Study of the cholestatic potential of relevant concentrations of cyclosporine A in primary human hepatocyte spheroids

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2020, Universidade de Lisboa, Faculdade de Farmácia.O fígado é o principal órgão responsável pelo metabolismo e excreção de compostos endógenos e exógenos, sendo o órgão mais exposto a compostos tóxicos. A colestase induzida por fármacos é causada pela acumulação tóxica de ácidos biliares nos hepatócitos, podendo a sua manifestação ter um início retardado. A colestase é uma patologia onde a secreção da bílis para o duodeno está reduzida, quer por reduzida funcionalidade dos hepatócitos, quer por obstrução da via secretória da bílis. O principal objetivo desta investigação era o estudo dos efeitos da ciclosporina A a longo termo em culturas de esferóides de hepatócitos primários humanos. Foi efetuado um estudo de dose-resposta durante 28 dias, onde as células eram expostas a diversas concentrações de fármaco. A viabilidade celular era medida pela quantificação do conteúdo total de ATP. Foi observado que a ciclosporina A era tóxica de forma dependente da concentração e tempo. Para além disso, foi estudado os efeitos sinérgicos entre a ciclosporina A e os ácidos biliares. Os esferóides eram expostos a ciclosporina A na presença e na ausência de ácidos biliares durante 28 dias. Os resultados sugeriram que os esferóides ficavam sensibilizados pela ciclosporina A na presença de ácidos biliares, confirmando o seu sinergismo. O índice colestático foi calculado para determinar que concentrações de ciclosporina A tinham o potencial de causar colestase. O índice colestático correspondia ao rácio entre o conteúdo de ATP dos hepatócitos na presença de ácidos biliares e o conteúdo de ATP dos hepatócitos na ausência de ácidos biliares. Valores inferiores ou iguais a 0.8 indicam que o composto tem o potencial de causar colestase. Os resultados demonstraram valores de índice colestático inferiores a 0.8, quando os esferóides estavam expostos a ciclosporina A a 5 μM, após 14 dias de exposição. De acordo com os resultados, a ciclosporina A foi considerada como exibindo potencial para induzir colestase. Mais estudos podem ser realizados nesta área, de forma a descobrir por quais mecanismos a ciclosporina A induz colestase, dado que estes ainda não são totalmente compreendidos. Ensaios de RT-qPCR e Western Blot podem ser executados para estudar a expressão génica e proteica, respetivamente.The liver is the major organ responsible for the metabolism and excretion of endogenous and exogenous compounds, being more exposed to toxic compounds than any organ. Drug-induced cholestasis is attributed to the toxic accumulation of bile acids inside the hepatocytes and its manifestation can be delayed in onset. Cholestasis is a pathology where bile secretion to duodenum is reduced, whether by reduced functionality of the hepatocytes or obstruction of the excretory pathway of bile. The main objective of this research was to study the long-term effects of cyclosporine A in primary human hepatocytes spheroid cultures. A dose-response assay was conducted for 28 days, where the cells were exposed to several drug concentrations. Cell viability was measured by quantification of the total ATP content. It was assessed that cyclosporine A was toxic in a dose and time dependent manner. Furthermore, it was studied the synergistic effects between cyclosporine A and bile acids. Spheroids were exposed to cyclosporine A in the presence and in the absence of bile acids for 28 days. Findings suggested that spheroids were sensitized to cyclosporine A in the presence of bile acids, therefore confirming their synergism. Cholestatic index was calculated to determine which cyclosporine A concentrations had the potential to cause cholestasis. Cholestatic index was the ratio between the ATP content of the hepatocytes in the presence of bile acids and the ATP content of the hepatocytes in the absence of bile acids. Values lower than or equal to 0.8 indicated that the compound had the potential to cause cholestasis. Results showed cholestatic index values lower than 0.8, when spheroids were exposed to 5 μM cyclosporine A after 14 days of drug exposure. According to the results, cyclosporine A was suggested to demonstrate potential to develop cholestasis. More studies should be done in this area to discover by which mechanisms cyclosporine A induces cholestasis, since it is not yet fully understood. This can be achieved by performing RT-qPCR and Western Blot assays to study gene and protein expression, respectively.Com o patrocínio da Vrije Universiteit Brussel

