Inhibition of L-type amino acid transport with non-physiological amino acids in the Pahenu2 mouse model of phenylketonuria

Phenylketonuria, an autosomal recessive Mendelian disorder, is one of the most common inborn errors of metabolism. Although currently treated by diet, many suboptimal outcomes occur for patients. Neuropathological outcomes include cognitive loss, white matter abnormalities, and hypo- or demyelination, resulting from high concentrations and/or fluctuating levels of phenylalanine. High phenylalanine can also result in competitive exclusion of other large neutral amino acids from the brain, including tyrosine and tryptophan (essential precursors of dopamine and serotonin). This competition occurs at the blood brain barrier, where the L-type amino acid transporter, LAT1, selectively facilitates entry of large neutral amino acids. The hypothesis of these studies is that certain non-physiological amino acids (NPAA; DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), α-aminoisobutyrate (AIB), and α-methyl-aminoisobutyrate (MAIB)) would competitively inhibit LAT1 transport of phenylalanine (Phe) at the blood-brain barrier interface. To test this hypothesis, Pah-/- mice (n=5, mixed gender; Pah+/-(n=5) as controls) were fed either 5% NL, 0.5% NB, 5% AIB or 3% MAIB (w/w 18% protein mouse chow) for 3 weeks. Outcome measurements included food intake, body weight, brain LNAAs, and brain monoamines measured via LCMS/MS or HPLC. Brain Phe values at sacrifice were significantly reduced for NL, NB, and MAIB, verifying the hypothesis that these NPAAs could inhibit Phe trafficking into the brain. However, concomitant reductions in tyrosine and methionine occurred at the concentrations employed. Blood Phe levels were not altered indicating no effect of NPAA competitors in the gut. Brain NL and NB levels, measured with HPLC, verified both uptake and transport of NPAAs. Although believed predominantly unmetabolized, NL feeding significantly increased blood urea nitrogen. Pah-/-disturbances of monoamine metabolism were exacerbated by NPAA intervention, primarily with NB (the prototypical LAT inhibitor). To achieve the overarching goal of using NPAAs to stabilize Phe transport levels into the brain, a specific Phe-reducing combination and concentration of NPAAs must be found. Our studies represent the first in vivo use of NL, NB and MAIB in Pah-/- mice, and provide proof-of-principle for further characterization of these LAT inhibitors. Our data is the first to document an effect of MAIB, a specific system A transport inhibitor, on large neutral amino acid transport.

EVALUATING THE OIL SANDS RECLAMATION PROCESS: ASSESSING POLICY CAPACITY AND STAKEHOLDER ACCESS FOR GOVERNMENT AND NON-GOVERNMENTAL ORGANIZATIONS OPERATING IN ALBERTA’S OIL SANDS

By employing interpretive policy analysis this thesis aims to assess, measure, and explain policy capacity for government and non-government organizations involved in reclaiming Alberta's oil sands. Using this type of analysis to assess policy capacity is a novel approach for understanding reclamation policy; and therefore, this research will provide a unique contribution to the literature surrounding reclamation policy. The oil sands region in northeast Alberta, Canada is an area of interest for a few reasons; primarily because of the vast reserves of bitumen and the environmental cost associated with developing this resource. An increase in global oil demand has established incentive for industry to seek out and develop new reserves. Alberta's oil sands are one of the largest remaining reserves in the world, and there is significant interest in increasing production in this region. Furthermore, tensions in several oil exporting nations in the Middle East remain unresolved, and this has garnered additional support for a supply side solution to North American oil demands. This solution relies upon the development of reserves in both the United States and Canada. These compounding factors have contributed to the increased development in the oil sands of northeastern Alberta. Essentially, a rapid expansion of oil sands operations is ongoing, and is the source of significant disturbance across the region. This disturbance, and the promises of reclamation, is a source of contentious debates amongst stakeholders and continues to be highly visible in the media. If oil sands operations are to retain their social license to operate, it is critical that reclamation efforts be effective. One concern non-governmental organizations (NGOs) expressed criticizes the current monitoring and enforcement of regulatory programs in the oil sands. Alberta's NGOs have suggested the data made available to them originates from industrial sources, and is generally unchecked by government. In an effort to discern the overall status of reclamation in the oil sands this study explores several factors essential to policy capacity: work environment, training, employee attitudes, perceived capacity, policy tools, evidence based work, and networking. Data was collected through key informant interviews with senior policy professionals in government and non-government agencies in Alberta. The following are agencies of interest in this research: Canadian Association of Petroleum Producers (CAPP); Alberta Environment and Sustainable Resource Development (AESRD); Alberta Energy Regulator (AER); Cumulative Environmental Management Association (CEMA); Alberta Environment Monitoring, Evaluation, and Reporting Agency (AEMERA); Wood Buffalo Environmental Association (WBEA). The aim of this research is to explain how and why reclamation policy is conducted in Alberta's oil sands. This will illuminate government capacity, NGO capacity, and the interaction of these two agency typologies. In addition to answering research questions, another goal of this project is to show interpretive analysis of policy capacity can be used to measure and predict policy effectiveness. The oil sands of Alberta will be the focus of this project, however, future projects could focus on any government policy scenario utilizing evidence-based approaches.