Clonal culture is crucial for experimental protocols that require growth or selection of pure populations of cells. Currently, there is no method for deriving neural stem cells (NSCs) clonally from single human embryonic stem cells (hESCs). Bulk derivation of neural progenitors from hESCs for cell therapies can lead to a host of problems including incomplete differentiation leading to proliferation of tumorigenic clusters in vivo. Clonal derivation allows for the screening and selection of only the most suitable cells for culture and expansion. We have developed a clonal, serum free method of generating NSCs and their progenitors directly from hESCs with an efficiency of 1.6%. The NSC colony-forming cell was identified as a TRA-1-60-/SSEA4- cell whose fate becomes specified in maintenance conditions by inhibition of bone morphogenic protein (BMP) signalling. This clonal culture method can be scaled up to produce vast quantities of NSCs for differentiation and use in cell therapies.

Septins are an evolutionarily conserved family of proteins that have been implicated in a multitude of cellular processes. Septin 5 is mainly expressed in the nervous system and it has been linked to regulated secretion through its binding to the SNARE protein syntaxin 1. However, the exact mechanism of septin 5 function in localized exocytosis remains unknown. Over-expression of septin 5 is known to lead to lower levels of secretion in HIT-T15 cells. Interestingly, in the current study, the knock-down of septin 5 also results in reduced levels of regulated secretion in PC12 cells, suggesting a more complex role of septin 5 that includes both negative and positive effects on exocytosis. Septin 5 knock-down data point to a possibility of septin 5 facilitating formation of a tether between the vesicles and their site of secretion.

Tracheal epithelium is organized along two axes: apicobasal, seen through apical ciliogenesis, and planar seen through organized ciliary beating, which moves mucus out of the airway. Diseased patients with affected ciliary motility have serious chronic respiratory infections. The standard method to construct epithelium is through air liquid interface culture which creates apicobasal polarization, not planar organization. Nanogrooved surface topography created in diffusible substrates for use in air liquid interface culture will induce planar organization of the cytoskeleton. We have created a nanogrooved gelatin device which allows basal nutrient diffusion. Multiple epithelial cells have been found to align in the direction of the nanogrooves in both sparse and confluent conditions. This device is also congruent with ALI culture as seen through formation of tight junctions and ciliogenesis. Thus, we have created nanogrooved surface topography in a diffusible substrate that induces planar alignment of epithelial cells and cytoskeleton.

Sarcolemmal chloride channels and associated cell volume regulatory pathways have been shown to be important in local ischemic preconditioning (IPC) induced protection against myocardial ischemia/reperfusion injury. Similarities between intracellular pathways in remote (rIPC) and classic IPC suggest that these mechanisms may also play an important role in rIPC. rIPC protected cultured rabbit ventricular cardiomyocytes against necrosis caused by 75 minutes simulated ischemia followed by 60 minutes simulated reperfusion. The protective effect was abolished by chloride channel blockade using 50 μM indanyloxyacetic acid 94 (IAA-94). rIPC also reduced peak cardiomyocyte swelling during exposure to 200 mOsm hypo-osmotic buffer. The reduction in peak swelling was also abolished by IAA-94. These results suggest that the protective effect of rIPC is achieved, at least in part, by enhancing cell volume regulation and that this effect is dependent on the availability of chloride channels in a similar fashion to local IPC.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a foodborne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the interferon gamma (IFNγ) pro-inflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this thesis was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants, stimulated with IFNγ, and then protein extracts were analyzed by immunoblotting. The data showed that IFNγ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using 2D-Difference Gel Electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated, complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFNγ-mediated cell activation. I conclude that E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.

Chromosomal toxin-antitoxin (TA) modules have been proposed to function as regulators of cell growth in response to environmental perturbations. The objective of this study was to characterize the MazEF TA system of the human pathogen Streptococcus mutans. Our data showed that the mazEF genes form a bicistronic operon. MazF toxin had a toxic effect on cells and this effect can be neutralized by coexpression of its cognate antitoxin MazE. Furthermore, we demonstrated that MazE and MazF proteins interact with each other in vivo, confirming the nature of this TA as a type II addiction system. We also demonstrated that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a protein complex. Our results suggest that the MazEF TA might represent a cell growth modulator facilitating the persistence of S. mutans in the oral cavity.

