Comprendre l’imperméabilité cutanée : étude spectroscopique de mélanges modèles de la phase lipidique du stratum corneum

Le stratum corneum (SC), la couche la plus externe de l'épiderme des mammifères, agit comme une barrière dictant le taux d'absorption des molécules exogènes à travers la peau et empêchant la perte d'eau du corps. Le SC est principalement composé de cellules mortes aplaties et entièrement kératinisées (cornéocytes), noyées dans une matrice lipidique constituée fondamentalement de céramides, d'acides gras libres et de cholestérol. La diffusion transdermique des molécules est principalement liée aux lipides intercellulaires qui forment des membranes multilamellaires en phase solide. L'objectif principal de cette thèse est de mieux comprendre la relation entre la structure de la matrice lipidique et l'imperméabilité de la peau. La phase lipidique du SC comprend plus de 400 lipides différents. En raison de cette composition très complexe, des membranes modèles sont souvent utilisées pour l'étude des propriétés physico-chimiques des membranes, du comportement des lipides et de la perméabilité de différents produits chimiques. Nous avons d'abord déterminé comment la longueur des chaînes acyles d’acides gras libres influence le comportement de phase d'une matrice lipidique composée de céramide NS24, d'acide lignocérique (FFA24) ou palmitique (FFA16) et de cholestérol. Les propriétés structurales des membranes ont été examinées par 2H RMN et par spectroscopie infrarouge. Cette étude a montré que le comportement de phase de ces mélanges ternaires est fortement affecté par la longueur de l’acide gras. Nous avons trouvé que l'acide lignocérique avec le céramide NS24 et le cholestérol conduit à la formation d'un mélange plus homogène que celui qui inclut l'acide palmitique. De plus, le mélange ternaire contenant de l'acide lignocérique a montré une transition de la phase solide vers la phase gel lorsqu'il a été chauffé au-dessus de 37 °C, une caractéristique inhabituelle pour ce type de membranes modèles. La combinaison de lipides ordonnés et membranes homogènes est proposée comme un élément critique pour l’imperméabilité du SC. Deuxièmement, nous avons étudié un mélange plus complexe contenant du cholestérol, une série d'acides gras libres variant de 16 à 24 atomes de carbone et deux types de céramides : le céramide NS24 et le céramide EOS. Ce dernier est considéré comme un composant clé pour la formation de la phase de périodicité longue dans les membranes du SC native et modèles. Les résultats de spectroscopie 2H RMN, infrarouge, et Raman ont montré que l'acide gras libre et la chaîne acyle du céramide NS24 restent en phase solide à la température physiologique tandis que la chaîne oléate du céramide EOS entraîne la formation de domaines hautement désordonnés. Ces nanogouttelettes restent à l'état liquide jusqu'à -30 °C. La contrainte stérique imposée par la matrice lipidique cristalline est proposée être à l’origine de la difficulté de cristallisation des chaînes oléate des céramides EOS. Cette découverte modifie substantiellement la description structurale du SC et propose un nouveau rôle physiologique du céramide EOS, ce lipide étant un puissant modulateur de l'équilibre solide/liquide du SC. Ces travaux conduisent à réexaminer le mécanisme présentement proposé pour expliquer la perméabilité du SC, ainsi que l’effet d’agents transdermiques. Finalement, nous avons étudié l'interaction d’acides gras à très longue chaîne avec des membranes de 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphocholine (POPC) afin de déterminer comment s’adaptent ces acides gras aux contraintes spatiales. Trois acides gras différents avec une chaîne acyle variant de 16 à 24 atomes de carbone ont été utilisés : l’acide palmitique (FFA16), arachidique (FFA20) et lignocérique (FFA24). L'épaisseur d’un feuillet d’une bicouche de POPC correspond à la longueur de la partie hydrophobe de FFA16, et donc inférieure à la longueur de FFA20 et FFA24. La façon dont ces acides gras s'adaptent à la bicouche de POPC a été étudiée par 2H RMN et par simulations de dynamique moléculaire. Nous avons trouvé que la partie inférieure de la chaîne acyle de FFA24 protoné est désordonnée d’une façon similaire à ce qui a été observé pour la chaîne oléate du céramide EOS, sa chaîne acyle interagit avec la partie la plus fluide du feuillet opposé. Cette interdigitation de la fin de la chaîne acyle provoque un deuxième plateau observé dans les profils d’ordre (SC-D), une caractéristique qui est inhabituelle dans les systèmes lipidiques. Dans ce cas, le groupe carboxyle protoné de FFA24 était situé légèrement sous la tête polaire de la POPC. La déprotonation du FFA24 déplace la molécule vers l'interface aqueuse. Cette translation diminue la contrainte spatiale, augmente l'ordre de la chaîne acyle et entraîne la disparition du plateau correspondant au bout de la chaîne désordonnée. Les résultats présentés dans cette thèse contribuent à mieux comprendre comment la structure de la matrice lipidique du SC dicte l'imperméabilité de la peau. Nous avons montré des preuves spectroscopiques du comportement de phase de certains lipides importants du SC et suggéré un nouveau mécanisme pour la régulation de la diffusion transdermique des molécules.The stratum corneum (SC), the outermost layer of mammal epidermis, acts as a barrier dictating the rate of absorption of exogenous molecules through the skin and preventing water loss from the body. SC is mainly composed of flattened and fully keratinized dead cells (corneocytes), embedded in a lipid matrix, which is mostly constituted of ceramides, free fatty acids, and cholesterol. The transdermal diffusion of molecules is mainly related to the intercellular lipids, which form multilamellar membranes in the solid-crystalline phase. The main goal of this thesis is to better understand the relationship between the structure of the lipid matrix and the skin impermeability. SC lipid phase includes more than 400 different lipid species. Due to this very complex composition, model membranes are often used for the study of the physicochemical properties of membranes, the lipid behavior, and of the permeability of different chemicals. First, we determined how the length of the free fatty acid acyl chains influences the phase behavior of a lipid matrix composed of ceramide NS24, lignoceric (FFA24) or palmitic (FFA16) acid, and cholesterol. The structural properties of membranes were examined by 2H NMR and infrared spectroscopy. This study revealed that the phase behavior of these ternary mixtures is strongly affected by the length of the FFA. We found that lignoceric acid led to the formation of a more homogeneous mixture with ceramide NS24 and cholesterol, than the palmitic acid/ceramide NS24/cholesterol mixture. Also, the tertiary mixture containing lignoceric acid showed a transition from solid to gel phase when heated above 37 oC, an unusual feature for this type of model membranes. The combination of ordered lipids and homogeneous membranes is proposed as a critical element for SC impermeability. Second, we studied a more complex mixture containing cholesterol, a series of free fatty acids varying from 16 to 24 carbon atoms, and two types of ceramides: ceramide NS24 and EOS. The latter is considered a key component for the formation of the long periodicity phase in native and model SC membranes. The 2H NMR, infrared and Raman spectroscopy results showed that both the free fatty acid and the ceramide NS24 acyl chain remained in the solid-crystalline phase at physiological temperature while the oleate chain in ceramide EOS led to the formation of highly disordered domains. These liquid nanodrops remained in the liquid state down to -30 °C. The steric constraint imposed by the crystalline lipid matrix is proposed to prevent the crystallization of ceramide EOS oleate chains. This finding modifies the structural description of the SC substantially and proposes a novel physiological role of ceramide EOS as this lipid is a strong modulator of SC solid/liquid balance. The work leads to a re-examination of the mechanism currently proposed to explain the permeability of SC, as well as the effect of transdermal agents. Finally, we studied the interaction of very long-chain fatty acids with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes to determine how these fatty acids adapt to spatial constraints. Three different FFAs with acyl chain varying from 16 to 24 carbon atoms were used: palmitic (FFA16), arachidic (FFA20) and lignoceric (FFA24) acid. The leaflet thickness of a POPC bilayer corresponds to the length of the hydrophobic part of FFA16, and therefore is smaller than the length of FFA20 and FFA24. The way in which these fatty acids structurally adapt in POPC bilayers was study by 2H NMR and molecular dynamics simulations. We found that the lower part of the protonated FFA24 acyl chain was disordered in a manner similar to that observed for the oleate chain of the EOS ceramide, its acyl chain interacts with the more fluid part of the opposite leaflet. This interdigitation of the end of the acyl chain caused a second plateau observed in the order profiles (SC−D), an unusual feature in lipid systems. In this case, the protonated carboxyl group of FFA24 was located slightly below the polar head of the POPC. The deprotonation of the FFA24 shifted the molecule toward the aqueous interphase. This movement reduces the spatial constraints, increases the order of the acyl chain and causes the disappearance of the plateau at the end of the chain. The results presented in this thesis contributed to better understand how the structure of the SC lipid matrix dictates the skin impermeability. We showed spectroscopic evidences of the distinct phase behavior of some of the most important SC lipids. Furthermore, we suggested a novel mechanism for the regulation of transdermal diffusion of molecules

In Vitro Maturation and Fertilization of Oocytes: From Laboratory Bench to Clinical Practice

Retrieval of immature oocytes from non-stimulated ovaries, followed by in vitro maturation (IVM), was initially proposed in order to avoid side effects of gonadotropin administration. The goal is to eradicate or significantly decrease the risk of ovarian hyperstimulation syndrome (OHSS) in patients with polycystic ovary syndrome (PCOS) and to reduce drug cost and burden of patients. This technology was also proposed for treatment of normal ovulatory women, fertility preservation, or infrequent conditions as failure of oocyte to mature or repeated development of poor-quality embryos. There is no downregulation, and only a small amount of hormones are injected if at all. In vitro maturation of the oocyte procedure obtained up to 35% clinical pregnancy rate in young women, compared with in vitro fertilization (IVF) in many programs. The obstetric and perinatal outcomes of IVM cycles are comparable with IVF/ICSI cycles; therefore it may gradually substitute IVF in certain cases, as the technique continues to develop and pregnancy rates continue to increase. IVM holds great promises as an alternative to assisted reproductive technologies and may be the procedure of choice not only for infertile patients but also for obtaining oocytes for donation or fertility preservation

El desarrollo de la habilidad “resanar” en la especialidad Albañilería

Resumen El artículo tuvo como propósito, fundamentar el desarrollo de las habilidades profesionales en la especialidad Albañilería, así como la estrategia didáctica a seguir para desarrollar la habilidad “resanar” en los estudiantes de primer año de esa especialidad. Los resultados del diagnóstico revelaron insuficiencias, lo que incide de manera negativa en la formación de los obreros calificados para integrarse al mundo del trabajo, con los requerimientos que la sociedad demanda. Se emplearon métodos de investigación teóricos, empíricos y estadísticos que, con la determinación de dimensiones e indicadores, facilitaron la valoración de la estrategia didáctica aplicada. La significación práctica es aportada desde la estrategia didáctica, con cada una de las acciones propuestas en ella en cada una de las etapas, que le permitan a los estudiantes desarrollar la habilidad profesional “resanar” acorde a las exigencias del modelo del profesional

Composición de las disoluciones y parámetros de los procesos empleados en la aplicación de recubrimientos biomiméticos sobre titanio. Estado del arte

En años recientes, muchos investigadores han fijado su atención en procesos que ocurren en organismos vivos debido a que en algunos casos resulta posible reproducirlos in vitro. Basado en dicho comportamiento, Tadashi Kokubo y cols. desarrollaron un método que permite obtener recubrimientos apatíticos en superficies bioactivas a partir de su inmersión en un fluido corporal simulado. En el trabajo se presenta una actualización del estado del arte del tema, haciendo énfasis en la composición de las disoluciones y en los parámetros del proceso empleados durante el tratamiento del titanio y de sus aleaciones. Como resultado del estudio, se constató que existe la tendencia a incrementar el contenido de iones calcio y fosfato en las disoluciones con el objetivo de acelerar el proceso de formación y posterior crecimiento del recubrimiento y que se utilizan concentraciones de dichos iones que superan hasta en 10 veces las presentadas por el fluido corporal simulado. La inclusión de cloruro de sodio, de sales portadoras de magnesio y de hidrógenocarbonato en las disoluciones también puede incidir favorablemente sobre la velocidad de deposición del recubrimiento y paralelamente afectar la cristalinidad de las estructuras apatíticas resultantes del tratamiento. Elevados contenidos de iones hidrógenocarbonato y magnesio en las disoluciones pueden generar estructuras amorfas en los recubrimientos. El pH y la temperatura de exposición también pueden influir significativamente sobre la deposición de los recubrimientos, contribuyendo a acelerarla o a retardarla

Two Approaches to the Study of a Controversial Relationship: Cutaneous Photosensitivity and Anti-Ro/SS-A Autoantibodies

