Atherosclerosis is a chronic inflammatory disease of arterial blood vessels, characterized by deposition of lipoproteins in the arterial wall. Atherosclerotic plaques form preferentially in distinct regions of the vasculature such as branch points, curvatures and bifurcations, suggesting that local hemodynamic forces may contribute to disease susceptibility. Shear stress imparted on endothelial cells (ECs) by the flowing blood has been shown to modulate gene expression and remodelling of the artery. In this thesis, an in vitro model was established to recreate the contrasting environments found in atherosclerosis-prone and atherosclerosis-resistant regions of the vasculature to demonstrate a direct causal-relationship between shear stress and expression of endothelial nitric oxide synthase (eNOS) and p65 in ECs. In vitro assessment of cell shape and expression patterns of these anti- and atherogenic genes demonstrated that shear stress can induce cell morphology and gene expression patterns that are similar to ECs in atherosclerosis-prone and atherosclerosis-resistant regions of the mouse vasculature. Regulation of eNOS transcription by shear stress was demonstrated using a transgenic mouse model and in vitro heterogeneous nuclear RNA (hnRNA) quantification. Similar to ECs in atherosclerosis-prone regions, epithelial cells lining the small intestine lumen express high levels of p65. To investigate the effects of p65 expression levels on constitutive and tumour necrosis factor α (TNFα)-induced nuclear factor-κB (NF-κB) signalling, p65 expression was suppressed in HeLa cells by RNA interference. Lower p65 expression resulted in reduced TNFα-induced expression of NF-κB target genes, including many subunits of inhibitor of nuclear factor κB (IκB), demonstrating modulation of NF-κB priming by p65 expression levels. Suppression of p65 also affected constitutive expression levels of IκB, and resulted in re-setting of the NF-κB/IκB equilibrium. Experiments using inhibitors of canonical NF-κB signalling found that basal expression of NF-κB components is independent of nuclear factor κB kinase β (IKKβ) activity and proteasome-mediated degradation of IκBα. Together, these studies elucidate the mechanism of flow-mediated gene regulation and the effect of resulting changes in p65 expression on NF-κB signalling.

Bladder obstruction (neurogenic or anatomic) induces strain injury in detrusor smooth muscle cells. Signaling via strain injury in other systems has been highly studied, while in bladder obstruction, it has been quite limited to a small number of pathways. In our study we have examined the effects of strain injury using a combination of in vivo, ex vivo and in vitro models, with the aim of understanding disease pathogenesis in the bladder. Using a combination of literature searches, phospho-protein screens and pathway analysis, we uncovered three pathways activated by mechanical strain, ERK, STAT3 and mTOR, with potential for changing not only the way we understand but also the way we treat obstructive myopathies of the bladder. We found that not only were these pathways activated in response to strain and distension injury of BSMC, but they were also responsible for proliferation and sometimes de-differentiation. Included herein are three chapters, published in 2006 and 2010, on the role of ERK, STAT3 and mTOR pathways in bladder smooth muscle cell proliferation and differentiation, 8 Appendices containing the first pages of other papers and reviews published during the course of my studies.

Perturbations of the complex intrinsic and extrinsic factors that contribute to cellular differentiation can have many consequences ranging from dedifferentiation to cell death. The overall objective of my research is to investigate the factors that contribute to the maintenance of mature midbrain neurons and glia. In order to address this objective, I first carried out a detailed immunocytochemical analysis to demonstrate that histone deacetylase inhibitor (HDACI) treatment of differentiated midbrain neurons in culture results in an overall destabilization of neuronal phenotype, which leads to caspase-independent cell death. GFAP positive astrocytes are refractory to the effects of HDACI treatment, suggesting that inhibition of HDACs has differential effects on neurons and glia. HDACI treatment alone was not sufficient to induce neuronal dedifferentiation as evidenced by RT-PCR analysis of stem/progenitor markers, and recovery experiments. Finally, I demonstrate that cortical neurons do not undergo cell death in response to HDACI treatment, suggesting that there may be microenvironmental factors that promote the susceptibility of midbrain neurons to the neurotoxic effects of HDACI. In the second part of this thesis I determined the molecular mechanism that was at least partly responsible for the effects of HDACI treatment on midbrain neurons. Gene expression profiling of HDACI treated midbrain cultures revealed a strong down-regulation of immune related factors. This observation is supported by the loss of microglia in HDACI treated midbrain cultures. I also provide evidence that Toll-like receptor (TLR) signaling, likely through the activation of Interleukin-6 (IL-6) expression, mediates HDAC-dependent neuronal survival. These data provide new evidence that the neuroimmune system is an extrinsic regulator for the homeostasis and survival of neurons.

