La métastase des cellules cancéreuses est un processus caractérisé par un comportement de la cellule anormal. Les propriétés mécaniques particulières des cellules cancéreuses est l'une des caractéristiques pathologiques principales. Ces propriétés sont liées à leurs capacités à envahir les tissus avoisinants, à transmigrer et à proliférer vers de nouveaux sites. La progression du cancer est caractérisée par la perturbation et la réorganisation du cytosquelette d'actine ainsi que par des changements des propriétés mécaniques des cellules, probablement liés à la capacité dupliquée des cellules cancéreuses à migrer et à s'adapter à l'environnement.Les propriétés mécaniques sont essentielles pour la régulation des fonctions cellulaires comme la migration, l'adhésion, la prolifération, et la différentiation, et les anomalies sont associées aux pathologies, en particulier le cancer. Les propriétés mécaniques sont aussi dépendantes du micro-environnement de la cellule, et la rigidité des substrats modifie les propriétés mécaniques internes des cellules, ainsi que la structure du cytosquelette. Ainsi, comprendre les processus impliqués dans les variations des propriétés viscoélastiques est essentiel pour l'étude de la progression des tumeurs.La microscopie à force atomique (AFM) a prouvé être un outil fiable pour sonder les propriétés mécaniques statiques et dynamiques (sur de grande gamme de fréquence) de matériaux mous, comme les spécimens biologiques, à de petites échelles et grande résolution.Dans cette étude, nous proposons de nouveaux marqueurs du cancer basé sur une approche mécanobiologique. Les propriétés viscoélastiques de cellules cancéreuses de la vessie sont mesurées par des expériences d'indentation dynamiques par AFM. Cette méthode est validée en utilisant des gels de polyacrylamide et un modèle à fractions dérivées est proposé pour décrire le comportement mécanique de ces gels. Ensuite, le module de cisaillement complexe de trois lignées cellulaires à potentiel métastatique différent est mesuré à trois positions différentes de la cellule: le noyau, le périnoyau et la périphérie de la cellule. En utilisant des drogues d'inhibition de l'actine, les propriétés mécaniques sont corrélées à la microstructure de l'actine obtenue par microscopie confocale. Nous proposons un modèle simplifié pour décrire le comportement des modules élastiques G' et visqueux G''. Une relation entre l'invasivité des cellules cancéreuses et leur propriétés mécaniques est aussi mis en avant. En particulier, nous trouvons que le plateau élastique et la fréquence de transition (quand G'=G'') peuvent être utilisés comme marqueurs d'invasivité. Enfin, nous mesurons le module de cisaillement complexe de cellules cancéreuses adhérentes sur des environnements mécaniques et biologiques différents, et des propriétés intrigantes de la périphérie des cellules cancéreuses sont reportées. / Cancer cell metastasis is a multi-stage process characterized by cell malfunctional behavior. Some of the major pathological characteristics of cancer cells are their particular mechanical properties which are linked to their ability to invade surrounding tissues, transmigrate and proliferate at new sites. There are evidences that cancer progression is characterized by disruption and reorganization of the actin cytoskeleton as well as changes in the mechanical properties. This change is probably associated with the enhanced capability of cancer cells to migrate and adapt to changing environments.The mechanical properties are essential for the regulation of cell functions like migration, adhesion, proliferation and differentiation, and abnormalities are connected with pathologies, in particular cancer metastasis. The mechanical properties are also dependent on the micro-environment of the cell, as substrate stiffness changes cell internal mechanical properties, as well as the cytoskeleton structure. Thus, the understanding of the mechanics involved in the variation of the viscoelastic properties is crucial for the study of tumor progression.Atomic force microscopy (AFM) has proved to be a reliable tool to probe static and frequency–dependent mechanical properties of soft materials, like biological specimens, at small scale with high resolution.In this study, we propose new markers of cancer metastasis based on a cell mechanics approach. We report on the viscoelastic properties of human bladder cancer cells measured by dynamic indentation experiments using AFM. This method is first calibrated using polyacrylamide gels and a fractional model is proposed to describe the behavior of such gels. We then investigate the complex shear modulus of three different cell lines with different metastatic potential. We probe the elastic G' and viscous G'' modulus at three different locations across the cell: nucleus, perinucleus and the cell periphery. With the use of actin inhibitory drugs, we correlate mechanical properties and the actin microstructure obtained by confocal microscopy imaging. We propose a simplified power-law model to describe the behavior of the elastic and viscous moduli. We also report a relationship between the malignancy of cancer cells and their viscoelastic properties. In particular, we find that the elastic plateau modulus and the transition frequency (frequency at which G' = G'' ) can be used as markers of invasiveness. Then, we probe the complex shear modulus of cancer cells on different mechanical and biological environments and we report intriguing properties of the periphery of cancer cells.

