<div>Mesenchymal stem cells (MSCs) are responsive to mechanical stimuli that play an essential role in directing their differentiation to the chondrogenic lineage. A better</div><div>understanding of the mechanisms that allow MSCs to respond to mechanical stimuli is important to improving cartilage tissue engineering and regenerative medicine. Hydrostatic pressure (HP) in particular is known to be a primary mechanical force in joints. However, little is known about the underlying mechanisms that facilitate HP</div><div>mechanotransduction. Understanding the signaling pathways in MSCs in transducing HP to a beneficial biologic response and their interrelationship were the focus of this thesis. Studies used porcine marrow-derived MSCs seeded in agarose gel. Calcium ion Ca++ signaling, focal adhesion kinase (FAK) involvement, and sirtuin1 activity were investigated in conjunction with HP application.</div><div><br></div><div><div>Intracellular Ca++ concentration was previously shown to be changed with HP application. In our study a bioreactor was used to apply a single application of HP to the MSC-seeded gel structures and observe Ca++ signaling via live imaging of a fluorescent calcium indicator in cells. However, no fluctuations in Ca++ concentrations were observed with 10 minutes loading of HP. Additionally a problem with the biore actor design was discovered. First the gel was floating around in the bioreactor even without loading. After stabilizing the gel and stopping it from floating, there were still about 16 µm of movement and deformation in the system. The movement and deformation was analyzed for the gel structure and different parts of the bioreactor. </div><div><br></div><div>Furthermore, we investigated the role of FAK in early and late chondrogenesis and also its involvement in HP mechanotransduction. A FAK inhibitor was used on MSCs from day 1 to 21 and showed a dose-dependent suppression of chondrogenesis. However, when low doses of FAK inhibitor added to the MSC culture from day 21 to 42, chondrogenesis was not inhibited. With 4 hour cyclic HP, FAK phosphorylation increased. The beneficial effect of HP was suppressed with overnight addition of the</div></div><div><div>FAK inhibitor to MSC medium, suggesting FAK involvement in HP mechanotransducation by MSCs.</div></div><div><br></div><div>Moreover, sirtuin1 participation in MSC chondrogenesis and mechanotransduction was also explored. The results indicated that overnight sirtuin1 inhibition increased chondrogenic gene expression (Agc, Col2, and Sox9) in MSCs. Additionally, the activity of sirtuin1 was decreased with both 4 hour cyclic hydrostatic pressure and inhibitor application. These two together demonstrated that sirtuin1 inhibition enhances chondrogenesis.</div><div><br></div><div><div>In this research we have investigated the role of Ca++ signaling, FAK involvement, and sirtuin1 activity in the mechanotransduction of HP in MSCs. These understandings about the mechanisms regulating the chondrogenesis with respect to HP could have important implications for cartilage tissue engineering and regenerative studies.</div></div>

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O estudo das propriedades eletrÃnicas, mecÃnicas e vibracionais dos nanotubos de carbono com poucas camadas à importante para desenvolver e aprimorar os modelos que descrevem o comportamento dos nanotubos de parede mÃltipla, que à o tipo de nanotubo mais usado em aplicaÃÃes tecnolÃgicas. Diversos estudos teÃricos e experimentais tÃm sido realizados visando o entendimento do comportamento dos nanotubos de carbono de paredes simples e dupla (SWNT e DWNT, respectivamente) submetidos a altas pressÃes hidrostÃticas. Entretanto, o comportamento de