Aristaless-like homeobox 4 is a homeodomain transcription factor that has important functions during mouse development. A recent report demonstrated that Alx4 expression is required in periductal stromal cells in the mouse mammary gland for normal mammary morphogenesis. To test the hypothesis that ALX4 is expressed in the normal human breast, and this expression is altered in breast cancer, immunohistochemistry was performed on normal and breast cancer tissue and breast tissue microarrays. In the normal breast, ALX4 was expressed in stromal fibroblasts and luminal epithelial cells, but not in myoepithelial cells. Expression was lost in breast cancer in both cell compartments. Upon global demethylation induced by 5-aza-2’-deoxycytidine, normal and breast cancer cell lines expressed ALX4, suggesting that hypermethylation may repress expression of ALX4 during malignant transformation of the breast. These results demonstrate that ALX4 may be used as a biomarker for breast cancer, and may act as a tumour suppressor.

Previous studies reported that matrix protein supplementation (fibronectin/fibrinogen, FN/FG) of agarose gel microcapsules enhances survival and pulmonary retention of syngeneic rat multipotent stromal cells (MSCs). I hypothesized that additional supplementation of microcapsules with osteopontin (OPN) and transglutaminase 2 (TG2) would enhance cell survival, while stabilizing the provisional matrix. Using monomeric OPN or OPN polymerized with TG2, I examined human MSC adhesion, morphology, focal contact formation and apoptosis. Polymeric OPN induced greater adhesion than monomeric OPN (84.5±10.7 vs. 44.3±10.0cells/field), and also significantly enhanced focal contact formation (351.5±21.2 vs. 45.6±17.6 focal contact sites/cell) and cell spreading (2.7x103±0.20x103μm2 vs. 1.2x103±0.26x102μm2) while preserving MSC pluripotency. Microcapsules supplemented with FN/FG, polymeric OPN and TG2 demonstrated significantly less apoptotic cells than FN/FG microcapsules (14.0±2.34% vs. 28.2±3.22%). Reduced apoptosis was attributed to matrix stabilization by TG2 and the synergistic activity of matrix proteins. It is anticipated that this enhanced survival will maximize the therapeutic potential of MSCs.

Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing through highly regulated pathways that involve the phagocytosis of bacteria and the generation of reactive oxygen species by the NADPH oxidase complex. The Rac small GTPases have prominent roles in the regulation of neutrophil signaling pathways but the research strategies used to analyze their functions in live cells have been limited, since neutrophils are terminally differentiated and difficult to manipulate genetically. In this thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely new way, in real time. Using this technique, I show that a bacterial protein known to inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis iii based on their different aminoacid residues in the polybasic domain. This mechanism depends on lipid metabolism and the accumulation of negatively charged lipids at cellular membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the lipid metabolism that occurs at the phagosome membrane decreases its negativity and selectively recruits Rac2. All together, this thesis describes the study of primary neutrophil functions from a new angle and adds some valuable information to the comprehension of effective neutrophil activation based on the analysis of Rac isoforms.

