O carcinoma espinocelular de boca (CEB) é a neoplasia maligna mais comum da cavidade oral, correspondendo à aproximadamente 94% dos casos dessa região. Apesar dos diversos estudos moleculares e celulares do CEB, a taxa de sobrevida dos pacientes é de aproximadamente 50%, devido principalmente ao tamanho do tumor, metástase em linfonodos regionais, grau de diferenciação das células e sítio anatômico. O microambiente tumoral do CEB, é extremamente complexo e diversificado, tendo como característica principal um estado inflamatório crônico imunossupressivo. Este microambiente é sustentado pela liberação de diferentes citocinas inflamatórias, como IL-6, TNF- - atividades exercidas tanto pelas células tumorais quanto pelas estromais. Dentre essas atividades, tem sido relatado na literatura que as citocinas inflamatórias são capazes de aumentar a migração e a capacidade de invasão das células tumorais. Entre as células estromais, os macrófagos são as mais abundantes e participam da manutenção do microambiente tumoral. De acordo com o estímulo, podem ser polarizados M1, com papel pró-inflamatório e antitumoral, e M2, com papel anti-inflamatório e pró-tumoral. O objetivo desta tese foi compreender o papel dos macrófagos no prognóstico de CEB e das citocinas inflamatórias IL-6, TNF- - linhagens celulares de CEB. Para verificar o papel dos macrófagos no prognóstico, foi realizada uma revisão sistemática na qual foram incluídos apenas os estudos que utilizavam amostra de pacientes com CEB e avaliavam o prognóstico com marcadores para macrófagos. Foi observado que maiores concentrações de macrófagos CD68+ e CD163+ estavam relacionados com pior prognóstico de pacientes com CEB, embora não tenha sido possível concluir qual região tumoral a presença destas células seja mais importante 7 para o desfecho. Para analisar o papel das citocinas inflamatórias IL-6, TNFILensaios in vitro utilizando duas linhagens celulares, SCC25 e Cal27, em condições promotoras de migração sob a influência dessas citocinas. Foi observado que a citocina IL-6 foi capaz de aumentar a velocidade de migração e a direcionalidade tanto da SCC25 quanto da Cal 27 e que esta melhora na capacidade migratória ocorreu através de um crosstalk entre a via de sinalização relacionada a IL6 (STAT3) e a via reguladora de migração celular, Rho GTPase Rac1. Estes dados reforçam o papel do microambiente tumoral no processo de progressão tumoral e sugerem potenciais alvos terapêuticos como a modulação do perfil da população de macrófagos e o papel de interleucinas no controle de invasão tecidual e metástase. / Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity, corresponding to approximately 94% of the cases in this region. Despite the diverse molecular and cellular studies of OSCC, the patient survival rate is approximately 50%, mainly due to tumor size, regional lymph node metastasis, cell differentiation and anatomic site. The OSCC tumor microenvironment is extremely complex and diverse, with the main characteristic being an immunosuppressive chronic inflammatory state. This microenvironment is supported by the release of different inflammatory cytokines, such as IL-6, TNF- - and enhance the activities of both tumor and stromal cells. Among these activities, it has been reported in the literature that inflammatory cytokines are capable of increasing migration and invasiveness of tumor cells. Among stromal cells, macrophages are the most abundant and participate in the maintenance of the tumor microenvironment. According to the stimulus, macrophages can be polarized in M1, with pro-inflammatory and anti-tumoral role, and M2, with antiinflammatory and pro-tumoral role. Thus, the aim of this thesis was to evaluate the role of macrophages in the prognosis of OSCC and the influence of inflammatory cytokines IL-6, TNF- - OSCC cell lines. To assess the role of macrophages in the prognosis, a systematic review was conducted in which only studies using a sample of OSCC patients were evaluated and the prognosis was evaluated with macrophage markers. It was observed that higher concentrations of CD68 + and CD163 + macrophages were related to worse prognosis in patients with OSCC, although it was not possible to conclude which tumor region the presence of these cells is more important for the outcome. In order to analyze the role of the inflammatory cytokines IL-6, TNF- - atory 9 behavior of OSCC cells, in vitro assays using two cell lines, SCC25 and Cal27, were performed in migration-promoting conditions under the influence of these cytokines. It was observed that IL-6 was able to increase the speed migration and directionality of both SCC25 and Cal 27 and that this improvement in migratory capacity occurred through a crosstalk between the IL6-related signaling pathway (STAT3) and cell migration-related pathway, RhoGTPase Rac1. These data reinforce the role of the tumor microenvironment in the tumor progression process and suggest potential therapeutic targets such as the modulation of the profile of the macrophages population and the role of interleukins in the control of tissue invasion and metastasis.

No description available.

No description available.