nanotubos de carbono de parede tripla (TWNTs) sob tais condiÃÃes à ainda desconhecido. Neste trabalho, apresentamos um estudo de espectroscopia Raman ressonante em TWNTs em funÃÃo da pressÃo hidrostÃtica. Utilizamos Ãleo de parafina (Nujol) e uma mistura de metanol:etanol na proporÃÃo de 4:1 como meios transmissores de pressÃo (PTM) e uma cÃlula de pressÃo de bigorna de diamante (DAC-Diamond Anvil Cell) para a aplicaÃÃo da pressÃo. A evoluÃÃo dos modos radiais (RBM) e tangenciais (Banda G) em funÃÃo da pressÃo foi analisada de forma detalhada e comparada com os resultados existentes para os nanotubos de parede simples e dupla. Os dados de espectroscopia Raman mostram que os efeitos de blindagem internos dos TWNTs sÃo mais acentuados que nos DWNTs. As modificaÃÃes nas intensidades dos modos Raman foram interpretadas como sendo associadas as mudanÃas nas condiÃÃes de ressonÃncia dos modos RBM em funÃÃo da pressÃo. / The study of the electronic, mechanical and vibrational properties of the carbon nanotubes with few layers is important for developing and improving models that could describe the behavior of multi-walled carbon nanotubes (MWNTs), which is the kind of nanotube most used in technological applications. Several theoretical and experimental studies have been conducted for understanding the behavior of single and double walled carbon nanotubes (SWNT and DWNT, respectively) under high hydrostatic pressures. However, the behavior of triple walled carbon nanotubes (TWNTs) under such conditions is still unknown. Here, we present a study of resonance Raman spectroscopy in TWNTs as function of hydrostatic pressure. We used paraffin oil and a mixture of methanol:ethanol in 4:1 ratio as pressure transmitting media (PTM) and a diamond anvil cell (DAC) for applying pressure. The evolution of the radial breathing modes (RBM) and the tangential modes (G band) as a function of pressure was analyzed in detail and compared with existing results for SWNTs and DWNTs. The Raman spectroscopy data show that the shielding effects of inner tubes in TWNTs are more pronounced than in DWNTs. The changes in the intensities of Raman modes were interpreted as being associated with pressure induced changes in the resonance conditions.

A produção de proteínas recombinantes é uma ferramenta essencial para a indústria biotecnológica e suporta a expansão da pesquisa biológica moderna. Uma variedade de hospedeiros pode ser utilizada para produzir estas proteínas e dentre eles, as bactérias E. coli são as hospedeiras mais utilizadas. No entanto, a expressão heteróloga de genes em E. coli frequentemente resulta em um processo de enovelamento incompleto que leva ao acúmulo de agregados insolúveis, conhecidos como corpos de inclusão (CI). Altas pressões hidrostáticas são capazes de desfavorecer interações intermoleculares hidrofóbicas e eletrostáticas, levando à dissociação dos agregados e por isso são úteis para solubilizar e renaturar proteínas agregadas em CI. O presente trabalho teve como objetivo o estudo do processo de desagregação dos CI e de renaturação das proteínas oligoméricas subunidade B da toxina colérica (CTB) e região globular da fibra adenoviral (RGFA) utilizando altas pressões hidrostáticas. A toxina colérica (CT) é composta por uma subunidade A e cinco subunidades B combinadas em uma holotoxina AB5. A CTB é a porção pentamérica não tóxica da CT, responsável pela ligação da holotoxina ao receptor gangliosídeo GM1. A fibra do adenovírus é uma proteína homotrimérica que forma parte do capsídeo viral, organizada em três regiões: a cauda N-terminal, a haste central e a região C-terminal (região globular). A RGFA se liga à proteína de membrana CAR nas células hospedeiras e promove a internalização