Tesis Doctorales
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Browsing Tesis Doctorales by Author "Klein Posternack, Andrés David"
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Item Identification of modifier genes/networks of lysosomal biology(Universidad del Desarrollo. Facultad de Medicina, 2023) Durán Mojica, Anyelo Alberto; Klein Posternack, Andrés David; Calderón, Juan FranciscoLysosomal storage diseases (LSDs) are a heterogeneous group of ~70 rare inherited metabolic diseases caused by loss-of-function variants in genes encoding for lysosomal enzymes, their activators, or transport proteins. Clinical symptoms manifest during early childhood or adolescence, causing varying degrees of disability and short life expectancies. At a cellular level, LSD cells show a progressive accumulation of undegraded substrates. In a subset of LSDs, called sphingolipidosis, the primary buildup material corresponds to lipids. These diseases can affect several organs, including the liver, brain, heart, peripheral nervous system, haematologic, skeletal, gastrointestinal system, lung, muscle, and others. On the other hand, defects in processing sphingolipids have also been observed in patients with common diseases such as neurodegenerative disorders and cancer. To date, the phenotypic variability observed in monogenic conditions is thought to be influenced by genomic loci variation other than in the primary disease locus. These genes are called modifiers. We harnessed the natural genetic variation between different strains of healthy mice to identify modifier genes/networks of lysosomal biology. We used a systems genetics approach. We measured the hepatic activity of 12 lysosomal enzymes and several of their natural substrates in livers derived from a panel of inbred mouse strains, followed by genetic regulators mapping by genome-wide association studies (GWAS), transcriptome associations, Bayesian integration, and pathway enrichment analysis. The GWA study identified 137 non-redundant genes associated with changes in lysosomal enzyme activities and 1744 modifiers for GSLs. Among these genes, 30 are shared between the enzymes and lipid groups. They are clustered into three pathways and associated with other nonLSD diseases. Surprisingly, they are under the regulation of ten common transcription factors, suggesting a common regulation of sphingolipid metabolism. In summary, we have identified novel regulators of lysosomal enzymes and GSL levels that may serve as therapeutic targets for LSD and implicated GSL metabolism in other diseases.Item Saccharomyces cerevisiae as a model for the identification of modifier genes of paraquat toxicity(Universidad del Desarrollo. Facultad de Medicina, 2024) Rubilar Espinoza, Juan Carlos; Klein Posternack, Andrés David; Cubillos Riffo, Francisco AlbertoParaquat (PQ) is a potent herbicide that induces oxidative stress and mitochondrial dysfunction. In humans, it is highly toxic, and it can induce Parkinson's Disease (PD). PD is a chronic and progressive neurodegenerative disease, with a worldwide prevalence of 315 per 100,000 people of all ages. Recent studies have revealed that neurons from PD patients exhibit stress responses, mitochondrial dysfunction, and metabolic deficits involving ATP and nicotinamide adenine dinucleotide (NAD+). While studies have demonstrated the potential of nicotinamide riboside (NR) to mitigate age- and disease-related metabolic decline in PD, the specific effects of NR on PD induced by pesticide exposure, such as paraquat, have not been extensively explored. Saccharomyces cerevisiae is a model organism that has allowed the study of relevant biological processes over time. It exhibits remarkable genetic diversity, making it an ideal model for studying the genetic basis of phenotypic variation. In this study, we hypothesized that genetically diverse Saccharomyces cerevisiae strains exposed to paraquat exhibit cellular and molecular responses that reveal potential modifier genes associated with its toxicity. We treated 1,011 isolates and 96 segregants from the cross of SA x WE founder Saccharomyces cerevisiae strains with PQ (75 μg/mL). We measured their growth curves and calculated the specific growth rate (μMax), used as a phenotypic trait for the genome-wide association studies (GWAS) and quantitative trait loci (QTL) mapping. We performed mixed-model association analysis using FaST-LMM for GWAS and linkage analysis using R/qtlsoftware, calculating LOD scores with a nonparametric model. We used diploid strains (603 isolates) for GWAS and 96 segregants for QTL mapping, identifying variants and markers that exceeded the significance threshold. Using these variants and markers, we identified candidate genes for validation. We validated NRT1 in a yeast mutant and subsequently showed that NR treatment provided significant protection against PQ-induced damage in both S288C yeast and iPSC-derived dopaminergic neurons from GBA-PD patients. In conclusion, this study provides valuable insights into the genetic and metabolic factors underlying PQ resistance in S. cerevisiae. The findings highlight the importance of NAD+ metabolism and mitochondrial function in mitigating the toxic effects of PQ. The identification of Nrt1 as a key transporter of NR, suggests potential therapeutic targets for interventions aimed at preventing or treating PQ-induced toxicity in PD.