Sistema de apoio à gestão da segurança e saúde no trabalho na cadeia de abastecimento alimentar – sistema SHIELD

Dissertação para obtenção do Grau de Mestre em Engenharia e Gestão IndustrialUma das principais missões das Empresas Focais das Cadeias de Abastecimento é assegurar a promoção da segurança e da saúde dos seus operadores. Sendo a Empresa Focal a entidade que regula ou domina a Cadeia de Abastecimento, compete-lhe criar mecanismos e soluções com o objectivo de promover a Segurança e Saúde no Trabalho ao longo da sua Cadeia de Abastecimento. A promoção da SST ao longo da Cadeia de Abastecimento passa por desenvolver medidas correctivas, tais como, criar processos de trabalho seguros nas outras entidades da cadeia ou garantir um fluxo de produtos seguro e saudável. Os Sistemas de Gestão da Segurança e Saúde no Trabalho desempenham um papel fundamental na promoção da segurança e da saúde nos locais de trabalho, na melhoria contínua e no acompanhamento e avaliação das medidas implementadas. Este trabalho apresenta o design de ferramentas de controlo operacionalizadas sob a forma de Checklists, desenvolvidas com base na recolha de dados realizada numa Cadeia de Abastecimento Alimentar. Estas ferramentas servirão para o desenvolvimento de um Sistema de Apoio à Gestão da SST aplicado à Cadeia de Abastecimento Alimentar. – Sistema SHIELD. Este sistema tem o objectivo de fornecer à Empresa Focal conhecimento técnico/empírico para promover a SST na sua cadeia. O Sistema SHIELD integra vários métodos, tais como a contratação de novos Suppliers e Contractors e a melhoria das condições no local de trabalho da Empresa Focal. O Sistema SHIELD integra igualmente, uma proposta para a monitorização das entidades que compõem a Cadeia de Abastecimento Alimentar e um método de melhoria contínua das ferramentas de controlo

A Selective Sweep on a Deleterious Mutation in CPT1A in Arctic Populations.

Arctic populations live in an environment characterized by extreme cold and the absence of plant foods for much of the year and are likely to have undergone genetic adaptations to these environmental conditions in the time they have been living there. Genome-wide selection scans based on genotype data from native Siberians have previously highlighted a 3 Mb chromosome 11 region containing 79 protein-coding genes as the strongest candidates for positive selection in Northeast Siberians. However, it was not possible to determine which of the genes might be driving the selection signal. Here, using whole-genome high-coverage sequence data, we identified the most likely causative variant as a nonsynonymous G>A transition (rs80356779; c.1436C>T [p.Pro479Leu] on the reverse strand) in CPT1A, a key regulator of mitochondrial long-chain fatty-acid oxidation. Remarkably, the derived allele is associated with hypoketotic hypoglycemia and high infant mortality yet occurs at high frequency in Canadian and Greenland Inuits and was also found at 68% frequency in our Northeast Siberian sample. We provide evidence of one of the strongest selective sweeps reported in humans; this sweep has driven this variant to high frequency in circum-Arctic populations within the last 6-23 ka despite associated deleterious consequences, possibly as a result of the selective advantage it originally provided to either a high-fat diet or a cold environment.This research was supported by ERC Starting Investigator grant (FP7 - 261213) to T.K. http://erc.europa.eu/. CTS, YX, QA and MS were supported by the Wellcome Trust (098051). TA was supported by The Wellcome Trust (WT100066MA). M.M and R.V. were supported by EU ERDF Centre of Excellence in Genomics to EBC; T.K, M.M and R.V. by Estonian Institutional Research grant (IUT24-1), and M.M by Estonian Science Foundation (grant 8973).This is the accepted manuscript. The final version is available from Cell/Elsevier at http://www.cell.com/ajhg/abstract/S0002-9297%2814%2900422-4

BioTIME: A database of biodiversity time series for the Anthropocene.

