Development and application of quantitative proteomics strategies to analyze molecular mechanisms of neurodegeneration

Neurodegenerative disorders such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis or Prion diseases are chronic, incurable and often fatal. A cardinal feature of all neurodegenerative disorders is the accumulation of misfolded and aggregated proteins. Depending on the disease, these aggregated proteins are cell type specific and have distinct cellular localizations, compositions and structures. Despite intensive research, the contribution of protein misfolding and aggregation to the cell type specific toxicity is not completely understood. In recent years, quantitative proteomics has matured into an exceptionally powerful technology providing accurate quantitative information on almost all cellular proteins as well as protein interactions, modifications, and subcellular localizations, which cannot be addressed by other omics technologies. The aim of this thesis is to investigate key features of neurodegeneration such as misfolded proteins and toxic protein aggregates with cutting edge proteomics, presenting a technological “proof of concept” and novel insights into the (patho)physiology of neurodegeneration

Development and application of quantitative proteomics strategies to analyze molecular mechanisms of neurodegeneration

Neurodegenerative disorders such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis or Prion diseases are chronic, incurable and often fatal. A cardinal feature of all neurodegenerative disorders is the accumulation of misfolded and aggregated proteins. Depending on the disease, these aggregated proteins are cell type specific and have distinct cellular localizations, compositions and structures. Despite intensive research, the contribution of protein misfolding and aggregation to the cell type specific toxicity is not completely understood. In recent years, quantitative proteomics has matured into an exceptionally powerful technology providing accurate quantitative information on almost all cellular proteins as well as protein interactions, modifications, and subcellular localizations, which cannot be addressed by other omics technologies. The aim of this thesis is to investigate key features of neurodegeneration such as misfolded proteins and toxic protein aggregates with cutting edge proteomics, presenting a technological “proof of concept” and novel insights into the (patho)physiology of neurodegeneration

Cross-Platform Comparison of Untargeted and Targeted Lipidomics Approaches on Aging Mouse Plasma.

Lipidomics - the global assessment of lipids - can be performed using a variety of mass spectrometry (MS)-based approaches. However, choosing the optimal approach in terms of lipid coverage, robustness and throughput can be a challenging task. Here, we compare a novel targeted quantitative lipidomics platform known as the Lipidyzer to a conventional untargeted liquid chromatography (LC)-MS approach. We find that both platforms are efficient in profiling more than 300 lipids across 11 lipid classes in mouse plasma with precision and accuracy below 20% for most lipids. While the untargeted and targeted platforms detect similar numbers of lipids, the former identifies a broader range of lipid classes and can unambiguously identify all three fatty acids in triacylglycerols (TAG). Quantitative measurements from both approaches exhibit a median correlation coefficient (r) of 0.99 using a dilution series of deuterated internal standards and 0.71 using endogenous plasma lipids in the context of aging. Application of both platforms to plasma from aging mouse reveals similar changes in total lipid levels across all major lipid classes and in specific lipid species. Interestingly, TAG is the lipid class that exhibits the most changes with age, suggesting that TAG metabolism is particularly sensitive to the aging process in mice. Collectively, our data show that the Lipidyzer platform provides comprehensive profiling of the most prevalent lipids in plasma in a simple and automated manner

Emotive Captioning and Access to Television

Closed captioning has been enabling access to television for people who are deaf and hard of hearing since the early 1970s. Since that time, technology and people’s demands have been steadily improving and increasing. Closed captioning has not kept up with these changes. We present the results of a study that used graphics, colour, icons and animation as well as text, emotive captions, to capture more of the sound information contained in television content. deaf and hard of hearing participants compared emotive and conventional captions for two short video segments. The results showed that there was a significant difference between deaf and hard of hearing viewers in their reaction to the emotive captions. Hard of hearing viewers seemed to enjoy them and find them interesting. deaf viewers had a strong dislike for them although they did see some potential for intermittent use of emotive captions or for use with children’s programs

Proteomics and C9orf72 neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity

Frontotemporal dementia and amyotrophic lateral sclerosis patients with C9orf72 mutation show cytoplasmic poly-GR and poly-PR aggregates. Short poly-(Gly-Arg) and poly-(Pro-Arg) (poly-GR/PR) repeats localizing to the nucleolus are toxic in various model systems, but no interactors have been validated in patients. Here, the neuronal interactomes of cytoplasmic GFP-(GR)(149) and nucleolar (PR)(175)-GFP revealed overlapping RNA-binding proteins, including components of stress granules, nucleoli, and ribosomes. Overexpressing the poly-GR/PR interactors STAU1/2 and YBX1 caused cytoplasmic aggregation of poly-GR/PR in large stress granule-like structures, whereas NPM1 recruited poly-GR into the nucleolus. Poly-PR expression reduced ribosome levels and translation consistent with reduction of synaptic proteins detected by proteomics. Surprisingly, truncated GFP-(GR)(53), but not GFP-(GR)(149), localized to the nucleolus and reduced ribosome levels and translation similar to poly-PR, suggesting that impaired ribosome biogenesis may be driving the acute toxicity observed in vitro. In patients, only ribosomes and STAU2 co-aggregated with poly-GR/PR. Partial sequestration of ribosomesmay chronically impair protein synthesis even in the absence of nucleolar localization and contribute to pathogenesis

EMPREGABILIDADE

INTRODUÇÃOSegundo a “Pesquisa Nacional por Amostra de Domicílios” (PNAD), as taxas de desemprego no Brasil vem crescendo desde 2012, devido à fatores como a baixa qualificação do trabalhador, industrialização e até a Crise econômica que vem sendo enfrentada desde então. Inclusive, foi registrado um aumento de 7,9% para 11,3% na taxa de desemprego no Brasil desde início de 2012 até o final do 2° trimestre de 2016.Gráfico 1 – Taxa de ocupação. O breve interesse para dar início ao trabalho surgiu quando os noticiários e jornais declararam que o país estava em uma situação deplorável de crise econômica e política, com isso foram feitos diversos estudos através de periódicos e portais de informações, com o intuito de adquirir informações sobre o assunto principal do projeto. Inicialmente seria desemprego o foco principal, porém, como o desemprego é consequência da alta evolução industrial e tecnológica do mercado, a empregabilidade tornou-se mais relevante no projeto, sendo assim o desemprego tornou-se uma das vertentes no projeto definitivo.Com isso, o objetivo do trabalho é, através de uma palestra, auxiliar o público alvo (especialmente os jovens), com orientações, informações e dicas, para entrar, manter-se e evoluir no mercado de trabalho. MATERIAIS E MÉTODOSPara a elaboração da palestra, foi feita uma revisão bibliográfica sobre empregabilidade, desemprego e formação profissional (cursos técnicos), sendo ela feita em livros obtidos na biblioteca do campus, em sites e periódicos que tratam do assunto. Os materiais que foram necessários para a execução do projeto foram:  ITEMDESCRIÇÃOQTD.Projetor MultimídiaProjetor multimídia disponibilizado pelo IFC1Local da apresentaçãoInstituto Federal Catarinense1ComputadorComputador usado para a apresentação multimídia1Caixa de somÀudio da apresentação1 RESULTADOS E DISCUSSÃOComo fruto de estudos aprofundados sobre o assunto, existem índices de empregabilidade relatando que a maioria de uma quantia de pessoas está presente no mercado, e isso é influência do período acadêmico em que as pessoas permanecem. O gráfico logo abaixo (fonte em “referências”) apresenta a porcentagem total de um público-alvo da pesquisa, aproximadamente 60% dos entrevistados estão efetivados em seus respectivos empregos, sendo quase 40% efetivados em escritório 15,42% em empresas como funcionários (não incluindo estagiários em período de aprendizagem) e quase 5% possuem ou fundaram seu próprio negócio. Gráfico 2 – Índice de empregabilidadeFonte:FGV Diretório Rio, Empregabilidade1.Desse mesmo total, 35,25% estão presentes na área pública, aproximadamente 26% estudando ou concluindo concurso público, 10% com cargo em comissão e no restante, que corresponde a quase 5% estão em área acadêmica. Isso deixa explícito que a formação acadêmica exerce grande influência no mercado assim como na carreira de futuros empregados ou empregadores. A formação, empregabilidade e cursos técnicos assim como o desemprego que é uma das vertentes do trabalho se mostram complexos para o grupo e os discentes que serão o foco do projeto. Mas o objetivo principal é repassar tudo o que estudamos e concluímos sobre o projeto da forma mais clara e compreensível possível. Foi realizada a estruturação da palestra, decidindo a ordem dos temas abordados, começando por desemprego, sucedido por empregabilidade e cursos de profissionalização, além da separação dos temas entre os apresentadores da palestra. A palestra encontra-se em elaboração final. CONCLUSÃOCom os resultados obtidos até o momento, pode-se dizer que assuntos que envolvem e dependem de fatos político-econômicos (como os que têm sido estudados) dependem de um grande esforço para ser compreendido e bem interpretado, e de um maior ainda para repassar isso para outros.1Disponível em: http://direitorio.fgv.br/graduacao/indices/empregabilidade   

Circulating Glucagon 1-61 Regulates Blood Glucose by Increasing Insulin Secretion and Hepatic Glucose Production

Glucagon is secreted from pancreatic a cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in b cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion

Deep Proteomic Evaluation of Primary and Cell Line Motoneuron Disease Models Delineates Major Differences in Neuronal Characteristics*

The fatal neurodegenerative disorders amyotrophic lateral sclerosis and spinal muscular atrophy are, respectively, the most common motoneuron disease and genetic cause of infant death. Various in vitro model systems have been established to investigate motoneuron disease mechanisms, in particular immortalized cell lines and primary neurons. Using quantitative mass-spectrometry-based proteomics, we compared the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a, as well as to non-neuronal control cells, at a depth of 10,000 proteins. We used this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicated substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton, and receptor signaling, whereas common metabolic pathways were more similar. The proteins associated with amyotrophic lateral sclerosis also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how mass-spectrometry-based proteomics can be used to evaluate disease model systems