Investigation of MAT2A dependency in Acute Myeloid Leukaemia: a preliminary study in Acute Myeloid Leukaemia cell line

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2017Acute Myeloid Leukemia (AML) is a disease of dismal prognosis, with a 5-year survival of under 40% that has remained unmodified for the last 3 decades. Sequencing analyses of patient samples have identified recurrent mutations and highlighted associated common disease mechanisms, which have putative therapeutic value. Additionally, loss-of-function genetic or expression knockdown screens can be used to identify individual factors required for disease maintenance that may not be targeted by mutation. A recent drop-out screen based on Clustered Regularly Interspaced Short Palindromic Repeats with the Cas9 endonuclease technology (CRISPR/Cas9) identified MAT2A (methionine adenosyltransferase2A) as a susceptibility gene in at least some forms of AML. In the work reported in this thesis, I aimed to validate the results of the screen by confirming dependency of cultured AML cells on MAT2A expression. I selected AML cell lines representing distinct common subtypes of human leukaemia, and evaluated the effects of knocking-out MAT2A through CRISPR/Cas9 gene editing technology. As a complementary strategy, I developed and validated RNA interference (RNAi) tools for MAT2A expression knockdown, which are also amenable for testing in AML patient samples. I observed that MAT2A loss resulted in reduced proliferation and survival of cultured AML cells. These results are currently being pursued mechanistically with a view to establishing novel therapeutic strategies in AML.A Leucemia mieloide aguda (LMA) é uma doença com um prognóstico bastante reservado, na qual menos de 40% dos doentes sobrevivem até 5 anos após diagnóstico da doença, sendo que esta percentagem permaneceu inalterada desde as últimas 3 décadas. As análises de sequenciação de amostras de pacientes identificaram mutações recorrentes e destacaram mecanismos associados, que têm valor terapêutico putativo. Além disso, fazer um screen para avaliar perdas de função genética ou mesmo knockdown de expressão de genes, pode ser útil na medida em que certos fatores individuais podem ser necessários para a manutenção da doença sem que estes sejam alvo de mutação. Um recente drop-out screen, que tem como base ir avaliando as consequências da perda individual de todos os fatores, baseada na tecnologia CRISPR, que consiste em pequenas porções do DNA bacteriano compostas por repetições de nucleotídeos, associada à endonuclease Cas9 (CRISPR/Cas9) identificaram o gene MAT2A (metionina adenosiltransferase2A) como um gene susceptível em pelo menos algumas formas de LMA. No trabalho exposto nesta tese, pretendi validar os resultados desse screen, confirmando a dependência das células com LMA cultivadas sobre a expressão de MAT2A. Selecionei linhas celulares com LMA que representavam subtipos comuns distintos de leucemia humana e avaliei os efeitos do knockout de MAT2A através da tecnologia de edição de genes CRISPR / Cas9. Como uma estratégia complementar, desenvolvi e legitimei ferramentas de RNA interferência (RNAi) para o knockdown de expressão de MAT2A, que também pode ser testado em amostras de pacientes com LMA. Observei que a perda de MAT2A resultou numa diminuição da proliferação e da sobrevivência nas células cultivadas com LMA. Estes resultados estão atualmente a ser seguidos mecanicamente com vista a estabelecer novas estratégias terapêuticas em LM

Estimation of mineral dust direct radiative forcing at the European Aerosol Research Lidar NETwork site of Lecce, Italy, during the ChArMEx/ADRIMED summer 2013 campaign: Impact of radiative transfer model spectral resolutions

