Bennett, Rachael (2012)
M.Res. thesis, University of Birmingham.
Project 1: The regenerative capacities between the peripheral and central nervous systems are contrasting; with axons in the peripheral nervous system being able to regenerate, unlike those in the central nervous system (CNS). Upon injury, the CNS responds by releasing myelin breakdown products, which are inhibitory to axon regrowth, and the formation of the glial scar which forms a physical barrier to regeneration. Whilst the formation of the glial scar takes days or weeks to form, the myelin breakdown products, which include myelin associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and Nogo, exert their inhibitory effects immediately. These products bind to the Nogo Receptor (NgR) complex which consists of the Nogo receptor (NgR), p75 neurotrophin receptor (p75NTR)/ (or an alternative protein known as TROY) and Lingo-1 which work together to activate RhoA, resulting in actin depolymerisation and growth cone collapse. The specific mechanisms by which this complex exhibits its effects remain unclear, particularly as recent unpublished data suggest that specific members of the Amigo family of proteins may be critical mediators of the inhibitory signaling pathway in vivo. This suggestion prompted us to clone NgR, p75NTR and Amigos 1, 2 and 3 in mammalian expression vectors and perform co-immunoprecipitation experiments with these constructs. These experiments suggest that Amigo-1 and -3 bind to NgR and/or p75NTR both independently or when expressed together. Although the approach was only semiquantitative at best, the experiments also suggest that Amigo 2 may bind to NgR and p75NTR independently and together, but to a much lesser extent. The results indicate that the Amigo family of proteins may form a component of the axon growth inhibitory complex, and potentially forming novel therapeutic targets for CNS injury.
Project 2: Mesenchymal stem cells (MSCs) are non haematopoietic cells capable of differentiating into adipocytes, chondrocytes and osteoblasts they are positive for CD105, CD90 and CD73 and negative for CD34 and CD45 amongst others. MSCs are not immunogenic and do not stimulate alloreactivity and this characteristic along with their ability to differentiate into such a range of cell types make them attractive for use in regenerative therapies by systemic infusion of ex vivo expanded cells. Its not known how MSCs are recruited from the circulation, however they may employ mechanisms similar to those used by leukocytes and haematopoietic stem cells when they are transported from the circulation such as the employment of selectins and via the aid of platelets. We therefore wanted to determine if MSCs can bind to collagen or P selectin coated microslides under flow conditions and in parallel observe if platelets as a whole can bind to MSCs in a variety of conditions by staining MSC and blood samples with CD105 (MSC marker) and CD42b (platelet marker) and use flow cytometry to looking at the effects of anticoagulant, time and platelet activation. The results demonstrated that MSCs do not bind to collagen or P selectin under flow conditions and demonstrated that a small proportion of platelets can bind to MSCs under appropriate conditions. Further, the binding experiments revealed that platelet activation reduced the level of binding between MSCs and platelets.
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