<p>The actin cytoskeleton is involved in many essential cellular events such as mitosis, cell migration, control of epithelial barrier function, and adherence of immune cells. Given the essential roles of the actin cytoskeleton, it is not surprising that it is a common target for bacterial virulence factors. <em>Legionella pneumophila</em>, the causative agent of Legionnaire’s pneumonia, establishes a replicative compartment using effectors secreted by its Dot/Icm secretion system. At least four Dot/Icm effectors, VipA, Ceg14, LegK2 and RavK have been shown to modulate the host actin cytoskeleton. Here, we identified RavJ (lpg0944) as an additional effector that interferes with the actin cytoskeleton in mammalian cells. We demonstrated that RavJ is a transglutaminase that functions to induce crosslink between actin and members of the Motin protein family, leading to inhibition of the binding between actin and ADF/cofilin. We also found that LegL1 (lpg0945) is a metaeffector of RavJ, which suppresses the transglutaminase activity of RavJ by blocking its enzymatic domain. </p>

Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for human genital and ocular infections. Species of Chlamydia utilize a type-III secretion system to deliver bacterial effector proteins into the host cell in order to promote invasion and establish residence within a parasitophorous vacuole called an inclusion. The effector protein Tarp has been previously implicated as an important effector for promoting invasion during Chlamydia trachomatis infection by directing the formation of new actin filaments and bundles. Intriguingly, the significance of Tarp mediated cytoskeletal changes has not been fully explored in vivo. Host-pathogen interaction studies that replicate the human infection can be performed with mouse adapted Chlamydia, Chlamydia muridarum. However, the genetic tools to create gene deletions in C. muridarum have been lacking. Recently, our collaborators in the Fields and Wolf Laboratories developed a novel genetic tool for creating Tarp deletion mutants and complement clones in Chlamydia muridarum. Through the use of this tool, we were able to study the significance of Tarp in a murine infection model. In addition to Tarp, two other early effectors TmeA and TmeB are hypothesized to play a role in invasion, but a full account of their involvement remained unknown. In our studies, we were able to determine the roles of TmeA and TmeB in remodeling the host cytoskeleton. Using biochemical crosslinking assays, and actin polymerization studies, we discovered that TmeA has the ability to activate host protein N-Wasp in order to increase Arp2/3-dependent actin polymerization, while TmeB can in turn inhibit Arp2/3-directed actin polymerization via direct interactions with Arp2/3. Collectively, these are important findings as our studies have revealed how a collection of early chlamydial effectors work to modulate the host cytoskeleton to facilitate Chlamydia infections.

The Cx43 CT mimetic peptide, αCT1, originally designed to bind to ZO1 and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing(ECIS), a TRITC-dextran transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, αCT1 was found to protect the endothelium from thrombin-induced breakdown in cell-cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular orientation distribution. In line with observations of increased cortical F-actin, αCT1 upregulated cell-cell border localization of endothelial VE-cadherin, the Tight Junction protein Zonula Occludens 1 (ZO1) , and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1-binding-incompetent variant of αCT1, αCT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of αCT1 in the treatment of vascular edema. / Doctor of Philosophy / Endothelial cells make up blood vessels within the heart and regulate the exchange of fluids between the circulation and heart tissue. In many forms of heart disease, the cardiac endothelium is disrupted, resulting in a damaging leakage and buildup of fluids within the heart. This work explores how a small peptide, derived from a naturally occurring molecule, may help to prevent fluid-associated damage to the heart by stabilizing the blood endothelium.

Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.

Endocytosis is both an ancient and a diverse feature of the eukaryotic cell. Studying how it evolved can provide insight into the nature of the last common eukaryotic ancestor, and the diversification of eukaryotes into the known extant lineages. In this thesis, I present two studies on the evolution of endocytosis. In the first part of the thesis I report results from a large-scale, phylogenetic and comparative genomic study of clathrin-mediated endocytosis (CME). The CME pathway has been studied to a great level of detail in yeast to mammal model organisms. Several protein families have now been identified as part of the complex set of protein-protein and protein-lipid interactions which mediate endocytosis. To investigate how such complexity evolved, first, I defined the modular nature of the CME interactome (CME-I) by literature review, and then I carried out a systematic phylogenetic and protein domain architecture analysis of the proteins involved. These data were used to construct a model of the evolution of the CME-I network, and to map the expansion of the network's complexity to the eukaryotic tree of life. In the second part of the thesis, I present results from evolutionary and functional studies of the eisosome, a protein complex which has been proposed to regulate the spatial distribution of endocytosis in S. cerevisiae. The phylogeny of eisosomes components Pil1 and Lsp1 reported here, suggests that eisosomes are likely to have originated at the base of the fungi, and then diversified significantly via multiple gene duplications. I thus studied the localisation and function of Pil1 and Lsp1 homologues in Magnaporthe oryzae to investigate the role of eisosomes in filamentous fungi. Results suggests that eisosomes are linked with septal formation and integrity in M. oryzae, and that the septal specific Pil2 paralogue was lost in budding yeasts. Together, the data presented in this thesis describe the evolutionary history of a complex biological system, but also highlights the problem of asymmetry in the understanding of endocytic diversity in the eukaryotes.