© 2016. American Geophysical UnionA field campaign took place in the western and central Mediterranean basin on June–July 2013 in the framework of the ChArMEx (Chemistry-Aerosol Mediterranean Experiment, http://charmex.lsce.ipsl.fr/)/ADRIMED (Aerosol Direct Radiative Impact on the regional climate in the MEDiterranean region, http://adrimed.sedoo.fr/) project to characterize the aerosol direct radiative forcing (DRF) over the Mediterranean. This work focuses on the aerosol DRF estimations at Lecce (40.33°N; 18.11°E; 30¿m above sea level) during the Saharan dust outbreak that affected southern Italy from 20 to 24 June 2013. The Global Atmospheric Model (GAME) and the Two-Stream (TS) model were used to calculate the instantaneous aerosol DRF in the short-wave (SW) and long-wave (LW) spectral ranges, at the surface and at the top of the atmosphere (TOA). The main differences between the two models were due to the different numerical methods to solve the radiative transfer (RT) equations and to the more detailed spectral resolution of GAME compared to that of TS. 167 and 115 subbands were used by GAME in the 0.3–4 and 4–37¿µm spectral ranges, respectively. Conversely, the TS model used 8 and 11 subbands in the same spectral ranges, respectively. We found on 22 June that the SW-DRFs from the two models were in good agreement, both at the TOA and at the surface. The instantaneous SW-DRFs at the surface and at the TOA varied from -50 to -34¿W¿m-2 and from -6 to +8¿W¿m-2, respectively, while the surface and TOA LW-DRFs ranged between +3.5 and +8.0¿W¿m-2 and between +1.7 and +6.9¿W¿m-2, respectively. In particular, both models provided positive TOA SW-DRFs at solar zenith angles smaller than 25° because of the mixing of the desert dust with anthropogenic pollution during its transport to the study site. In contrast, the TS model overestimated the GAME LW-DRF up to about 5 and 7.5 times at the surface and at the TOA, respectively, when the dust particle contribution was largest. The low spectral resolution of the real (n) and imaginary (k) refractive index values was mainly responsible for the LW-DRF overestimates of the TS model. However, we found that the “optimization” of the n and k values at 8.75 and 11.5¿µm was sufficient in this study to obtain a satisfactory agreement between the LW-DRFs from the two models, both at the TOA and at the surface. The impact of the spectral dependence of the water vapor absorption coefficients on the estimation of the flux without aerosol has also been addressed. Paper results did not reveal any significant impact due to the different numerical methods used by the two models to solve the RT equations.Peer ReviewedPostprint (published version

The effect of FK506 on transforming growth factor beta signaling and apoptosis in chronic lymphocytic leukemia B cells.

Background Loss of response to transforming growth factor-beta (TGF-beta) is thought to contribute to the progression of chronic lymphocytic leukemia. Recent findings of over-activation of the TGF-beta signal in FKBP12-knockout mouse prompted us to investigate whether FK506, the canonical ligand of FKBP, can activate the TGF-beta signal in chronic lymphocytic leukemia. Design and Methods We studied 62 chronic lymphocytic leukemia samples from patients with Rai/Binet stage 0 to 4 disease. The TGF-beta signal was investigated by western blotting and flow cytometry. The levels of Bcl2-family members and death-associated-protein kinase were also investigated by western blotting, whereas apoptosis was studied in flow cytometry. Down-modulation of FKBP12 was obtained by gene silencing with short interfering RNA. Results Twenty-two out of 62 chronic lymphocytic leukemia samples were sensitive to TGF-beta-induced apoptosis. All but two of the responsive samples underwent apoptosis also when cultured with FK506, but not with cyclosporine. Thirteen samples that were not sensitive to TGF-beta were sensitive to FK506. Overall, response to FK506 occurred in 33 samples. FK506 induced Smad2 phosphorylation and nuclear translocation. Accordingly, death-associated-protein kinase, a transcriptional target of Smad, was induced. At the same time, Bcl-2 and Bcl-xL levels decreased whereas the levels of Bim and Bmf increased. A loss of mitochondrial membrane potential preceded caspase activation and cell death. FK506 removed FKBP12 from its binding to the TGF-beta-receptor. FKBP12 release activated the receptor-kinase activity as suggested by the enhanced levels of phospho-Smad found in cells depleted of FKBP12. Conclusions Our study shows that most chronic lymphocytic leukemia cells escape the homeostatic control of TGF-beta and that FK506 restores the TGF-beta signal in a proportion of non-responsive samples. We demonstrated that FK506 activates TGF-beta receptor I kinase activity in chronic lymphocytic leukemia, which transduces apoptosis by a mitochondrial-dependent pathway

Impact of phosphate factory on the biological characteristics of North Lebanon surface sediments (Levantine Basin)

11 p.This study aims to analyse the surface sediment of Batroun coastal area in North Lebanon in order to reveal the integrating impact of Selaata chemical plant on its biological properties. Sediment samples were collected with cores by diving at 12 stations in summer period between mid July and beginning of August 2003. The values of the hydobiological parameters in the first 3 cm were ranging respectively between: 4.17 to 7.9 for pH, 0 to 171 mV for Eh,, 0 to 0.94 µg.g-1 for chlorophyll a and 24 to 4166 ind.10 cm-2 for meiofauna. The results showed that there was a reverse relationship between chlorophyll a and meiofauna and that Batroun marine area could be separated in two main areas of influence The first area includes the stations located in front of the main north-western emissary and where the sediments were more acid and less oxygenated with strong odour of hydrogen sulphide accompanied with high chlorophyll a contents and low numbers of meiofauna. The second area is consisted of stations located to the south or far offshore from the plant and where the sediments are characterized by lower acidity and higher redox potential with low chlorophyll a concentrations and higher population of meifauna. Exception could be found in some stations like M8 and M 13 that presented particular cases. The impact on the biological indices was limited to plant proximity where conditions were more perturbing and the variability in the concentrations of chlorophyll a and the numbers of meiofauna were not depending on sediments compositions but on the nature and toxicity of particulate matters rejected out of plant's emissaries such as the phosphogypsum