Le gène CDKN1A a été initialement décrit comme une cible transcriptionnelle de la protéineoncosuppressive p53. Son produit, p21Cip1 (p21), supprime l’activité des kinases dépendantes descyclines et de la protéine PCNA, ce qui en fait un puissant inhibiteur du cycle et de la proliférationcellulaires. En outre, p21 est fréquemment inactivée dans les cancers épithéliaux. Or, la progressionde ces tumeurs est associée à l’altération de l’organisation tissulaire, au processus invasif et à ladissémination métastatique. Ces phénomènes résultent des modifications cytosquelettiquesconduisant à la transformation des propriétés d’adhésion et de migration cellulaires. Pourtant, le rôlede p21 dans la dynamique cytosquelettique des cellules épithéliales humaines n’a jamais été adressé.Nous montrons ici que p21 contribue à l’adhésion et la migration normale des cellules épithélialesmammaires non transformées. Nos résultats montrent également que l’inactivation de p21 provoquela suppression de l’adhésion focale et des fibres de stress. Ce phénotype est caractérisé parl’inactivation de la GTPase Rho et l’activation de la cofiline, facteur de dépolymérisation de l’actine. Enoutre, la suppression de p21 provoque une désacétylation des microtubules associée à unedéstabilisation microtubulaire globale. La réduction de l’instabilité dynamique, par inhibition de ladésacétylase HDAC6, restaure partiellement l’étalement cellulaire et l’adhésion focale altérés parl’inactivation de p21. L’ensemble de nos données démontre que la régulation de la dynamiquecytosquelettique par p21 est nécessaire au contrôle de l’adhésion des cellules épithéliales humainesnon tumorales. / CDKN1A gene was initially identified as a target of the tumor suppressor p53. The ability to inhibitcyclin-dependant kinase and PCNA activities confers to its product, p21Cip1 (p21), strong antiproliferativeproperties. Moreover, p21 is frequently inactivated in epithelial cancers. Progression ofthese malignancies is also associated with disorganized tissue architecture, invasion and metastaticdissemination. These processes involve major cytoskeletal reorganization associated withtransformation of adhesion and migration properties. Still, the role of p21 in cytoskeletal dynamics innormal epithelial cell has never been addressed. Here we show that p21 contributes to normaladhesion and migration in untransformed human mammary epithelial cells (HMEC). We alsodemonstrate that p21 inactivation in HMEC suppresses focal adhesion and stress fiber assembly. p21depletion is also associated with inactivation of Rho GTPase and activation of the F-actin severingfactor cofilin. In addition, p21 silencing provokes microtubule hypoacetylation associated withincreased dynamic instability. We find that forced microtubule stabilization by HDAC6 inhibitionpartially restore cell spreading and focal adhesion in p21-depleted cells. Collectively, our data showthat regulation of cytoskeletal dynamics by p21 is required for adhesion control in untransformedhuman epithelial cells.

