The parental effect in the immune system

Tese de mestrado, Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2019Durante a gestação e a amamentação, diversos fatores maternos podem influenciar o sistema imunitário dos filhos, levando a diversas alterações que terão um impacto para o resto da vida. Populações de células imunitárias, que residam nos tecidos, formam uma interface materno-recém-nascido são primordiais na transferência vertical de imunidade. As células T γδ encontram-se em diversas mucosas, uma da qual é a mucosa do aparelho reprodutor feminino, onde estas perfazem a maioria das células T residentes no tecido. Por outro lado, as células B perfazem uma fração substancial de linfócitos residentes na maioria dos tecidos linfoides e não-linfoides, sendo responsáveis pela produção de anticorpos naturais e adaptativos. No desenvolvimento desta tese, demonstraremos a importância que o sistema imunitário materno tem no sistema imunitário pulmonar das crias. Quando comparámos os pulmões de ratinhos filhos de mães TCRδ+/- com mães TCRδ-/- no estado estacionário, observámos uma tendência do ambiente imunitário para respostas imunitárias do tipo-2 nas cria de mães TCRδ-/-, tendo um aumento na concentração de citocinas relacionadas com este tipo de resposta imunológica, nomeadamente IL-33 e IL-4, havendo também um aumento de citocinas pro-inflamatórias como o TNF-α e a IL-1α. Além do mais, os pulmões das crias de mães TCRδ-/- apresentavam um aumento de certas populações de células imunitárias produtoras de citocinas de respostas imunitários tipo-2, IL-13+ ILC2s e IL-5+ mastócitos. Após infetarmos com Nippostrongylus brasiliensis os ratinhos de mães TCRδ-/- apresentaram diferenças significativas em populações de células imunitárias no dia 6 pós-infeção (sem haver diferenças no dia 2 pós-infeção), tendo aumento do número total de leucócitos, com um aumento de células que estão associadas com respostas imunitários do tipo-2, nomeadamente macrófagos e DCs que expressam PD-L2. Baseando-nos nos nossos resultados, pusemos a hipótese de que o sistema imunitário materno, pudendo estar a influenciar o sistema imunitário das crias através da transferência de microbiota e/ou anticorpos. Para testar a primeira hipótese, administramos um cocktail de antibióticos aos pais, antes serem estabelecidos os cruzamentos, mantendo-os até ao desmame. Consistente com a hipótese, as diferenças observadas relativamente à expressão de citocinas foram abolidas. Por outro lado, a transferência de anticorpos maternos não demonstrou ter influência neste processo, não existindo diferenças nos níveis das diferentes classes de IgG nas mães TCRδ+/- or TCRδ-/-, nem nas suas crias. Contundo, usando um modelo complementar, as crias de mães deficientes em células B apresentaram uma tendência para respostas imunitários do tipo-1, existindo um aumento de citocinas relacionadas com respostas imunitárias do tipo-1, como a IL-12p40 e a IL-12p70, havendo também um aumento células NK e neutrófilos, enquanto macrófagos alveolares e CD11b- DCs estavam diminuídos, quando comparados com crias de mães suficientes em células B. Em suma, os nossos resultados demonstram que o sistema imunitário materno tem um grande impacto no sistema imunitário das crias, onde diferenças na composição da microbiota ou a incapacidade de produzir anticorpos poderão ser causadas por deficiências de células imunitárias, tais como os linfócitos T γδ analisados nesta tese, levando a disfunções no sistema imunitário da descendência.During gestation and nursing, different maternal factors may influence the immune system of the offspring, leading to several alterations that will have an impact throughout life. Immune cell populations that reside in tissues forming the maternal-newborn interface are paramount in the vertical transfer of immunity. γδ T cells can populate several mucosal sites, including the female reproductive tract, where they represent the majority of tissue-resident T cells. B cells on the other hand, make up a substantial fraction of resident lymphocytes in almost all lymphoid and non-lymphoid tissues, being responsible for the production of natural and adaptive antibodies. In this thesis we show the importance of the maternal immune system in the pulmonary immune system of the offspring. When comparing the lungs of mice born from TCRδ+/- or TCRδ-/- dams at steady state, we observed a type-2 biased immune environment in the pups born from TCRδ-/- dams, having an increase in type-2 cytokines, such as IL-33 and IL-4, and of pro-inflammatory cytokines, such as TNF-α and IL-1α. Also, the lungs of pups born from TCRδ-/- dams had an increase in cells that produce type-2 cytokines, such as IL-13+ ILC2s and IL-5+ mast cells. Upon Nippostrongylus brasiliensis infection mice born from TCRδ-/- dams showed significant differences in lung immune populations at day 6 p.i. (but no differences at day 2 p.i.), displaying a greater number of total leukocytes, with an increase in cells associated with type-2 immune responses, such as eosinophils and PD-L2-expressing macrophages and DCs. Based on these results we hypothesized that the maternal immune system might be influencing the offspring immune system through the transfer of microbiota and antibodies. To test the first hypothesis, we gave a cocktail of antibiotics to the parents prior to the establishment of the breedings and maintained until fostering. Interestingly, the differences previously observed in the levels of pulmonary cytokine were abolished. Maternal transfer of antibodies, on the other hand, seemed to not be playing a role in this process, as both TCRδ+/- or TCRδ-/- dams and their progenies showed no differences in the circulating levels of different IgG subclasses. However, using a complementary model, pups born from B cell-deficient dams showed a bias towards type-1 immune responses, having an increase in the type-1 cytokines IL-12p40 and IL-12p70, with an increase in lung NK cells and neutrophils, while displaying a decrease in alveolar macrophages and CD11b- DCs, when compared to pups born from B cell-sufficient dams. Overall, our results demonstrate the great impact of the maternal immune system on offspring immunity. Thus, immune cell deficiencies, such as γδ T cells, may result in differences in the microbiota composition or the inability to produce antibodies, which in turn leads to dysfunctions in the offspring immune system

Estrelas jovens e nebulosas de reflexão no "bordo" inferior da galáxia

Tese de mestrado, Astronomia e Astrofísica, Universidade de Lisboa, Faculdade de Ciências, 2009Este trabalho apresenta a caracterização de uma região de formação de estrelas, associada a nebulosas de reflexão, na região da fonte IRAS 07383 − 3325 localizada no “bordo” inferior do disco Galáctico, na zona da Galáxia exterior. Para o estudo desta região, foram usadas observações de infravermelho-próximo (JHKS) obtidas usando o telescópio VLT/ISAAC, observações no milímetro de espectros de CO obtidos usando o telescópio SEST, e uma imagem óptica na banda V do telescópio YALO. Os espectros de CO foram reduzidos usando o programa XS. Foi feito o folding e a subtração das baselines, seguindo-se a extracção de parâmetros dos espectros. Estes espectros revelaram a presença de uma nuvem molecular na região estudada e levaram à determinação das propriedades da nuvem, como a sua velocidade (radial), a sua massa e a extinção que produz. A zona de maior densidade da nuvem revelou também ser a zona mais dinâmica e coincide espacialmente com uma nebulosa de reflexão descoberta nas nossas imagens de infravermelho. As imagens de infravermelho foram reduzidas usando o programa IRAF, tendo-se corrigido os efeitos do brilho do céu, da não-uniformidade do detector (flatfield) e a presença de bad-pixeis. Foi efectuada a detecção das fontes presentes nas imagens finais seguida da sua fotometria de abertura. Os resultados da fotometria foram analisados de modo a procurar identificar as estrelas jovens embebidas na nuvem e distingui-las das estrelas de campo. Detectou-se assim, um novo enxame de estrelas jovem, composto por algumas dezenas de estrelas, com cerca de um milhão de anos. Demonstrou-se assim a ocorrência de formação de estrelas nesta região fronteira do disco Galáctico.This work contains a characterization of a star formation region, associated with reflection nebulae, towards the region of IRAS 07383-3325 located at the lower side of the Galactic disc, in the outer Galaxy. To study this region, we used near-infrared (JHKS) images obtained with the VLT/ISAAC telescope, millimeter CO lines obtained with the SEST telescope, and an optical V-band image from the YALO telescope. The CO spectra were reduced using the XS software. Folding and baseline subtraction were carried out, followed by extraction of spectral parameters. The spectra revealed the presence of a molecular cloud in this region, and led to the determination of the cloud properties, such as its (radial) velocity, mass and extinction. The densest region of the cloud turned out to be also its most dynamic region, spatially coincident with a reflection nebula discovered through our near-infrared images. Infrared images have been reduced using the IRAF software, and the effects of the sky brightness, of the detector variable pixel-to-pixel sensitivity (flatfield) and of the presence of bad pixels were corrected. The sources present in the final images were detected, and the aperture photometry of these detections were preformed. The photometric results were analyzed in order to identify the young stars embedded in the cloud and separate them from field stars. In this manner, a new young embedded stellar cluster was detected, composed of tens of stars, with an age of about a million years. The occurrence of star formation at this border region of the Galactic disc has thus been demonstrated

Molecular gas and a new young stellar cluster in the far outer Galaxy

We investigate the star-formation ocurring in the region towards IRAS07527-3446 in the molecular cloud [MAB97]250.63-3.63, in the far outer Galaxy. We report the discovery of a new young stellar cluster, and describe its properties and those of its parent molecular cloud. Near-infrared JHKS images were obtained with VLT/ISAAC, and millimetre line CO spectra were obtained with the SEST telescope. VLA archive date were also used. The cloud and cluster are located at a distance of 10.3 kpc and a Galactocentric distance of 15.4 kpc, in the far outer Galaxy. Morphologically, IRAS 07527-3446 appears as a young embedded cluster of a few hundred stars seen towards the position of the IRAS source, extending for about 2-4 pc and exhibiting sub-clustering. The cluster contains low and intermediate-mass young reddened stars, a large fraction having cleared the inner regions of their circumstellar discs responsible for (H-Ks) colour excess. The observations are compatible with a < 5 Myr cluster with variable spatial extinction of between Av = 5 and Av = 11. Decomposition of CO emission in clumps, reveals a clump clearly associated with the cluster position, of mass 3.3 x 10^3 M(solar). Estimates of the slopes of the Ks-band luminosity function and of the star-formation efficiency yield values similar to those seen in nearby star-formation sites. These findings reinforce previous results that the distant outer Galaxy continues to be active in the production of new and rich stellar clusters, with the physical conditions required for the formation of rich clusters continuing to be met in the very distant environment of the outer Galactic disc.Comment: 11 pages, 13 figure

Fontes bibliográficas para a compreensão do vocabulário geométrico tardio de Almada Negreiros.

UIDB/00657/2020 UIDP/00657/2020O presente artigo revela novos dados atinentes à pesquisa de Almada Negreiros sobre arte e geometria. A partir de vários livros da biblioteca pessoal do autor, um manuscrito e um desenho, até hoje inéditos, reúnem-se uma série de novas conclusões a propósito dos referentes de Almada. Os autores que o marcaram, as imagens que cita explicitamente e até relações pessoais com outros autores, servem de base para uma melhor compreensão do percurso da sua investigação geométrica. Este novo conhecimento contribuirá ainda para datações precisas de múltiplas obras do autor. This article reveals new data regarding Almada Negreiros’ research on art and geometry. From several books in the author’s personal library, a previously unpublished manuscript, and drawings, we present several new conclusions regarding Almada’s referents. The au-thors who inspired him, the images he explicitly quotes, and even personal relationships with other authors, are the basis for a better understanding of how his geometric research developed. This new knowledge will also contribute to an accurate dating of many of the author’s works.publishersversionpublishe

Redefining the role of ectoderm in somitogenesis: a player in the formation of the fibronectin matrix of presomitic mesoderm

The absence of ectoderm impairs somite formation in cultured presomitic mesoderm (PSM) explants, suggesting that an ectoderm-derived signal is essential for somitogenesis. Here we show in chick that the standard enzymatic treatments used for explant isolation destroy the fibronectin matrix surrounding the anterior PSM, which fails to form somites when cultured for 6 hours. By contrast, explants isolated with collagenase retain their fibronectin matrix and form somites under identical culture conditions. The additional presence of ectoderm enhances somite formation, whereas endoderm has no effect. Furthermore, we show that pancreatin-isolated PSM explants cultured in fibronectin-supplemented medium, form significantly more somites than control explants. Interestingly, ectoderm is the major producer of fibronectin (Fn1) transcripts, whereas all but the anterior-most region of the PSM expresses the fibronectin assembly receptor, integrin alpha5 (Itga5). We thus propose that the ectoderm-derived fibronectin is assembled by mesodermal alpha5beta1 integrin on the surface of the PSM. Finally, we demonstrate that inhibition of fibronectin fibrillogenesis in explants with ectoderm abrogates somitogenesis. We conclude that a fibronectin matrix is essential for morphological somite formation and that a major, previously unrecognised role of ectoderm in somitogenesis is the synthesis of fibronectin.Fundação para a Ciência e a Tecnologia (FCT)/FEDER projects POCTI/BCI/40754/2001 and POCI/BIA-BCM/59201/2004 and the FP6/EU Network of Excellence ‘Cells into Organs’ of which P.R., C.L., R.P.A., G.R., I.P. and S.T. are members. L.C. was supported by the European Social Fund contract 1/3.2/PRODEP/2001. R.P.A. was supported by FCT (SFRH/BPD/9432/2002)

Bird extinctions and introductions are causing taxonomic and functional homogenization in oceanic islands