Axon degeneration is a selective elimination of axons, which plays a crucial role during development, injury, and maintenance of neuronal connections. The p75 neurotrophin receptor (NTR) is responsible for maintaining the specificity of neuronal connectivity in parts of the adult brain by inducing the degeneration of aberrantly growing axons into myelinated tracts. The objective of this study is to identify and characterize the signaling pathways used by p75NTR to mediate axon degeneration on myelin. Since p75NTR signals via JNK/Bax/caspase pathway to cause apoptosis, I asked whether this pathway might also be involved in axon degeneration. I observed that inhibition of JNK or Bax significantly decreased myelin-induced axonal degeneration, while depolarization of axons with potassium chloride prevented axonal degeneration on myelin. Together, these results suggest that p75NTR-dependent, myelin-mediated axon degeneration occurs via JNK/BAX signaling, and that neural activity is important for the prevention of myelin-induced axonal degeneration.

Calcific aortic valve disease (CAVD) is associated with increased presence of myofibroblasts, osteoblastic cells and, occasionally, adipocytes and chondrocytes in lesions. The ectopic cell types in diseased valves may be elaborated by an unidentified multipotent progenitor subpopulation within the valve interstitial cells (VICs) that populate the valve interstitium. Notably, lesions form preferentially in the fibrosa layer, the stiffer layer of the valve leaflet. It has been shown that differentiation of VICs to myofibroblasts and osteoblasts is modulated by matrix stiffness. However, the molecular mechanisms involved in mediating stiffness-dependent mechanotransduction remain obscure. The objectives of this thesis were: (1) to determine whether VICs contain a subpopulation of multipotent mesenchymal progenitor cells and to measure the frequencies of the mesenchymal progenitors and osteoprogenitors; (2) to determine the role of β-catenin and matrix stiffness in transforming growth factor-β1 (TGF-β1)-induced myofibroblast differentiation of VICs; and (3) to preliminarily investigate the involvement of four and a half LIM domains protein 2 (FHL2) in CAVD and stiffness-dependent mechanotransduction downstream of RhoA in VICs. Firstly, VICs were found to contain a subpopulation of mesenchymal progenitors that are inducible to osteogenic, myofibroblastic, adipogenic, and chondrogenic lineages. The frequencies of mesenchymal progenitors and osteoprogenitors were significantly higher than other reported sources. Secondly, it was demonstrated that β-catenin is required in TGF-β1-induced, matrix stiffness-regulated myofibroblast differentiation. Notably, TGF-β1 was only able to induce β-catenin nuclear translocation and myofibroblast differentiation on matrices with fibrosa-like stiffness, but not on matrices with ventricularis-like stiffness. Thirdly, FHL2 was found to be upregulated and colocalized with runt-related transcriptional factor 2 (Runx2) in lesions in the fibrosa layer of diseased valves, suggesting its role in osteogenic processes in CAVD. Notably, increasing matrix stiffness increased FHL2 nuclear translocation and RhoA activity in VICs. Preliminary data showed that matrix stiffness regulates FHL2 nuclear translocation via RhoA activity. These results suggest that differentiation of the rich valve progenitor subpopulation, regulated by both mechanical and biochemical cues, may contribute to the preferential occurrence of ectopic cell types in the fibrosa in CAVD. More broadly, these results highlight the critical role of mechanical environment in modulating cellular biochemical signaling.

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.

Medulloblastoma is the most common malignant pediatric brain tumour. Although survival rates have improved in recent years, long-term survivors exhibit a significantly diminished quality of life complicated by neurological, endocrine, intellectual, and social sequelae as a result of conventional therapies. In order to improve the current outlook for patients with medulloblastoma, rational, targeted therapies that are more efficient and less toxic are required. Despite insight gained from the study of hereditary tumour syndromes and candidate gene approaches, the molecular basis of medulloblastoma remains poorly defined, with more than half of all cases remaining unaccounted for at the genetic level. The intent of my PhD research program was to use high-resolution genomics in an attempt to gain an improved understanding of the medulloblastoma genome and potentially uncover novel genes and pathways driving its pathogenesis. By applying a combination of single nucleotide polymorphism (SNP) arrays, exon arrays, and microRNA arrays to a large cohort of primary medulloblastoma samples, we have identified novel oncogenes and tumour suppressors, implicated deregulation of the histone code as an important event in the pathogenesis of medulloblastoma, and refined the definition of medulloblastoma subgroups. This thesis demonstrates the extent of heterogeneity that exists in the medulloblastoma genome, showing that relatively few genomic aberrations are common when studying medulloblastoma as a single disease. In spite of this heterogeneity, we have identified novel candidate genes and processes that may serve as potential targets for future therapies. Importantly, we have established an improved method of classifying medulloblastomas into distinct molecular variants, showing that certain genomic changes are enriched and occasionally restricted to a specific subgroup. Finally, in addition to genomic differences, we have confirmed that medulloblastoma subgroups differ in their demographics and clinical behavior, and propose that medulloblastoma subgroup affiliation should become an integral component of patient stratification in the future.