Pendant la morphogenèse, la réparation des tissus et le cancer, les cellules peuvent migrer en manière mésenchymateuse et collective. Le cytosquelette est essentiel pour la migration, mais alors que l'actine et les microtubules ont été largement étudiés, le rôle des filaments intermédiaires (FIs) est encore largement inconnu. La déplétion des FI diminue souvant la vitesse de migration et les FI sont fréquemment surexprimé dans les tumeurs invasives. Pour ces propriétés, nous supposons que les FIs peuvent jouer un rôle clé dans la mécanique cellulaire pendant la migration.Pour étudier le rôle des FI dans la migration collective, nous avons utilisé des astrocytes, les principales cellules gliales du système nerveux central. Les astrocytes migrent collectivement pendant le développement et l'astrogliose en réponse à des signaux pathologiques ou traumatiques. Les astrocytes expriment trois principales FI cytoplasmiques: nestine, GFAP (protéine acide fibrillaire fibreuse) et vimentine, qui forment un réseau dense. Les FI sont surexprimé pendant l'astrogliose et dans les glioblastomes, des tumeurs cérébrales hautement invasives et létales. On ignore si la surexpression des FI est responsable de l'invasion du glioblastome.Au cours de la migration collective dans un test de blessure, les FI contrôlent le positionnement du noyau, la polarité et la migration. On montre que les FI régulent la migration collective dirigée de manière dépendante de la rigidité. Ils agissent avec la protéine connecteur cytoplasmique plectine pour contrôler les point focaux et les jonctions adhérentes. Les FI contrôlent la dynamique et l'organisation de l'actine et régulent la distribution des tractions cellulaires et des contraintes dans la monocouche migrante. Ces résultats démontrent le rôle crucial des FI dans les propriétés mécaniques des cellules migrantes. / During morphogenesis, tissue repair and cancer, cells can migrate in a mesenchymal and collective manner. The cytoskeleton is essential for migration, but whereas actin and microtubules have been extensively studied, the role of intermediate filaments (IFs) is still largely unknown. IF depletion generally decreases migration speed and IF proteins are frequently found upregulated in invasive tumours. Because of IF properties, we hypothesise that they may be key players in cell mechanics during migration. To study the role of IFs in collective migration we used astrocytes, the main glial cells of the central nervous system. Astrocytes migrate collectively during development and astrogliosis in response to pathological or traumatic signals. Astrocytes express three main cytoplasmic IFs: nestin, GFAP (Glial Fibrillary Acidic Protein) and vimentin, which form a dense network. IF proteins are upregulated during astrogliosis and glioblastomas, highly invasive and lethal brain tumours. Whether upregulation of IFs is responsible for glioblastoma invasion is still unknown. During wound-induced collective migration, IFs control nuclear positioning, polarisation and migration. We found that IFs regulate collective directed migration in a stiffness-dependent manner. They act in concert with the cytolinker protein plectin to control focal adhesions and adherens junctions. IFs control actin dynamics and organisation and regulate the distribution of cell tractions and stresses across the migrating cell monolayer. These results demonstrate the crucial role of IFs in the mechanical properties of migrating cells.

Les cellules exerçert des forces sur la matrice extra cellulaire sur laquelle elles adhérent impliquant une boulle de régulation biomécanique. Cependant les sous-jacents de par lesquelles les cellules sentent et transmettent les forces restent encore m^echaniemes à elucider. Dans la premiére partie nous allons comment le modulations géométrique les forces de tractions et la distributions des tensions dans le cytosquelette d'actine ainsi que localisation des adhésions focales sur une cellule unique en combinant l'utilisation de la technique de micropatterning et de microscopie à traction de force. Nous avons mesuré la force de traction cellulaire sur différentes formes géométriques mocropatterns (comme un U, flieche ou H) recouvert de protéines sur des gels mous de polyacrilamide 2D dans lequl sont intégrés des nanobilles fluorescenes. Nous avons montré que la géométrie influencait la distribution des forces de tractions localement tandis que l'aire projetée des differentes formes restait conservée (pour une celule). Puis nous avons comparé la force de traction cellulaire développée quand les cellules sont sur des motif contirees circulaire 2D avec des motifs discrets en forme de micropiliers 3D de la me^me rigidité . Nous avons aussi étudie comment les forces varies en fonction de la rigidité de la matrice extracellulaire dans les deux cas précédents une mesure quantitative a été faite sur la localisation spatiale des protéines d'adhésion sur les formes circulaires et aussi sur l'organisation de l'actine. Afin d'ovoir une compréhension systématique de la distribution des forces nous nous sommes concentré sur la localisation et l'orientation des forces sur différentes géométrics de motifs (V, T, Tripod et plus). Nous avons corrélé la distribution avec celle des dibers de stesses et la localisation des adhésions focales. Ensuite nous nous sommes intéressés la distribution des centrosome en corrélation avec la forces et l'organisation d'autres eléments internes. Nous avons égolement essayé de prédire la force de traction en utilisant un modele théorique. Pour finir, nous avons developpé une novelle méthode de micropatterning fabriqué à partir de brosses de polymeres de PNIPAM qui sont thermosensibles. La fonctionalisation de surface et l'adhésion des cellules sur la surface sont aussi décrites. Nous discutons également de la dépendance en température des propriétés du PNIPAm et de l' utilisation desbrosses de polymeres comme actuateur pour induire les détachments des cellules. Nous avons aussi regardé la distribution des fibres de stress quand la cellule est cultivée sur différent types de motif thermosensible. / It has grown a great interest among biophysicists that adherent cell senses substrate stiffness and geometry by the process of mechanotransduction. Cells exert force on the extra cellular matrix on which it is subjected to adhere by active mechanism, which involves biomechanical regulatory feedback loop. It is still unclear whether biomechanical,biochemical or geometrical stimuli dominates invivo. Underlying mechanism behind the way cell senses, redistributes and transmits force still needs to elucidate. In the first part we show how geometrical modulation influences traction force and tension distribution in the actin cytoskeleton, and also localization of focal adhesion at single cell level by combining use of Micropatterning and Traction force microscopy technique. We measure cell traction force seeded on different micropattened shapes(like U,arrow and H) coated with protein on 2D soft polyacrilamide gel embedded with nano beads. We show that geometrical cue redistributes traction force locally while projected area designed for a single cell is conserved. we compare cell traction force developed when cells are on a continuous 2D circular array pattern with discrete 3D micropillar array of same stiffness. We also have investigated how forces are varied with rigidity modulation of the extra cellular matrix in both these two cases. A quantitative measurement has been done on the spatially localized adhesion proteins on the circular dots and also actin re-organization. In the sencod part to achieve more systematic understanding of force distribution we have consider more on force localization and orientation on different patterned geometry( V, T, Tripod, Plus). We correlate force distribution with stress fiber and focal adhesion localization. Finally we look into centrosome distribution in correlation with force and other internal organization. An alternate approach has been made towards the development of thermoresponsive micropattern, made of poly(N-isopropyla crylamide) brushes, grafted at high surface density. Surface functionalization and cell attachment on the surface are bescribed. We discuss temperature-dependent swelling properties of PNIPAM and the polymerbrush as a microactuator which induces cell detachment. We also have looked into stress fiber distribution when cell is cultured on different thermoresponsive pattern geometries.

Introdução: A Síndrome de Kindler é uma doença autossômica recessiva que se caracteriza por surgimento de bolhas acrais na infância que diminuem com o passar dos anos, fotossensibilidade, poiquilodermia persistente e progressiva e atrofia cutânea. Desde o primeiro relato, mais de 100 casos já foram descritos, mas a associação com Poiquilodermia Acroceratótica e Epidermólise Bolhosa tem freqüentemente dificultado o diagnóstico. Exames moleculares nunca foram realizados em paciente brasileiros com Síndrome de Kindler. Objetivos: O objetivo deste estudo é a identificação de alterações genéticas e estruturais nos pacientes com Síndrome de Kindler do sul do Brasil. Pacientes e métodos: Este estudo é uma série de casos de sete pacientes acompanhados no Hospital de Clínicas de Porto Alegre durante o período de janeiro de 2004 a dezembro de 2005. Os pacientes com SK foram examinados clinicamente e o registro foi realizado em um questionário estruturado. As amostras de pele e sangue foram coletadas após consentimento informado com a aprovação pelo comitê de ética. Resultados: Os achados clínicos mais consistentes para o diagnóstico foram bolhas na infância, fotossensibilidade e poiquilodermia. A ocorrência de carcinomas de pele não melanomas em dois pacientes jovens sugere que a SK pode ser um fator de risco no desenvolvimento de malignidades cutâneas. Uma mutação no exon 5 foi identificada nas duas famílias distintas que após o heredograma mostraram ter parentes em comum que vieram da mesma região da Itália há três gerações. Conclusões: Estes achados são muito importantes para a descrição de uma síndrome tipicamente caracterizada e para incrementar a estratégia de detecção. A elucidação das mutações existentes é igualmente importante para o desenvolvimento de novos tratamentos e manejos dos pacientes afetados. / Background: Kindler syndrome (KS) is a rare autossomal recessive disease characterized by acral bullae in infancy that diminished with time, photosensitivity, persistent progressive poikiloderma and severe cutaneous atrophy. Since the first report more than 100 cases have already been described but the association with acrokeratotic poikiloderma and epidermolysis bullosa has often misled the diagnosis. The molecular study of Brazilian patients with Kindler syndrome was never performed. Objectives: To asses the clinical features and the molecular basis of KS in patients from South Brazil and to examine molecular evidence for a mutation. Patients and methods: This study is a series of seven patients atending to Hospital de Clinicas de Porto Alegre during the period of January 2004 to December 2005. The KS affected individuals were examined clinically and a structured recording sheet was completed. Blood and skin samples were collected after informed consent and with the Ethics committee approval. Results: The most consistent clinical signs for a diagnosis were blisters in childhood, photosensitivity and poikiloderma. The occurrence of non-melanoma skin cancers in two of the affected individuals at a relatively young age suggests that patients with KS may be prone to cutaneous malignancy. A mutation in exon 5 was identified in the two distinct families that after family tree showed to have relatives in common that came from the same region in Italy more than three generations ago. Conclusions: These findings are very important to report a rare but well characterized syndrome and to increment detection strategies. The elucidation of the existent mutations is equally important in the developing of new treatments and in the management of the affected patients.

The basis of this study are mutant plants with ARP2/3 complex lacking in one of its subunits (arpc5 and arp2). These plants also express CSC subunit CESA6 of primary cell wall tagged by YFP. Thanks to modern imaging technologies, it is possible to observe the movement of tagged cellulose synthase complexes in vivo at plasmatic membrane. Kymograph analyses was used to measure the velocity of CESA complexes. In addition to observing CESA complexes directly on the plasma membrane, experiments were made to regenerate cell walls of protoplasts of Arabidopsis thaliana plants arpc5 and WT. It was found, that observed mutants arpc5 and arp2 have reduced velocity of CESA complexes in comparison to WT and arpc5 protoplasts regenerate cellulose mesh of cell wall slower. Keywords: Cellulose synthesis, ARP2/3 complex, CESA, CSC velocity, arpc5, arp2, Arabidopsis thaliana.

xii, 107 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Non-muscle myosin II generates contractile forces for processes such as cytokinesis, motility, and polarity. Contractility requires assembly of myosin molecules into bipolar mini-filaments through electrostatic interactions between coiled-coil tail domains of the heavy chains. Analyses of myosin II from various organisms have revealed "assembly domains" within the C-terminal portion of the tail domain that mediate filament formation. However, it has been unclear precisely how assembly domains interact with one another, or otherwise contribute to tail-tail interactions, to form the bipolar mini-filament structure. To understand tail domain interactions, we first identified a 90-residue region (1849-1940) of the Drosophila non-muscle myosin II tail domain that was necessary and sufficient for filament formation, using salt-dependent solubility and a novel fluorescence energy transfer assay. We identified residues within this "assembly domain" that were critical for filament assembly by analyzing the effect of point mutations. We found that single point mutations in specific positively charged regions completely disrupt filament assembly. Surprisingly, none of the negatively charged regions within the assembly domain are required for assembly. Most of the mutations in positively charged residues that disrupted filament assembly clustered within a 15-residue segment (1880-1894) that appears to form a critical interaction surface. Using this information, along with known geometrical constraints and electrostatic calculations, we constructed a structural model of the bipolar mini-filament. This model features one favored anti-parallel tail overlap and multiple slightly less stable alternative overlaps. The ability of the positive segment to interact with multiple negative regions explains the lack of required negatively charged residues in the assembly domain. To our knowledge, this structural model of the non- muscle myosin II bipolar filament is consistent with all physical observations and provides a framework for understanding the detailed mechanism by which this fundamental cellular structure is generated. This dissertation contains previously published and unpublished co-authored material. / Committee in charge: Tom Stevens, Chairperson, Chemistry; Kenneth Prehoda, Advisor, Chemistry; J. Andrew Berglund, Member, Chemistry; Christopher Doe, Member, Biology; Karen Guillemin, Outside Member, Biology

