Ion channels play a critical role in maintaining homeostasis by moving various ions in and out of cells. The Na+-K+-2Cl- or NKCC1 ion channel is involved in the regulation of Na+, K+, and Cl- across cell membranes, and plays a key role in many forms of cellular physiology. In the cochlea, NKCC1 is involved in endolymph production and maintenance of the endocochlear potential. Our hypothesis is that blocking NKCC1 channels should directly impact auditory sensitivity causing hearing loss. Our lab has also shown that the hormone aldosterone (ALD) can upregulate NKCC1 protein expression in vitro and in vivo. In the present investigation, we use electrophysiology and molecular biology techniques to study the biophysical mechanisms underlying the action of ALD in vitro on NKCC1 in the SH-SY5Y cell line. Our initial protein expression studies using RT-PCR found that proteins specific to NKCC1channels were present in SH-SY5Y neuronal cells. Whole cell currents measured using patch clamp methodology, were used to analyze the effects of various compounds on NKCC1 in the SH-SY5Y cell line. Control data were collected under perfusion of extracellular solution (ECS), then ECS containing 10µM bumetanide was applied, and, finally a washout condition completed the experiment. Similar experiments were conducted using ALD, and we observed an increase in K+ currents when bumetanide as well as when ALD was applied. This is the first report that indicates that ALD can directly regulate K+ channels in SH-SY5Y cells.

In an effort to help understand the mechanical properties of human platelets, their deformations were measured using micropipette experiments over an aspiration pressure range of 1-5 cmH2O, in steps of 1 cmH2O. The experiments confirmed the previously reported linear relationship between deformation and pressure. The experimental results were used to determine the material constants of a thin-axisymmetric shell model based on a strain-energy constitutive relation to describe the platelet deformations under aspiration. The model was successful in capturing the experimental deformations. It also suggested that the mechanical properties of human platelets are not significantly influenced by their volumes, but do vary depending on the platelets’ undeformed shape ratios. In addition, the model suggested that platelet membrane ruptures due to micropipette aspiration may be strain-related. The limitations of the experimental methods arising from direct contact with reactive cells such as platelets are highlighted, prompting the need for developing new methods which will not require the use of inhibition agents that alter the platelets’ mechanical properties. Afin d’approfondir les connaissances des propriétés mécaniques des plaquettes humaines, leurs déformations ont été mesurées lors d’expériences avec des micropipettes pour des pressions d’aspiration de 1-5 cmH2O, par intervalles de 1 cmH2O. Les expériences ont confirmé la relation linéaire entre les déformations et la pression d’aspiration telle que précédemment publié. Les données expérimentales ont été utilisées pour déterminer les constantes matérielles d’un modèle de membrane mince axisymétrique basé sur une loi de comportement caractérisant l’énergie de déformation. Le modèle simule bien les déformations des plaquettes sous aspiration; il suggère également que les propriétés mécaniques des plaquettes humaines ne sont pas influencées significativement leur volume, mais varient en fonction de leurs formes avant déformation. De plus, le modèle suggère que les ruptures de la membrane des plaquettes sous aspiration seraient reliées aux déformations. Les limites des méthodes expérimentales utilisées, du fait du contact direct avec des cellules aussi réactives que les plaquettes sont soulignées, et mettent l’emphase sur le besoin de mettre au point de nouvelles méthodes ne requérant pas d’agents d’inhibitions qui altèrent les propriétés mécaniques des plaquettes.

No description available.

No description available.

