In vitro pharmacological inhibition of myofibroblast differentiation and force generation in a collagen-GAG matrix

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005.Includes bibliographical references (leaves 35-39).Induced regeneration studies from three organs in the adult mammal (skin, peripheral nerves, and the conjunctiva) suggest an antagonistic relationship between myofibroblast-mediated contraction of wounds and induced regeneration. In each instance of induced regeneration, contraction blocking was accomplished using regeneration templates, or scaffolds of highly specific structural and chemical properties that mainly control the environment or the density of contractile cells (myofibroblasts). In addition to scaffolds, diffusible factors could be used to inhibit specific components of the intracellular pathway in an effort to further evaluate the relationship between contraction and induced regeneration. The crucial role that Rho- associated coiled-coil forming protein serine/threonine kinase (ROCK) seems to play in cell-mediated contraction and cytoskeletal remodeling behavior of fibroblasts make it an attractive target for pharmacological inhibition. A preliminary in vitro study was conducted to evaluate the effect of Y-27632, a specific pharmacological inhibitor of ROCK, on the contraction of highly porous, three-dimensional type I collagen- glycosaminoglycan (CG) matrices over time by attached NR6WT fibroblasts treated with TGF-[beta]1, a known upregulator of both fibroblast contraction and expression of the contractile filament [alpha]-smooth muscle actin ([alpha]-SMA), a hallmark of the myofibroblast phenotype. NR6WT fibroblasts were seeded into free-floating cylindrical CG matrices (8 mm in diameter, n=6) and treated with serum-containing media supplemented with TGF-[beta]1 (3 ng/ml) or both TGF-[beta]1 (3 ng/ml) and Y--27632 (10 [mu]M) for 12 days.(cont.) Control cells (untreated) were cultured as well as unseeded matrix controls. The diameter reduction of matrices was determined daily by visual comparison to circles of known diameter (...). Contraction was calculated as the change in matrix diameter from the day 0 value divided by the day 0 diameter and is a measure of radial strain in the substrate. Cell-mediated contraction (CMC) was determined by subtracting the contraction of the unseeded matrices from the contraction of the seeded matrices. The average number of attached fibroblasts per matrix for each experimental group was determined at the end of the 12 day contraction experiment. At a dosage of 10 [mu]M, Y-27632 significantly attenuated TGF-[beta]1-stimulated cell-mediated contraction of free-floating CG matrices by NR6WT fibroblasts. While Y-27632 treatment also decreased the cellular content of the matrices compared to control cells, the relative magnitudes of cell-mediated contraction (C(MC) normalized o the attached number of fibroblasts indicate a statistically significant difference between mean values for TGF-[beta]1 treated cells and cells treated with both TGF-[beta]1 and Y-27632 (p<0.001). The data suggest that cellular de-adhesion alone does not account for the inhibitory effect of Y-27632 on TGF-[beta]1 stimulated cell-mediated contraction of CG matrices. A likely explanation is that the inhibitor is blocking the ability of fibroblasts to express an agonist-induced contractile phenotype and instead encouraging the development of a more migratory one.(cont.) The observed effect of the inhibitor on contraction could have been through inhibition of the contractile filament [alpha]-SMA, although expression of this protein was not assayed in this study. The demonstrated ability of Y-27632 to inhibit TGF-[beta]l1-stimulated force generation support its use in a future in vivo study that would evaluate the relationship between contraction blocking using pharmacological inhibitors and induced regeneration in a peripheral nerve wound model.by Eric C. Soller.S.M

Cell-mediated contraction and induced regeneration of the injured peripheral nerve

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2011.Cataloged from PDF version of thesis.Includes bibliographical references.Cell-mediated mechanical forces drive closure of severe wounds in adult mammalian organs, including the sciatic nerve following neurotmesis. Without experimental intervention the defect closes rapidly via contraction of transected nerve stumps by a thick, cohesive capsule of myofibroblasts (MFB) and subsequent collagen synthesis (scar), leading to a painful neuroma. Despite considerable progress in regenerating injured peripheral nerves with biomaterials, adequate recovery is generally limited to inter-stump gap lengths of about 20-30 mm in humans. Observations of successful induced regeneration in adults coincide with reduced MFB formation, yet the direct effect of the MFB capsule on nerve regeneration is unknown. According to the pressure cuff theory, a transected peripheral nerve could heal by regeneration, rather than MFB-mediated contraction and scar formation, provided the MFB capsule size (and corresponding cellular forces applied) are reduced. A well-characterized library of type I collagen tubular scaffolds with identical chemical composition and pore size, and varying degradation rate was used to evaluate the ability of a porous scaffold to block MFB contraction after injury in a demanding model of peripheral nerve regeneration in the adult rat. At 9 weeks post-neurotmesis, the MFB capsule thickness, 5, around the regenerating nerve was measured and the correlation with several quantitative measures of the quality of nerve regeneration, Q, was evaluated. A negative, statistically significant association was observed between the contractile capsule thickness, 6, and the quality of axonal regeneration, Q, (consisting of measures of regenerate area, the number of myelinated fibers, and the number of largediameter fibers) at 9 weeks post-sciatic neurotmesis. This constitutes the strongest evidence to date that capsules of contractile MFB antagonize induced regeneration of severely injured peripheral nerves in the adult mammal. Collagen devices of intermediate degradation rate minimized 5 and maximized Q. Reduced contractile cell presence and disrupted organization inside moderately cross-linked scaffolds that consequently degraded at an intermediate rate, but not in highly cross-linked scaffolds that degraded at very low rate, support the hypothesis of cell escape from the wound (making use of scaffold permeability) as a mechanism for scaffold regenerative activity.by Eric C. Soller.Ph.D

Efeito do armazenamento a baixa temperatura na viabilidade do ovo de Thaumastocoris peregrinus (Hemiptera, Thaumastocoridae).

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Avaliação de Cleruchoides noackae em Laboratório e Campo em Minas Gerais, Brasil.

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Influência da densidade de fêmeas no parasitismo de Cleruchoides noackae (Hymenoptera, Mymaridae) sobre ovos de Thaumastocoris peregrinus (Hemiptera, Thaumastocoridae).

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An integrated approach for analysing and assessing the performance of virtual learning groups

Collaborative distance learning involves a variety of elements and factors that have to be considered and measured in order to analyse and assess group and individual performance more effectively and objectively. This paper presents an approach that integrates qualitative, social network analysis (SNA) and quantitative techniques for evaluating online collaborative learning interactions. Integration of various different data sources, tools and techniques provides a more complete and robust framework for group modelling and guarantees a more efficient evaluation of group effectiveness and individual competence. Our research relies on the analysis of a real, long-term, complex collaborative experience, which is initially evaluated in terms of principled criteria and a basic qualitative process. At the end of the experience, the coded student interactions are further analysed through the SNA technique to assess participatory aspects, identify the most effective groups and the most prominent actors. Finally, the approach is contrasted and completed through a statistical technique which sheds more light on the results obtained that far. The proposal draws a well-founded line toward the development of a principled framework for the monitoring and analysis of group interaction and group scaffolding which can be considered a major issue towards the actual application of the CSCL proposals to real classrooms.Peer ReviewedPostprint (author's final draft

Plasmid-based gap-repair recombineered transgenes reveal a central role for introns in mutually exclusive alternative splicing in Down Syndrome Cell Adhesion Molecule exon 4

Alternative splicing is a key feature of human genes, yet studying its regulation is often complicated by large introns. The Down Syndrome Cell Adhesion Molecule (Dscam) gene from Drosophila is one of the most complex genes generating vast molecular diversity by mutually exclusive alternative splicing. To resolve how alternative splicing in Dscam is regulated, we first developed plasmid-based UAS reporter genes for the Dscam variable exon 4 cluster and show that its alternative splicing is recapitulated by GAL4-mediated expression in neurons. We then developed gap-repair recombineering to very efficiently manipulate these large reporter plasmids in Escherichia coli using restriction enzymes or sgRNA/Cas9 DNA scission to capitalize on the many benefits of plasmids in phiC31 integrase-mediated transgenesis. Using these novel tools, we show that inclusion of Dscam exon 4 variables differs little in development and individual flies, and is robustly determined by sequences harbored in variable exons. We further show that introns drive selection of both proximal and distal variable exons. Since exon 4 cluster introns lack conserved sequences that could mediate robust long-range base-pairing to bring exons into proximity for splicing, our data argue for a central role of introns in mutually exclusive alternative splicing of Dscam exon 4 cluster

Acute thiamethoxam toxicity in honeybees is not enhanced by common fungicide and herbicide and lacks stress-induced changes in mRNA splicing

Securing food supply for a growing population is a major challenge and heavily relies on the use of agrochemicals to maximize crop yield. It is increasingly recognized, that some neonicotinoid insecticides have a negative impact on non-target organisms, including important pollinators such as the European honeybee Apis mellifera. Toxicity of neonicotinoids may be enhanced through simultaneous exposure with additional pesticides, which could help explain, in part, the global decline of honeybee colonies. Here we examined whether exposure effects of the neonicotinoid thiamethoxam on bee viability are enhanced by the commonly used fungicide carbendazim and the herbicide glyphosate. We also analysed alternative splicing changes upon pesticide exposure in the honeybee. In particular, we examined transcripts of three genes: (i) the stress sensor gene X box binding protein-1 (Xbp1), (ii) the Down Syndrome Cell Adhesion Molecule (Dscam) gene and iii) the embryonic lethal/abnormal visual system (elav) gene, which are important for neuronal function. Our results showed that acute thiamethoxam exposure is not enhanced by carbendazim, nor glyphosate. Toxicity of the compounds did not trigger stress-induced, alternative splicing in the analysed mRNAs, thereby leaving dormant a cellular response pathway to these man-made environmental perturbations

Kondo effect and spin-active scattering in ferromagnet-superconductor junctions

We study the interplay of superconducting and ferromagnetic correlations on charge transport in different geometries with a focus on both a quantum point contact as well as a quantum dot in the even and the odd state with and without spin-active scattering at the interface. In order to obtain a complete picture of the charge transport we calculate the full counting statistics in all cases and compare the results with experimental data. We show that spin-active scattering is an essential ingredient in the description of quantum point contacts. This holds also for quantum dots in an even charge state whereas it is strongly suppressed in a typical Kondo situation. We explain this feature by the strong asymmetry of the hybridisations with the quantum dot and show how Kondo peak splitting in a magnetic field can be used for spin filtering. For the quantum dot in the even state spin-active scattering allows for an explanation of the experimentally observed mini-gap feature.Comment: 14 pages, 7 figures, accepted by PR