Beta cell mass and growth after syngeneic islet cell transplantation in normal and streptozocin diabetic C57BL/6 mice

In islet transplantation, nonimmunological factors such as limited growth capacity or increased death rate could reduce the beta cell mass in the graft and lead to failure of the transplant. We studied the evolution of beta cell replication and mass after transplantation of insufficient, minimally sufficient, or excessive islet tissue. Streptozocin diabetic C57BL/6 mice received 150 or 300 syngeneic islets under the kidney capsule and normal mice received 300 islets. In streptozocin diabetic mice 300 islets restored normoglycemia; beta cell replication in transplanted islets was similar to replication in normal pancreas and beta cell mass in the graft remained constant. In contrast, 150 islets were insufficient to achieve normoglycemia; beta cell replication was increased initially but not by 18 or 30 d despite persistent hyperglycemia, and beta cell mass fell progressively. When islets were transplanted into normal recipients, beta cell replication remained normal but beta cells underwent atrophy and mass in the graft was substantially reduced. Therefore, with a successful islet transplant, in diabetic mice beta cell replication and mass remain constant. In contrast, when insufficient islet tissue is transplanted an initial increase in beta cell replication can not compensate for a decline in beta cell mass. When excessive islet tissue is transplanted, beta cell mass is reduced despite normal beta cell replication.

Análisis comparativo de tres técnicas de derivación de células madre

Las células madre embrionarias (Embryonic Stem Cells; ESC) son células pluripotentes que presentan la capacidad de dividirse indefinidamente a la vez que mantienen la habilidad para diferenciarse a cualquier tipo celular. Aunque de manera rutinaria se derivan a partir de la masa celular interna de embriones en estadio de blastocisto, también pueden derivarse a partir de embriones en estadios precompactacionales y de embriones reconstruidos por procesos de transferencia nuclear. Debido a que durante el desarrollo embrionario temprano, momento en el que se derivan las ESC, tienen lugar profundos cambios de metilación en el genoma, tanto la derivación como el cultivo se consagran como técnicas que pueden alterar los patrones de metilación en genes regulados por impronta genómica. Con el objetivo de analizar la estabilidad epigenética de embriones preimplantacionales y ESC murinas, en este trabajo se ha optimizado un protocolo de anàlisis de los niveles de metilación mediante pirosecuenciación. Para ello se han seleccionado tres genes regulados por impronta genómica (H19/Igf2, Snrpn and Peg3), dos genes relacionados con el mantenimiento de pluripotencia en ESC (Oct4, Nanog y Sox2) y dos genes marcadores de diferenciación temprana (Cdx2 y Gata6). Nuestros resultados muestran que algunos grupos de embriones preimplantacionales presentan una hipo e hipermetilación en las regiones diferencialmente metiladas (Differentially Methylated Regions, DMRs) de los genes Snrpn y Peg3. Además, la línea de ESC analizada presentó anomalías en los tres genes regulados por impronta genómica. No obstante, el hecho de que esta línea fuera inestable a nivel cariotípico no permite establecer una relación entre el cultivo in vitro o la técnica de derivación y la inestabilidad epigenética demostrada. Por todo esto, parece pertinente analizar tanto la integridad epigenética como la estabilidad cromosómica de ESC antes de proceder a realizar ensayos clínicos en humanos.

Transplanted beta cell response to increased metabolic demand. Changes in beta cell replication and mass

We determined the capacity of transplanted beta cells to modify their replication and mass when stimulated by changes in metabolic demand. Five groups of Lewis rats were studied: group 1 (Tx-Px) had a 95% pancreatectomy 14 d after transplantation of 500 islets; group 2 (Px-Tx) had a 95% pancreatectomy 14 d before transplantation of 500 islets; group 3 (Tx) was transplanted with 500 islets; group 4 (Px) had a 95% pancreatectomy; and group 5 (normal) was neither transplanted nor pancreatectomized. Blood glucose was normal in Tx-Px and Tx groups at all times. Px-Tx and Px groups developed severe hyperglycemia after pancreatectomy that was corrected in Px-Tx group in 83% of rats 28 d after transplantation. Replication of transplanted beta cells increased in Tx-Px (1.15 +/- 0.12%) and Px-Tx (0.85 +/- 0.12%) groups, but not in Tx group (0.64 +/- 0.07%) compared with normal pancreatic beta cells (0.38 +/- 0.05%) (P < 0.001). Mean beta cell size increased in Tx-Px (311 +/- 14 microns2) and Px-Tx (328 +/- 13 microns2) groups compared with Tx (252 +/- 12 microns2) and normal (239 +/- 9 microns2) groups (P < 0.001). Transplanted beta cell mass increased in Tx-Px (1.87 +/- 0.51 mg) and Px-Tx (1.55 +/- 0.21 mg) groups compared with Tx group (0.78 +/- 0.17 mg) (P < 0.05). In summary, changes in transplanted beta cells prevented the development of hyperglycemia in Tx-Px rats. Transplanted beta cells responded to increased metabolic demand increasing their beta cell mass.

Study of inner ear and lateral line hair cell regeneration

Death of sensory hair cells in the inner ear results in two global health problems that millions of people around the world suffer: hearing loss and balance disorders. Hair cells convert sound vibrations and head movements into electrical signals that are conveyed to the brain, and as a result of aging, exposure to noise, modern drugs or genetic predisposition, hair cells die. In mammals, the great majority of hair cells are produced during embryogenesis, and hair cells that are lost after birth are not replaceable. However, in the last decades, researches have shown some model organisms that retain the ability to regenerate hair cells damaged after embryogenesis, such as Zebrafish and chicken, providing clues as to the cellular and molecular mechanisms that may block hair cell regeneration in mammals. This discovery initiated a search for methods to stimulate regeneration or replacement of hair cells in mammals, a search that, if fruitful, will revolutionize the treatment of hearing loss and balance disorders. One aim of my project is to study the role of retinoic acid in adult Zebrafish and in mice, which is a metabolite of vitamin A known as an essential molecule to activate hair cell regeneration after cells damaged in Zebrafish embryo. We want to study important genes involved in retinoic acid pathway, such as Aldh1a3 and RARs genes, to check what their role is in the inner ear of adult Zebrafish and compare result obtained in the inner ear of mice. On the other hand, Zebrafish lateral line contains neuromast, which are formed by the same structure than the inner ear: hair cells surrounded by supporting cells and neurons. The lateral line is a structure below the skin's surface that makes easier to damage hair cells to study their regeneration. For that reason, another aim of my project is to study how Sox2 and Atoh1, essential genes during the inner ear development, change their expression during hair cell regeneration in the lateral line. In my project, the most important concepts related to Zebrafish world are explained in order to understand why we have studied this animal and these essential genes. Then, techniques that we used are explained, with their protocol attached in the annexes. Finally, results of my project are shown, but many of them were not expected and they would be needed to follow studying.

Insertion of a malE B-Galactosidase fusion protein into the envelope of Escherichia coli disrupts biogenesis of outer membrane proteins and processing of inner membrane proteins

The synthesis of a membrane-bound MalE ,B-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%o. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.

Could cell membranes produce acoustic streaming? Making the case for synechococcus self-propulsion

Sir James Lighthill proposed in 1992 that acoustic streaming occurs in the inner ear, as part of the cochlear amplifier mechanism. Here we hypothesize that some of the most ancient organisms use acoustic streaming not only for self-propulsion but also to enhance their nutrient uptake. We focus on a motile strain of Synechococcus, a yanobacteria whose mechanism for self-propulsion is not known. Molecular motors could work like piezoelectric transducers acting on the crystalline structure surrounding the outer cell membrane. Our calculations show that a traveling surface acoustic wave (SAW)could account for the observed velocities. These SAW waves will also produce a non-negligible Stokes layer surrounding the cell: motion within this region being essentially chaotic. Therefore, an AS mechanism would be biologically advantageous, enhancing localized diffusion processes and consequently, chemical reactions. We believe that acoustic streaming, produced by nanometer scale membrane vibrations could be widespread in cell biology. Other possible instances are yeast cells and erythrocytes. Flows generated by acoustic streaming may also be produced by silica coated diatoms along their raphe. We note that microelectromechanical (MEMS) acoustic streaming devices were first introduced in the 1990’s. Nature may have preceded this invention by 2.7 Gyr.

The Coordinated Radio and Infrared Survey for High-Mass Star Formation (The CORNISH Survey). I. Survey Design

We describe the motivation, design, and implementation of the CORNISH survey, an arcsecondresolution radio continuum survey of the inner galactic plane at 5 GHz using the Very Large Array (VLA). It is a blind survey coordinated with the northern SpitzerGLIMPSE I region covering 10° massive star formation. A sensitivity around 2 mJy will allow the automatic distinction between radio-loud and radio-quiet midIR sources found in the Spitzersurveys. This survey has many legacy applications beyond star formation, including evolved stars, active stars and binaries, and extragalactic sources. The CORNISH survey for compact ionized sources complements other Galactic plane surveys that target diffuse and nonthermal sources, as well as atomic and molecular phases to build up a complete picture of the interstellar medium in the Galaxy.

Radial displacement of a fluid annulus in a rotating Hele¿Shaw cell

The radial displacement of a fluid annulus in a rotating circular Hele-Shaw cell has been investigated experimentally. It has been found that the flow depends sensitively on the wetting conditions at the outer interface. Displacements in a prewet cell are well described by Darcy's law in a wide range of experimental parameters, with little influence of capillary effects. In a dry cell, however, a more careful analysis of the interface motion is required; the interplay between a gradual loss of fluid at the inner interface, and the dependence of capillary forces at the outer interface on interfacial velocity and dynamic contact angle, result in a constant velocity for the interfaces. The experimental results in this case correlate in the form of an empirical scaling relation between the capillary number Ca and a dimensionless group, related to the ratio of centrifugal to capillary forces, which spans about three orders of magnitude in both quantities. Finally, the relative thickness of the coating film left by the inner interface, alpha i, is obtained as a function of Ca.

Low viscosity contrast fingering in a rotating Hele-Shaw cell

We study the fingering instability of a circular interface between two immiscible liquids in a radial Hele-Shaw cell. The cell rotates around its vertical symmetry axis, and the instability is driven by the density difference between the two fluids. This kind of driving allows studying the interfacial dynamics in the particularly interesting case of an interface separating two liquids of comparable viscosity. An accurate experimental study of the number of fingers emerging from the instability reveals a slight but systematic dependence of the linear dispersion relation on the gap spacing. We show that this result is related to a modification of the interface boundary condition which incorporates stresses originated from normal velocity gradients. The early nonlinear regime shows nearly no competition between the outgrowing fingers, characteristic of low viscosity contrast flows. We perform experiments in a wide range of experimental parameters, under conditions of mass conservation (no injection), and characterize the resulting patterns by data collapses of two characteristic lengths: the radius of gyration of the pattern and the interface stretching. Deep in the nonlinear regime, the fingers which grow radially outwards stretch and become gradually thinner, to a point that the fingers pinch and emit drops. We show that the amount of liquid emitted in the first generation of drops is a constant independent of the experimental parameters. Further on there is a sharp reduction of the amount of liquid centrifugated, punctuated by periods of no observable centrifugation.

Analyzing peptides and proteins by mass spectrometry: principles and applications in proteomics

The study of proteins has been a key element in biomedicine and biotechnology because of their important role in cell functions or enzymatic activity. Cells are the basic unit of living organisms, which are governed by a vast range of chemical reactions. These chemical reactions must be highly regulatedin order to achieve homeostasis. Proteins are polymeric molecules that havetaken on the evolutionary process the role, along with other factors, of controlthese chemical reactions. Learning how proteins interact and control their up anddown regulations can teach us how living cells regulate their functions, as well asthe cause of certain anomalies that occur in different diseases where proteins areinvolved. Mass spectrometry (MS) is an analytical widely used technique to studythe protein content inside the cells as a biomarker point, which describesdysfunctions in diseases and increases knowledge of how proteins are working.All the methodologies involved in these descriptions are integrated in the fieldcalled Proteomics.

Spectral analysis and limit behaviours in a spring-mass system

We consider a model for a damped spring-mass system that is a strongly damped wave equation with dynamic boundary conditions. In a previous paper we showed that for some values of the parameters of the model, the large time behaviour of the solutions is the same as for a classical spring-mass damper ODE. Here we use spectral analysis to show that for other values of the parameters, still of physical relevance and related to the effect of the spring inner viscosity, the limit behaviours are very different from that classical ODE

