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.

Assessment of dietary background exposure of the Irish adult population to dioxins and PCBs particularly taking into account additional exposure due to the 2008 Irish dioxin food contamination incident

Irish monitoring data on PCDD/Fs, DL-PCBs and Marker PCBs were collated and combined with Irish Adult Food Consumption Data, to estimate dietary background exposure of Irish adults to dioxins and PCBs. Furthermore, all available information on the 2008 Irish pork dioxin food contamination incident was collated and analysed with a view to evaluate any potential impact the incident may have had on general dioxin and PCB background exposure levels estimated for the adult population in Ireland. The average upperbound daily intake of Irish adults to dioxins Total WHO TEQ (2005) (PCDD/Fs & DLPCBs) from environmental background contamination, was estimated at 0.3 pg/kg bw/d and at the 95th percentile at 1 pg/kg bw/d. The average upperbound daily intake of Irish adults to the sum of 6 Marker PCBs from environmental background contamination ubiquitous in the environment was estimated at 1.6 ng/kg bw/d and at the 95th percentile at 6.8 ng/kg bw/d. Dietary background exposure estimates for both dioxins and PCBs indicate that the Irish adult population has exposures below the European average, a finding which is also supported by the levels detected in breast milk of Irish mothers. Exposure levels are below health based guidance values and/or Body Burdens associated with the TWI (for dioxins) or associated with a NOAEL (for PCBs). Given the current toxicological knowledge, based on biomarker data and estimated dietary exposure, general background exposure of the Irish adult population to dioxins and PCBs is of no human health concern. In 2008, a porcine fat sample taken as part of the national residues monitoring programme led to the detection of a major feed contamination incidence in the Republic of Ireland. The source of the contamination was traced back to the use of contaminated oil in a direct-drying feed operation system. Congener profiles in animal fat and feed samples showed a high level of consistency and pinpointed the likely source of fuel contamination to be a highly chlorinated commercial PCB mixture. To estimate additional exposure to dioxins and PCBs due to the contamination of pig and cattle herds, collection and a systematic review of all data associated with the contamination incident was conducted. A model was devised that took into account the proportion of contaminated product reaching the final consumer during the 90 day contamination incident window. For a 90 day period, the total additional exposure to Total TEQ (PCDD/F &DL-PCB) WHO (2005) amounted to 407 pg/kg bw/90d at the 95th percentile and 1911 pg/kg bw/90d at the 99th percentile. Exposure estimates derived for both dioxins and PCBs showed that the Body Burden of the general population remained largely unaffected by the contamination incident and approximately 10 % of the adult population in Ireland was exposed to elevated levels of dioxins and PCBs. Whilst people in this 10 % cohort experienced quite a significant additional load to the existing body burden, the estimated exposure values do not indicate approximation of body burdens associated with adverse health effects, based on current knowledge. The exposure period was also limited in time to approximately 3 months, following the FSAI recall of contaminated meat immediately on detection of the contamination. A follow up breast milk study on Irish first time mothers conducted in 2009/2010 did not show any increase in concentrations compared to the study conducted in 2002. The latter supports the conclusion that the majority of the Irish adult population was not affected by the contamination incident.