MotivationThe BioTIME database contains raw data on species identities and abundances in ecological assemblages through time. These data enable users to calculate temporal trends in biodiversity within and amongst assemblages using a broad range of metrics. BioTIME is being developed as a community-led open-source database of biodiversity time series. Our goal is to accelerate and facilitate quantitative analysis of temporal patterns of biodiversity in the Anthropocene.Main types of variables includedThe database contains 8,777,413 species abundance records, from assemblages consistently sampled for a minimum of 2 years, which need not necessarily be consecutive. In addition, the database contains metadata relating to sampling methodology and contextual information about each record.Spatial location and grainBioTIME is a global database of 547,161 unique sampling locations spanning the marine, freshwater and terrestrial realms. Grain size varies across datasets from 0.0000000158 km2 (158 cm2) to 100 km2 (1,000,000,000,000 cm2).Time period and grainBioTIME records span from 1874 to 2016. The minimal temporal grain across all datasets in BioTIME is a year.Major taxa and level of measurementBioTIME includes data from 44,440 species across the plant and animal kingdoms, ranging from plants, plankton and terrestrial invertebrates to small and large vertebrates.Software format.csv and .SQL

Selective sweep on human amylase genes postdates the split with Neanderthals

Humans have more copies of amylase genes than other primates. It is still poorly understood, however, when the copy number expansion occurred and whether its spread was enhanced by selection. Here we assess amylase copy numbers in a global sample of 480 high coverage genomes and find that regions flanking the amylase locus show notable depression of genetic diversity both in African and non-African populations. Analysis of genetic variation in these regions supports the model of an early selective sweep in the human lineage after the split of humans from Neanderthals which led to the fixation of multiple copies of AMY1 in place of a single copy. We find evidence of multiple secondary losses of copy number with the highest frequency (52%) of a deletion of AMY2A and associated low copy number of AMY1 in Northeast Siberian populations whose diet has been low in starch content

Local selection in the presence of high levels of gene flow: Evidence of heterogeneous insecticide selection pressure across Ugandan Culex quinquefasciatus populations

Background: Culex quinquefasciatus collected in Uganda, where no vector control interventions directly targeting this species have been conducted, was used as a model to determine if it is possible to detect heterogeneities in selection pressure driven by insecticide application targeting other insect species. Methodology/Principal findings: Population genetic structure was assessed through microsatellite analysis, and the impact of insecticide pressure by genotyping two target-site mutations, Vgsc-1014F of the voltage-gated sodium channel target of pyrethroid and DDT insecticides, and Ace1-119S of the acetylcholinesterase gene, target of carbamate and organophosphate insecticides. No significant differences in genetic diversity were observed among populations by microsatellite markers with HE ranging from 0.597 to 0.612 and low, but significant, genetic differentiation among populations (FST = 0.019, P = 0.001). By contrast, the insecticide-resistance markers display heterogeneous allelic distributions with significant differences detected between Central Ugandan (urban) populations relative to Eastern and Southwestern (rural) populations. In the central region, a frequency of 62% for Vgsc-1014F, and 32% for the Ace1-119S resistant allele were observed. Conversely, in both Eastern and Southwestern regions the Vgsc-1014F alleles were close to fixation, whilst Ace1-119S allele frequency was 12% (although frequencies may be underestimated due to copy number variation at both loci). Conclusions/Significance: Taken together, the microsatellite and both insecticide resistance target-site markers provide evidence that in the face of intense gene flow among populations, disjunction in resistance frequencies arise due to intense local selection pressures despite an absence of insecticidal control interventions targeting Culex

Evolutionary applications of population genetics with a focus on malaria : a computational approach

