique des précurseurs protéiques acheminés vers la voie de sécrétion, et Yps1p, une aspartyl protéase dont la fonction est encore peu connue puisque aucun de ses substrats biologiques n'a encore été identifié. Par conséquent, notre objectif principal était de découvrir un ou plusieurs substrats naturels de l'endoprotéase Yps1 dans le but de lui associer une fonction précise. Puisque certaines évidences semblaient indiquer une implication possible de Yps1p dans la régulation des propriétés de la paroi cellulaire via le clivage endoprotéolytique de la chitine synthétase I, nous avons premièrement tenté de démontrer un lien direct entre Yps1p et l'activation de cette enzyme. Toutefois, nos résultats ont plutôt démontré que Yps1p était impliquée dans l'activation d'une ou plusieurs autres protéines que Chs1p. Nous avons donc opté pour une approche plus globale en caractérisant les phénotypes des mutants yps1[delta], kex2[delta] et yps1[delta]kex2[delta]. C'est alors que nous avons fait un premier lien entre Yps1p et les mannoprotéines de la paroi cellulaire. Par la suite, une comparaison entre les profils électrophorétiques des mannoprotéines sécrétées dans le milieu de culture nous a permis d'identifier quelques candidats potentiels que nous avons ensuite caractérisés par séquençage en N-terminal. En plus de confirmer que Yps1p joue un rôle important dans la régulation des propriétés de la paroi cellulaire chez S. cerevisiae, nos résultats ont démontrés que cette endoprotéase est impliquée dans le clivage endoprotéolytique de Gas1p, une [bêta] 1,3-glucanosyltransférase responsable de l'élongation des chaînes latérales de [bêta] 1,3-glucans. Par conséquent, cette découverte fait de Gas1p le premier substrat naturel de l'endoportéase Yps1 à être identifié.

Introduction : La protéase High Temperature Requirement Factor A1 (HtrA1) pourrait être associée au pronostic du cancer de l’ovaire (CO). Objectif : Évaluer l’effet de l’expression de HtrA1 dans des tissus tumoraux sur le pronostic des femmes avec un CO séreux. Méthodes : Une étude de cohorte a été menée chez 122 femmes ayant un CO séreux traitées au CHU de Québec entre 1993-2006. Par immunohistochimie, l’expression de HtrA1 a été mesurée de façon visuelle et informatisée (% noyaux positifs). Des risques relatifs (HR) de progression et de décès ont été estimés avec le modèle de Cox multivarié. Résultats: Un faible pourcentage de noyaux marqués par HtrA1 était associé à une diminution des risques de progression (visuel HR=0,66, p=0,04, informatique HR=0,63, p=0,03) et de décès (visuel HR=0,69, p=0,09, informatique HR=0,56, p=0,01). Conclusion : La sous-expression de HtrA1 était associée à un meilleur pronostic des femmes avec un CO séreux. / Background: The protease High Temperature Requirement Factor A1 (HtrA1) might be associated with prognosis in ovarian cancer (OC). Objective: To evaluate the effect of HtrA1 expression in tumoral tissues on prognosis of women with serous OC. Methods: A cohort study was conducted among 122 women with a serous OC treated at the CHU de Québec between 1993-2006. Tissue microarrays were immunostained for HtrA1. HtrA1 expression was assessed visually and by digital image analysis (% of positive nuclei). Cox regression multivariate models taking into account standard prognostic factors were used to estimate adjusted hazard ratios (HR) of progression and death. Results: Low percentage of HtrA1 marked nuclei was associated with lower risks of progression (visual HR=0.66, p=0.04, digital HR=0.63, p=0.03) and death (visual HR=0.69, p=0.09, digital HR=0.56, p=0.01). Conclusion: Nuclear downregulation of HtrA1 was associated with a better prognosis in women with serous OC.

La nemosis est un processus d’activation cellulaire déclenché in vitro par la mise en culture tridimensionnelle (sphéroïde) de cellules mésenchymateuses saines et qui se termine inéluctablement par une mort cellulaire de type nécrose programmée. Les fibroblastes sont les entités cellulaires les plus représentées dans la pulpe dentaire. Le premier objectif de ce travail était de confirmer l’existence de la réaction némotique dans ces cellules pulpaires et de la comparer à celle du fibroblaste pulmonaire pris comme référence. La caractérisation de ce phénomène a permis de mettre en évidence des similitudes mais aussi des différences marquées entre ces deux types cellulaires, en termes de libération de cytokines, de production d’enzymes protéolytiques et d’expressions géniques. Le second objectif de cette étude était de différencier la réaction némotique d’une autre nécrose programmée, la nécroptose. La finalité de ce travail était d’objectiver si cette activation fibroblastique peut se retrouver in vivo lors de pathologies pulpaires. / Nemosis is a cell activation process – induced in vitro when sound mesenchymal cells are forced to cluster (spheroids) – which inevitably leads to a programmed necrotic cell death. Fibroblasts are the most commonly found cells in the dental pulp. The first aim of this study was to confirm that nemotic reaction could be observed in these cells and to compare their behaviour with that of sound lung fibroblasts, employed as a reference model for nemosis. This process displayed not only important similarities but also differences between the two cell types studied regarding cytokines release, proteolytic enzymes production and gene expressions. The second purpose of this work was to discriminate between nemosis and necroptosis, which is a programmed necrotic cell death. Finally, this study aimed to highlight whether this nemotic cell activation can occur in vivo during dental pulp diseases.