Calcific aortic valve disease (CAVD) is associated with significant cardiovascular morbidity. While late-stage valve disease is well-described, there remains an unmet scientific need to elucidate early pathobiological processes. In CAVD, pathological differentiation of valvular interstitial cells (VICs) and lesion formation occur focally in the fibrosa layer. This VIC pathological differentiation has been shown to be influenced by matrix stiffness in vitro. However, little is known about the focal layer specific mechanical properties of the aortic valve in health and disease and how these changes in matrix moduli may influence VIC pathological differentiation in vivo. In this thesis, micropipette aspiration (MA) was shown to be capable of measuring the mechanical properties of a single layer in multilayered biomaterial or tissue such as the aortic valve, if the pipette inner diameter was less than the top layer thickness. With MA, the fibrosa of normal porcine aortic valves was significantly stiffer than the ventricularis; stiffer locations found only within the fibrosa were comparable to stiffnesses shown in vitro to be permissive to VIC pathological differentiation. Early CAVD was induced in a porcine model, which developed human-like early CAVD lesion onlays. Extracellular matrix remodeling occurred in the absence of lipid deposition, macrophages, osteoblasts, or myofibroblasts, but with significant proteoglycan-rich onlays and chondrogenic cell presence. These early onlays were softer than the collagen-rich normal fibrosa, and their proteoglycan content was positively correlated with Sox9 chondrogenic expression, suggesting that soft proteoglycan-rich matrix may be permissive to chondrogenic VIC differentiation. The findings from this thesis shed new light on early disease pathogenesis and improve the fundamental understanding of aortic valve mechanics in health and disease.

Dans la partie "adhésion", nous avons montré que la dynamique d'adhésion<br />de vésicules induite par des ligands spécifiques était gouvernée soit par la diffusion<br />de ligands dans la membrane, soit par le temps de réaction entre le ligand et le<br />récepteur, dépendant de la préparation chimique des surfaces. Au contraire, les<br />premières étapes de l'adhésion de cellules semblent être contrôlées par la<br />dissipation visqueuse à l'intérieur de la cellule.<br />Dans la partie "tubes de membrane", nous avons étudié la formation et<br />l'élongation de tubes de membrane. Tout d'abord, en formant des tubes à partir de<br />vésicules adhérées, nous avons montré que l'élongation des tubes s'accompagne<br />d'un étirement élastique de la membrane. Ensuite, en analysant expérimentalement<br />et théoriquement l'interaction et la coalescence de deux tubes membranaires, nous<br />avons proposé une méthode pour déterminer la rigidité de courbure de vésicules<br />lipidiques. Enfin, nous avons revisité la description mécanique de tubes extraits de<br />globules rouges et nous avons mis en évidence un comportement rhéofluidifiant de<br />la membrane durant l'élongation, indiquant l'influence du squelette de spectrine.

Calcific aortic valve disease (CAVD) is associated with significant cardiovascular morbidity. While late-stage valve disease is well-described, there remains an unmet scientific need to elucidate early pathobiological processes. In CAVD, pathological differentiation of valvular interstitial cells (VICs) and lesion formation occur focally in the fibrosa layer. This VIC pathological differentiation has been shown to be influenced by matrix stiffness in vitro. However, little is known about the focal layer specific mechanical properties of the aortic valve in health and disease and how these changes in matrix moduli may influence VIC pathological differentiation in vivo. In this thesis, micropipette aspiration (MA) was shown to be capable of measuring the mechanical properties of a single layer in multilayered biomaterial or tissue such as the aortic valve, if the pipette inner diameter was less than the top layer thickness. With MA, the fibrosa of normal porcine aortic valves was significantly stiffer than the ventricularis; stiffer locations found only within the fibrosa were comparable to stiffnesses shown in vitro to be permissive to VIC pathological differentiation. Early CAVD was induced in a porcine model, which developed human-like early CAVD lesion onlays. Extracellular matrix remodeling occurred in the absence of lipid deposition, macrophages, osteoblasts, or myofibroblasts, but with significant proteoglycan-rich onlays and chondrogenic cell presence. These early onlays were softer than the collagen-rich normal fibrosa, and their proteoglycan content was positively correlated with Sox9 chondrogenic expression, suggesting that soft proteoglycan-rich matrix may be permissive to chondrogenic VIC differentiation. The findings from this thesis shed new light on early disease pathogenesis and improve the fundamental understanding of aortic valve mechanics in health and disease.