do vírus. Os estudos apresentados neste trabalho demonstraram que a alta pressão hidrostática foi eficaz na desagregação dos CI da CTB e da RGFA. As condições de renaturação foram otimizadas utilizando-se diferentes proporções do par redox glutationa oxidada e reduzida, concentrações de agentes caotrópicos, presença de aditivos e esquemas diferenciados de compressão/descompressão daqueles previamente descritos na literatura. CTB solúvel e pentamérica foi obtida pela compressão da suspensão de CI a 2,4 kbar por 16 horas em tampão TrisHCl 50 mM pH 8,5, 1 mM de tween 20 e descompressão direta seguida de incubação em pressão atmosférica. O rendimento de renaturação da CTB solúvel e pentamérica foi de até 45 % e 288 mg de CTB/litro de cultura bacteriana. Esta proteína apresentou estrutura regular e atividade biológica. RGFA trimérica foi obtida pela compressão da suspensão de CI em tampão TrisHCl 50 mM pH 8,0 e 0,5 M de L-arginina a 2,4 kbar por 1,5 horas e 0,4 kbar por 16 horas antes da completa descompressão. O rendimento de proteína solúvel trimérica da RGFA foi de 4 %, porém não foi possível obter a atividade biológica desta proteína. / The production of recombinant proteins is an essential tool for the biotechnology industry and supports the expansion of modern biological research. Recombinant proteins can be produced by a variety of hosts and among them the bacteria E. coli is the most commonly used. However, the expression of heterologous genes in E. coli often results in an incomplete folding process that leads to the accumulation of insoluble aggregates known as inclusion bodies (IB). The application of high hydrostatic pressure impairs intermolecular hydrophobic and electrostatic interactions of proteins in solution, leading to dissociation of aggregates and is therefore useful tool to solubilize and refold aggregated proteins in IB. This work aimed to study the process of disaggregation of IB and refolding of oligomeric proteins the B subunit of cholera toxin (CTB) and the globular region of the adenoviral fiber (RGFA) using high hydrostatic pressure. The cholera toxin (CT) comprises one A subunit and five B subunits, combined in the AB5 holotoxin. The pentameric CTB is non-toxic moiety of CT which is responsible for binding to the receptor ganglioside GM1 holotoxin. The adenovirus fiber is a homotrimeric protein wich forms part of the viral capsid and it is organized into three regions: the N-terminal tail, the central rod and the C-terminal region (globular region). The RGFA binds to membrane protein CAR in host cells and promotes the internalization of virus. The studies presented here demonstrate that high hydrostatic pressure was effective in the disaggregation of the CTB and RGFA IB. The refolding conditions were optimized using different proportions of the redox couple oxidated and reduced glutathione, concentrations of chaotropic agents, presence of additives and pressure/decompression schemes distinguished from the previously described in the literature. Soluble pentameric CTB was obtained when the suspension of IB were compressed at 2.4 kbar for 16 hours in 50 mM of Tris-HCl buffer pH 8.5, 1 mM of tween 20, followed by direct decompression and incubation at atmospheric pressure. The yield of refolded soluble pentameric CTB was up to 45 % and 288 mg of CTB/ liter of bacterial culture. This protein was shown to presented regular structure and biological activity. Trimeric RGFA was obtained by compression of the suspension of IB in 50 mM of Tris-HCl buffer pH 8.0, 0.5M L-arginine at 2.4 kbar for 1.5 hours and at 0.4 kbar for 16 hours prior to the complete decompression. The yield of soluble trimeric RGFA was 4 %, however this protein did not present biological activity.