The search for more effective treatment strategies in melanoma led to many new innovative approaches aiming at different molecular targets. Chemotherapy still remains the most effective treatment and many efforts are put in order to improve targeting and delivery of the chemotherapeutic agents. Among these, peptide conjugates of anticancer drugs were designed to increase stability, cell penetration, specificity and accumulation in cancer cells. We as well as others evaluated such a conjugate, termed PSF (L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine-ethylester) in terms of its cytotoxicity in vitro and in vivo using a human melanoma tumor as a model, its stability, transport, and metabolisation. <p>By comparing the cytotoxicity of PSF and melphalan towards different cancer primary melanoma cell cultures, we noticed some interesting observations: PSF displayed the same toxicity pattern both in short (2h) and long term (24h) cell exposures whereas melphalan and m-sarcolysin needed long term exposure to reach the same toxicity. This could indicate that PSF very quickly penetrates the cells in accordance with what has been shown with red blood cells (RBCs). PSF has shown a much better and quicker penetration into the cells in vitro as compared to melphalan. <p>In this present work, the cytotoxic effect of PSF was further evaluated in vivo using a standardized nude mice tumor model bearing a human melanoma. First, the acute toxicity in rats and mice and the maximum tolerated dose were determined. After a dose-escalation study one dose was singled out and tested as a single dose and as a fractionated dose. PSF was able to reach the tumor site and a dose-response relationship was observed. The IP administration of fractionated doses of PSF had significantly better effect on tumor growth inhibition, regression and regrowth than single dose administration and this without any evidence for general toxicity monitored by animal weight loss. We also compared the efficacy of PSF to its parent drug m-sarcolysin, melphalan and cyclophosphamide and observed that PSF was much more active than both melphalan and m-sarcolysin at the same molar doses.<p>Body distribution of the 14C-labelled PSF revealed ratios of 2.4 and 1.5 compared to muscle tissue for the two melanoma tumors evaluated with no significant and stable accumulation in any vital organ. The amount of tracer was still high in the blood after 24 hours explaining the high radioactivity in the kidney and partly in the liver. Interestingly, the spleen had an unusual high radioactivity uptake reflecting the exceptional binding of the tracer to blood cells (BC), while the pancreas very high load was an indicator of protease-mediated specific delivery and strongly support our hypothesis elaborated on the basis of in vitro results. <p><p>Our in vitro data point to a particular mechanism of action of PSF based on the transport of PSF through the body by the rapid binding to blood cells and the delivery at the tumor site by the subsequent release of its active metabolites due to cleavage by tumor-associated proteases.<p>Concerning the binding of PSF to membranes and its transport the following observations were made: while PSF was stable in human plasma, it disappeared very quickly in whole blood along with the generation of a main metabolite: m-sarcolysin. The presence of BC membranes was required for both binding and generating the metabolites. Binding to natural or artificial membranes was achieved and only competition with melanoma cells or proteolytic enzymes such as dispase, led to the generation of active metabolites. The different metabolites were isolated using preparative LC and were then identified using Electrospray Ionisation Mass Spectrometry (ESI). Three metabolites, of which m-sarcolysin was the main one, were identified all bearing the chloroethyl alkylating group. <p>Enzymatic catalysis was further supported by a set of experiments where the enzymatic activity was non-specifically and specifically inhibited. In order to look at the effect of extracellular matrix proteases on PSF, three representatives of ECM proteases were incubated with PSF: collagenase A had no effect, but both dispase and trypsine were able to process PSF. <p>The following data indicate the higher processing of PSF in the presence of cells with a higher proteolytic activity and thus the delivery of the blood cell-bound PSF. When comparing BC with melanoma cells (MC), the latter showed a higher ability to bind and process PSF both by membrane-associated and most interestingly soluble proteases. A lot of families of enzymes are reported to be overexpressed by melanoma cells including: metalloproteases, cysteine cathepsins, serine proteases and aminopeptidases. All the melanoma cells and cell lines evaluated were able to generate PSF active metabolites. <p>To identify the families of enzymes expressed on the membrane of melanoma cells that might be involved in the mechanism of action of PSF, we performed 2D-gel electrophoresis on their membrane extracts. The 2D-gels experiments revealed the presence of proteins compatible with enzymes known to be important in melanoma and further work is needed to identify the individual enzymes involved by using mass spectrometry and Western blotting. <p><p>Both our in vitro and in vivo findings strongly suggest that not only melanoma tumor cells and tumor sites but other types of tumors as well may be targets for the toxic activity of PSF owing to their much higher load in proteolytic enzymes that are closely related to their invasive potential. The transport of PSF by the blood cells and the release of its metabolites at the tumor site result in a low amount of drug in its free soluble form within the blood and this may explain the relatively lower side-effects observed. PSF is thus expected to have a much better therapeutic index than conventional alkylating agents. This original mechanism of drug delivery may well be extended to other cancer and non-cancer drugs than alkylating agents.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished