Development of ex vivo organ culture models to mimic human corneal scarring

PURPOSE: To develop ex vivo organ culture models of human corneal scarring suitable for pharmacological testing and the study of the molecular mechanisms leading to corneal haze after laser surgery or wounding. METHODS: Corneas from human donors were cultured ex vivo for 30 days, either at the air-liquid interface (AL) or immersed (IM) in the culture medium. Histological features and immunofluorescence for fibronectin, tenascin C, thrombospondin-1, and α-smooth muscle actin were graded from 0 to 3 for control corneas and for corneas wounded with an excimer laser. The effects of adding 10 ng/ml transforming growth factor-β1 (TGF-β1) to the culture medium and of prior complete removal of the epithelium and limbus, thus preventing reepithelialization, were also analyzed on wounded corneas. Collagen III expression was detected with real-time PCR. RESULTS: Wounding alone was sufficient to induce keratocyte activation and stromal disorganization, but it was only in the presence of added TGF-β1 that intense staining for fibronectin and tenascin C was found in the AL and IM models (as well as thrombospondin-1 in the AL model) and that α-smooth muscle actin became detectable. The scar-like appearance of the corneas was exacerbated when TGF-β1 was added and reepithelialization was prevented, resulting in the majority of corneas becoming opaque and marked upregulation of collagen III. CONCLUSIONS: THE MAIN FEATURES OF CORNEAL SCARRING WERE REPRODUCED IN THESE TWO COMPLEMENTARY MODELS: the AL model preserved differentiation of the epithelium and permits the topical application of active molecules, while the IM model ensures better perfusion by soluble compounds

Identification and characterisation of the Pex3-Inp1 complex as the first peroxisome-plasma membrane tether

Eukaryotic cells have evolved molecular mechanisms that control organelle size, number and position. Molecular tethers are required for organelle positioning, multiplication and establishment of interorganellar contact sites. The balance between organelle tethering and motility determines the intracellular distribution of organelles and their segregation during cell division. In Saccharomyces cerevisiae, correct peroxisome distribution is achieved by the opposing processes of cortical anchoring in the mother cell and Myosin-dependent transport towards the bud. The Inp1-Pex3 tethering complex is required for peroxisome retention during cell division and for peroxisome positioning along the mother cortex. As has been postulated for other organelles, yeast peroxisomes interact with many cellular structures including the plasma membrane, ER, vacuole, mitochondria and lipid bodies. Components of some interorganellar peroxisomal contact sites have recently been identified whereas others are still completely uncharacterised including the plasma membrane- peroxisome (PM-PER) contact site. The work presented in this thesis identifies Inp1 as the first known plasma membrane-peroxisome (PM-PER) tether by demonstrating that Inp1 meets the predefined criteria which a contact site tether protein must adhere to. This thesis first describes a conserved Pex3 binding motif in the C-terminal region of Inp1. This motif bears a striking resemblance to the Pex3 binding site present on Pex19 and both in vitro and in vivo evidence is presented which illustrates that Pex19 and Inp1 can compete for binding to Pex3. In addition, the N-terminal 100 amino acids of Inp1 are shown to localise to the plasma membrane, bind to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and, when artificially attached to the peroxisomal membrane, restore retention by relocating peroxisomes to the cell periphery in inp1Δ cells. In this study, Inp1 is shown to be present in the correct sub-cellular location to interact with both the plasma membrane and peroxisomal membrane and the data illustrates the structural and functional capacity of Inp1 to be a PM-PER tether. Through detailed analysis of the molecular function of Inp1, the work in this thesis identifies a novel role for Inp1 as a PM- PER tether and concludes that tethering of peroxisomes to the plasma membrane is required for peroxisome retention. This is the first molecular characterisation of the PM-PER tether and has allowed for the proposal of a new model for peroxisome retention

Cereal density and N-fertiliser effects on the flora and biodiversity value of arable headlands

