From the simplest unicellular organisms to complex metazoans, cell polarity is a widespread characteristic that is essential for almost every aspect of biology. Proper polarization of cells is crucial for the establishment and maintenance of higher order structures such as tissue and organs. Cell polarity refers to the asymmetric distribution of various macromolecules and cellular structures, resulting in polarized architecture and function of the cell. Defects in cell polarity lead to various phenotypes, ranging from aberrant signaling, proliferation, cell adhesion and migration, cell fate determination and pluripotency, as well as embryonic lethality, neoplasia and cancer. Given the various roles for cell polarity in development and disease, the characterization of the components involved in polarity and their mechanisms of function is of great importance. My thesis work has encompassed three major projects, each of which is focused on understanding the role of cell polarity in development and disease. Although genetic screens in invertebrates have led to the identification of a number of cell-polarity proteins, similar systematic approach have not been undertaken in mammalian systems. The goal of my first project was to design and implement a high-throughput screen to systematically knockdown individual genes using siRNA, and then assess cell junction integrity as a measure of cell polarity. Given the importance of cell polarity to signaling pathways, I next sought to determine the mechanism by which cell polarity affects TGFβ and Hippo pathways, two important signaling pathways involved in development and disease. Lastly, by studying the acquisition of pluripotency by somatic cells, I uncovered a central role for cell polarity in the establishment and maintenance of pluripotency. Here I will present and discuss our discovery pertaining to the role of cell polarity in cell signaling and pluripotency.

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.

Activation of muscarinic acetylcholine receptors (mAChRs) has pronounced effects on GABAergic interneurons, including depolarization of their resting membrane potential, and increasing their action potential and vesicular release frequency. Moreover, postsynaptic mAChR activation in hippocampal pyramidal neurons reduces the expression of the K+-Cl- cotransporter (KCC2). However, whether mAChR activation modulates the expression of disinhibition-mediated synaptic plasticity has not been examined. I induced inhibitory long-term potentiation (LTP) by applying coincident pre/postsynaptic stimulation in the hippocampus. This plasticity was characterized by an increase in the postsynaptic potential (PSP) amplitude and a depolarization in the inhibitory postsynaptic potential (IPSP) reversal potential; characteristics of disinhibition-mediated LTP (dmLTP). Activation of mAChRs during this plasticity induction protocol prevented the expression of dmLTP via a presynaptic downregulation of transmitter release. This was concluded from evidence that the PSP amplitude and IPSP reversal potential were unaltered, and paired-pulse depression occurred following plasticity induction in the presence of mAChR activation.

From the simplest unicellular organisms to complex metazoans, cell polarity is a widespread characteristic that is essential for almost every aspect of biology. Proper polarization of cells is crucial for the establishment and maintenance of higher order structures such as tissue and organs. Cell polarity refers to the asymmetric distribution of various macromolecules and cellular structures, resulting in polarized architecture and function of the cell. Defects in cell polarity lead to various phenotypes, ranging from aberrant signaling, proliferation, cell adhesion and migration, cell fate determination and pluripotency, as well as embryonic lethality, neoplasia and cancer. Given the various roles for cell polarity in development and disease, the characterization of the components involved in polarity and their mechanisms of function is of great importance. My thesis work has encompassed three major projects, each of which is focused on understanding the role of cell polarity in development and disease. Although genetic screens in invertebrates have led to the identification of a number of cell-polarity proteins, similar systematic approach have not been undertaken in mammalian systems. The goal of my first project was to design and implement a high-throughput screen to systematically knockdown individual genes using siRNA, and then assess cell junction integrity as a measure of cell polarity. Given the importance of cell polarity to signaling pathways, I next sought to determine the mechanism by which cell polarity affects TGFβ and Hippo pathways, two important signaling pathways involved in development and disease. Lastly, by studying the acquisition of pluripotency by somatic cells, I uncovered a central role for cell polarity in the establishment and maintenance of pluripotency. Here I will present and discuss our discovery pertaining to the role of cell polarity in cell signaling and pluripotency.

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder which results from fetal platelet destruction by maternal antibodies against platelet antigens, including GPIIbIIIa (αIIbβ3 integrin) and GPIbα. β3 integrin is also expressed by angiogenic endothelial cells (ECs) and is required for angiogenesis. Therefore, we investigated whether anti-β3 antibodies in FNIT cross-react with blood vessels of the fetus/neonate and contribute to pathogenesis. Antibodies to GPIbα were used as controls. To mimic human FNIT, β3 integrin- or GPIbα-deficient female mice were immunized with wild-type platelets and bred with wild-type male mice. Pups in both groups had thrombocytopenia but intracranial hemorrhage was only observed in anti-β3-mediated FNIT. Anti-β3-mediated FNIT pups had increased apoptosis in the brain and impaired vascularization of the brain and retina. In addition, anti-β3 sera inhibited proliferation and vascular-like tube formation by ECs in vitro. Therefore, anti-β3 antibodies in FNIT likely impair angiogenesis in the developing fetus/neonate.

