Le travail de cette thèse s’est articulé autour de l’étude de CRMP5 au cours du développement du système nerveux central. Nous avons mis en évidence une interaction directe entre CRMP5 et la tubuline, conduisant à une inhibition de la pousse neuritique dans différentes lignées cellulaires, ainsi qu’à une inhibition d’élongation uniquement au niveau des dendrites et non de l’axone, dans des cultures primaires de neurones de l’hippocampe. De plus, nous avons montré que CRMP5 pouvait annuler l’action de CRMP2, connue pour promouvoir la pousse neuritique, de façon dominante mais dépendante de la présence sur CRMP5 du site de fixation à la tubuline. Contrairement à CRMP2, l’expression de CRMP5 étant transitoire pendant la différentiation neuronale, elle permettrait de restreindre de façon spatio-temporelle l’effet de CRMP2 sur la pousse neuritique, régulant ainsi la polarité neuronale. D’autre part, nous avons également rapporté la présence de CRMP5 au niveau mitochondrial où elle pourrait jouer un rôle dans le processus d’autophagie des mitochondries. Enfin, nous nous sommes intéressés à l’étude de la CRMP5 exprimée en conditions pathologiques, et nous avons observé une nouvelle localisation nucléaire de la protéine dans certaines cellules cancéreuses. Etant localisée dans plusieurs compartiments subcellulaires et impliquée dans différents mécanismes moléculaires, l’ensemble de ce travail décrit donc la protéine CRMP5 comme une protéine « multi-fonctionnelle ». / The purpose of this work is to focus on the study of CRMP5 during development of the central nervous system. We have demonstrated a direct interaction between CRMP5 and tubulin, leading to inhibition of neurite outgrowth in different cell lines, and inhibition of growth only at dendritic but not axonal level, in hippocampal neurons. Furthermore, we showed that CRMP5 could counteract the previously described CRMP2 effect on neurite outgrowth. The CRMP5 acted as a dominant signal to counteract CRMP2 outgrowth promotion and this function is also dependent on the tubulin-binding capacity of CRMP5.Unlike CRMP2, the CRMP5 expression being transient during neuronal differentiation, it would imply in the spatiotemporal regulation of the CRMP2 effect on neurite outgrowth, thereby regulating neuronal polarity. In another part, we also reported the presence of CRMP5 at mitochondrial level in vivo where it could play a role in mitochondrial autophagic process.Finally, we were interested in the study of CRMP5 expressed in pathological conditions, and we discovered a new nuclear localization of the protein in some cancer cells. Being localizedin several subcellular compartments and involved in different molecular mechanisms, this work describes CRMP5 as a "multi-functional" protein.

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

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

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The growth of a neuronal dendritic tree depends on the neuron’s internal state and the environment within which it is situated. Different types of neuron develop dendritic trees with specific characteristics, such as the average number of terminal branches and the average length of terminal and intermediate segments. A key aspect of the growth process is the construction of the microtubule cytoskeleton within the dendritic tree. Neurite elongation requires assembly of microtubules from free tubulin at the growth cone. The stability of microtubule bundles is an important factor in determining how likely it is for a growth cone to split to form new daughter branches. Microtubule assembly rates and bundle stability are controlled by microtubule-associated proteins, principally MAP2 in dendrites. Extending previous work (Hely et al, J. Theor. Biol. 210:375-384, 2001) I have developed a mathematical model of neurite outgrowth in which elongation and branching rates are determined by the phosphorylation state of MAP2 at the tips of each terminal branch. Tubulin and MAP2 are produced in the cell body and transported along the neurite by a combination of diffusion and active transport. Microtubule (dis)assembly at neurite tips is a function of tubulin concentration. The rate of assembly depends on the amount of unphosphorylated MAP2 bound to the microtubules and linking them together. Phosphorylation of MAP2 destroys its linking capability and destabilises the microtubule bundles. Each terminal has a probability of branching that depends on the phosphorylation of MAP2 which, in turn, is a function of calcium concentration. Results from this model show that changes in the (de)phosphorylation rates of MAP2 affect the topology of the final dendritic tree. Higher phosphorylation promotes branching and results in trees with many short terminal branches and relatively long intermediate segments. Reducing phosphorylation promotes elongation and inhibits branching.