Cerebral palsy (CP) as a neurodevelopmental disorder that affects individuals’ movement, posture, and balance and occurs in every 2-3 out of 1,000 live births. Symptoms of CP can include seizures, hydrocephalus, impairment of the limbs, and learning disabilities. External contributors to CP are well known, but there are 80% of CP cases are idiopathic and in which no brain injury is reported. Recently, several genetic studies have shown that deleterious de novo mutations in CP patients may be implicated in CP pathogenesis. One such potentially deleterious de novo mutation of RhoB was identified in two CP patients. RhoB encodes for RHOB protein, a Rho GTPase that regulates the actin cytoskeleton. Biochemical and structural analyses of RhoB (S73F) protein suggested that the RhoB mutation generates a hyperactive form of RhoB. However, how the RhoB (S73F) protein may interfere with brain development and can contribute to CP is unknown. To determine whether RhoB (S73F) expression affects cortical development, we used in utero electroporations in mice to study the effect of RhoB (S73F) expression on cellular morphology, polarity, migration, and Golgi localization in the embryonic mouse model at E15.5 and P0, comparing it to a RhoB overexpression model as well as control. To address changes in cell morphology, we examined the cell size, shape, and volume of RhoB expressing cells using Imaris software. We show that RhoB overexpression and RhoB (S73F) expression cause detrimental changes in cell shape, polarity, and neuritogenesis. Furthermore, RhoB (S73F) expressing cells migrate less compared to RhoB overexpressing cells and control. Interestingly, we found that RhoB (S73F) expressing cells that did not migrate away from the ventricular surface still became neurons. To determine the effect of RhoB (S73F) expression on the subcellular environment, we examined the localization of the Golgi apparatus, and found the Golgi to be mislocalized and fragmented when RhoB (S73F) was expressed. Overall, this study shows that overexpression of RhoB is sufficient to cause changes in cell morphology, polarity, migration, and subcellular localization of the Golgi. Importantly, expression of RhoB (S73F) is distinct and unique from RhoB overexpression, causing more severe changes in cell size, shape, polarity, cell process number, and Golgi localization that result in failed neuronal migration. This data suggests the potential for genetic mutations to enact changes within the structure and function of cortical cells, which may contribute to the pathogenesis of CP. / Cell Biology

No description available.

No description available.

The Wnts, a family of secreted glycoprotein ligands, act through the frizzled (Fz) receptor, a family of seven-transmembrane (7TM) receptor proteins, to mediate intracellular signaling pathways that regulate cell fate determination, cell migration, or both. Whereas many molecular components of the Wnt signal transduction cascade have been identified, it remains unclear how the signal is transduced from the Fz receptors to the cytoplasm. To address this important question, a membrane-based yeast two-hybrid (MbY2H) screen was performed to identify potential Fz-interacting proteins. For this screen, the Frizzled7 (Fz7) protein was used as the bait and a mouse brain library was used the prey. This screen identified resistance to inhibitors of cholinesterase 8 homolog A (Ric8A), a 542–amino acid cytoplasmic protein, along with other proteins as putative Fz7-binding proteins. Ric8A had been studied previously in C. elegans and D. melanogaster for its function in regulating asymmetric cell division as a receptor-independent guanine nucleotide exchange factor (GEF) for Gα proteins. Additional studies in M. musculus and X. laevis further uncovered a role for this protein during gastrulation and neurulation; however, the mechanisms by which Ric8A regulated these processes remained unclear. In this thesis, I show Ric8A to be a bona fide binding partner for both Fz7; that Ric8A can also bind to the phosphoprotein Dishevelled (Dvl); and that both its interaction with Fz7 and Dvl is Wnt-regulated. The spatial and temporal mRNA expression pattern of the Xenopus homologue of Ric8A suggests a potential role in regulating Wnt signaling. The Xenopus homologue of Ric8A was cloned and gain-of-function and loss-of-function approaches in Xenopus uncovered a role for Ric8A in gastrulation and neural tube closure. Additionally, we found inhibition of Ric8A function mechanistically prevents activation of Rac1 which is required downstream of Wnt/Fz signaling during gastrulation. Overall, this study uncovers a novel regulator of Wnt signaling during early development / Biology

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

No description available.

Rho GTPases and their regulators such as guanosine exchange factors (GEFs) and GTPase activating proteins (GAPs) represent an important class of molecules controlling dendritic spine plasticity. Although they are typically described as cytoskeletal modulators, roles for the GTPases in endocytosis and cell polarity establishment have also been defined. The neuronal proteins a1- and a2-chimaerins belong to a group of Rac and Cdc42 GAPs that inactivate these GTPases; in addition to a GAP domain, the a-chimaerins share a phosphokinase C (PKC)-like C1 domain but have distinct N-terminal domains (NTDs). My project has explored the importance of specific domains of a1-chimaerin both in induction of a morphological cellular protrusion collapse phenotype ('circularisation') and in interactions with partner proteins that may help to explain the phenotype. The results described in my thesis show that a1-chimaerin possesses a previously undescribed C-terminal domain (CTD) that is indispensable for the ability of the protein to induce collapse of protrusions, and consequent circularisation, in various cell types; moreover, an intact CTD is also important for association of a1-chimaerin with its known effector EphA4, and potentially with other undefined membrane proteins, in a C1-domain- dependent manner. In addition, my results show that a1-chimaerin associates via its NTD with the Src kinase Fyn, and via its C1 domain with the NR2A subunit of the NMDA receptor. Further experiments explored a1-chimaerin effects on EphA4 and NMDA receptor cell surface expression, as well as binding to other putative partners - including the adaptor protein p35 and the polarity protein PAR6. Finally, I have shown that inhibition of a pathway involving the Rho-associated coiled-coil containing protein kinase (ROCK) reverts circularisation induced by a1- chimaerin, and that a blocking peptide based on the CTD may be employed to partially counteract the phenotype. These results uncover a novel domain in a1-chimaerin that may have a crucial importance for the induction of cellular process collapse by a1-chimaerin with a potential relevance to the EphA4-induced dendritic spine retraction, EphA4 receptor endocytosis, and cell surface expression of NR2A-containing NMDA receptors. This suggests a model of a multi-protein signalling complex involving a1-chimaerin that coordinates cellular process remodelling, and that is likely to be important both for adult neuronal circuit plasticity and for neurodegenerative diseases.