N-myc controls proliferation, morphogenesis, and patterning of the inner ear

Myc family members play crucial roles in regulating cell proliferation, size, and differentiation during organogenesis. Both N-myc and c-myc are expressed throughout inner ear development. To address their function in the mouse inner ear, we generated mice with conditional deletions in either N-myc or c-myc. Loss of c-myc in the inner ear causes no apparent defects, whereas inactivation of N-myc results in reduced growth caused by a lack of proliferation. Reciprocally, the misexpression of N-myc in the inner ear increases proliferation. Morphogenesis of the inner ear in N-myc mouse mutants is severely disturbed, including loss of the lateral canal, fusion of the cochlea with the sacculus and utriculus, and stunted outgrowth of the cochlea. Mutant cochleas are characterized by an increased number of cells exiting the cell cycle that express the cyclin-dependent kinase inhibitor p27Kip1 and lack cyclin D1, both of which control the postmitotic state of hair cells. Analysis of different molecular markers in N-myc mutant ears reveals the development of a rudimentary organ of Corti containing hair cells and the underlying supporting cells. Differentiated cells, however, fail to form the highly ordered structure characteristic for the organ of Corti but appear as rows or clusters with an excess number of hair cells. The Kölliker's organ, a transient structure neighboring the organ of Corti and a potential source of ectopic hair cells, is absent in the mutant ears. Collectively, our data suggest that N-myc regulates growth, morphogenesis, and pattern formation during the development of the inner ear.

Id Gene regulation and function in the prosensory domains of the chicken inner ear: a link between Bmp Signaling and Atoh1

Bone morphogenetic proteins (Bmps) regulate the expression of the proneural gene Atoh1 and the generation of hair cells in the developing inner ear. The present work explored the role of Inhibitor of Differentiation genes (Id1-3) in this process. The results show that Id genes are expressed in the prosensory domains of the otic vesicle, along with Bmp4 and Bmp7. Those domains exhibit high levels of the phosphorylated form of Bmp-responding R-Smads (P-Smad1,5,8), and of Bmp-dependent Smad transcriptional activity as shown by the BRE-tk-EGFP reporter. Increased Bmp signaling induces the expression of Id1-3 along with the inhibition of Atoh1. Conversely, the Bmp antagonist Noggin or the Bmp-receptor inhibitor Dorsomorphin elicit opposite effects, indicating that Bmp signaling is necessary for Id expression and Atoh1 regulation in the otocyst. The forced expression of Id3 is sufficient to reduce Atoh1 expression and to prevent the expression of hair cell differentiation markers. Together, these results suggest that Ids are part of the machinery that mediates the regulation of hair cell differentiation exerted by Bmps. In agreement with that, during hair cell differentiation Bmp4 expression, P-Smad1,5,8 levels and Id expression are downregulated from hair cells. However, Ids are also downregulated from the supporting cells which contrarily to hair cells exhibit high levels of Bmp4 expression, P-Smad1,5,8, and BRE-tk-EGFP activity, suggesting that in these cells Ids escape from Bmp/Smad signaling. The differential regulation of Ids in time and space may underlie the multiple functions of Bmp signaling during sensory organ development.

High mass microquasars and low latitude gamma-ray sources

Population studies of unidentified EGRET sources suggest that there exist at least three different populations of galactic gamma-ray sources. One of these populations is formed by young objects distributed along the galactic plane with a strong concentration toward the inner spiral arms of the Galaxy. Variability, spectral and correlation analysis indicate that this population is not homogeneous. In particular, there is a subgroup of sources that display clear variability in their gamma-ray fluxes on timescales from days to months. Following the proposal by Kaufman Bernad\'o et al. (2002), we suggest that this group of sources might be high-mass microquasars, i.e. accreting black holes or neutron stars with relativistic jets and early-type stellar companions. We present detailed inhomogeneous models for the gamma-ray emission of these systems that include both external and synchrotron self-Compton interactions. We have included effects of interactions between the jet and all external photon fields to which it is exposed: companion star, accretion disk, and hot corona. We make broadband calculations to predict the spectral energy distribution of these objects from radio up to GeV energies. The results and predictions can be tested by present and future gamma-ray instruments like INTEGRAL, AGILE, and GLAST.

Radial displacement of a fluid annulus in a rotating Hele-Shaw cell

The radial displacement of a fluid annulus in a rotating circular HeleShaw cell has been investigated experimentally. It has been found that the flow depends sensitively on the wetting conditions at the outer interface. Displacements in a prewet cell are well described by Darcy"s law in a wide range of experimental parameters, with little influence of capillary effects. In a dry cell, however, a more careful analysis of the interfacemotion is required; the interplay between a gradual loss of fluid at the inner interface, and the dependence of capillary forces at the outer interface on interfacial velocity and dynamic contact angle, result in a constant velocity for the interfaces. The experimental results in this case correlate in the form of an empirical scaling relation between the capillary number Ca and a dimensionless group, related to the ratio of centrifugal to capillary forces, which spans about three orders of magnitude in both quantities. Finally, the relative thickness of the coating film left by the inner interface is obtained as a function of Ca.