The non-receptor tyrosine kinase Src has been found to be overexpressed and activated in many human cancers, where it has been implicated in changes in cellular proliferation, adhesion, migration, apoptosis, angiogenesis, and tumour growth. In addition, several other members of the Src family have also been implicated in various cancer phenotypes. Our examination of a wide panel of colon, breast, and lung cancer cell lines revealed that not only Src, but also Yes, Fyn, Lyn, and Lck, were expressed at both the mRNA and protein levels in different combinations, and at varying levels, between cell lines. When examined for kinase activity, it was discovered that only a subset of the expressed Src family members had detectable kinase activity within a given cell line. To investigate the involvement of the Src family members in the proliferation, adhesion, migration, and colony forming ability of four selected cancer cell lines, both Src family kinase inhibitors, which inhibit the kinase activity of multiple Src family members, and RNA interference, which selectively decreases the expression of individual proteins, were used. It was found that the involvement of these proteins in all of the cellular processes investigated was cell line-specific, with the greatest effects observed in HT29 cells, which have relatively high Src protein levels and kinase activity. Furthermore, the consequences of Src family member inhibition were also inhibitor specific, as treatment with PP2 and SKI I generally had greater effects on the cellular processes examined than did treatment with SU6656 or SKI II. It was also found that the inhibition of multiple Src family kinases by at least one of the inhibitors generally resulted in greater effects on the cancer cell phenotypes investigated than were observed when the expression of Src, Fyn, or Yes was decreased using RNA interference. This suggests that multiple Src family members may need to be targeted in order to inhibit the increased proliferation, cell-matrix adhesion, migration, and colony forming ability exhibited by cancer cells. The identification of the cancer cell phenotypes in which particular Src family members are involved is of interest, as these proteins are attractive targets for cancer therapy.

La mitochondrie est une organelle très importante vu son implication dans plusieurs processus cellulaires. Elle produit notamment la majeure partie de l'énergie qui est consommée par la cellule, grâce aux processus d'oxydation phosphorylante (OXPHOS). La phosphorylation des enzymes impliquées dans les OXPHOS apparait comme une voie de régulation importante de la production énergétique. L'objectif de ce thèse était donc de comprendre comment les phosphorylations, et plus particulièrement, les phosphorylations sur tyrosine induites par la Src kinase influencent les OXPHOS. Il a donc été démontré qu'il existe, à l'intérieur des mitochondries, des voies de régulation de ces processus de phosphorylation induits par la Src kinase. Ces processus pouvant induire la phosphorylation de plusieurs enzymes mitochondriales, notamment plusieurs sous-unités des complexes du système des électrons et ainsi, grandement influencer les OXPHOS. Il a aussi été démontré que la Src kinase semble aussi présente dans les mitochondries de cellules cancéreuses, induisant la phosphorylation d'une sous-unité de la NADH-oxidoréductase et une augmentation du métabolisme énergétique mitochondrial. Cette régulation des OXPHOS dans les cellules cancéreuses par la Src kinase pourrait participer à l'établissement du phénotype hautement prolifératif de ces cellules. / Mitochondria are implicated in several key cellular processes. They are producing most part of the energy that is consumed by the cell via oxidative phosphorylation processes (OXPHOS). Phosphorylation of different components implicated in OXPHOS are known to constitute an important regulation pathway of energetic production. The objective of this thesis was to understand how tyrosine phosphorylation induced by the Src kinase could influence OXPHOS. First, it was shown that Src kinase mediated phosphorylation can be regulated directly in mitochondria, inducing phosphorylation of several mitochondrial proteins and different effects on OXPHOS. I also demonstrated that Src kinase is also present in mitochondria of cancer cells where it can lead to phosphorylation of NADH-oxidoreductase. This phosphorylation site is associated with increase of OXPHOS which could be implicated in the establishment of global phenotype of cancer cells.

