Elastin is an essential extracellular protein that is a key component of elastic fibres, providing elasticity to cardiac, dermal, and arterial tissues. During the development of the human cardiovascular system, elastin self-assembles before being integrated into fibres, undergoing no significant turnover during the human lifetime. Abnormalities in elastin can adversely affect its self-assembly, and may lead to malformed elastic fibres. Due to the longevity required of these fibres, even minor abnormalities may have a large cumulative effect over the course of a lifetime, leading to late-onset vascular diseases. This thesis project has identified important, over-represented repetitive elements in elastin which are believed to be important for the self-assembly and elastomeric properties of elastin. Initial studies of single nucleotide polymorphisms (SNPs) from the HapMap project and dbSNP resulted in a set of genetic variation sites in the elastin gene. Based on these studies, glycine to serine and lysine to arginine substitutions were introduced in elastin-like polypeptides. The self-assembly properties of the resulting elastin-like polypeptides were observed under microscope and measured using absorbance at 440nm. Assembled polypeptides were also cross-linked to form thin membranes whose mechanical and physical properties were measured and compared. These mutations resulted in markedly different behavior than wild-type elastin-like proteins, suggesting that mutations in the repetitive elements of the elastin sequence can lead to adverse changes in the physical and functional properties of the resulting protein. Using next-generation sequencing, patients with thoracic aortic aneurysms are being genotyped to discover polymorphisms which may adversely affect the self-assembly properties of elastin, providing a link between genetic variation in elastin and cardiovascular disease.

Elastin is an essential extracellular protein that is a key component of elastic fibres, providing elasticity to cardiac, dermal, and arterial tissues. During the development of the human cardiovascular system, elastin self-assembles before being integrated into fibres, undergoing no significant turnover during the human lifetime. Abnormalities in elastin can adversely affect its self-assembly, and may lead to malformed elastic fibres. Due to the longevity required of these fibres, even minor abnormalities may have a large cumulative effect over the course of a lifetime, leading to late-onset vascular diseases. This thesis project has identified important, over-represented repetitive elements in elastin which are believed to be important for the self-assembly and elastomeric properties of elastin. Initial studies of single nucleotide polymorphisms (SNPs) from the HapMap project and dbSNP resulted in a set of genetic variation sites in the elastin gene. Based on these studies, glycine to serine and lysine to arginine substitutions were introduced in elastin-like polypeptides. The self-assembly properties of the resulting elastin-like polypeptides were observed under microscope and measured using absorbance at 440nm. Assembled polypeptides were also cross-linked to form thin membranes whose mechanical and physical properties were measured and compared. These mutations resulted in markedly different behavior than wild-type elastin-like proteins, suggesting that mutations in the repetitive elements of the elastin sequence can lead to adverse changes in the physical and functional properties of the resulting protein. Using next-generation sequencing, patients with thoracic aortic aneurysms are being genotyped to discover polymorphisms which may adversely affect the self-assembly properties of elastin, providing a link between genetic variation in elastin and cardiovascular disease.

Les protéines de type prion, contenant des Séquences en acides aminés de Faible Complexité (SFC), ont tendance à s’agréger et à former des compartiments non-membranaires dans la cellule. Ces derniers ont des propriétés physiques communes à celles des liquides, telles que la capacité de mouiller les surfaces, de s’écouler et de fusionner avec d’autres corps liquides. Dans cette étude, nous avons démontré que la protéine Hrp1 forme, in vitro, des gouttes de différentes tailles via une transition de phase liquide à liquide, et ce, uniquement lorsqu’elle est exposée à un milieu chargé négativement. Exclusivement dans ce même milieu, nous avons aussi observé que le domaine SFC de Hrp1 s’assemble et forme une matière de type gel. Sur la base de ces observations, nous avons émis l’hypothèse que la tendance des systèmes moléculaires à former des compartiments liquides in vivo peut être influencée par la présence, dans le cytosol, de polyélectrolytes chargés négativement tels que l'ADN, l'ARN et les polyphosphates (PolyP). En utilisant la levure comme modèle cellulaire et des techniques de microscopie à fluorescence, nous nous sommes focalisés sur l’étude du rôle des PolyP dans l'assemblage des P-bodies. Les P-bodies ont été choisis comme système moléculaire de référence in vivo, étant des corps qui, après une transition de phase, se trouvent dans le cytosol sous forme de gouttes. Nous avons démontré que la déplétion du phosphate et la délétion du gène vtc4, responsable de la synthèse des PolyP dans la levure, n’ont pas d’influence dans la formation des P-bodies. Nous avons aussi remarqué que les PolyP et la protéine Edc3, une des composantes principales des P-bodies, ne sont pas co-localisés dans la cellule. Cette étude préliminaire nous suggère un manque de corrélation entre la formation des P-bodies et la présence de PolyP dans la cellule. Cependant, pour confirmer nos observations, des expériences complémentaires doivent être envisagées, en considérant d’autres composantes des P-bodies, tel que Lsm4, ou en analysant, in vivo, les effets des PolyP sur d’autres systèmes moléculaires de nature liquide. / Prion-like proteins containing Low Complexity Sequences (LCSs) have the propensity to aggregate and form membrane-less compartments in the cell. These proteins form droplets that have liquid features such as wetting, dripping and fusion. In this study, we demonstrated that the prion domain-containing protein Hrp1 forms droplets of different sizes in the presence of negatively charged polymers via liquid-liquid phase separation, whereas under the same conditions, the prion-like domain PolyQ/N of Hrp1 forms a gel-like material. Based on these findings, we hypothesize that droplets in vivo could be modulated by negatively charged polyelectrolytes found in the cell such as DNA, RNA and polyphosphate (PolyP). My goal was to examine the role of the polyanionic nature of PolyP on the assembly of P-bodies using Saccharomyces cerevisiae as a cellular model and fluorescence microscopy. We chose to study processing (P)- bodies, based on previous findings that these cellular subcompartments are formed by liquid-liquid phase separation of component proteins in the cytoplasm. We found that depleting phosphate from the media and deleting vtc4 gene, which is responsible for PolyP synthesis, did not have any effect on P-body formation. In addition, we demonstrated that PolyP and the protein Edc3, a core component of P-bodies, do not colocalize. Our data suggest that PolyP does not affect P-body formation. However, further and complementary studies have to be performed to confirm that PolyP have no effects on other membrane-less organelles.