No presente trabalho estudamos a renaturação sob alta pressão hidrostática de uma forma mutante da proteína verde fluorescente (enhanced GFP, eGFP), a qual somente emite fluorescência característica quando enovelada na sua forma nativa. A abordagem do presente estudo foi focada no controle da bioatividade da proteína recombinante, a fluorescência, como alternativa à determinação de solubilidade da proteína, fator que não é um indicador ideal de enovelamento proteico adequado. A ação da alta pressão na solubilização dos corpos de inclusão (CI) de eGFP produzidos em bactérias E. coli recombinantes e no enovelamento da proteína foi estudada. A compressão dos CI de eGFP em 2,4 kbar durante 30 minutos promoveu a dissociação dos agregados. No entanto, a incubação nesta condição não favoreceu o enovelamento da eGFP. O processo de renaturação foi avaliado em diversas condições de descompressão após a dissociação em 2,4 kbar. Durante a descompressão gradual, o aumento da fluorescência foi obtido em pressões que variaram entre a pressão atmosférica e 1,38kbar. Os níveis mais elevados de fluorescência de eGFP foram obtidos por incubação durante várias horas a níveis de pressão entre 0,35 e 0,69 kbar. Esta condição de pressão se mostrou favorável à renaturação de eGFP e é possível que também possa ser utilizada para favorecer o enovelamento de outras proteínas monoméricas. Ainda utilizando a eGFP como modelo, verificamos que os CI desta proteína produzidos por bactérias cultivadas em menor temperatura (37ºC) possuem maior quantidade de proteína recombinante apresentando a fluorescência característica em 509 nm, ou seja, na sua forma nativa, do que os CI expressos em temperaturas mais elevadas (42ºC e 47ºC). A análise realizada por espectroscopia de infravermelho (FT-IR) também demonstrou que os CI produzidos em temperaturas mais brandas possuem maior grau de estruturas secundárias semelhantes às da proteína na sua forma nativa. Além disso, os CI produzidos a 37ºC também são mais facilmente solubilizados pela ação da alta pressão do que aqueles produzidos em maior temperatura. Conforme esperado, a renaturação da eGFP a partir de CI produzidos a 37ºC foi 25 vezes mais eficiente do que a obtida utilizando CI produzidos a 47ºC. No presente estudo demonstramos também que a dissociação dos agregados exercida pela ação da alta pressão (2,4 kbar) pode ser amplificada quando em associação com a incubação em baixa temperatura (-9ºC) e que a combinação destas duas propriedades físicas eleva a solubilização dos agregados em CI, com a consequente elevação dos rendimentos de renaturação de eGFP. Mostramos ainda no presente estudo que a cinética de renaturação de eGFP em 0,69 kbar é proporcional à temperatura de incubação (entre 10ºC e 50ºC). O nível mais elevado de fluorescência foi obtido quando a renaturação de eEGP foi realizada a 20ºC. A taxa de maturação do cromóforo da eGFP é mais fortemente afetada pela temperatura do que a taxa de enovelamento da proteína. Em conclusão, a temperatura de produção dos CI, a temperatura de dissociação dos agregados e a temperatura de enovelamento podem afetar muito o rendimento e a cinética da renaturação de eGFP em alta pressão. Os resultados do presente estudo podem abrir novas perspectivas para melhorias no processo de enovelamento de proteínas a partir de CI utilizando alta pressão. Também neste trabalho descrevemos a renaturação das proteínas de Xac, PilB e os produtos dos genes XAC2810 e XAC3272 nunca antes obtidas na forma solúvel. Os rendimentos de solubilização destas três proteínas foram muito altos, entre 75% e 89%. A proteína PilB renaturada em alta pressão apresentou atividade ATPasica elevada, o que nunca antes foi demonstrado para a PilB de Xac. / In the present work we studied the refolding under high hydrostatic pressure of a mutant form of the green fluorescent protein (eGFP), which only emits the green characteristic fluorescence when in the native folded state. The approach of the present study was focused on controlling the bioactivity of the recombinant protein, the fluorescence, as an alternative for the determination of protein solubility, which is not an ideal indicator of proper protein folding. We studied the action of high pressure in the solubilization of the inclusion bodies (IB) of eGFP produced in bacteria E. coli and in the folding of this protein. The compression of a suspension of eGFP IB at 2.4 kbar for 30 minutes promoted dissociation of aggregates. However, the eGFP folding, monitored by the fluorescence at 509 nm, does not occur in this pressure level. The process of eGFP refolding was evaluated under various decompression conditions after dissociation of the IB at 2.4 kbar. During the gradual decompression, the increase in fluorescence was achieved at pressures ranging between atmospheric pressure and 1.38 kbar. The higher levels of eGFP fluorescence were obtained by incubation for several hours at pressure levels between 0.35 and 0.69 kbar. It is possible that the pressure condition that