Les cellules détectent et intègrent une multitude de signaux d'instruction provenant de leur microenvironnement via un ensemble de récepteurs transmembranaires. Ces informations sont ensuite collectées au niveau des nœuds de signalisation intracellulaires pour être ensuite dispersées en cascades de signalisation afin de déterminer la destinée cellulaire. La manière dont un nœud de signalisation peut interpréter plusieurs stimuli et transmettre de manière spatio-temporelle les informations appropriées restent incomprises. Le proto-oncogène c-Src est une tyrosine kinase pléiotrope, un nœud signalisation essentiel au pilotage de nombreux processus cellulaires, tels que la migration, l'invasion, la dégradation et la division cellulaire. Nous avons développé une approche synthétique pour explorer la relation entre la structure de la SRC et la multiplicité des processus cellulaires qu’elle régule. Notre approche a abouti au découplage des différents modules composant la protéine SRC afin de comprendre l’impact de chacun d’eux sur son activité dans l’espace et dans le temps. Notre approche pour contrôler plusieurs états de la conformation SRC était la conception d’un OptoSrc capable à la fois de former des oligomères et d’être recruté à la membrane plasmique. Pour ce faire, nous avons modifié la structure de la SRC afin qu'elle soit potentiellement active dans le noir et nous l'avons fusionnée avec le CRY2 sensible à la lumière. La stimulation lumineuse induit l'hétérodimérisation CRY2 avec un CIBN ancré à la membrane plasmique et son homo-oligomérisation et déclenche une relocalisation de l’OptoSrc à la membrane plasmique ou son oligomérisation. Ce double système a permis de générer deux types de mobilitéz différentes au sein des adhérences focales à deux destins différents, la formation de lamellipodes dans un cas et la formation d’invadosomes dans l’autre. / Cells sense and integrate a multitude of instructional signals from their microenvironment through a diverse set of transmembrane receptors. This information is then collected at intracellular signaling nodes to later disperse down signaling cascades to drive cell fate. How one signaling node can interpret multiple stimuli and spatio-temporally transmit the appropriate information remains poorly understood. The proto-oncogene c-Src is a pleiotropic tyrosine kinase, is one such node essential for driving many cellular processes, such as migration, invasion, degradation, and cell division. We developed a synthetic approach to explore the relationship between SRC structure and the multiplicity of cellular processes it regulates. Our approach resulted in the decoupling of the different modules composing SRC protein to understand how each of them impacts its activity in space and time. Our approach to control multiple state of SRC conformation was the design of an OptoSrc both capable of forming oligomers and to be recruited at the plasma membrane. To do so, we modified SRC structure to be potentially active in the dark and fused it with light sensitive CRY2. Light stimulation induces CRY2 hetero-dimerization with a CIBN anchored at the plasma membrane and its homo-oligomerisation triggering relocalization of OptoSrc at the plasma and/or its oligomerization. This system generated two different type of mobility of OptoSrc inside focal adhesion inducing two different adhesion fates, the formation of invadosome in one case and the formation of lamellipodia on the other.

La polyarthrite rhumatoïde (PR) est une maladie inflammatoire auto-immune qui affecte environ 1% de la population. Il s’agit d’une maladie douloureuse et débilitante, caractérisée par la déformation des articulations touchées, un processus secondaires à la destruction du cartilage et de l’os. À ce jour, il n’existe aucun traitement ciblant le processus de dégradation du cartilage articulaire. Notre laboratoire a observé précédemment que les synoviocytes de type fibroblastique (FLS), provenant de patients atteints de PR (RA-FLS) ou isolés de rats souffrants d’arthrite induite au collagène (CIA) (A-FLS), présentent une augmentation de l’activation de Src se traduisant par une augmentation de la formation d’invadosomes et de la dégradation d’une matrice de gélatine in vitro. De plus, l’inhibition de Src chez des rats atteints de CIA permet de diminuer la dégradation du cartilage articulaire comparativement aux rats contrôles. Chez les cellules cancéreuses, des études indiquent que les protéines adaptatrices Tyrosine Kinase Substrate 4 et 5 (Tks4,Tks5) sont essentielles à la formation de l’invadosome ainsi qu’à sa maturation menant à la dégradation de la matrice extracellulaire. L’objectif de cette étude est de déterminer si l’activation de Src chez les A-FLS et les RA-FLS médie la formation d’invadosomes et la dégradation du cartilage en faisant intervenir les protéines Tks4 et Tks5. Nous avons déterminé que l’expression de Tks4 et Tks5 est augmentée chez les A-FLS et les RA-FLS comparativement aux FLS sains. De plus, l’inhibition de Tks4 ou Tks5 résulte en une diminution significative du pourcentage de cellules formant des invadosomes chez les A-FLS et les RA-FLS, ainsi que de leur capacité à dégrader une matrice de gélatine. Nous avons aussi déterminé que les protéines Tks4 et Tks5 sont phosphorylées par la protéine kinase Src, et qu’il y a formation d’un complexe entre la MT1-MMP et Tks4 et/ou Tks5. Finalement, nous avons démontré que l’inhibition de la Tks4 chez des rats CIA permettait de diminuer significativement le nombre et l’ampleur des zones de dégradations du cartilage par les cellules composant la membrane synoviale ainsi que la dégradation du collagène de type II, une composante majeure du cartilage articulaire. Tks4 et Tks5 sont donc des cibles thérapeutiques prometteuses afin d’empêcher la dégradation du cartilage dans la polyarthrite rhumatoïde et ainsi diminuer sa progression.