Humans are quickly reshaping species assemblages through the loss and gain of species at multiple scales. Extinctions and introductions are non-random events known to be contributing to taxonomic homogenization. However, it is not yet clear if they also promote functional homogenization. Here, we assess whether extinctions and introductions are leading to taxonomic and functional homogenization of 64 oceanic island bird assemblages, belonging to 11 archipelagos. Based on island lists of extinct and extant, native and introduced species and on species traits, we use probabilistic hypervolumes in trait space to calculate functional beta-diversity before and after extinctions and introductions. Bird extinctions and introductions promoted taxonomic and functional homogenization on most oceanic islands. These results follow our expectations, considering previous studies on taxonomic homogenization, the predictable link between taxonomic and functional diversity, and the trait similarity of many introduced species, often adapted to anthropogenic habitats, linked to the non-randomness of bird introductions on islands. Taxonomic homogenization was more common across than within archipelagos, also corroborating previous studies describing stronger homogenization on islands that are further apart and thus had distinctive native assemblages. Surprisingly, the widespread loss of species with similar traits, namely large flightless birds, often led to functional differentiation across archipelagos. However, this differentiation effect tended to be offset by the homogenizing effect of introductions. Functional homogenization increases the vulnerability to global changes, by reducing the variability of responses to disturbance and thus the resilience of ecosystem services, posing a threat to human societies on islands. Our results highlight subtle variations in taxonomic and functional beta-diversity of bird assemblages in oceanic islands, providing important insights to allow a better assessment of how anthropogenic changes might alter ecosystem functioning, which is vital to develop effective long-term conservation strategies. Read the free Plain Language Summary for this article on the Journal blog.Peer reviewe

Combined effects of bird extinctions and introductions in oceanic islands : Decreased functional diversity despite increased species richness

Aim We analyse the consequences of species extinctions and introductions on the functional diversity and composition of island bird assemblages. Specifically, we ask if introduced species have compensated the functional loss resulting from species extinctions. Location Seventy-four oceanic islands (> 100 km(2)) in the Atlantic, Pacific and Indian Oceans. Time period Late Holocene. Major taxa studied Terrestrial and freshwater bird species. Methods We compiled a species list per island (extinct and extant, native and introduced), and then compiled traits per species. We used single-trait analyses to assess the effects of past species extinctions and introductions on functional composition. Then, we used probabilistic hypervolumes in trait space to calculate functional richness and evenness of original versus present avifaunas of each island (and net change), and to estimate how functionally unique are extinct and introduced species on each island. Results The net effects of extinctions and introductions were: an increase in average species richness per island (alpha diversity), yet a decline in diversity across all islands (gamma diversity); an average increase in the prevalence of most functional traits, yet an average decline in functional richness and evenness, associated with the fact that extinct species were functionally more unique (when compared to extant natives) than introduced species. Main conclusions Introduced species are on average offsetting (and even surpassing) the losses of extinct species per island in terms of species richness, and they are increasing the prevalence of most functional traits. However, they are not compensating for the loss of functional richness due to extinctions. Current island bird assemblages are becoming functionally poorer, having lost unique species and being composed of functionally more redundant species. This is likely to have cascading repercussions on the functioning of island ecosystems. We highlight that taxonomic and functional biodiversity should be assessed simultaneously to understand the global impacts of human activities.Peer reviewe

Optimizing sampling design to deal with mist-net avoidance in Amazonian birds and bats

Mist netting is a widely used technique to sample bird and bat assemblages. However, captures often decline with time because animals learn and avoid the locations of nets. This avoidance or net shyness can substantially decrease sampling efficiency. We quantified the day-to-day decline in captures of Amazonian birds and bats with mist nets set at the same location for four consecutive days. We also evaluated how net avoidance influences the efficiency of surveys under different logistic scenarios using re-sampling techniques. Net avoidance caused substantial declines in bird and bat captures, although more accentuated in the latter. Most of the decline occurred between the first and second days of netting: 28% in birds and 47% in bats. Captures of commoner species were more affected. The numbers of species detected also declined. Moving nets daily to minimize the avoidance effect increased captures by 30% in birds and 70% in bats. However, moving the location of nets may cause a reduction in netting time and captures. When moving the nets caused the loss of one netting day it was no longer advantageous to move the nets frequently. In bird surveys that could even decrease the number of individuals captured and species detected. Net avoidance can greatly affect sampling efficiency but adjustments in survey design can minimize this. Whenever nets can be moved without losing netting time and the objective is to capture many individuals, they should be moved daily. If the main objective is to survey species present then nets should still be moved for bats, but not for birds. However, if relocating nets causes a significant loss of netting time, moving them to reduce effects of shyness will not improve sampling efficiency in either group. Overall, our findings can improve the design of mist netting sampling strategies in other tropical areas.We thank the Brazilian CNPq and Ministério do Meio Ambiente for research permits. João Valsecchi, Helder Queiróz, Miriam Marmontel, Pedro Santos, Jerry Tavares dos Santos, several field assistants, and the staff of the Instituto de Desenvolvimento Sustentável Mamirauá provided invaluable help with planning, logistics and field work. We also thank L. Reino, C. Miranda and N. Castanheira for help during field work. We are grateful to the communities of ASDR for their hospitality. Monique MacKenzie kindly helped with the GEE analyses. Dr. Mark Brigham made valuable comments and suggestions that helped to improve an earlier version of the manuscript.publishe