Yersinia pestis is a human pathogen and the causative agent of bubonic plague, responsible for causing three massive pandemics, resulting in hundreds of millions of deaths in the 14th century alone. Yersinia’s virulence stems from its ability to overcome host immune defences by the injection of six Yersinia outer proteins (Yops) into the host cells via its Type III secretion system. One of these Yops, YopO specifically disables the actin polymerization machinery, leading to the crippling of phagocytosis. YopO consists of a GDI domain which sequesters Rac and Rho, and a kinase domain, the activity of which is dependent on host actin. Little is known about the targets of the kinase domain and the mechanism of function of YopO remains incomplete. In this work, YopO was crystallized in complex with actin, revealing that YopO binds to actin on subdomain 4, away from the 'hotspot’ between subdomains 1 and 3 which is involved in binding most actin-binding proteins. The structure reveals how recruitment of YopO-bound actin monomers stalls actin polymerization by steric hindrance. The structure also demonstrates how YopO uses actin for self-activation and suggests that actin is being used by YopO as bait for recruitment into actin machineries. Using SILAC mass spectrometry, actin cytoskeletal machineries within macrophages that recruit YopO are identified and these include, amongst others: VASP family proteins, gelsolin family proteins, formins and WASP. Of these, VASP, EVL, diaphanous1, WASP and gelsolin have been identified to be phosphorylated by YopO and were validated by in vitro phosphorylation. This work demonstrates that YopO uses actin as a scaffold for selection of kinase substrates, enabling targeted phosphorylation of the actin machinery and provides insight into the regulation of the actin cytoskeleton by phosphorylation under non-pathogenic conditions.

Mon travail fut de développer différents projets en vue de mieux comprendre la dynamique et l'organisation des réseaux d'actine et les mécanismes moléculaires à l'origine de la production de force, cela en systèmes reconstitués bio-mimétiques. Dans un premier temps je me suis intéressée à l'étude de l'organisation spatio-temporelle des réseaux d'actine et de ses protéines associées durant la motilité de particules recouverte de promoteurs de nucléation (Achard et al, Current Biology, 2010 et Reymann et al, sous presse à MBOC). J'ai suivi en temps réel l'incorporation de deux régulateurs de l'actine (capping protein et ADF/cofiline) et montré que leur contrôle biochimique sur l'actine gouverne également ces propriétés mécaniques. Afin de mieux caractériser les propriétés mécaniques de ces réseaux d'actine en expension, j'ai ensuite développé un système biomimétique novateur utilisant un set-up de micro-patterning permettant un contrôle spatial reproductible des sites de nucléation d'actine. Cela m'a permis de montrer comment des barrières géométriques, semblables à celles trouvées dans les cellules, peuvent influencer la formation dynamique de réseaux organisés d'actine et ainsi contrôler la localisation de la production de forces. (Reymann et al, Nature Materials, 2010). De plus l'addition de moteurs moléculaires sur ce système versatile nous a permis d'étudier la contraction induite par des myosines. En particulier les myosines VI-HMM interagissent de manière sélective sur différentes architectures d'actine (organisation parallèle ou antiparallèle, réseau enchevêtré), aboutissant à un processus en trois phase : tension puis déformation des réseaux d'actine fortement couplé à un désassemblage massif des filaments. Ce phénomène est intimement dépendant de l'architecture du réseau d'actine et pourrait donc jouer un rôle essentiel dans la régulation spatiale des zones d'expansion et de contraction du cytosquelette in vivo. (Travail en cours d'écriture). / I have developed different projects in order to tackle the problem of actin network dynamics and organization as well as the molecular mechanism at the origin of force production in biomimetic reconstituted systems. My first interest concerned the spatiotemporal organization of actin networks and actin-binding proteins during actin based motility of nucleation promoting factor-coated particles (Achard et al, Current Biology, 2010 and Reymann et al, in press at MBOC). I tracked in real time the incorporation of two actin regulators and showed that their biochemical control of actin dynamics also governs its mechanical properties. To further characterize mechanical properties of expanding actin networks, I used an innovative micro-patterning set-up allowing a reproducible spatial control of actin nucleation sites. It allowed me to show that geometrical boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures and hence control the location of force production (Reymann et al, Nature Materials, 2010). Finally the addition of molecular motors on this tunable system allowed me to study implications for myosin-induced contractility. In particular, HMM-MyosinVI selectively interact with the different actin network architectures (parallel, anti-parallel organization or entangled networks) and leads to a selective three-phase process of tension, deformation of actin networks tightly coupled to massive filament disassembly. This phenomenon being highly dependent on actin network architecture could therefore play an essential role in the spatial regulation of expanding and contracting regions of actin cytoskeleton in cells. (Work in writing process).

Introdução: As células do músculo liso vascular (CMLV) são quiescentes nos vasos adultos, com baixa capacidade de migração e de secreção de matriz extracelular, caracterizando fenótipo contrátil. Evidências apontam para a heterogeneidade fenotípica das CMLV ao longo da árvore arterial: há distribuição heterogênea de doenças e de resposta a determinadas drogas nos diferentes vasos, além de variabilidade de expressão dos genes de proteínas contráteis de músculo liso entre eles. O papel das CMLV, em fase adulta, é classicamente descrito como restrito à regulação do tônus de pequenos vasos, sendo insignificante a contribuição da mecânica das CMLV para a complacência das artérias elásticas. Existe a hipótese de que a viscoelasticidade das CMLV contribua para a mecânica final das artérias, sendo o enrijecimento dessas células associado à rigidez arterial. Objetivo: Estudar a variabilidade das propriedades mecânicas e de expressão proteica das CMLV, ao longo da árvore arterial, buscando identificar moduladores regionais para esse fenótipo. Avaliar se situações clínicas sabidamente associadas à rigidez arterial (envelhecimento, sexo feminino pós-menopausa, ancestralidade genética africana, diabetes mellitus e tabagismo) cursam com enrijecimento de CMLV. Métodos: 1) Estudou-se a composição e a organização da camada média de diferentes artérias. As CMLV desses vasos foram avaliadas quanto à viscoelasticidade de citoplasma (G), por meio do ensaio de Citometria Magnético Ótica de Oscilação e, quanto à expressão proteica global, usando cromatografia multidimensional e espectrometria de massas em tandem de alta resolução (Proteômica Shotgun). Os dados mecânicos obtidos foram correlacionados com as características da matriz extracelular (MEC) dos vasos de origem (porcentagem de elastina e quantidade de MEC). Em paralelo, foi realizado experimento de estiramento cíclico (10%/1Hz) das CMLV das diferentes artérias por 24 e 48h, seguido pela mensuração de rigidez de citoplasma. 2) Foram isoladas as CMLV de fragmentos de artéria mamária de 80 pacientes submetidos à cirurgia de revascularização do miocárdio, células essas que foram avaliadas quanto à viscoelasticidade de citoplasma (G, G\' e G\'\'). Elaborou-se modelo estatístico para avaliar se as variáveis clínicas idade, sexo feminino, ancestralidade africana, tabagismo e diabetes mellitus estavam associadas a alterações de mecânica celular. Resultados: 1) A viscoelasticidade das CMLV variou significativamente entre as artérias. As CMLV provenientes de artérias distais (artérias femoral e renal) mostraram-se significativamente mais rígidas que as CMLV de aorta torácica (p < 0,001). Identificou-se correlação negativa entre rigidez de CMLV e quantidade de MEC / elastina na camada média vascular. O regime de estiramento cíclico por 48h reduziu globalmente a rigidez das CMLV. As CMLV provenientes da aorta torácica expressaram maior quantidade de proteínas relacionadas com a estrutura e a organização do citoesqueleto em relação às CMLV da artéria femoral. 2) Constatou-se variabilidade interindividual de viscoelasticidade de CMLV e associação entre tabagismo e sexo feminino com enrijecimento de CMLV. Conclusões: As CMLV são heterogêneas quanto às propriedades mecânicas, à organização do citoesqueleto e à expressão proteica ao longo da árvore arterial, reforçando o conceito de plasticidade fenotípica das CMLV. A mecânica das CMLV é modulada pelas características da MEC e pela tensão circunferencial cíclica aplicada às paredes vasculares pelo fluxo sanguíneo. Mulheres pós-menopausa e tabagistas exibem enrijecimento de CMLV, sendo esse fato um provável contribuinte para a rigidez arterial associada a essas condições e um possível alvo terapêutico a ser avaliado futuramente / Rational: Vascular smooth muscle cells (VSMC) lose their ability to migrate and secrete extracellular matrix (ECM) with the end of vascular development, condition known as contractile phenotype and reversible in the presence of vascular injury. There is evidence of heterogeneity of VSMC phenotype along arterial tree, as the distribution of diseases (atherosclerosis) and the response to drugs vary between different vessels, as well as the expression of smooth muscle-contractile protein genes. The role played by VSMC mechanics on determining large arteries\' compliance was always considered irrelevant. It has been hypothesized that the VSMC mechanical properties are important for vascular mechanics, especially in the pathological scenario, where VSMC stiffening may be associated with arterial rigidity. Goals: Study the variation of VSMC mechanics and protein expression along arterial tree, identifying regional modulators of this phenotype. Evaluate if clinical situations associated with arterial rigidity (ageing, post-menopausal women, African ancestry, diabetes mellitus and smoking) concur with VSMC stiffening. Methods: 1) Different arteries were studied in terms of composition and organization of their media layer. VSMC isolated from these arteries were evaluated regarding cytoplasm viscoelasticity, measured using Optical Magnetic Twisting Cytometry Assay (OMTC), and protein expression, using two-dimensional liquid chromatography and tandem mass spectrometry (Shotgun Proteomics). Mechanical data were correlated with ECM characteristics (percentage of elastin and ECM amount) of the vessels of origin. In parallel, VSMC of different arteries were subjected to cyclic stretching (10%/1Hz) during 24 and 48h, followed by the measurement of their cytoplasm rigidity. 2) VSMC were isolated from fragments of mammary artery of 80 patients subjected to coronary bypass surgery and evaluated regarding their viscoelasticity (G, G\' e G\'\'). A statistic model was elaborated to address if the clinical variables age, female sex, African ancestry, smoking and diabetes mellitus were associated with changes of VSMC mechanics. Results: 1) VSMC viscoelasticity varied significantly amongst the studied arteries. VSMC from heart-distant arteries (femoral and renal arteries) were stiffer than VSMC from thoracic aorta (p < 0,001). There was a negative correlation between VSMC rigidity and the amount of ECM / percentage of elastin within the media layer. 48h-cyclic stretching was associated with a global reduction of VSMC rigidity. VSMC of thoracic aorta expressed significantly more proteins associated with cytoskeleton structure and organization than VSMC of femoral artery. 2) There was a significant inter-individual variation of VSMC viscoelasticity. Smoking and female sex were associated with VSMC stiffening. Conclusion: VSMC mechanics, cytoskeleton organization and protein expression are heterogeneous along arterial tree. VSMC mechanical properties are modulated by ECM characteristics and by regional mechanical forces. This reinforces the concept of phenotypic heterogeneity of VSMC. Post-menopausal women and smokers exhibit stiffer VSMC, representing an important factor for the understanding of the arterial rigidity associated with these conditions and also a possible future therapeutic target

Regulation of the actin cytoskeleton is key to cellular function and underlies processes including cell migration, mitosis and endocytosis. Motile cells send out dynamic actin protrusions that enable them to sense and interact with their environment, as well as generating physical forces. Linking of the actin cytoskeleton to the cell membrane is essential for the formation of these protrusions. The proteins that are thought to fulfil such a role have a membrane interacting domain (such as the PH domain in lamellipodin, or I-BAR protein in IRSp53) and a domain which interacts with actin regulatory proteins (such as the SH3 domain of IRSp53, which binds Ena and VASP). I investigated the contribution of the F-BAR protein Toca-1 in linking actin polymerisation to membranes, by characterising a new protein-protein interaction and the interaction of Toca-1 with giant unilamellar vesicles. FBP17, a homologue of Toca-1, can oligomerise to form 2D flat lattices and 3D tubules on membranes. Proteins of the Toca-1 family have previously been implicated in actin polymerisation in cell-free systems and during endocytosis. However, there is emerging evidence that Toca-1 family proteins could also be involved in the formation of outward facing protrusions, lamellipodia and filopodia. In an in vitro system that recapitulates the formation of filopodia-like structures (FLS) on supported lipid bilayers, Toca-1 is recruited early, suggesting a Toca-1 scaffolding mechanism could precede the recruitment of other actin regulators. One prediction of this model is that Toca-1 would bind proteins previously implicated in filopodia formation, such as formins. I found that extracts depleted of Toca-1 binding partners no longer forms filopodia-like structures and subsequently optimised pull-down assays to identify Toca-1 binding partners by mass-spectrometry. I identified four formins, Diaph1, Diaph3, FHOD1 and INF2, and as well as the actin elongation factors and filopodia proteins, Ena and VASP. I further characterised these interactions and found that Toca-1 binds Ena and VASP via its SH3 domain. The interaction is direct and is strongly reduced if the proline-rich region in Ena is deleted. VASP was still able to bind without its proline rich region, suggesting there could be additional binding sites. I discovered that the binding of Ena and VASP was dependent on the clustering state of Toca-1, whilst the binding of the previously identified Toca-1 binding partner N-WASP was not. This further supports the importance of Toca-1 oligomerisation in actin polymerisation. I tested these interactions in the FLS system and found that increasing Toca-1 concentration leads to increased recruitment of N-WASP, as well as the novel binding partner Ena to the structures, whereas an increase in VASP was not observed. SH3-domain mediated interactions are required for Toca-1 recruitment to FLS, suggesting that its membrane and protein binding activities act cooperatively. I showed that unlike N-WASP, which promotes the formation of branched actin, Ena and VASP are not required for actin polymerisation on supported lipid bilayers, suggesting that they are redundant with other factors in the elongation step of FLS formation. Ena and VASP are known to be important for the formation of neuronal filopodia and so I began to further test the role of these interactions in a cellular context using a neuronal cell culture system. As well as recruiting protein binding partners, F-BAR family proteins are implicated in stabilising lipid microdomains and can induce the clustering of phosphoinositides. I investigated the role of Toca-1 in actin polymerisation on PI(4,5)P2-rich giant unilamellar vesicles (GUVs). Actin-rich tails formed on the GUVs only when excess Toca-1 was supplemented into the extracts, and I propose that this is due to lipid organisation by Toca-1. In summary, my work suggests a model in which Toca-1 clusters, stabilises the membrane lipids and recruits regulators of actin polymerisation, such as Ena. This mechanism could be used to link actin polymerisation to the membrane in cellular protrusions, such as filopodia.

Introdução: As células do músculo liso vascular (CMLV) são quiescentes nos vasos adultos, com baixa capacidade de migração e de secreção de matriz extracelular, caracterizando fenótipo contrátil. Evidências apontam para a heterogeneidade fenotípica das CMLV ao longo da árvore arterial: há distribuição heterogênea de doenças e de resposta a determinadas drogas nos diferentes vasos, além de variabilidade de expressão dos genes de proteínas contráteis de músculo liso entre eles. O papel das CMLV, em fase adulta, é classicamente descrito como restrito à regulação do tônus de pequenos vasos, sendo insignificante a contribuição da mecânica das CMLV para a complacência das artérias elásticas. Existe a hipótese de que a viscoelasticidade das CMLV contribua para a mecânica final das artérias, sendo o enrijecimento dessas células associado à rigidez arterial. Objetivo: Estudar a variabilidade das propriedades mecânicas e de expressão proteica das CMLV, ao longo da árvore arterial, buscando identificar moduladores regionais para esse fenótipo. Avaliar se situações clínicas sabidamente associadas à rigidez arterial (envelhecimento, sexo feminino pós-menopausa, ancestralidade genética africana, diabetes mellitus e tabagismo) cursam com enrijecimento de CMLV. Métodos: 1) Estudou-se a composição e a organização da camada média de diferentes artérias. As CMLV desses vasos foram avaliadas quanto à viscoelasticidade de citoplasma (G), por meio do ensaio de Citometria Magnético Ótica de Oscilação e, quanto à expressão proteica global, usando cromatografia multidimensional e espectrometria de massas em tandem de alta resolução (Proteômica Shotgun). Os dados mecânicos obtidos foram correlacionados com as características da matriz extracelular (MEC) dos vasos de origem (porcentagem de elastina e quantidade de MEC). Em paralelo, foi realizado experimento de estiramento cíclico (10%/1Hz) das CMLV das diferentes artérias por 24 e 48h, seguido pela mensuração de rigidez de citoplasma. 2) Foram isoladas as CMLV de fragmentos de artéria mamária de 80 pacientes submetidos à cirurgia de revascularização do miocárdio, células essas que foram avaliadas quanto à viscoelasticidade de citoplasma (G, G\' e G\'\'). Elaborou-se modelo estatístico para avaliar se as variáveis clínicas idade, sexo feminino, ancestralidade africana, tabagismo e diabetes mellitus estavam associadas a alterações de mecânica celular. Resultados: 1) A viscoelasticidade das CMLV variou significativamente entre as artérias. As CMLV provenientes de artérias distais (artérias femoral e renal) mostraram-se significativamente mais rígidas que as CMLV de aorta torácica (p < 0,001). Identificou-se correlação negativa entre rigidez de CMLV e quantidade de MEC / elastina na camada média vascular. O regime de estiramento cíclico por 48h reduziu globalmente a rigidez das CMLV. As CMLV provenientes da aorta torácica expressaram maior quantidade de proteínas relacionadas com a estrutura e a organização do citoesqueleto em relação às CMLV da artéria femoral. 2) Constatou-se variabilidade interindividual de viscoelasticidade de CMLV e associação entre tabagismo e sexo feminino com enrijecimento de CMLV. Conclusões: As CMLV são heterogêneas quanto às propriedades mecânicas, à organização do citoesqueleto e à expressão proteica ao longo da árvore arterial, reforçando o conceito de plasticidade fenotípica das CMLV. A mecânica das CMLV é modulada pelas características da MEC e pela tensão circunferencial cíclica aplicada às paredes vasculares pelo fluxo sanguíneo. Mulheres pós-menopausa e tabagistas exibem enrijecimento de CMLV, sendo esse fato um provável contribuinte para a rigidez arterial associada a essas condições e um possível alvo terapêutico a ser avaliado futuramente / Rational: Vascular smooth muscle cells (VSMC) lose their ability to migrate and secrete extracellular matrix (ECM) with the end of vascular development, condition known as contractile phenotype and reversible in the presence of vascular injury. There is evidence of heterogeneity of VSMC phenotype along arterial tree, as the distribution of diseases (atherosclerosis) and the response to drugs vary between different vessels, as well as the expression of smooth muscle-contractile protein genes. The role played by VSMC mechanics on determining large arteries\' compliance was always considered irrelevant. It has been hypothesized that the VSMC mechanical properties are important for vascular mechanics, especially in the pathological scenario, where VSMC stiffening may be associated with arterial rigidity. Goals: Study the variation of VSMC mechanics and protein expression along arterial tree, identifying regional modulators of this phenotype. Evaluate if clinical situations associated with arterial rigidity (ageing, post-menopausal women, African ancestry, diabetes mellitus and smoking) concur with VSMC stiffening. Methods: 1) Different arteries were studied in terms of composition and organization of their media layer. VSMC isolated from these arteries were evaluated regarding cytoplasm viscoelasticity, measured using Optical Magnetic Twisting Cytometry Assay (OMTC), and protein expression, using two-dimensional liquid chromatography and tandem mass spectrometry (Shotgun Proteomics). Mechanical data were correlated with ECM characteristics (percentage of elastin and ECM amount) of the vessels of origin. In parallel, VSMC of different arteries were subjected to cyclic stretching (10%/1Hz) during 24 and 48h, followed by the measurement of their cytoplasm rigidity. 2) VSMC were isolated from fragments of mammary artery of 80 patients subjected to coronary bypass surgery and evaluated regarding their viscoelasticity (G, G\' e G\'\'). A statistic model was elaborated to address if the clinical variables age, female sex, African ancestry, smoking and diabetes mellitus were associated with changes of VSMC mechanics. Results: 1) VSMC viscoelasticity varied significantly amongst the studied arteries. VSMC from heart-distant arteries (femoral and renal arteries) were stiffer than VSMC from thoracic aorta (p < 0,001). There was a negative correlation between VSMC rigidity and the amount of ECM / percentage of elastin within the media layer. 48h-cyclic stretching was associated with a global reduction of VSMC rigidity. VSMC of thoracic aorta expressed significantly more proteins associated with cytoskeleton structure and organization than VSMC of femoral artery. 2) There was a significant inter-individual variation of VSMC viscoelasticity. Smoking and female sex were associated with VSMC stiffening. Conclusion: VSMC mechanics, cytoskeleton organization and protein expression are heterogeneous along arterial tree. VSMC mechanical properties are modulated by ECM characteristics and by regional mechanical forces. This reinforces the concept of phenotypic heterogeneity of VSMC. Post-menopausal women and smokers exhibit stiffer VSMC, representing an important factor for the understanding of the arterial rigidity associated with these conditions and also a possible future therapeutic target