Mouse embryonic stem cells (ESCs) are known to possess an “open” global chromatin architecture characterized by dispersed chromatin fibres throughout the nucleus. This is in contrast to differentiated cell types, where chromatin generally congregates into numerous compact domains. Core transcription factors in ESCs regulate many genes involved in maintaining pluripotency and previous research has hinted a connection between these factors and chromatin organization. My hypothesis is that Nanog, one of the core transcription factors, functions in maintaining an “open” chromatin organization in mouse ESCs. In this study, the chromatin organization in ESCs expressing varying levels of Nanog was examined at the sub-micron level through electron spectroscopic imaging. An inverse correlation was identified between Nanog expression level and the chromatin fibre density in constitutive heterochromatic regions. Furthermore, global chromatin in the more differentiated epiblast stem cells became less compact upon Nanog overexpression. Altogether, these findings support the idea that Nanog plays a role in maintaining dispersed chromatin in mouse ESCs.

Ubiquitin fold modifier 1 (Ufm1) is a member of the ubiquitin like protein (UBL) family. Like other UBLs, Ufm1 can be conjugated to protein substrates via specific E1 (Uba5), E2 (Ufc1) and E3 (Ufl1) enzymes, and removed from these substrates via the action of Ufm1-specific proteases. While Ufm1 has been implicated in endoplasmic reticulum (ER) function, its biological roles remain poorly understood. By identifying; (a) Ufm1 binding proteins, (b) protein interactors of the Ufm1 conjugation/deconjugation system, (c) Ufm1 conjugates, as well as (d) the intracellular localization of Ufm1 and its main interactors, I aimed to better characterize the biological role(s) of this poorly understood UBL.

Transient Receptor Potential Melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation channel implicated as a key regulator of neuronal cell death in stroke. Our research group has previously shown that TRPM7 dependent cytoskeletal regulation particularly via cofilin mediates neuronal death in oxygen glucose deprivation (in vitro stroke model). LIMK1 phosphorylation was also shown to decrease downstream of TRPM7 activation during anoxia. In the present study we investigated the effects of TRPM7 activation during anoxia, on three regulators of LIMK and cofilin; Rho-associated kinase 2 (ROCK2), P-21 activated kinase 3 (PAK3) and Slingshot family phosphatase 1 (SSH1). Our findings suggest that PAK3 activity is downregulated during OGD through TRPM7 independent mechanisms. However, SSH1 activity appears to be regulated downstream of TRPM7 in a manner that is consistent with LIMK and cofilin regulation. Overall, our work suggests that SSH1 is a new link between anoxia-induced TRPM7activity and cofilin hyperactivation.

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder which results from fetal platelet destruction by maternal antibodies against platelet antigens, including GPIIbIIIa (αIIbβ3 integrin) and GPIbα. β3 integrin is also expressed by angiogenic endothelial cells (ECs) and is required for angiogenesis. Therefore, we investigated whether anti-β3 antibodies in FNIT cross-react with blood vessels of the fetus/neonate and contribute to pathogenesis. Antibodies to GPIbα were used as controls. To mimic human FNIT, β3 integrin- or GPIbα-deficient female mice were immunized with wild-type platelets and bred with wild-type male mice. Pups in both groups had thrombocytopenia but intracranial hemorrhage was only observed in anti-β3-mediated FNIT. Anti-β3-mediated FNIT pups had increased apoptosis in the brain and impaired vascularization of the brain and retina. In addition, anti-β3 sera inhibited proliferation and vascular-like tube formation by ECs in vitro. Therefore, anti-β3 antibodies in FNIT likely impair angiogenesis in the developing fetus/neonate.

Mouse embryonic stem cells (ESCs) are known to possess an “open” global chromatin architecture characterized by dispersed chromatin fibres throughout the nucleus. This is in contrast to differentiated cell types, where chromatin generally congregates into numerous compact domains. Core transcription factors in ESCs regulate many genes involved in maintaining pluripotency and previous research has hinted a connection between these factors and chromatin organization. My hypothesis is that Nanog, one of the core transcription factors, functions in maintaining an “open” chromatin organization in mouse ESCs. In this study, the chromatin organization in ESCs expressing varying levels of Nanog was examined at the sub-micron level through electron spectroscopic imaging. An inverse correlation was identified between Nanog expression level and the chromatin fibre density in constitutive heterochromatic regions. Furthermore, global chromatin in the more differentiated epiblast stem cells became less compact upon Nanog overexpression. Altogether, these findings support the idea that Nanog plays a role in maintaining dispersed chromatin in mouse ESCs.