proved favorable for refolding of eGFP can also be used to favor the folding of other monomeric proteins. Using eGFP as a model, we also found that the IB produced by bacteria grown in a relatively low temperature (37ºC) is more fluorescent, presenting a higher amount of recombinant protein with the characteristic fluorescence at 509 nm, i.e., in its native form, than the IB expressed at higher temperatures (42ºC and 47ºC). The analysis by infrared spectroscopy (FT-IR) also demonstrated that the IB produced at milder temperatures have a higher degree of secondary structure similar to the protein in its native form. Furthermore, the IB produced at 37ºC are also more readily solubilized by the action of high pressure than those produced at the higher temperatures. As expected, the folding of eGFP from IB produced at 37ºC was 25 times more efficient than that obtained using IB produced at 47ºC. In this study we demonstrated that the dissociation of aggregates exerted by the action of high pressure (2.4 kbar) can be amplified by combination with incubation at low temperature (-9ºC) and the association of these two physical properties can be used to increase the solubilization of the aggregates in IB, with a consequent increase in the yield of eGFP refolding. In the present study we also showed that the kinetics of refolding of eGFP is proportional to temperature (10ºC 50ºC). The higher level of fluorescence was obtained when the refolding of eGFP was performed at 20°C. The rate of maturation of the eGFP chromophore is more strongly affected by temperature than the rate of folding of the protein. In conclusion, the temperature of production of IB, the temperature of dissociation of aggregates and the folding temperature can greatly affect the yield and kinetics of refolding of eGFP at high pressure. The results of this study may open new perspectives for improvements in the process of protein folding from IB using high pressure. In this paper we also describe the refolding of the proteins of Xac, PilB and the gene products XAC2810 and XAC3272, which have never before been achieved in soluble form. The yields of solubilization/refolding of these three proteins were very high, between 75% and 89%. The protein PilB refolded at high pressure presented high ATPase activity, which has never been shown for the PilB of Xac.

Les modèles d'ascendance convective utilisés actuellement dans les paramétrisations de la convection ne prennent pas en compte explicitement le terme de pression non-hydrostatique, bien que son rôle fondamental soit documenté et expliqué depuis les années 70. Pour palier cette limitation, un modèle anélastique pronostique à deux colonnes d'une ascendance convective,prenant en compte explicitement le terme de pression non-hydrostatique, a été développé avec une géométrie 2D et 3D. Des fonctions de forme sont introduites pour réduire l'impact de la basse résolution horizontale. Trois paramètres doivent être prescrits : la hauteur et le rapport d'aspect de l'ascendance ainsi que la taille totale de la cellule convective. Le modèle est évalué en utilisant des profils de flottabilité idéalisés et constants, puis en s'appuyant sur une simulation LES de développement diurne de la convection profonde. Le comportement du modèle est en accord avec notre connaissance de la pression non hydrostatique à l'intérieur des nuages et de son rôle.L'ascendance simulée atteint rapidement un état stationnaire (5 min) en réponse au champ de flottabilité. Ainsi une version diagnostique a été développée, confirmant les résultats de la version pronostique. Sous le cœur de l'ascendance, un gradient de pression dirigé vers le bas est simulé permettant à l'ascendance de franchir la barrière d'inhibition convective. La future implémentation de ce modèle dans un schéma de convection, pour remplacer les formulations des modèles d'ascendance actuelles, devrait permettre d'augmenter la durée des évènements convectifs. / A simple anelastic 2-columns pronostic model of convective updraft accounting explicitly for thenon-hydrostatic pressure term is developed with 2D and 3D geometries. Shape fonctions areintroduced to overcome the low horizontal resolution. Three parameters must be prescribed :updraft depth, aspect ratio and total cell size. The model is tested for constant idealized buoyancyprofiles and successfully evaluated against a LES simulation of daytime development of deepconvection. The model behaviour agrees with our understanding of the non-hydrostatic pressurewithin clouds. The simulated updraft quickly reaches a steady state (5 min) in response to thebuoyancy field. So a diagnostic version has also been developped, confirming results of thepronostic version. Below an updraft core, a downward pressure gradient is simulated allowing theupdraft to overcome a barrier of convective inhibition. The further implementation of this modelwithin a convection scheme to replace a drag formulation of the updraft model, is expected toincrease the duration of convective events.

