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Many researchers, especially those new to the field, need to determine which criteria are essential for demonstrating autophagy, either for the purposes of their own research, or in the capacity of a manuscript or grant review [1,2]. Acceptable standards are an important issue, particularly considering that each of us may have her/his own opinion regarding the answer. Furthermore, as science progresses and the field evolves, the answer is in part a “moving target” [3]. This can be extremely frustrating for researchers who may think they have met those criteria, only to find out that the reviewers of their work disagree. Conversely, as a reviewer, it is tiresome to raise the same objections repeatedly, wondering why researchers have not fulfilled some of the basic requirements for establishing the occurrence of an autophagic process. In addition, drugs that potentially modulate autophagy are increasingly being used in clinical trials, and screens are being carried out for new drugs that can modulate autophagy for therapeutic purposes. Clearly, it is important to determine whether these drugs are truly affecting autophagy, and which step(s) of the process/es is/are affected, based on a set of accepted criteria. To this aim, we describe here a basic set of updated guidelines that can be used by researchers to plan and interpret their experiments, by clinicians to evaluate the literature with regard to autophagy-modulating therapies, and by both authors and reviewers to justify or criticize an experimental approach. Several fundamental points must be kept in mind as we establish guidelines for the selection of appropriate methods to monitor autophagy [2]. Importantly, there are no absolute criteria for determining autophagic status that are applicable in every single biological or experimental context. This is because some assays are nsuitable, problematic or may not work at all in particular cells, tissues or organisms [1–4]. For example, autophagic responses to drugs may be different in transformed versus nontransformed cells, in confluent versus nonconfluent cells, or in cells grown with or without glucose [5]. These guidelines are likely to evolve as new methodologies are developed and current assays are superseded. info:eu-repo/semantics/publishedVersion

Machado-Joseph Disease / Spinocerebellar Ataxia Type 3

O estudo da divergência entre espécies relacionadas, utilizando dados de vários loci é uma das formas de estudar a especiação e permite distinguir as forças que actuam em todo o genoma daquelas, como por exemplo a selecção natural, que afectam apenas os loci individualmente. Assim sendo, um dos grandes objectivos deste trabalho foi estudar a história da divergência entre duas espécies relacionadas, Drosophila madeirensis e D. subobscura , utilizando dados de sequências de DNA. O polimorfismo das inversões cromossómicas é uma característica comum do genoma do género Drosophila , no qual cerca de 60% das espécies são polimórficas para as inversões pericêntricas em populações naturais. O outro grande objectivo deste trabalho foi estudar o nível de variação nucleotídica em duas diferentes ordenações do cromossoma X de populações de D. subobscura (A2 e Ast). Assim, a análise da variação nucleotídica ao longo da inversão pode indicar o papel desempenhado pela selecção natural na formação e manutenção dos polimorfismos cromossómicos. Para atingir estes dois principais objectivos foram estudadas cinco regiões (que se distribuíam ao longo da inversão A2) do cromossoma X. De uma forma geral, o nível de polimorfismo nucleotídico encontrado foi similar nas duas espécies, apesar de D. madeirensis ser uma espécie endémica da ilha da Madeira e as populações de D. subobscura estudadas serem também insulares, e por tal se espera que possuam um tamanho populacional efectivo pequeno. Estimou-se o tempo de divergência entre D. subobscura e D. madeirensis em 640.000 e 1.400.000 anos e o modelo de isolamento sem fluxo génico após a divergência como causa de especiação não foi rejeitado. Em relação ao estudo das ordenações cromossómicas de D. subobscura , verificou-se que existe uma grande diferenciação genética em todas as regiões estudadas entre as duas ordenações. Os dados apontam para a existência de fenómenos de conversão génica e pouca evidência de troca de material genético na parte central da inversão. An approach to analyze the genetic changes that occur in populations during speciation is to examine the level and pattern of DNA sequence variation in species that recently shared a common ancestor. This approach becomes even more informative when data from multiple loci are available and thus they can be used to distinguish forces that act on all genes from those, like natural selection or gene flow, that affect individual loci. Therefore, one of the main goals of this study was shed some light into speciation divergence process between D. subobscura and D. madeirensis , using data from five gene regions of X chromosome. Chromosomal inversion polymorphism is a common feature of the genome in the Drosophila genus. About 60% of the Drosophila species are polymorphic for paracentric inversions in natural populations. D. subobscura is a species with a very rich inversion polymorphism, thus, another objective of this study was to analyze the levels of nucleotide variation in two different chromosomal arrangements of this species: A2 and Ast. The levels of nucleotide polymorphism found were very similar between the two species, although D. madeirensis is expected to have a much lower effective population size. A divergence process in allopatry without gene flow could be a likely explanation for the speciation between D. madeirensis and D. subobscura , as the isolation model without migration was not rejected. A strong genetic differentiation in all studied regions was detected between the two chromosomal arrangements of D. subobscura , and evidence of gene conversion was also detected. These data, with those previously reported for the O3 inversion, if extended to other inversions of D. subobscura might indicate that the genome of this species is highly structured.