Livro Vermelho dos Vertebrados de Portugal

Os Livros Vermelhos têm sido reconhecidos, pelas entidades responsáveis pela conservação da natureza, as organizações não-governamentais, a co- munidade científica e os decisores de projectos com incidência no ordena- mento e gestão do território, como elementos de consulta e instrumento de apoio à tomada de decisão de inegável utilidade. Neles se indica o esta- tuto de ameaça das espécies selvagens, de acordo com critérios quantitati- vos para avaliar níveis de risco de extinção e, ainda, informação sobre as populações, causas de ameaça e medidas de conservação. Estes são documentos em permanente actualização, reflectindo cada edição o melhor conhecimento científico disponível, e a sua elaboração deve ser considerada como uma tarefa de interesse público e mobilizadora de todos os que disponham de informação relevante e actualizada para a avaliação do estatuto das diferentes espécies. Um Livro Vermelho é ainda uma chamada de atenção e uma tomada de consciência perante a diminuição da diversidade biológica à escala global. Desde 1500, registou-se a extinção de 92 espécies de peixes, 5 de anfíbios, 22 de répteis, 131 de aves e 87 de mamíferos (Hylton-Taylor 2000). Regularmente, a União Mundial para a Conservação (IUCN) actualiza e alarga a avaliação do risco de extinção à escala mundial a um ainda maior número de espécies. Assim, de acordo com o exercício realizado em 2004 (IUCN 2004a) verifica-se o seguinte: para os mamíferos, a avaliação revela 23% de espécies ameaçadas, correspondente a 20% do total das espécies co- nhecidas; nas aves, a avaliação perfaz a quase totalidade das espécies conhecidas e atinge os 12% de espécies ameaçadas; os répteis avaliados apresentam uma percentagem muito elevada de espécies ameaçadas (61%), muito embora esta percentagem corresponda apenas a 4% do to- tal das espécies conhecidas; os anfíbios ameaçados são cerca de 31% das espécies conhecidas; nos peixes, grupo cuja proporção de avaliações está muito aquém das espécies conhecidas, os resultados revelam 46% de ameaçadas, correspondentes a 3% do total de espécies conhecidas. Nesta edição são listadas as espécies de peixes dulciaquícolas e migradores, anfíbios, répteis, aves e mamíferos que ocorrem em Portugal. Mas este é sobretudo o livro das espécies ameaçadas que enfrentam risco de extinção e a que, por isso, foram atribuídas as categorias de .Vulnerável., .Em Perigo. ou .Criticamente em Perigo.; é também o livro de algumas espécies às quais foi atribuída a categoria de .Quase Ameaçado. e ainda daquelas com .Informação Insuficiente. sobre a sua distribuição, requisitos de habitat, dimensão ou tendência populacional (entre outros aspectos) o que impediu a avaliação do risco de extinção. Atribuído um estatuto, identificam-se ainda os factores de ameaça, devendo estes ser entendidos como os fenómenos ou processos que sendo contí- nuos, esporádicos ou recorrentes, localizados ou generalizados, afectam as populações ou alteram a estrutura ou o funcionamento dos sistemas na- turais em que elas se integram, de um modo que afecta a sua reprodução ou mesmo sobrevivência. Estes factores são alterações das características físicas, químicas ou biológicas dos habitats das espécies ou acções que causam mortalidade intencional ou acidental ou, ainda, a redução do sucesso reprodutor das espécies. Com esta edição, alcançou-se uma meta no processo de avaliação da fau- na de vertebrados de Portugal, remetendo-se para uma segunda etapa a avaliação das espécies de peixes marinhos e estuarinos. Tendo a clara noção da efemeridade da avaliação do risco de extinção suportada pelas condições e conhecimentos de um momento, espera-se que o Livro Vermelho seja um documento periodicamente actualizado. Finalmente, é elevada a expectativa de todos os que participaram na sua elaboração, de que este venha a contribuir decisivamente para um reforço da conservação das espécies ameaçadas em Portugal

Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis

Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis