The actual role of programmed cell death (PCD) in embryonic processes and the extrinsic signals that define the death fate in developing cells are still poorly understood. Here, we show that during secondary palate shelf fusion in the mouse, PCD appeared in the medial edge epithelia (MEE) of the anterior region only after shelf contact. Contact was necessary for efficient cell death activation in the MEE. However, exogenous all-trans-retinoic acid (RA) increased cell death independently of contact. Competence to induce cell death by contact or by RA exposure was obtained when the MEE were close to touch. Endogenous RA is a relevant regulator of the secondary palate PCD since this was reduced by a retinol dehydrogenase inhibitor and an RAR specific antagonist. Bmp-7 expression was positively regulated by RA. However, BMP-7 was unable to activate cell death within the palate tissue and NOGGIN, a natural BMP antagonist, did not block PCD. Reduction of PCD at the MEE directly with a caspase inhibitor or by inhibiting retinol dehydrogenase resulted in unfused palate shelves, but adhesion was not affected. In contrast, exogenous RA also blocked fusion, but in this situation the increased cell death within the MEE appeared to affect adhesion, thereby causing cleft palate in vivo.

Programmed cell death (PCD) is considered one of the most important cellular processes in the morphogenesis of organs and tissues during animal development. Although the embryonic limb has been established as a classic model for the study of PCD, detailed studies on this process' contribution to morphogenesis are still lacking. In the present work, using modern computer-aided techniques, we estimated the contribution of PCD to mouse limb morphogenesis. For the detection of apoptotic cell death, we stained whole embryonic limbs with acridine orange or, in some instances, used the TUNEL technique, and visualized the tissues by confocal laser scanning microscopy. We found that cell death patterns are dynamic during limb development, and occur in gradients oriented with the main limb axes, anteroposterior, dorsoventral and distoproximal. Interdigital apoptosis in the autopod was initially detected at the most distal region, and then more proximally as development proceeded. Interestingly, we found that digit separation is more pronounced on the dorsal side, contrary to what is expected from the apoptotic cell distribution, which shows more abundant cell death in the ventral region. Using 2-D and 3-D models, we found that most digit individualization occurs rather by digit growth than by interdigital cell death. Therefore, digits do not mainly individualize by degeneration of preformed interdigital tissue, but probably by a dynamic balance between proliferation and cell death, reducing interdigital growth, which results in protrusion of digits. We determined the expression pattern of fgf-8 during the period of digit individualization, as the product of this gene could participate in defining the limb growth pattern. Initially, fgf-8 expression was coincident with the apical ectodermal ridge, but when cell death was first detected in the interdigits, fgf-8 expression became restricted to the tip of the growing digits. Therefore, FGF-8 could be one of the factors responsible for differential digit-interdigit growth, and might also act as a survival factor on interdigital tissue. We also found that the expression patterns of rar-␤, bmp-2, bmp-4, bmp-7, msx-1, and msx-2 genes, proposed to be involved in the activation of interdigital cell death, did not overlap with, or were not highly expressed in the major zones of cell death in the developing limb.

The effects that both soap concentration and base oil viscosity exert on the rheology of lubricating greases and its relationship with grease microstructure are discussed in this work. With this aim, different lubricating grease formulations were manufactured by modifying the concentration of lithium 12-hydroxystearate and the viscosity of the base oil, according to an RSM statistical design. These lubricating greases were rheologically characterized through small-amplitude oscillatory shear (SAOS) and viscous flow measurements. In addition to these, scanning electronic microscopy (SEM) observations and mechanical stability tests were also carried out. It has been found that the structural skeleton (size and shape of the disperse phase particles) was highly influenced by the base oil viscosity. In this sense, the values of the viscoelastic functions in the linear viscoelastic region and the mechanical stability of the lubricating greases increase as the viscosity of the base oil decreases. An opposite tendency was observed during viscous flow tests at high shear rates, when the grease microstructure was mostly destroyed. On the other hand, the microstructural network of these greases becomes stronger as soap concentration increases. These results have been explained taking into account the balance between the solvency of the thickener in the base oil and the level of entanglements formed by soap fibers, which influence the lubricating grease network.

Thermal-induced changes in the viscous and viscoelastic responses of lubricating greases have been investigated through different rheological techniques in a temperature range of 0-175°C. Small-amplitude oscillatory shear and viscous flow measurements were carried out on a model conventional lithium lubricating grease prepared by inducing the in situ saponification reaction between 12-hydroxystearic acid and hydrated lithium hydroxide. The linear viscoelasticity functions dramatically decrease above 110°C, but not below this critical temperature, which determines the maximum recommended operating temperature in relation to its durability and resistance under working conditions. Two different regions, below and above this critical temperature, in the plateau modulus versus temperature plot have been detected. From this thermal dependence, a much larger thermal susceptibility of the lubricating grease at temperatures above 110°C is apparent. The thermo-mechanical reversibility of this material has been studied by applying different combined stress-temperature protocols. Regarding the viscous flow, a minimum in the shear stress versus shear rate plots appeared at temperatures above 60°C, more pronounced as temperature increases, resulting from material instabilities. The experimental results obtained have been explained on the basis of the thermo-mechanical degradation of the lubricating grease microstructure.

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In this work, non-perturbed microstructures of several commercial and model lubricating greases, differing in nature and concentration of the thickener agent, were examined using the atomic force microscopy (AFM) technique. Grease microstructure mainly depends on the nature of the thickener employed and, also, on thickener concentration and viscosity of the base oil. Thermal-induced changes in the viscoelastic response of lubricating greases have been investigated by using different rheological techniques in a temperature range of 0-175°C. Small-amplitude oscillatory shear (SAOS) measurements were carried out to determine the mechanical spectra of the different samples studied. Lubricating grease rheological thermal susceptibility was analysed by following the evolution of the plateau modulus with temperature. SAOS functions dramatically decrease, in most cases, above a characteristic temperature, which depends on nature and/or concentration of the thickener used and therefore on grease microstructure. The thermo-mechanical reversibility of grease microstructure has been studied by carrying out triple-step-shear stress tests (shear stresses inside and outside of the linear viscoelasticity range), at different temperatures. The degree of lubricating grease non-reversible structural breakdown, which increases with temperature, depends on the shear stress applied above the linear viscoelasticity limits.