Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

Impact of mutated KRAS signalling on autophagy regulation in colorectal carcinoma

Programa Doutoral em Biologia Molecular e Ambiental

“PhenoWorld”: a new multidimensional strategy for studying behaviour in rodents

Tese de Doutoramento em Ciências da Saúde

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.

Development of computational and experimental methods for biomass composition and evaluation of its impact in genome-scale models prediction

The use of genome-scale metabolic models has been rapidly increasing in fields such as metabolic engineering. An important part of a metabolic model is the biomass equation since this reaction will ultimately determine the predictive capacity of the model in terms of essentiality and flux distributions. Thus, in order to obtain a reliable metabolic model the biomass precursors and their coefficients must be as precise as possible. Ideally, determination of the biomass composition would be performed experimentally, but when no experimental data are available this is established by approximation to closely related organisms. Computational methods however, can extract some information from the genome such as amino acid and nucleotide compositions. The main objectives of this study were to compare the biomass composition of several organisms and to evaluate how biomass precursor coefficients affected the predictability of several genome-scale metabolic models by comparing predictions with experimental data in literature. For that, the biomass macromolecular composition was experimentally determined and the amino acid composition was both experimentally and computationally estimated for several organisms. Sensitivity analysis studies were also performed with the Escherichia coli iAF1260 metabolic model concerning specific growth rates and flux distributions. The results obtained suggest that the macromolecular composition is conserved among related organisms. Contrasting, experimental data for amino acid composition seem to have no similarities for related organisms. It was also observed that the impact of macromolecular composition on specific growth rates and flux distributions is larger than the impact of amino acid composition, even when data from closely related organisms are used.

Análise de fragmentos arqueológicos: um diálogo entre a Química, Biologia e Arqueologia

Dissertação de mestrado em Técnicas de Caracterização e Análise Química

Dos efeitos da Procriação Medicamente Assistida no Direito Sucessório

Dissertação de mestrado em Direito das Crianças, Família e Sucessões

The role of macroH2A1 in prostate carcinogenesis

Dissertação de mestrado em Genética Molecular