In structural concrete, hooked bars are used to shorten anchorage length when the requirements for straight bar anchorage cannot be provided within the available dimensions of elements. The objective of this study was to provide an overview of hooked bar anchorage. Design examples and structural details are based on Building code requirements for structural concrete (ACI 318-08) and commentary. Examples of standard hooks in exterior beam-column joint and hooked bar anchorage details for reinforced concrete beam-SRC column joints are discussed. The general behavior of anchorage of hooked reinforcing bars is summarized from a review of previous studies. Then, design requirements for the development length of standard hook are discussed and used in an example. An example of the use of hooked bars in reinforced concrete beam-SRC column joint is provided. Four options for short development length are presented and compared: Adding more reinforcement, welding bars, confinement by steel column flanges, and anchorage by plate welded between flanges. / text

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Head and neck squamous cell carcinoma (HNSCC) is a type of cancer that begins in the epithelial cells that line the mucosal surfaces of the head and neck, including the oral cavity, pharynx, larynx, paranasal sinuses, nasal cavity, and salivary glands. Head and neck cancer is the sixth most common type of cancer with a 5-year survival rate of 60% for all cases. Over the past few years, a subset of cells with stem-like properties, called cancer stem cells, are believed to have tumor-initiation capabilities and are responsible for maintaining on-going tumor growth. Previous data from our lab suggested that cells grown in suspension, called spheroids, may have stem cell like properties. We employed a model system where a primary HNSCC cell line, HN4, was used to set up spheroids. We found that expression of EPS8 and its downstream targets, FOXM1 and CXCL5, was increased in HN4 spheroids. In addition, we measured the expression of Nanog, as it is a transcription factor involved in the self-renewal of human embryonic stem cells. We also used a metastatic HNSCC cell line, HN12, to see how it compared to spheroids. We wanted to investigate the hypothesis that activation of Src potentiates EPS8 function to deregulate downstream signaling pathways. We used a small molecule tyrosine kinase inhibitor, Dasatinib, on HN4 spheroids and HN12 cells. We found that when Src is inhibited, EPS8 expression is decreased in HN4 spheroids and it also interferes with spheroid formation. The results of the current study were also able to show that the proliferation capability of HN12 cells is greatly diminished when treated with Dasatinib, due to G1 arrest in the cell cycle. When we measured for FOXM1, which is a cell cycle regulator, we found the levels were reduced in Dasatinib treated cells, preventing the cells from completing mitosis. With all of the data taken together, it suggests that Src does in fact play a role in regulating the downstream signaling pathways of EPS8, and its inhibition leads to the loss of cell proliferation. Additional studies need to be performed to discover whether Src inhibition will stop the proliferation of cancer stem cells, which are believed to be more resistant to cytotoxic therapies.