The objective of this work is to optimize high-power low-frequency sonication (US) pretreatment of sludge, and especially to investigate for the first time possible improvements by higher pressure and audible frequency. After a preliminary examination of regular process conditions (sludge conditioning, sludge type, prior alkalization, temperature control, etc), effects of US parameters (power -PUS, intensity -IUS, specific energy input -ES, frequency -FS, etc.) and of hydrostatic pressure (Ph) were specifically looked into, separately and in combination, first under cooling at constant temperature (28°C), then under the progressive temperature rise provoked by sonication. First, it was confirmed that specific energy input (ES) plays a key role in sludge US disintegration (i.e. solubilisation of organic matter) and that temperature rise during adiabatic-like sonication is beneficial through additional effects of thermal hydrolysis and cavitation. At a given ES value, low FS (12 kHz vs. 20 kHz) and high PUS enhance soluble chemical oxygen demand (SCOD) due to more violent cavitation, while hydrostatic pressure gives rise to an optimum value due to its opposite effects on cavitation threshold and intensity. One major result is that optimal pressure depends on IUS (P¬US) as well as temperature profile, but not on ES, FS, nor sludge type. Setting the other parameters at the most favorable conditions expected, i.e. 12 kHz, 360 W , 28 gTS/L, and adiabatic conditions, final optimization was achieved by searching for this pressure optimum and examining sequential procedure to avoid too high temperature dampening cavitation intensity and damaging the transducer. Such conditions with sequential mode and Ph of 3.25 bar being selected succeeded in achieving very high SCOD, but only marginally improved subsequent methanization yield.

The objective of this work is to develop an innovative and quantitative method to study cell failure under fluidic pressure to understand cell membrane mechanical properties. Due to lack of experimental data related to cell failure property, the current research focuses on investigating the cell failure using a micro pipette aspiration experiment method to elaborate gradually increasing hydrostatic pressure to the cell causing the membrane to deform and eventually rupture. Based on our observation, the prostate cancer cells (PC-3) deformed into a deflated and flattened shape under higher hydrostatic pressure (249 Pa) while prostate epithelial cells (PrEC LH) cells generate a spherical and rounded shape. The stress along the cell membrane was estimated from the curvature data captured from the 2D microscopic images for each pressure magnitude to quantify the damage before rupture state. From the results, non-transformed prostate epithelial cells (PrEC LH) presented a stiffer and rupture resilient property compared to transformed prostate cancer cells (PC-3) which presented a softer and vulnerable property. Besides, the alteration of shape of the aspirated membrane directly affected the stress distribution over the membrane and as a result, provoked membrane failure. Multiple pieces of research have shown a higher stiffness of healthy cells compared to cancer cells including one of the previous studies done by our group which have also found that cancer cell tends to become stiffer after exposing to fluid shear stress. The discovery of this cellular behavior and novel numerical quantification method of cell failure could advance the study of cancer cell membrane failure, cellular matrix structure, response to mechanical loadings and potentially foundation in developing new treatment for cancer other than destructive chemical treatment.

No description available.

The aim of this study was to investigate the effect of high hydrostatic pressure (HHP) treatment (200, 300, 400 MPa / 5

The objective of this thesis is to predict lining thickness inside circular mine shafts. A numerical study with different rock mass strengths and different in-situ non-hydrostatic stresses are carried out in 2D shaft section models to predict pressures that develop on lining support. An iterative process of applying support pressure until observing no failure zone around shaft is used to simulate lining support pressure for each individual model. Later, regression and fuzzy logic analyses are carried out to find a pressure equation for all of the models. Finally, the pressure equation derived is used in elastic &ldquo / thick-walled cylinder&rdquo / equation to calculate the lining thickness required to prevent the development of a failure zone around shafts. At the end of this research, a computer program &ldquo / Shaft 2D&rdquo / is developed to simplify the lining thickness calculation process.