La maladie d'Alzheimer est une pathologie neurodégénérative caractérisée par une perte progressive des fonctions cognitives. Cette perte des fonctions cognitives est directement liée à une atteinte neuronale et plus particulièrement synaptique. Deux caractéristiques histopathologiques en lien avec des dérégulations protéiques sont retrouvées chez les patients atteints de la MA : les plaques séniles extracellulaires composées de peptides β-amyloïdes (Aβ) fibrillaires et la dégénérescence neurofibrillaire constituée d'agrégats intracellulaires de protéines tau hyper et anormalement phosphorylées. Les formes agrégées de ces protéines ont longtemps été considérées comme neurotoxiques, cependant, il est maintenant avéré que les formes solubles de ces protéines dérégulées étaient à l'origine de la pathologie. Les synapses excitatrices situées au niveau des épines dendritiques sont les cibles du peptide Aβ sous forme soluble et oligomèrique (Aβo). Ce dernier en altère la fonction et induit leurs pertes. Récemment, il a été montré que cette action synaptotoxique de l'Aβo est dépendante de la protéine tau. De plus, dans un autre modèle de tauopathie, la démence fronto-temporale avec syndrome parkinsonien liée au chromosome 17 (FTDP-17), la synaptotoxicité de tau s'est révélée dépendante de son état de phosphorylation. Ainsi, il émerge le concept de tau synaptique dans un contexte pathologique. Cependant, des études plus récentes ont montré que, en condition physiologique, une petite portion de tau se retrouve au niveau de la synapse. Au regard de ces nouvelles données, il est possible que tau, en plus d'être une protéine axonale, nucléaire et membranaire, soit aussi synaptique. Dans ce contexte, les travaux présentés dans cette thèse visent à étudier l'implication de la protéine tau dans la fonction synaptique et les perturbations induites par la présence d'Aβo. Ces travaux ont été effectués sur un modèle cellulaire de cultures primaires de neurones corticaux et sur tranche d'hippocampe de souris par des méthodes biochimiques et d'analyse dynamique en microscopie confocale sur cellules vivantes. Afin d'étudier l'impact d'une activation synaptique sur un système de culture neuronal, l'utilisation combinée de la bicuculline, antagoniste des récepteurs gabaergique GABAa et de 4-amino pyridine, bloqueur de canaux potassique, permet d'établir une potentialisation à long terme sur les synapses. Grâce à un protocole d'extraction permettant d'isoler le compartiment post-synaptique (fraction contenant la densité post synaptique dont le marqueur protéique PSD-95), nous avons montré que l'activation synaptique enrichit la fraction PSD en protéine tau suggérant son implication dans les phénomènes de plasticité synaptique. L'étude du cytosquelette d'actine prépondérant au niveau synaptique a révélé que l'actine filamenteuse est un partenaire de tau. Dans un contexte pathologique, l'incubation d'Aβo induit le recrutement de tau à la synapse et perturbe l'organisation du cytosquelette d'actine. Ce changement structurel du cytosquelette d'actine pourrait être à l'origine des perturbations de la plasticité et du maintien synaptique induit par Aβo. En conclusion, l'ensemble des résultats de cette thèse suggère que tau exerce une fonction physiologique au sein de la synapse impliquant une interaction avec le cytosquelette d'actine et qu'en conditions pathologiques (induites par Aβo), on observe une altération fonctionnelle du rôle de tau à la synapse qui pourrait participer aux perturbations cognitives caractéristiques de la MA. / Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive functions. This loss of cognitive functions is directly related to neuronal impairment, and more specifically, synaptic dysfunction. Two histopathological features found in AD patients' brains are related to protein deregulation: extracellular neuritic plaques composed of fibrillar β-amyloid peptide (Aβ) and intracellular aggregates composed of hyper-phosphorylated tau, named neurofibrillary tangles. Aggregated forms of these peptides have been considered neurotoxic, however, it is now recognized that soluble forms of these deregulated proteins are causal to the pathology. Soluble, oligomeric forms of Aβ peptide (Aβo) target excitatory synapses where they diminish synaptic function and cause loss of dendritic spines. Recently, it has been shown that the Aβo synaptotoxicity is tau-dependent. Another tauopathy, fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), exhibits synaptotoxicity, which has proved to be dependent on the phosphorylation state of tau. Thus, emerged the concept of synaptic tau in a pathological context. Recent studies have shown that a small quantity of tau is present in synapses under physiological conditions. These new data suggest that tau is a synaptic protein, in addition to being axonal, nuclear and membrane-associated. In this context, the work presented in this thesis characterizes the involvement of tau in synaptic function and its Aβo-induced disturbances. This work was conducted using primary cortical neurons cultured from mice and hippocampus slices and employed biochemical methods and confocal live-cell imaging. To study the impact of a synaptic activation on synaptic tau, we combined bicuculline, an antagonist of GABAa receptors and 4-amino-pyridine, potassium channel blocker, to establish long-term synaptic potentiation. By isolating the post-synaptic compartment (i.e. the fraction containing the post synaptic density and its marker PSD-95), we have shown that synaptic activation induced an enrichment of tau in PSD. This suggests its involvement in synaptic plasticity. The study of the actin cytoskeleton, which is specifically enriched in dendritic spines, revealed that filamentous actin is a molecular partner of tau, which may provide a means of recruiting tau to the synapse. Turning our attention to a pathological context, exposure to Aβo induced tau recruitment to the synapse and disrupts the actin cytoskeleton organization without exogenous synaptic stimulation. This structural modification of the actin cytoskeleton could underlie the disturbance of plasticity and synaptic maintenance induced by Aβo. In conclusion, this thesis provides evidence that tau performs a physiological synaptic function that involves an interaction with the actin cytoskeleton. Further, the synaptic function of tau is altered in pathological conditions (i.e. exposure to Aβo), and may contribute to the cognitive disturbances in AD.

Introdução: A Síndrome de Kindler é uma doença autossômica recessiva que se caracteriza por surgimento de bolhas acrais na infância que diminuem com o passar dos anos, fotossensibilidade, poiquilodermia persistente e progressiva e atrofia cutânea. Desde o primeiro relato, mais de 100 casos já foram descritos, mas a associação com Poiquilodermia Acroceratótica e Epidermólise Bolhosa tem freqüentemente dificultado o diagnóstico. Exames moleculares nunca foram realizados em paciente brasileiros com Síndrome de Kindler. Objetivos: O objetivo deste estudo é a identificação de alterações genéticas e estruturais nos pacientes com Síndrome de Kindler do sul do Brasil. Pacientes e métodos: Este estudo é uma série de casos de sete pacientes acompanhados no Hospital de Clínicas de Porto Alegre durante o período de janeiro de 2004 a dezembro de 2005. Os pacientes com SK foram examinados clinicamente e o registro foi realizado em um questionário estruturado. As amostras de pele e sangue foram coletadas após consentimento informado com a aprovação pelo comitê de ética. Resultados: Os achados clínicos mais consistentes para o diagnóstico foram bolhas na infância, fotossensibilidade e poiquilodermia. A ocorrência de carcinomas de pele não melanomas em dois pacientes jovens sugere que a SK pode ser um fator de risco no desenvolvimento de malignidades cutâneas. Uma mutação no exon 5 foi identificada nas duas famílias distintas que após o heredograma mostraram ter parentes em comum que vieram da mesma região da Itália há três gerações. Conclusões: Estes achados são muito importantes para a descrição de uma síndrome tipicamente caracterizada e para incrementar a estratégia de detecção. A elucidação das mutações existentes é igualmente importante para o desenvolvimento de novos tratamentos e manejos dos pacientes afetados. / Background: Kindler syndrome (KS) is a rare autossomal recessive disease characterized by acral bullae in infancy that diminished with time, photosensitivity, persistent progressive poikiloderma and severe cutaneous atrophy. Since the first report more than 100 cases have already been described but the association with acrokeratotic poikiloderma and epidermolysis bullosa has often misled the diagnosis. The molecular study of Brazilian patients with Kindler syndrome was never performed. Objectives: To asses the clinical features and the molecular basis of KS in patients from South Brazil and to examine molecular evidence for a mutation. Patients and methods: This study is a series of seven patients atending to Hospital de Clinicas de Porto Alegre during the period of January 2004 to December 2005. The KS affected individuals were examined clinically and a structured recording sheet was completed. Blood and skin samples were collected after informed consent and with the Ethics committee approval. Results: The most consistent clinical signs for a diagnosis were blisters in childhood, photosensitivity and poikiloderma. The occurrence of non-melanoma skin cancers in two of the affected individuals at a relatively young age suggests that patients with KS may be prone to cutaneous malignancy. A mutation in exon 5 was identified in the two distinct families that after family tree showed to have relatives in common that came from the same region in Italy more than three generations ago. Conclusions: These findings are very important to report a rare but well characterized syndrome and to increment detection strategies. The elucidation of the existent mutations is equally important in the developing of new treatments and in the management of the affected patients.