Orientador: Antonio Carlos Boschero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T03:25:29Z (GMT). No. of bitstreams: 1 Cunha_DanielAndradeda_D.pdf: 2004407 bytes, checksum: 40e252218ab6f8007c9bfb5afde02a52 (MD5) Previous issue date: 2006 / Resumo: Prolactina induz a maturação da resposta secretória das células B pancreáticas em ilhotas de ratos neonatos in vitro. Assim, o objetivo deste trabalho foi avaliar se a maturação na resposta a glicose, induzida pela prolactina, está associada a alterações na expressão, associação e fosforilação de proteínas envolvidas na mobilização e extrusão dos grânulos de insulina. Para isso, ilhotas pancreáticas de ratos neonatos foram cultivadas por cinco dias em presença de prolactina e após extração do RNA e proteína total foram realizados RT-PCR e western blot. Observamos aumento na expressão gênica e protéica da MAP-2 e cinesina em ilhotas cultivadas com prolactina. Analisamos também a associação e fosforilação através de imunoprecipitação seguido de western blot de proteínas SNARE e MAP-2/cinesina em ilhotas estimuladas agudamente (20 min) com prolactina. Prolactina aumentou a associação entre proteínas SNARE e MAP-2/cinesina e reduziu a ligação entre sintaxina IA/munc-I8. Fosforilação em resíduos serina das proteínas SNAP-25, sintaxina IA, munc-I8 e MAP-2 encontravam-se aumentadas enquanto que da cinesina foi diminuída, em ilhotas estimuladas com prolactina. Ainda, foi observado aumento na formação do complexo SNARE em ilhotas agudamente estimuladas com prolactina, 22 mM de glicose, 40 de mM K+, 200 J.lMde carbacol e I J.lMde PMA (ativador da PKC). A inibição da via da MAP cinase, por PD098059, bloqueou a formação do complexo SNARE e fosforilação da sintaxina induzida por prolactina. Desta forma, podemos concluir que prolactina auxilia na maturação das células B por aumentar a expressão, fosforilação e associação de proteínas que compõem a maquinaria de extrusão dos grânulos de insulina, provavelmente via MAP cinase/PKC / Abstract: Prolactin induces maturation of insulin secretion in cultured neonatal rat islets. In this study, we investigated whether the improved secretory response to glucose caused by prolactin involves alteration in the expression, association and phosphorylation of several proteins that participate in these processes. Messenger RNA was extracted from neonatal rat islets cultured for five days in the presence of prolactin and reverse transcribed. Gene expression was analyzed by semi-quantitative RT-PCR and by western blotting for proteins. The gene transcription and protein expression of kinesin and MAP-2 were increased in prolactin-treated islets compared to the controls. The association and phosphorylation of proteins was analyzed by immunoprecipitation followed by western blotting, after acute exposure to prolactin. Prolactin increased the association between SNARE proteins and kinesin/MAP-2 while the association of munc-I8/syntaxin IA was decreased. Serine phosphorylation of SNAP-25, syntaxin IA, munc-18, MAP-2 was significantly higher whereas kinesin phosphorylation was decreased in prolactintreated islets. There was an increase in SNARE complex formation in islets stimulated with prolactin, 22 mM glucose, 40 mM K+, 200 f.lMcarbachol and 1 f.lMPMA (pKC activator). The prolactin-induced increase in the formation of SNARE complex and syntaxin IA phosphorylation was inhibited by PD098059, a blocker of the MAPK pathway. These findings indicate that prolactin primes pancreatic B-cells to release insulin by increasing the expression and phosphorylation/association of proteins implicated in the secretory machinery, probably via the MAPKlPKC pathway / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

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