Enhanced DNA mixture deconvolution of sexual offense samples using the DEPArray™ system

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Airborne Bacteria structure and chemical composition relationships in winter and spring PM10 samples over southeastern Italy

Abstract The Redundancy Discrimination Analysis (RDA) and Spearman correlation coefficients were used to investigate relationships between airborne bacteria at the phylum and genus level and chemical species in winter and spring PM10 samples over Southeastern Italy. The identification of main chemical species/pollution sources that were related to and likely affected the bacterial community structure was the main goal of this work. The 16S rRNA gene metabarcoding approach was used to characterize airborne bacteria. Seventeen phyla and seventy-nine genera contributing each by mean within-sample relative abundance percentage > 0.01% were identified in PM10 samples, which were chemically characterized for 33 species, including ions, metals, OC, and EC (organic and elemental carbon, respectively). Chemical species were associated with six different pollution sources. A shift from winter to spring in both bacterial community structure and chemical species mass concentrations/sources and the relationships between them was observed. RDA triplots pointed out significant correlations for all tested bacterial phyla (genera) with other phyla (genera) and/or with chemical species, in contrast to correlation coefficient results, which showed that few phyla (genera) were significantly correlated with chemical species. More specifically, in winter Bacillus and Chryseobacterium were the only genera significantly correlated with chemical species likely associated with particles from soil-dust and anthropogenic pollution source, respectively. In spring, Enterobacter and Sphingomonas were the only genera significantly correlated with chemical species likely associated with particles from the anthropogenic pollution and the marine and soil-dust sources, respectively. The results of this study also showed that the correlation coefficients were the best tool to obtain unequivocal identifications of the correlations of phyla (genera) with chemical species. The seasonal changes of the PM10 chemical composition, the microbial community structure, and their relationships confidentially suggested that the seasonal changes of atmospheric particles may have likely contributed to seasonal changes of bacterial community in the atmosphere

NF-κB pathway activators as potential ageing biomarkers: targets for new therapeutic strategies.

Chronic inflammation is a major biological mechanism underpinning biological ageing process and age-related diseases. Inflammation is also the key response of host defense against pathogens and tissue injury. Current opinion sustains that during evolution the host defense and ageing process have become linked together. Thus, the large array of defense factors and mechanisms linked to the NF-κB system seem to be involved in ageing process. This concept leads us in proposing inductors of NF-κB signaling pathway as potential ageing biomarkers. On the other hand, ageing biomarkers, represented by biological indicators and selected through apposite criteria, should help to characterize biological age and, since age is a major risk factor in many degenerative diseases, could be subsequently used to identify individuals at high risk of developing age-associated diseases or disabilities. In this report, some inflammatory biomarkers will be discussed for a better understanding of the concept of biological ageing, providing ideas on eventual working hypothesis about potential targets for the development of new therapeutic strategies and improving, as consequence, the quality of life of elderly populatio

As ilhas oceânicas dos Açores por Vitorino Nemésio e Cecília Meireles: Paisagem, viagem e memória

O objetivo deste trabalho é realizar uma análise temática e comparativa, à luz dos conceitos de paisagem, espaço, lugar e memória, de seis textos de Vitorino Nemésio e de Cecília Meireles nos quais se representam as ilhas atlânticas do arquipélago dos Açores. Nestes textos, a paisagem se revela como manifestação da topografia ontológica de cada autor e resulta num processo subjetivo de construção e ressignificação dos lugares das ilhas. Em comum, os textos destes autores partilham os temas da simplicidade da vida dos ilhéus, do isolamento e da pobreza. Se estes traços os aproximam, outros há que os afastam. Se em Nemésio, as ilhas são transfiguradas em paisagem turística e espaço de emigração, espelhando a substância e os constrangimentos da açorianiedade, em Cecília, a ilha surge como um lugar na fronteira entre o real e o imaginário, construído pela fantasia da poeta, a partir da sua geografia emocional e das memórias de infância. Se em Nemésio, os textos manifestam imagens-reservatórios de memória individual e coletiva, em Cecília, o trabalho da memória facilita uma fuga ao real e uma viagem a uma realização do mito do paraíso perdido: a Ilha do Nanjainfo:eu-repo/semantics/publishedVersio