Introdução: A Síndrome de Kindler é uma doença autossômica recessiva que se caracteriza por surgimento de bolhas acrais na infância que diminuem com o passar dos anos, fotossensibilidade, poiquilodermia persistente e progressiva e atrofia cutânea. Desde o primeiro relato, mais de 100 casos já foram descritos, mas a associação com Poiquilodermia Acroceratótica e Epidermólise Bolhosa tem freqüentemente dificultado o diagnóstico. Exames moleculares nunca foram realizados em paciente brasileiros com Síndrome de Kindler. Objetivos: O objetivo deste estudo é a identificação de alterações genéticas e estruturais nos pacientes com Síndrome de Kindler do sul do Brasil. Pacientes e métodos: Este estudo é uma série de casos de sete pacientes acompanhados no Hospital de Clínicas de Porto Alegre durante o período de janeiro de 2004 a dezembro de 2005. Os pacientes com SK foram examinados clinicamente e o registro foi realizado em um questionário estruturado. As amostras de pele e sangue foram coletadas após consentimento informado com a aprovação pelo comitê de ética. Resultados: Os achados clínicos mais consistentes para o diagnóstico foram bolhas na infância, fotossensibilidade e poiquilodermia. A ocorrência de carcinomas de pele não melanomas em dois pacientes jovens sugere que a SK pode ser um fator de risco no desenvolvimento de malignidades cutâneas. Uma mutação no exon 5 foi identificada nas duas famílias distintas que após o heredograma mostraram ter parentes em comum que vieram da mesma região da Itália há três gerações. Conclusões: Estes achados são muito importantes para a descrição de uma síndrome tipicamente caracterizada e para incrementar a estratégia de detecção. A elucidação das mutações existentes é igualmente importante para o desenvolvimento de novos tratamentos e manejos dos pacientes afetados. / Background: Kindler syndrome (KS) is a rare autossomal recessive disease characterized by acral bullae in infancy that diminished with time, photosensitivity, persistent progressive poikiloderma and severe cutaneous atrophy. Since the first report more than 100 cases have already been described but the association with acrokeratotic poikiloderma and epidermolysis bullosa has often misled the diagnosis. The molecular study of Brazilian patients with Kindler syndrome was never performed. Objectives: To asses the clinical features and the molecular basis of KS in patients from South Brazil and to examine molecular evidence for a mutation. Patients and methods: This study is a series of seven patients atending to Hospital de Clinicas de Porto Alegre during the period of January 2004 to December 2005. The KS affected individuals were examined clinically and a structured recording sheet was completed. Blood and skin samples were collected after informed consent and with the Ethics committee approval. Results: The most consistent clinical signs for a diagnosis were blisters in childhood, photosensitivity and poikiloderma. The occurrence of non-melanoma skin cancers in two of the affected individuals at a relatively young age suggests that patients with KS may be prone to cutaneous malignancy. A mutation in exon 5 was identified in the two distinct families that after family tree showed to have relatives in common that came from the same region in Italy more than three generations ago. Conclusions: These findings are very important to report a rare but well characterized syndrome and to increment detection strategies. The elucidation of the existent mutations is equally important in the developing of new treatments and in the management of the affected patients.