Made available in DSpace on 2016-12-08T16:24:12Z (GMT). No. of bitstreams: 1 PGCV07MA018.pdf: 1132263 bytes, checksum: 6e10cf4471ceb93e5b44c3be1e5da2c8 (MD5) Previous issue date: 2007-02-17 / The main objective of Cryobiology is the optimization of an oocyte cryopreservation technique. It will permit to create germoplasm banks for maintenance of animal biodiversity, serving as an important instrument in transgenic, cloned or in vitro produced animals. It is consensus that the vitrification is the best method to cryopreserve oocytes and that high cooling rates improve the viability. To evaluate the use of carrier tools with different thermal conductivity in vitrification, 1.454 oocytes (Experiment 1) were 30 seconds exposed to 10% EG + 10% DMSO + 20% estrus mare serum (SEE) in TCM Hepes. Just after, groups of 5 or 6 oocytes were exposed (20 seconds) to vitrification solution (SV - 20% EG + 20% DMSO and 0.5M Sucrose). They were loaded in PM (stainless metallic straws, n=265), MV (glass micropipette, n=279), PB (straw bevel cuted, n=280), OPS (open pulled straw, n=272) or maintained without vitrification GC (control group n=358). Vitrification was obtained by plunging it in super cooled liquid nitrogen. In the experiment 2 (n=709) the aim was to evaluate bovine oocytes vitrification in reduced (25 and 50%) cryoprotectants concentration. Oocytes were 30 sec. exposed to the first cryoprotectant solution, and just after during 20 seconds to one of the allows vitrification solutions: SV100 (n=187), 20% EG + 20% DMSO + 0.5M Suc; SV75 (n=187), 15% of EG + 15% of DMSO and 0.375M of Suc; or SV50 (n=146), 10% of EG, 10% of DMSO + 0.25M Suc. A non vitrified group (n=189) was used as control. The re-warming was performed by exposure (5 minutes each) to decreasing sucrose solutions (0.30 and 0.15M), heated up to 35ºC. The oocytes were then maturated, fertilized, and the presumptive zygotes cultured in SOFaaci medium, at 39ºC, in saturated humidity. Cleavage, blastocysts and hatching rates were used as viability criteria. In the experiment 1 there was not differences in cleavage rates (p>0.05) among treatments, that were lower (P <0.05) than control group. In the vitrified groups, higher blastocyst rates were obtained with PM treatment (10.2%), that was lower (P <0.05) than the OPS (6.1%) and PB (6.1%) treatments, not differing from the MV group (8.0%), although with tendency of be higher (P <0.1). The treated groups had identical hatching rates, which were lower than the control group. In the second experiment, there was not difference in the cleavage rate among SV100 and SV75 treatments, being both higher (p <0.05) than the SV50 treatment. In the blastocyst rates, there was difference among all groups (P <0.05). The higher rate was obtained in control group (38.0%), followed by the SV100 (10.1%), SV75 (7.6%) and SV50 (0.5%) treatments, respectively. The hatching rates of SV100 and SV75 groups were similar, being lower (P <0.05) than control group. In the SV50 group none blastocyst hatched. We conclude that increasing cooling speed rates improves the viability of immature vitrified bovine oocytes, and that the metallic straws are more effective than recipients of lower conductivity (plastic straws), with a tendency of superiority when compared to the glass micropipette. Still, the reduction of the cryoprotectors in 25% or up does not allow an appropriate cryoprotection to vitrify bovine oocytes, in the conditions of this study / A otimização da técnica de vitrificação