Les vertébrés, du poisson à l'homme, possèdent un potentiel membranaire médié en partie par les ions chlorure (Cl-). L’une des premières formes d’activité neuronale lors du développement est la dépolarisation médiée par les ions chlorures extrudés par les canaux glycinergiques (GlyR) et GABAergiques. Cette dépolarisation est rendu possible grâce à l’expression retardée du co-transporteur d’ions chlorure et de potassium KCC2 lors du développement qui génère un gradient hyperpolarisant postnatalement chez les mammifères. Le rôle de cette dépolarisation précoce paradoxale durant le développement est inconnu. En injectant l’ARNm de KCC2 dans des embryons de poissons zébrés nouvellement fertilisé, nous avons devancé l’expression de ce co-transporteur rendant ainsi la glycine hyperpolarisante dans tous les neurones dès les premières phases du développement. Nous avons aussi ciblé le récepteur glycinergique directement en bloquant son activité et son expression à l’aide d’une drogue spécifique, la strychnine et d’un morpholino antisens (Knockdown). Dans les trois cas (KCC2, strychnine et GlyR KD), les perturbations de l’activité neuronale ont provoqués des erreurs dans la neurogenèse, en particulier une diminution du nombre d’interneurones sans avoir d’effets sur les motoneurones et les neurones sensoriels. De plus, en bloquant les canaux calciques activés à bas voltage dans le développement avec la drogue nifedipine, il y a des erreurs dans la neurogénèse semblables à celles remarquées dans les trois conditions précédentes. Nous concluons que la dépolarisation précoce par la glycine permet l’entrée du calcium et l’activation de la neurogénèse chez les interneurones. / Vertebrates, from fish to man, have a membrane potential mediated in part by chloride ions (Cl-). One of the first neuronal activity during development of the zebrafish spinal cord is cell depolarisation mediated by chloride extrusion via glycinergic receptors (GlyRs) and GABAergic receptors. This depolarisation is due to the absence of chloride-potassium cotransport channel KCC2, whose expression comes later in development, creating a hyperpolarising gradient. The role of this paradoxal depolarisation period during early stages of development is still unknown. By injecting KCC2 mRNA in newly fertilised zebrafish embryos, we expressed this co-transporter channel in neurons causing glycine to hyperpolarize in early phases of development. We also directly targeted the glycine receptor (GlyR) itself by blocking its activation with a chronic treatment of Strychnine, a specific drug, and by knocking down the expression of this receptor with an antisense morpholino injection. In those three conditions (KCC2, Strychnine and GlyR KD), perturbation of neuronal activity provoked major defects in neurogenesis, particulary in development of interneurons, without affecting other types of cells like motoneurons and sensory neurons. In addition, blocking low-voltage activated calcium channels with nifedipine provoked similar phenotypes. We conclude that the early glycine-mediated depolarisation allow calcium entry, thus activating certain aspects of interneurons neurogenesis.

Contexte: Le centrosome est un petit organite bien connu pour son rôle dans l'établissement du fuseau bipolaire pendant la division cellulaire. Les déficiences de la fonction du centrosome donnent souvent lieu à des maladies humaines, y compris le cancer et la formation de kystes rénaux. Nous sommes intéressés à étudier la fonction d'une nouvelle protéine centrosomale nommée CEP78, identifiée dans un criblage protéomique pour de nouveaux composants centrosomaux. Méthodes et résultats : Le traitement des cellules avec le nocodazole, un agent qui dépolymérise spécifiquement les microtubules cytoplasmiques mais pas les microtubules stabilisés du centrosome, a montré que CEP78 est un composant centrosomal stable. La colocalisation de cette protéine avec d'autres marqueurs centrosomaux tels que CEP164, SAS6, Centrine, tubuline polyglutamylée et POC5, à différentes phases du cycle cellulaire a indiqué que CEP78 est précisément à l'extrémité distale des centrioles, mères et filles. Il existe deux pointts CEP78 au cours de l’interphase et les cellules passent par la mitose, procentrioles maturent, et le nombre de points de CEP78 augmente à 4 par cellule et, à la fin de la télophase chaque cellule fille possède 2 points CEP78. La caractérisation des domaines fonctionnels de CEP78 a montré que des répétitions riches en leucine sont nécessaires pour la localisation centrosomale de la protéine. En outre, nous avons constaté que la surexpression de CEP78 ne change pas le nombre de mères/procentrioles mais diminue le nombre et l'intensité des points de CEP170 (protéine d'appendice sous-distal) sans diminution du niveau d'expression de cette protéine. D'autres études ont montré qu'il n'y a pas d'interaction entre ces deux protéines. Enfin, la surexpression de CEP78 protège des microtubules contre la dépolymérisation en présence de nocodazole, ce qui suggère qu'il possède la capacité de lier les microtubules. Conclusion : Nos résultats suggèrent que CEP78 est destiné à l'extrémité distale des centrioles matures par ses répétitions riche en lecuine, où il pourrait être impliqué dans la maturation ou la régulation de l'assemblage ou de la rénovation de l'appendice sous-distal centriolaire, une structure connue dans la nucléation des microtubules et d'ancrage. Comprendre la fonction de Cep78 contribuera à éclaircir le rôle du centrosome dans le cycle cellulaire. / Background: The centrosome is a tiny organelle well-known for its role in establishing the bipolar spindle during cell division. Defects in centrosome function often give rise to human diseases including cancer and kidney cyst formation. We are interested in studying the function of one novel centrosomal protein named CEP78, identified in a proteomic screen for novel centrosomal components. Methods and results: Treatment of cells with nocodazole, a microtubule-depolymerizing agent that specifically depolymerizes cytoplasmic microtubules but not the stabilized centrosome microtubules, showed that CEP78 is a stable centrosomal component. Colocalization of this protein with other centrosomal markers such as CEP164, SAS6, Centrin, Polyglutamylated tubulin and POC5 at different phases of the cell cycle indicated that CEP78 specifically localizes to the distal end of the mother and daughter centrioles. There are 2 CEP78 dots during the interphase and as the cells go through mitosis, procentrioles mature, and the number of CEP78 dots increases to 4 dots per cell and by the end of telophase each daughter cell has 2 CEP78 dots. Characterization of CEP78 functional domains showed that Leucine-rich repeats are necessary for centrosomal localization of the protein. In addition, we found that overexpression of CEP78 did not change the number of centrioles and centrosomes but decreased the number and intensity of CEP170 dots (sub-distal appendage protein) without a decrease in the expression level of this protein. Further studies showed that there is no interaction between these 2 proteins. Finally, overexpression of CEP78 protects microtubules from depolymerization in the presence of nocodazole, suggesting its ability to bind microtubules. Conclusion: Our findings suggest that CEP78 is targeted to the distal end of mature centrioles via its lecuine-rich repeats, where it could be involved in centriolar maturation or regulation of sub-distal appendage assembly and/or remodeling, a structure known to nucleate and anchor microtubules. Understanding the function of CEP78 will shed light on the role of the centrosome in cell cycle.

