The study of pexophagy (selective peroxisome degradation by autophagy) is relevant to other selective autophagy pathways that are implicated in human diseases. In this dissertation I explore the signaling of pexophagy by gamma;-aminobutyric acid (GABA), the protein-protein interactions of the pexophagy receptor and its regulation by phosphorylation and the putative role of a pexophagy receptor-associated protein in pexophagy. Yeast was used as a model organism to study the physiological effects of elevated levels of the inhibitory neurotransmitter, GABA, in mammalian disorders, such as succinic acid- semialdehydyde dehydrogenease (SSADH) deficiency. GABA blocked pexophagy and mitophagy specifically in yeast, but not general, or other selective autophagy pathways and increased intracellular levels of reactive oxygen species (ROS). Mechanistically, the GABA effect was mediated by elevation of Tor1 activity and the effects of GABA could be alleviated by rapamycin, a Tor1 inhibitor. These results were extended to studies in SSADH mice, suggesting the possibility of using rapamycin and its analogues to alleviate disorders caused by elevated GABA levels. In studies involving the Pichia pastoris pexophagy receptor, Atg30, we found that this protein is regulated by phosphorylation: determining the ability of Atg30 to interact with two key proteins of the core autophagy machinery, Atg8 and Atg11, thereby activating pexophagy. We also found that the phosphorylation state of the receptor is affected by its interaction with a component, Pex3 (of the peroxisome biogenesis machinery). We describe a new domain on, and a function for, Pex3 that regulates the phosphorylation, and therefore activation, of Atg30. Finally, by examining the protein-protein interactions of the Atg30 network to better understand the pexophagy signaling process, we identified using mass spectrometry, a potential interacting protein, Rvs167, that could also be involved in pexophagy. In summary, these studies shed light on the signaling and regulation of the important selective autophagy pathway of pexophagy

Pexophagy is a process that selectively degrades peroxisomes by autophagy. The Pichia pastoris pexophagy receptor Atg30 is recruited to peroxisomes under peroxisome proliferation conditions. During pexophagy, Atg30 undergoes phosphorylation, a prerequisite for its interactions with the autophagy scaffold protein Atg11 and the ubiquitin-like protein Atg8. Atg30 is subsequently shuttled to the vacuole along with the targeted peroxisome for degradation. Here, we defined the binding site for Atg30 on the peroxisomal membrane protein Pex3 and uncovered a role for Pex3 in the activation of Atg30 via phosphorylation and in the recruitment of Atg11 to the receptor protein complex. Pex3 is classically a docking protein for other proteins that affect peroxisome biogenesis, division, and segregation. We conclude that Pex3 has a role beyond simple docking of Atg30 and that its interaction with Atg30 regulates pexophagy in the yeast P. pastoris.

In addition to key roles in embryonic neurogenesis and myelinogenesis, γ-aminobutyric acid (GABA) serves as the primary inhibitory mammalian neurotransmitter. In yeast, we have identified a new role for GABA that augments activity of the pivotal kinase, Tor1. GABA inhibits the selective autophagy pathways, mitophagy and pexophagy, through Sch9, the homolog of the mammalian kinase, S6K1, leading to oxidative stress, all of which can be mitigated by the Tor1 inhibitor, rapamycin. To confirm these processes in mammals, we examined the succinic semialdehyde dehydrogenase (SSADH)-deficient mouse model that accumulates supraphysiological GABA in the central nervous system and other tissues. Mutant mice displayed increased mitochondrial numbers in the brain and liver, expected with a defect in mitophagy, and morphologically abnormal mitochondria. Administration of rapamycin to these mice reduced mTOR activity, reduced the elevated mitochondrial numbers, and normalized aberrant antioxidant levels. These results confirm a novel role for GABA in cell signaling and highlight potential pathomechanisms and treatments in various human pathologies, including SSADH deficiency, as well as other diseases characterized by elevated levels of GABA.

Abstract Background Deployment of highly effective artemisinin-based combination therapy for treating uncomplicated malaria calls for better targeting of malaria treatment to improve case management and minimize drug pressure for selecting resistant parasites. The Integrated Management of Malaria curriculum was developed to train multi-disciplinary teams of clinical, laboratory and health information assistants. Methods Evaluation of training was conducted in nine health facilities that were Uganda Malaria Surveillance Programme (UMSP) sites. From December 2006 to June 2007, 194 health professionals attended a six-day course. One-hundred and one of 118 (86%) clinicians were observed during patient encounters by expert clinicians at baseline and during three follow-up visits approximately six weeks, 12 weeks and one year after the course. Experts used a standardized tool for children less than five years of age and similar tool for patients five or more years of age. Seventeen of 30 laboratory professionals (57%) were assessed for preparation of malaria blood smears and ability to interpret smear results of 30 quality control slides. Results Percentage of patients at baseline and first follow-up, respectively, with proper history-taking was 21% and 43%, thorough physical examination 18% and 56%, correct diagnosis 51% and 98%, treatment in compliance with national policy 42% and 86%, and appropriate patient education 17% and 83%. In estimates that adjusted for individual effects and a matched sample, relative risks were 1.86 (95% CI: 1.20,2.88) for history-taking, 2.66 (95%CI: 1.60,4.41) for physical examination, 1.77 (95%CI: 1.41,2.23) for diagnosis, 1.96 (95%CI: 1.46,2.63) for treatment, and 4.47 (95%CI: 2.68,7.46) for patient education. Results were similar for subsequent follow-up and in sub-samples stratified by patient age. Quality of malaria blood smear preparation improved from 21.6% at baseline to 67.3% at first follow-up (p < 0.008); sensitivity of interpretation of quality control slides increased from 48.6% to 70.6% (p < 0.199) and specificity increased from 72.1% to 77.2% (p < 0.736). Results were similar for subsequent follow-up, with the exception of a significant increase in specificity (94.2%, p < 0.036) at one year. Conclusion A multi-disciplinary team training resulted in statistically significant improvements in clinical and laboratory skills. As a joint programme, the effects cannot be distinguished from UMSP activities, but lend support to long-term, on-going capacity-building and surveillance interventions.