de oócitos é um dos principais objetivos da criobiologia, pois possibilitará a formação de bancos de germoplasma para manutenção da biodiversidade animal, além de se constituir num importante instrumento na produção de animais transgênicos, clones ou programas de produção in vitro de animais. É consenso que a vitrificação é o método mais adequado para criopreservar oócitos e que elevadas taxas de resfriamento melhoram a viabilidade da técnica. Para avaliar o uso de materiais com diferentes condutividade térmica na vitrificação (Experimento 1), 1454 oócitos foram utilizados. Inicialmente os oócitos foram expostos a 10% EG + 10% DMSO + 20% soro de égua em estro (SEE), em TCM Hepes, por 30 segundos. Após, grupos de 5 ou 6 oócitos foram submetidos à solução de vitrificação (SV) composta de 20% EG + 20% DMSO e 0,5M Sacarose, durante 20 segundos, período em foram envasados e mergulhados em nitrogênio super-resfriado. O envase foi realizado em PM (palheta metálica inoxidável, n=265), MV (micropipeta de vidro, n=279), PB (palheta cortada em bisel, n=280), OPS (open pulled straw, n=272). Simultaneamente, um grupo não vitrificado serviu como controle, (GC n=358). No Experimento 2, 709 oócitos foram vitrificados com concentrações reduzidas (25 e 50%) de crioprotetores. Inicialmente os oócitos foram expostos, por 30 segundos, a uma solução de 10% EG + 10% DMSO + 20% SEE, em TCM Hepes. Logo após, foram expostos, por 20 segundos, a uma das soluções de vitrificação: SV100 (n=187), 20% EG + 20% DMSO + 0,5M Sacarose; SV75 (n=187), 15% de EG + 15% de DMSO e 0,375M de sacarose; ou SV50 (n=146), 10% de EG, 10% de DMSO + 0,25M de sacarose, além de um grupo controle não vitrificado (n=189). O reaquecimento foi realizado pela exposição (5 minutos cada) às soluções decrescentes de sacarose (0,30 e 0,15M), aquecidas a 35ºC. Os oócitos foram então maturados e fecundados, e os prováveis zigotos cultivados em meio SOFaaci, à 39ºC. As taxas de clivagem, de blastocistos e eclosão foram utilizadas com critérios de viabilidade. No experimento 1 não foram verificadas diferenças (P>0,05) nas taxas de clivagem dos tratamentos, que foram inferiores (P<0,05) ao grupo controle. A maior taxa de blastocistos nos grupos vitrificados foi obtida com o tratamento PM (10,2%), que foi superior (p<0,05) aos tratamentos OPS (6,1%) e PB (6,1%), não diferindo do grupo MV (8,0%), embora com tendência de ser superior (p<0,1). Também as taxas de eclosão nos grupos tratados foram semelhantes, sendo inferiores as do grupo controle. No experimento 2, não houve diferença na clivagem entre os tratamentos SV100 e SV75, sendo ambos superiores (P<0,05) ao tratamento SV50. Na taxa de blastocistos, houve diferença entre todos os tratamentos (p<0,05). A maior taxa foi obtida com o grupo controle (38,0%), seguido do tratamento SV100 (10,1%), SV75 (7,6%) e SV50 (0,5%), respectivamente. As taxas de eclosão dos grupos SV100 e SV75 foram semelhantes, sendo inferiores (p<0,05) ao controle. No grupo SV50 não houve eclosão. Conclui-se que o aumento da velocidade de resfriamento melhora a viabilidade pós vitrificação de oócitos bovinos imaturos, sendo que as palhetas metálicas são mais efetivas que os recipientes de menor condutividade (palhetas plásticas), com uma tendência de superioridade quando comparada à micropipeta de vidro. Ainda, a redução dos crioprotetores em percentual igual ou superior a 25% não permite uma adequada crioproteção na vitrificação de oócitos bovinos, com a metodologia proposta neste estudo