Background: Autoantibodies (Aabs) are the hallmark of numerous systemic autoimmune pathologies (SAPs), for instance anti-Ro/SS-A Aabs are usually found in Systemic Lupus Erythematosus (SLE) and Sjogren’s Syndrome. Cutaneous photosensitivity (CP) is found in most forms and subsets of LE and consists of a skin rash as a result of unusual reaction to sunlight. There are many theories which relate specifically the presence of circulating anti-Ro/SS-A Aabs with the CP phenomenon, though there are several studies which are in disagreement. Results: In this study we analyzed the relationship between CP and anti-Ro Aabs by means of two approaches. The first one included an in vitro model where we evaluated by flow cytometry the binding capacity of affinity-purified Aabs to autoantigens relocalized on apoptotic keratinocyte’s surface. We found that there was no relationship between the binding capacity of serum from 10 selected patients or their corresponding purified anti-Ro52 and anti-Ro60 Aabs, and the presence or absence of CP, neither with the SAPs. The in vivo model consisted of Hairless SKH:1 mice which were induced to produce anti-murine Ro52 and/or Ro60 Aabs and were subsequently irradiated with UVB light. We evaluated the skin histology and also the epidermal production of TNF-α. We found no differences between the groups in neither of the parameters evaluated. Conclusions: These results agree with some studies on the role of the Aabs in CP, considering anti-Ro Aabs not as the only responsible for the manifestation; and disagree with many other authors, who believe in the strong association between these two events.Fil: Paz, Mariela Laura. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Cela, Eliana Maiten. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Ferrari, Alejandro. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Friedrich, Adrian D.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Leoni, Juliana. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gonzalez Maglio, Daniel Horacio. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Semi-permeable Diffusion Barriers Enhance Patterning Robustness in the C. elegans Germline

Positional information derived from local morphogen concentration plays an important role in patterning. A key question is how morphogen diffusion and gene expression regulation shape positional information into an appropriate profile with suitably low noise. We address this question using a model system--the C. elegans germline--whose regulatory network has been well characterized genetically but whose spatiotemporal dynamics are poorly understood. We show that diffusion within the germline syncytium is a critical control of stem cell differentiation and that semi-permeable diffusion barriers present at key locations make it possible--in combination with a feedback loop in the germline regulatory network--for mitotic zone size to be robust against spatial noise in Notch signaling. Spatial averaging within compartments defined by diffusion barriers is an advantageous patterning strategy, which attenuates noise while still allowing for sharp transitions between compartments. This strategy could apply to other organs

Respuesta a la suplementación de microminerales a vaquillas en pastoreo de Zacate Estrella de África en la costa norte de Nayarit

En el Campo Experimental Pecuario "El Macho", Tecuala, Nay. México, con clima tropical Aw, fue realizado un estudio para medir la respuesta a la suplementación con microminerales a vaquillas que pastorearon en Estrella de Africa durante 280 días

Effect of chemical and thermochemical treatments on the surface properties of commercially pure titanium

Chemical and physical methods have been generally used for modifying the surface of titanium implants with the aim of achieving better osseointegration. In this work, an evaluation of the effect of different chemicals and thermochemical treatments on the surface modification of titanium was carried out. Plates of commercially pure titanium were polished and treated with four different methods. Scanning electron microscopy, atomic force microscopy, energy-dispersive X-ray spectroscopy, infrared spectroscopy, X-ray diffraction and drop shape analysis were used to characterize the modified titanium surface. Our results showed that the AEPTTT surface exhibited the highest Ra values followed by AEAT, AEPT, polished Ti and AE samples. In addition, the AEAT and AEPTTT treatments developed the largest surface area, while the AE treatment produced the lowest surface area. Furthermore, the AEAT treatment created the most hydrophilic surface, which is known to positively affect the osseointegration rate of dental implants.Keywords: Titanium, surface modification, hydrogen peroxide treatment, alkali treatment, thermochemical treatment.

Trade Liberalisation, Market Deregulation and Agricultural Performance in Central America

One frequently encounters the argument that trade liberalisation and deregulation of domestic markets in developing countries result in increased incentives for agriculture. This proposition is considered for the Central American countries, all of which passed through fundamental policy change either in the 1980s or 1990s. After characterising the policy regimes in each country over various periods, the analysis moves to an inspection of agricultural trade performance. The evidence indicates that liberalisation of foreign trade and deregulation of domestic markets has not been associated with improved agricultural performance. It is suggested that the failure of agriculture to respond positively to policy changes can be in part explained by an unfavourable trend in world prices of the region's major tradable commodities.