Melanin is the major component of skin-, hair-, and eye pigmentation in mammals. Synthesis of melanin takes place in specialized organelles in melanocytes, called melanosomes. As melanosomes mature during pigment synthesis, they are transported towards the tips of dendrites in the melanocyte, and eventually transferred to neighbouring keratinocytes to distribute pigment throughout the skin. A large number of proteins regulate melanin synthesis and distribution. Over one hundred genes have been associated with coat colour mutations in mice, and many of these genes have also been identified in human pigmentation disorders. Other proteins involved in pigmentation are part of pathways that are not unique to pigmentation alone, such as the Ras/ERK pathway. In mouse B16 cells, cAMP stimulation leads to the upregulation of melanin synthesis and dendrite extension. However, cAMP also activates the Ras/ERK pathway in these cells, which, upon prolonged stimulation, leads to an inhibition of melanin synthesis and dendrite extension. Here I show that the protein CNrasGEF, which was previously identified in our lab, is responsible for cAMP-dependent Ras activation in B16 cells, and therefore a part of the negative regulatory pathway of melanogenesis. In order to find other proteins involved in pigmentation pathways, I have developed a method to detect melanosomes using Cellomics KineticScan (KSR) high-content image analysis. This system could potentially be used in a high-throughput RNA interference screen to identify proteins that affect melanosome formation or transport. However, in a pilot study it appeared that knockdown levels achieved upon transient transfection of knockdown constructs from a mouse shRNAmir library against selected targets were in many cases not sufficient to detect an effect on melanocytes, either by confocal microscopy, or by Cellomics KSR analysis. Further reduction of expression levels is necessary before this system can be scaled up to high-content/high-throughput identification of proteins involved in pigmentation.

Vascular endothelial cell loss initiates directional migration of medial smooth muscle cells into the arterial intima contributing to in-stent restenosis, atherosclerosis and coronary arterial by-pass graft failure. N-cadherin is a cell-cell adhesion molecule that mediates the interaction between vascular endothelial cells and the innermost smooth muscle cells to stabilize the arterial wall. Upon injury, I reasoned that relocalization of N-cadherin on the inner most smooth muscle cells to the posterior-lateral borders stimulates cell polarization to enable directional migration. Using an in vitro scratch-wound model to stimulate cell polarity and locally remove cell-cell contacts at one pole of smooth muscle cells, I found that N-cadherin localization provides signaling cues via a Cdc42/GSK pathway that promote polarized reorganization of the cytoskeleton and directional cell migration. I also found that N-cadherin was important to functions of lamellipodia at the anterior of migrating cells. In lamellipodia, actin polymerization drives protrusion of the leading edge and coincident, but more posterior, actin depolymerization results in retrograde flow of actin and associated plasma membrane structures. Using live cell imaging, I found that clusters of N-cadherin-GFP repeatedly accumulated at the leading edge specifically at the neck of large pinocytotic vesicles called macropinosomes that were internalized and transported away from the leading edge. This localization is consistent with a role for N-cadherin in closure and scission of vesicles during macropinocytosis. These are the first studies to examine polarity in migrating vascular smooth muscle cells, and advance our understanding concerning cell-cell adhesions in controlling directional cell migration. My results suggest that N-cadherin may serve as a viable target for the treatment of arterial stenosis that would limit smooth muscle cell migration and stabilize the arterial wall. Furthermore, I report on a novel localization and function of N-cadherin in the biogenesis of macropinosomes in the lamellipodia that contribute to cell protrusion.