No description available.

Caveolae sind charakteristische Plasmamembraneinstülpungen, die in vielen Zelltypen vorkommen und deren biologische Funktion umstritten ist. Ihre besondere Form und ihre Häu gkeit in Zellen, die stets mechanischen Belastungen ausgesetzt sind, führten zu der Annahme, dass Caveolae die Plasmamembran vor mechanischen Belastungen schützen und als Membranreservoir dienen. Dies sollte mit dieser Dissertation experimentell geprüft werden. Zunächst wurde der Ein uss der Caveolae auf die Membranspannung von Zellen im Normalzustand untersucht. Dann wurden die Zellen mechanisch belastet. Mit Fluoreszensmikroskopie wurde das Verschwinden von Caveolae nach Strecken der Zellen oder nach einem hypo-osmotischen Schock beobachtet. Messungen der Membranspannung vor und unmittelbar nach dem hypo-osmotischem Schock zeigten, dass Caveolae einen Anstieg der Membranspannung verhindern, unabhängig von ATP und dem Cytoskelett. Die Erzeugung von Membranvesikel mit Caveolae erlaubte es, diesen Effekt der Caveolae in einem vereinfachten Membransystem zu beobachten. Schliesslich wurden Muskelzellen untersucht. Zellen, die genetisch bedingt weniger Caveolae haben und mit Muskelschwundkrankheiten in Verbingung stehen, waren mechanisch weniger belastbar als gesunde Zellen. Zusammenfassend wird mit dieser Dissertation die These bestärkt, dass Caveolae einem Anstieg der Membranspannungen entgegenwirken. Dass dies in Zellen und in Vesikeln unabhängig von Energie und Cytoskelett geschieht, lässt auf einen passiven, mechanisch getriebenen Prozess schliessen. Diese Erkenntnis trägt zum Verständnis der Rolle von Caveolae in Zellen bei und kann dem besseren Verständnis von Krankheiten bedingt durch Caveolin-Mutationen, wie z.B. Muskelschwundkrankheiten, dienen. / Caveolae, the characteristic plasma membrane invaginations present in many cells, have been associated with numerous functions that still remain debated. Taking into account the particular abundance of caveolae in cells experiencing mechanical stress, it was proposed that caveolae constitute a membrane reservoir and bu er the membrane tension upon mechanical stress. The present work aimed to check this proposition experimentally. First, the in uence of caveolae on the membrane tension was studied on mouse lung endothelial cells in resting conditions using tether extraction with optically trapped beads. Second, experiments on cells upon acute mechanical stress showed that caveolae serve as a membrane reservoir bu ering surges in membrane tension in their immediate, ATP- and cytoskeleton-independent attening and disassembly. Third, caveolae incorporated in membrane vesicles also showed the tension bu ering. Finally, in a physiologically more relevant case, human muscle cells were studied, and it was shown that mutations with impaired caveolae which are described in muscular dystrophies render muscle cells less resistant to mechanical stress. In Summary the present work provides experimental evidence for the hypothesis that caveolae bu er the membrane tension upon mechanical stress. The fact that this was observed in cells and membrane vesicles in an ATP and cytoskeleton independent manner reveals a passive, mechanically driven process. This could be a leap forward in the comprehension of the role of caveolae in the cell, and in the understanding of genetic diseases like muscular dystrophies. / Cavéoles sont des invaginations caractéristiques de la membrane plas- mique présents dans beaucoup de types cellulaires. Ils sont liées à plusieurs fonctions cellulaires, ce qui sont encore débattues. Prenant compte de l importance des cavéoles dans les cellules soumises au stress mécanique, les cavéoles sont proposées de constituer un réservoir membranaire et de tamponner la tension membranaire pendant des stresses mécaniques. Cette étude a eu le but de tester cette hypothèse expérimentalement. En premier, l in uence des cavéoles sur la tension membranaire au repos a été étudiée sur des cellules endothéliales du poumon de la souris. Puis, on a montré que les cavéoles tamponnent l augmentation de la tension membranaire après l application d un stress mécanique. En suite, la réalisation des vésicules membranaires contenant des cavéoles a permit de montrer leur rôle comme réservoir membranaire dans un système simpli é. Finalement, dans un contexte physiologiquement plus relevant, l étude des cellules musculaires a montrée que les mutations du cavéolin associées aux dystrophies musculaires rendent les cellules moins résistante aux stresses mécaniques. En conclusion, cette étude supporte l\'hypothèse que les cavéoles tamponnent la tension membranaire pendant des stresses mécaniques. Le fait que cela se passe dans les cellules et les vésicules indépendamment d ATP et du cytosquelette révèlent un processus passif et mécanique. Cela pourrait servir à une meilleure compréhension du rôle des cavéoles dans la cellule et les maladies génétiques comme les dystrophies musculaires.