Malaria is a major public health concern for the one-third of the human population esti- mated to be exposed to the threat of the most virulent species, Plasmodium falciparum. Modern molecular and computational tools from population genetics may help to better understand and fight the burden of drug resistant malaria. Malaria biology is substantially different from the underlying paradigm of standard population genetics models, most notably because malaria has both a asexual haploid phase and a sexual diploid phase where selfing (i.e. mating between genetically identical parasites) is possible. It is therefore fundamental to understand if commonly used population genetics methods are robust to the deviations from standard expectations imposed by the malaria life-cycle. We build novel models of malaria population genetics and provide guidelines to interpret empirical studies of the spread of drug resistance. Using realistic models of epistasis between genes involved in drug resistance we suggest that all signals of linkage disequilibrium (LD) are possible and that researchers should be confident in reporting a lack of statistical association between genes involved in resistance to antimalarials. We also suggest that researchers should be cautious in interpreting changes in the prevalence of drug resistance after control interventions as reductions in transmission can cause a change in prevalence without concomitant change in frequency of resistance. We provide guidelines to better design and interpret studies related to estimating effective population size (Ne). We use computational simulations to study scenarios that can approximate control and elimination interventions (i.e. where a significant part of the population is killed). For Ne estimation our results suggest that researchers must increase the number of individuals and loci genotyped in order to have sufficiently precise Ne estimates. LD-based Ne estimators are more appropriate for early detection of control and elimination interventions than temporal-based Ne estimators. Long- term estimators based on heterozygosity should not be used to make inferences about contemporary demographic processes. We also applied our analysis to disease vectors and we concluded that LD-based estimation is able to detect demographic seasonality patterns (i.e. changes in population size due to variations imposed by wet and dry seasons) whereas temporal estimators will provide averages over longer periods of time. We also studied selection detection using FsT-outlier approaches. Our results suggest that temporal FST might not be appropriate for early detection of genes involved in drug resistance (e.g. in the case of artesunate derivatives). We also provide software and guidelines to better design and interpret studies (also across other taxa) of selection based on FsT-outlier approaches. Most notably our results suggest that sampling only one or two SNPs per locus (as it is done in many empirical studies) might not be sufficient to detect areas of the genome under selection, and that at least 4 SNPs per loci should be genotyped.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

O espaço de habitar vernacular no barrocal algarvio

A presente dissertação pretende contribuir para a compreensão dos componentes morfológicos culturais da paisagem, em específico para o caso da unidade de paisagem predominantemente rural do Barrocal Algarvio. Constata-te que a paisagem do Barrocal é, na sua quase totalidade, uma construção, concebida em função de uma economia agrária, onde a acção contínua do Homem sobre solos pobres e acidentados procurou adaptar, em simbiose entre a actividade humana e a natureza, o espaço às necessidades agrícolas de tipo Mediterrânico que este território suporta. Assim, interessou estudar as edificações vernáculas em conjunto com a estrutura fundiária, isto é, com a propriedade privada sobre a terra, o suporte dos espaços domésticos e agrícolas, da qual se analisam os seguintes elementos: culturas agrícolas - relativamente às formas e compassos; tipos de limites - valados, valas, muros e sebes; e, por fim, as diferentes tipologias que constituem a rede de circulação; ### Abstract: This dissertation pretends to contribute to the comprehension of the morphological cultural components of the landscape, in particular to the case of the predominantly rural landscape unit of the Algarve´s Barrocal. Barrocal´s landscape is almost entirely a construction, designed in function of an agrarian economy, where the continuous action of man on poor soils and slopes tried to adapt, in a symbiosis between human activity and nature, the space to a Mediterranean type of agriculture that this territory supports. For the purpose, it mattered to study the vernacular edifications in the relationship with private ownership of land, the support of domestic and agricultural spaces, from which the following elements are analyzed: agricultural crops - for the forms and distance between trees; types of boundaries – earth banks, ditches, walls and hedges; and, finally, the typologies that constitute the circulation network

A Distributed Debugging Tool for a Parallel Software Engineering Environment

We discuss issues in the design and implementation of a flexible debugging tool and its integration into a parallel software engineering environment.