Haematopoietic differentiation of embryonic stem cells by aorta-gonad-mesonephros region co-culture

The piuripotential nature of embryonic stem (ES) cells in vitro has been used to develop model systems for the differentiation of tissue-specific lineages during organogenesis. The differentiation of ES cells into haematopoietic lineages in vitro has been particularly well characterised, but the successful generation of substantial numbers of haematopoietic stem cells (HSCs) able to engraft lethally irradiated mice in vivo has been limited. It is possible that the in vitro differentiation protocols used to date do not provide a suitable microenvironment for HSC development from ES cells. This thesis describes the effect of co-culturing differentiating ES cells in the presence of embryonic tissue that was predicted to provide a more appropriate microenvironment for haematopoietic commitment.The aorta-gonad-mesonephros (AGM) region is the earliest site in the embryo proper that is able to generate definitive, adult-type HSCs autonomously. Clusters of HSCs appear in the AGM region at day 10 of gestation in the mouse, where they proliferate before colonising the foetal liver to continue haematopoietic development. Long term repopulating HSC activity has been found to increase within AGM region explant cultures indicating that elements of the supporting microenvironment for definitive HSC expansion can be captured in vitro.In this study an explant culture system was developed to examine the inductive properties of the AGM region on differentiating ES cells. A highly significant increase in the number of primitive haematopoietic progenitors, as measured by in vitro colony assays, was observed after co-culture of ES cells with the AGM region from a 10.5 day embryo. However, engraftment in vivo of ES-derived progenitors after transplantation was not achieved. The effect of three stromal cell lines derived from the AGM region and foetal liver on the differentiation of ES cells in co-culture was also examined. Although the cell lines were similar to each other in terms of the expression of surface markers, they exhibited diverse effects on the differentiation of ES cells. Preliminary data also suggest that the AGM region-derived factor(s) responsible for the increase in haematopoietic differentiation of ES ceils is dependent on direct cell-ceii contact.The AGM region culture system was also used as a novel investigative tool in the analysis of a putative haematopoietic phenotype in a mouse mutant deficient in the murine homologue of the ubiquitin conjugating enzyme, Ubc7, generated previously by a gene-trapping technique. The numbers of haematopoietic progenitors in AGM regions from wildtype, heterozygous and homozygous embryos after culture were compared, but in this case no measurable haematopoietic defect was observed

Post cracking behaviour of fibre reinforced concrete with mineral wool fibers

The research concerns the feasibility of recycling fibers from mineral wool residues from construction and demolition waste, by incorporating them into a fine grained concrete. All the specimens of concrete with various types of fibers have been subjected to the mechanical tests of flexural and compressive strength. Also the pre-cracking and post-cracking softening behaviour of concrete with fiber residues has been investigated. The flexural load-CMOD and load-deflection relationships, obtained in three-point bending test, have been used to determine the fracture parameters: fracture energy, critical stress intensity factor, critical crack tip opening displacement, critical effective crack length. It was verified that the incorporation of mineral residues to a cement concrete is a viable alternative for their recycling; the new composites analyzed maintain adequate mechanical properties for different application

Effect of fibers reinforcement on shear capacity of double span reinforced concrete beams

Fifteen double-span reinforced concrete beams (RC) with the addition of dispersed reinforcement were examined to determine the effect of the reinforcement on the shear resistance at the support area. Asalt fibers, whose mechanical properties (especially high tensile strength) have a beneficial effect on the behavior of components under load were used. Beams were subjected to a constant load forces gathered in the middle of the span of each bay. The study aimed to determine the deflection of beams, measuring the crack width perpendicular and diagonal capacity and determination of shear and/or bending. Almost all the beams have reached a shear failure mode. The addition of basalt fiber in each of a series of research results in diverse growth destructive force beam and a corresponding increase in the lateral force on the supports. The maximum strength increase of at 54%, as compared to the reference beam without the fibers was observed for the beam A-III 50/50

Analyzing the effects of surface distribution of pores in cell electroporation for a cell membrane containing cholesterol

This paper presents a model and numerical analysis (simulations) of transmembrane potential induced in biological cell membrane under the influence of externally applied electric field (i.e., electroporation). This model differs from the established models of electroporation in two distinct ways. Firstly, it incorporates the presence of cholesterol (~20% mole-fraction) in biological membrane. Secondly, it considers the distribution of pores as a function of the variation of transmembrane potential from one region of the cell to another. Formulation is based on the role of membrane tension and electrical forces in the formation of pores in a cell membrane, which is considered as an infinitesimally thin insulator. The model has been used to explore the process of creation and evolution of pores and to determine the number and size of pores as a function of applied electric field (magnitude and duration). Results show that the presence of cholesterol enhances poration by changing the membrane tension. Analyses indicate that the number of pores and average pore radii differ significantly from one part of the cell to the other. While some regions of the cell membrane undergo rapid and dense poration, others remain unaffected. The method can be a useful tool for a more realistic prediction of pore formation in cells subjected to electroporation.Comment: 11 pages, 3 figures. v2: added new references, grammatical changes, corrected typo

Dynamics of lattice spins as a model of arrhythmia

We consider evolution of initial disturbances in spatially extended systems with autonomous rhythmic activity, such as the heart. We consider the case when the activity is stable with respect to very smooth (changing little across the medium) disturbances and construct lattice models for description of not-so-smooth disturbances, in particular, topological defects; these models are modifications of the diffusive XY model. We find that when the activity on each lattice site is very rigid in maintaining its form, the topological defects - vortices or spirals - nucleate a transition to a disordered, turbulent state.Comment: 17 pages, revtex, 3 figure

A simulation study of the reaction of human heart to biphasic electrical shocks

BACKGROUND: This article presents a study, which examines the effects of biphasic electrical shocks on human ventricular tissue. The effects of this type of shock are not yet fully understood. Animal experiments showed the superiority of biphasic shocks over monophasic ones in defibrillation. A mathematical computer simulation can increase the knowledge of human heart behavior. METHODS: The research presented in this article was done with different models representing a three-dimensional wedge of ventricular myocardium. The electrophysiology was described with Priebe-Beuckelmann model. The realistic fiber twist, which is specific to human myocardium was included. Planar electrodes were placed at the ends of the longest side of the virtual cardiac wedge, in a bath medium. They were sources of electrical shocks, which varied in magnitude from 0.1 to 5 V. In a second arrangement ring electrodes were placed directly on myocardium for getting a better view on secondary electrical sources. The electrical reaction of the tissue was generated with a bidomain model. RESULTS: The reaction of the tissue to the electrical shock was specific to the initial imposed characteristics. Depolarization appeared in the first 5 ms in different locations. A further study of the cardiac tissue behavior revealed, which features influence the response of the considered muscle. It was shown that the time needed by the tissue to be totally depolarized is much shorter when a biphasic shock is applied. Each simulation ended only after complete repolarization was achieved. This created the possibility of gathering information from all states corresponding to one cycle of the cardiac rhythm. CONCLUSIONS: The differences between the reaction of the homogeneous tissue and a tissue, which contains cleavage planes, reveals important aspects of superiority of biphasic pulses. ..

Second Harmonic Generation for time-resolved monitoring of membrane pore dynamics subserving electroporation of neurons

Electroporation of neurons, i.e. electric-field induced generation of membrane nanopores to facilitate internalization of molecules, is a classic technique used in basic neuroscience research and recently has been proposed as a promising therapeutic strategy in the area of neuro-oncology. To optimize electroporation parameters, optical techniques capable of delivering time and spatially-resolved information on electroporation pore formation at the nanometer scale would be advantageous. For this purpose we describe here a novel optical method based on second harmonic generation (SHG) microscopy. Due to the nonlinear and coherent nature of SHG, the 3D radiation lobes from stained neuronal membranes are sensitive to the spatial distribution of scatterers in the illuminated patch, and in particular to nanopore formation.We used phase-array analysis to computationally study the SHG signal as a function of nanopore size and nanopore population density and confirmed experimentally, in accordance with previous work, the dependence of nanopore properties on membrane location with respect to the electroporation electric field; higher nanopore densities, lasting < 5 milliseconds, are observed at membrane patches perpendicular to the field whereas lower density is observed at partly tangent locations. Differences between near-anode and near-cathode cell poles are also measured, showing higher pore densities at the anodic pole compared to cathodic pole. This technique is promising for the study of nanopore dynamics in neurons and for the optimization of novel electroporation-based therapeutic approaches

Organic-walled dinoflagellate cyst records from a prospective Turonian - Coniacian (upper Cretaceous) GSSP, Slupia Nadbrzezna, Poland

AbstractA river section at Słupia Nadbrzeżna, central Poland, has been proposed as a candidate Turonian – Coniacian (Cretaceous) GSSP, in combination with the Salzgitter-Salder quarry section of Lower Saxony, Germany. Results of a high-resolution (25 cm) palynological study of the boundary interval in the Słupia Nadbrzeżna section are presented. Terrestrial palynomorphs are rare; marine organic-walled dinoflagellate cysts dominate the palynological assemblage. The dinoflagellate cyst assemblage has a low species richness (5–11 per sample; total of 18 species recorded) and diversity (Shannon index H = 0.8–1.4), dominated by four taxa: Circulodinium distinctum subsp. distinctum; Oligosphaeridium complex; Spiniferites ramosus subsp. ramosus; Surculosphaeridium longifurcatum. Declining proportions of O. complex and S. ramosus subsp. ramosus characterise the uppermost Turonian, with an increased dominance of S. longifurcatum in the lower Coniacian. The Turonian – Coniacian boundary interval includes an acme of C. distinctum subsp. distinctum in the upper Mytiloides scupini Zone, a dinoflagellate cyst abundance maximum in the Cremnoceramus walterdorfensis walterdorfensis Zone, and the highest occurrence of Senoniasphaera turonica in the basal Coniacian lower Cremnoceramus deformis erectus Zone. Most previously reported Turonian – Coniacian boundary dinoflagellate cyst marker species are absent; a shallow-water oligotrophic epicontinental depositional setting, remote from terrestrial influence, likely limited species diversity and excluded many taxa of biostratigraphic value