Characterisation of the role of canonical BMP-Smad 1/5/8 signalling in the development of ventral midbrain dopaminergic neurons

Ventral midbrain (VM) dopaminergic (DA) neurons, which project to the dorsal striatum via the nigrostriatal pathway, are progressively degenerated in Parkinson’s disease (PD). The identification of the instructive factors that regulate midbrain DA neuron development, and the subsequent elucidation of the molecular bases of their effects, is vital. Such an understanding would facilitate the generation of transplantable DA neurons from stem cells and the identification of developmentally-relevant neurotrophic factors, the two most promising therapeutic approaches for PD. Two related members of the bone morphogenetic protein (BMP) family, BMP2 and growth/differentiation factor (GDF) 5, which signal via a canonical Smad 1/5/8 signalling pathway, have been shown to have neurotrophic effects on midbrain DA neurons both in vitro and in vivo, and may function to regulate VM DA neuronal development. However, the molecular (signalling pathway(s)) and cellular (direct neuronal or indirect via glial cells) mechanisms of their effects remain to be elucidated. The present thesis hypothesised that canonical Smad signalling mediates the direct effects of BMP2 and GDF5 on the development of VM DA neurons. By activating, modulating and/or inhibiting various components of the BMP-Smad signalling pathway, this research demonstrated that GDF5- and BMP2-induced neurite outgrowth from midbrain DA neurons is dependent on BMP type I receptor activation of the Smad signalling pathway. The role of glial cell-line derived neurotrophic factor (GDNF)-signalling, dynamin-dependent endocytosis and Smad interacting protein-1 (Sip1) regulation, in the neurotrophic effects of BMP2 and GDF5 were determined. Finally, the in vitro development of VM neural stem cells (NSCs) was characterised, and the ability of GDF5 and BMP2 to induce these VM NSCs towards DA neuronal differentiation was investigated. Taken together, these experiments identify GDF5 and BMP2 as novel regulators of midbrain DA neuronal induction and differentiation, and demonstrate that their effects on DA neurons are mediated by canonical BMPR-Smad signalling.

Biogas production from novel substrates

Biogas production is the conversion of the organic material into methane (CH4) and carbon dioxide (CO2) under anaerobic conditions. Anaerobic digestion (AD) is widely used in continental and Scandinavian communities as both a waste treatment option and a source of renewable energy. Ireland however lags behind this European movement. Numerous feedstocks exist which could be digested and used to fuel a renewable transport fleet in Ireland. An issue exists with the variety of feedstocks; these need to be assessed and quantified to ascertain their potential resource and application to AD. From literature the ideal C:N ratio is between 25 and 30:1. Low levels of C:N (<15) can lead to problems with ammonia inhibition. Within the digester a plentiful supply of nutrients and a balanced C:N is required for stable performance. Feedstocks were sampled from a range of over 100 different substrates in Ireland including for first, second and third generation feedstocks. The C:N ranged from 81:1 (Winter Oats) to 7:1 (Silage Effluent). The BMP yields were recorded ranging from 38 ± 2.0 L CH4 kg−1 VS for pig slurry (weaning pigs) to 805 ± 57 L CH4 kg−1 VS for used cooking oil (UCO). However the selection of the best preforming feedstock in terms of C:N ratio or BMP yield alone is not sufficiently adequate. A total picture has to be created which includes C:N ratio, BMP yield, harvest yield and availability. Potential feedstocks which best meet these requirements include for Grass silage, Milk processing waste (MPW) and Saccharina latissima. MPW has a potential of meeting over 6 times the required energy for Ireland’s 2020 transport in energy targets. S. Latissima recorded a yield of over 10,000 GJ ha-1 yr-1 which out ranks traditional second generation biofuels by a factor of more than 4.