Das maligne Melanom ist ein Hauttumor mit steigender Inzidenz und hohen Mortalitätsraten. Da die molekularbiologischen Ereignisse, die der Melanomentwicklung zugrundeliegen, nur unzureichend bekannt sind, gibt es kaum spezifische Therapieansätze. Zur Untersuchung der Melanomentwicklung eignet sich das Xiphophorus-Modell. In diesem System ist die Anwesenheit der RTK Xmrk ausreichend, um durch Aktivierung proliferativer und entdifferenzierender Signalwege und Apoptoseinhibition Melanome zu verursachen. Im Rahmen der vorliegenden Arbeit konnte gezeigt werden, dass Xmrk auch die Migration der Melanozytenzellinie Melan a-Hm induzieren kann. Die Migration der durch Xmrk transformierten Zellen ist amöboid und unabhängig von MAPK- und PI3K-Signalwegen. Eine Funktion bei der Migration haben jedoch die Kinasen FAK und Fyn. Sie bilden möglicherweise einen Proteinkomplex, der für FAK und Src aus zahlreichen anderen Systemen bekannt ist und als Signalplattform für die Zellmigration fungiert. Diese Erkenntnisse können dazu beitragen, das Xiphophorus-Modell weiterzuentwickeln und die Grundlagen der Melanomgenese besser zu verstehen. / Malignant melanoma is a tumor of the skin with increasing incidence and high mortality rates. As the biomolecular events underlying melanoma development are poorly understood, specific therapeutics for this disease are few. The Xiphophorus system is suitable for studying melanoma development. In this model, presence of the RTK Xmrk is sufficient to initiate melanoma formation by activating proliferative and anti-apoptotic pathways and interfering with differentiation. This work demonstrates that Xmrk is also able to induce migration of the melanocytic cell line Melan a-Hm. Cells transformed by Xmrk migrate in amoeboid fashion, independently of MAPK or PI3K pathways. However, kinases FAK and Fyn are involved in Melan a-Hm migration, possibly as a signal-integrating protein complex similar to the role that has been described for FAK and Src in various systems. The results from this work serve to extend the Xiphophorus model to other aspects of melanoma development and may help to better understand the molecular basis of melanoma.

Thesis (Ph.D.)--University of Toledo, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Title from title page of PDF document. Bibliography: pages 64-70, 104-108, 121-158.

Histone deacetylase inhibitors (HDIs) are a group of chemo-preventive and chemo-therapeutic agents that have generated significant attention in clinical trials, given their ability to selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Presently, these agents are proposed to function by altering gene expression levels, primarily by promoting histone hyperacetylation and gene transcription. However, in this thesis, HDIs are reported to control the expression of genes from the c-Src kinase family and p21WAF1 by means other than transcriptional activation. <p>Overexpression and activation of c-Src, a 60kDa non-receptor tyrosine kinase, has been implicated in the development, growth, progression, and metastasis of several human cancers, especially those of the colon. Butyrate and the more specific histone deacetylase inhibitor trichostatin A (TSA) were both found to effectively inhibit the expression of c-Src mRNA and protein in a number of tumor cell lines, including those of the colon, liver and breast. Expression of the SRC oncogene is alternatively regulated by the SRC1A and SRC1 promoters. HDIs were shown to repress c-Src expression by inhibiting transcription of both of these promoters, independent of any new protein synthesis. Furthermore, butyrate and TSA similarly regulated the expression of the c-Src family kinase (SFK) members Yes, Fyn, Lyn and Lck in human colon cancer cell lines. In addition, TATA binding protein (TBP) associated factor 1 (TAF1) was shown to be necessary for basal transcription of the SRC1A, YES and LYN promoters, but was not required for HDI mediated repression. <p>Induction of the potent cyclin dependent kinase inhibitor p21WAF1 has been identified to be a key feature of HDI mediated cell cycle arrest. The level of p21WAF1 expression has been extensively reported to be directly upregulated by HDIs in a p53 independent manner that requires Sp family binding sites in the p21WAF1 proximal promoter to induce transcription. However, HDIs were shown to be capable of inducing p21WAF1 gene expression, dependent on new protein synthesis, by increasing mRNA stability. To date, p21WAF1 mRNA stability has been extensively studied and a number of cis-acting elements in the 3 untranslated region (UTR) of the p21WAF1 mRNA have been implicated in the regulation of mRNA stability, such as AU rich elements (AREs) and a 42 nucleotide HuD/Elav binding element. Similarly, in this work, two novel cis-acting elements were identified in the 3 UTR of p21WAF1 and were shown to facilitate basal and HDI induced post-transcriptional regulation of p21WAF1 mRNA stability in HepG2 cells. Collectively, these studies highlight the intricacy of HDI mediated effects and challenge the preconceptions regarding the molecular mechanism of these anti-tumor agents.