Modern intensive farming caused pronounced changes to the European arable flora. Many species adapted to less intensive traditional farming declined severely, as did the potential of unsown arable vegetation to support higher trophic levels. To reverse these trends, various agri-environment measures were introduced. One such measure is to manage cereal headlands as conservation headlands, involving strict restrictions on pesticide and fertiliser use. An additional modification to management which could reduce crop competition and thus deliver benefits to arable plants is cereal sowing at reduced rates. However, little is known about its benefits to rare and declining arable plants, or to species of value to higher trophic levels, and whether it can be implemented without concomitant increase in undesirable weeds. We set up identical two-factorial experiments in winter wheat and spring barley, combining a nitrogen fertiliser versus no fertiliser treatment with cereal sowing at economic rates versus sowing at rates reduced by 75 %, with added sowing of a mixture of rare arable species. Both experiments also included an uncropped but cultivated control equivalent to another agri-environment measure. Our results show that reduced cereal sowing in conservation headlands can benefit rare and declining species, as well as arable plant diversity, without necessarily resulting in a concomitant increase in undesirable weeds. While such benefits tended to be larger in uncropped cultivated controls, conservation headlands have the advantage of not requiring land being taken out of production. Moreover, as shown in this study, their benefits to arable plants can be maximised by reduced sowing

Aversive Racism and Child Protection Practice with Gypsy, Roma and Traveller Children and Families

Reiterating the urgent need for the development of anti-racist practice with Gypsy, Roma and Traveller families, this discussion paper develops the conclusions presented by Allen and Riding (2018) in the Fragility of Professional Competence report. Viewing their findings through the lens of aversive racism, we aim to shed some light on a rarely seen paradox in child protection. A paradox that exists when child protection practitioners who, by nature of their professional status, publicly sympathise with Gypsy, Roma and Traveller communities as victims of injustice, support the principle of equality, and regard themselves as non-prejudiced, but simultaneously possess negative feelings, views, and beliefs about them. Emphasising the opportunity for children’s guardians, family court advisers, and independent social workers to identify racism and diversify power systems, we introduce three characteristics that represent important initial steps to address the intersecting oppressions that many Gypsy, Roma and Traveller children and families face. Concentrating on the opportunity for change, we end the discussion with a brief description of the Gypsy, Roma and Traveller Social Work Association; a group that aims to challenge racism and enable child protection professionals to stand with children and families at grassroots, and promote their right to live self-determined lives without fear, discrimination, or retaliation

Role of the Netrin-like Domain of Procollagen C-Proteinase Enhancer-1 in the Control of Metalloproteinase Activity

The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments

Failure of Mineralized Collagen Microfibrils Using Finite Element Simulation Coupled to Mechanical Quasi-brittle Damage

Bone is a multiscale heterogeneous materiel of which principal function is to support the body structure and to resist mechanical loading and fractures. Bone strength does not depend only on the quantity and quality of bone which is characterized by the geometry and the shape of bones but also on the mechanical proprieties of its compounds, which have a significant influence on its deformation and failure. This work aim to use a 3D nano-scale finite element model coupled to the concept of quasi-brittle damage with the behaviour law isotropic elasticity to investigate the fracture behaviour of composite materiel collagen-mineral (mineralized collagen microfibril). Fracture stress-number of cross-links and damping capacity-number of cross-links curves were obtained under tensile loading conditions at different densities of the mineral phase. The obtained results show that number of cross-links as well as the density of mineral has an important influence on the strength of microfibrils which in turn clarify the bone fracture at macro-scale.Comment: 6; http://www.sciencedirect.com/science/article/pii/S187770581100714

The Pex3–Inp1 complex tethers yeast peroxisomes to the plasma membrane

A subset of peroxisomes is retained at the mother cell cortex by the Pex3–Inp1 complex. We identify Inp1 as the first known plasma membrane–peroxisome (PM-PER) tether by demonstrating that Inp1 meets the predefined criteria that a contact site tether protein must adhere to. We show that Inp1 is present in the correct subcellular location to interact with both the plasma membrane and peroxisomal membrane and has the structural and functional capacity to be a PM-PER tether. Additionally, expression of artificial PM-PER tethers is sufficient to restore retention in inp1Δ cells. We show that Inp1 mediates peroxisome retention via an N-terminal domain that binds PI(4,5)P2 and a C-terminal Pex3-binding domain, forming a bridge between the peroxisomal membrane and the plasma membrane. We provide the first molecular characterization of the PM-PER tether and show it anchors peroxisomes at the mother cell cortex, suggesting a new model for peroxisome retention