Neurodegeneration, cell signaling and neuroreparative strategies

Funding Information: This work was partly supported by the grant UID/DTP/04138/2019-21 (iMed.ULisboa) as well as La Caixa Foundation and -Luzón Foundation through project HR21-00931. VP acknowledges financial support from the Spanish Ministry of Science through the Ramón y Cajal grant RYC2019-027489-I.publishersversionpublishe

Microbial pectin recognition and utilization of the mammalian gastrointestinal tract

PhD ThesisPlant cell wall polysaccharides represent one of the major nutrients available to the human microbiota, which has a significant impact on host nutrition and health. Pectins are one of the major plant cell wall components. Understanding the mechanism of protein recognition and enzymatic degradation of these polysaccharides can have significant implications, not only in promotion of human health, but also in an industrial context. In this thesis the founding member of a carbohydrate binding module (CBM) family that targets homogalacturonan (HG) was characterized. The mechanism of degradation of rhamnogalacturonan-I and II (RG-I and RG-II) by Bacteroides thetaiotaomicron, a member of human microbiota, was also explored. Efficient plant cell wall degradation requires a close association between catalytic modules with CBMs. In Chapter 3, the characterization of Ruminococcus flavefaciens CBM77PL1/9 revealed that this CBM specifically binds to non-methylated HG. The structural characterization disclosed a new binding mechanism where the positively charged residues (lysines) interact with the negative carboxyl groups present in the ligand. Additionally, a dimer of this CBM has shown to be a suitable probe to target pectins in a plant cell wall context. B. thetaiotaomicron is able to grow on pectins. In this study, a model for RG-I and RG-II degradation was described. The cleavage of RG-II is achieved by a hierchical exo mode of degradation. Chapter 4 showed that the degradation of Chain B requires eight enzymes where the -L-rhamnosidases, BT0986 and BT1019, specifically target the linkages 1,2 and 1,3-arabinopyranose, respectively. It was shown that the linkage between L-rhamnose and D-apiose is an -linkage, likely 1,3’. BT1001, which targets the rhamnose-apiose glycosidic bond requires the cleavage of side chain and backbone, suggesting that this is the last linkage cleaved in RG-II degradation. The structural and biochemical characterization of a GH106 enzyme (BT0986) revealed a calcium dependent enzyme with a catalytic apparatus consistent with an inverting mechanism. The structure of a GH127 enzyme (BT1003) involved in RG-II showed that the aceric acidase lacks the residue that is the proposed catalytic acid/base in GH127 -L-arabinofuranosidases. This may indicate that the aceric acidase cleaves glycosidic bonds by a mechanism that differs from other enzymes in the GH127 family. In Chapter 5, key enzymes in the RG-I degradation system were characterized. Three polysaccharide lyases (PLs) (BT4170, BT4175 and BT4183) revealed complementary specificities. BT4170, identified on the cell surface, was shown to be essential for RG-I backbone utilization. Four GH28 enzymes were characterized: BT4149 and BT4153 target the RG-I backbone displaying distinct substrate specificities based on the degree of polymerization of the oligosaccharide. BT4155 removes the HG remnants attached to RG-I and BT4146 is specific for a RG-I backbone disaccharide. The surface glycan binding protein BT4167 has shown to recognize RG-I. The structural characterization of BT4170 (PL9) revealed that the residues implicated in substrate recognition in this rhamnogalacturonan lyase are not conserved in PL9 pectate lyases. The proposed model of how 13 enzymes encoded by B. thetaiotaomicron mediate the degradation of RG-I contributes to our knowledge of pectin degradation

Neurodegeneration, cell signaling and neuroreparative strategies, Volume II

Funding Information: This work was partly supported by the grant UID/DTP/04138/2019-22 (iMed.ULisboa) as well as La Caixa Foundation and Francisco Luzón Foundation through project HR21-00931. VP acknowledges financial support from the Spanish Ministry of Science through the Ramón y Cajal grant RYC2019-027489-I.publishersversionpublishe

Minas de urânio : determinação dos efeitos genéticos em ambientes contaminados com metais e radionuclídeos

Mestrado em Biologia Molecular e CelularDurante a extração de urânio são gerados volumes consideráveis de resíduos compostos por metais e radionuclídeos. A exposição da população e animais, que habitam nos arredores das minas, a estes resíduos levanta sérias preocupações ambientais e de saúde pública. A população e animais residentes podem estar expostos à radiação e metais através dos próprios resíduos, poeiras radioativas, bem como através da ingestão de alimentos e água contaminados. A exposição a estes contaminantes está ligada a uma série de danos genéticos, como o caso das quebras da cadeia dupla do DNA. A radiação e os metais podem causar diretamente danos na molécula de DNA, como também o podem fazer indiretamente por formação de espécies reativas de oxigénio. Assim sendo, tornou-se crucial o uso de biomarcadores moleculares uma vez que, estes fornecem uma resposta rápida sobre os efeitos moleculares da exposição a estes contaminantes, mesmo que os efeitos sub-letais não se manifestem. Neste trabalho foram analisadas respostas ao nível molecular, em coelhos, com a finalidade de determinar o risco químico e/ou radiológico de águas e alimentos contaminados, provenientes de terrenos nos arredores da mina de urânio da Cunha Baixa. Este trabalho teve como objetivo principal a avaliação de potenciais riscos, associados ao consumo de alimentos e água contaminados, quer para os organismos quer para a população residente na Cunha Baixa. Para tal, foram feitos ensaios de exposição in situ com coelhos, sendo que, posteriormente, foram analisadas as alterações da expressão genética, de cinco supressores de tumor e dois oncogenes, como biomarcadores de exposição à radiação e a metais. Os resultados revelaram sobre-expressão não só de supressores de tumor (ATM, GADD45A, CDKN1A e RB) como também de oncogenes (SET e FGF2). Com isto verificou-se que o coelho (Oryctolagus cuniculus) é um bom bioindicador, uma vez que: este organismo foi sensível à contaminação, uma vez que foi possível detetar alterações moleculares após a exposição a metais e radionuclídeos; permitiu avaliar possíveis efeitos nos consumidores e extrapolar os possíveis riscos para a saúde humana e dos organismos residentes. Para além disto, este estudo contribuiu para a caracterização de potenciais riscos da exposição a alimentos e água contaminados, evidenciando que os contaminantes têm um grande potencial para causar instabilidade genómica e, consequentemente, aumentar o risco do desenvolvimento de doenças genéticas, como o cancro. Posto isto, o presente estudo aponta a mina de urânio da Cunha Baixa, bem como outras áreas onde se procede à extração de minério radioativo, como sendo um risco para a população e organismos residentes.During the extraction of uranium, considerable amounts of residues, made primarily of metals and radionuclides, are generated. The exposure of the local human population and wildlife to these dangerous residues are a serious public and environmental health issue. Living organisms can be exposed to radioactive dust clouds, ingestion of contaminated food and contaminated water. The exposure to these residues seems to be linked to several genetic damages such as, DNA double-strand breaks. The exposure to radiation and metals can cause direct and/or indirect damage to DNA molecules, through the formation of reactive oxygen species. This made imperative the use of molecular biomarkers, because of their rapid and sensitive response to the presence of a contaminant. In this work we examined the molecular response of rabbits with the goal of determining the chemical and/or radiological risk of water and food consumption grown in the soils in and around the Cunha Baixa uranium mine for the residents of the Cunha Baixa village. Tests were conducted in situ with rabbits, and showed that the genetic expression of five suppressor tumor genes and two oncogenes were altered. The results showed the over expression of, not only ATM, GADD45A, CDKN1A and RB, but also of the oncogenes SET and FGF2. These results show that the Oryctolagus cuniculus (rabbit) is a good bioindicator species for the effects of metal and radionuclide exposure, since it was sensitive to the contamination, evidencing molecular alterations after exposure to the residues, granting the possibility to evaluate the effects in consumers and extrapolate possible risks to humans. Furthermore, this study contributed for the characterization of potential risks in the consumption of contaminated food and water, since it showed the capability of these contaminants, in altering and creating genomic instability, increasing the chance of developing genetic diseases such as cancer. In conclusion this study shows that the Cunha Baixa uranium mine, as well as other uranium mining areas are a risk to the organisms and human populations

A coopetição nas pequenas e médias empresas: estudo do caso da Zea Partners

Mestrado em Contabilidade - AuditoriaNum mundo cada vez competitivo, as empresas têm que adoptar estratégias para ganharem vantagens competitivas. Para isso deverão procurar as melhores vias de desenvolvimento para implementarem as suas estratégias. Com a realização deste trabalho pretendeu-se dar a conhecer uma forma recente de implementar estratégias - a coopetição. Para tal, foi utilizada como metodologia a revisão de literatura. Quanto ao segundo objectivo procedemos ao estudo do caso prático: a Zea Partners. Para o estudo deste caso foi usado o estudo de Soekijad e Joode (2009:146) e o site pertencente à organização. Com este caso, conseguimos mostrar como funciona uma coopetição, que actualmente é bem sucedida, numa pequena e média empresa.In an increasingly competitive world, enterprises have to adopt strategies to gain competitive advantages. For this, they should seek the best routes to implement their development strategies. With this work we intended to get to know one way to implement new strategies - the coopetition. To do this, the methodology used was literature review. As to the second purpose, we proceeded to study the practical case: the Zea Partners. For this case study, we used to study Soekijad and Jood (2009:146) and the website belonging to the organization. In this case, we showed how it works a coopetition, which currently is successful, in a small and medium enterprise

Patrones de diversidad liquénica en las dunas arenosas de la costa del norte de Portugal

Coastal sand-dunes of northern Portugal are dynamic ecosystems under natural and human disturbances. Still, they constitute an interesting subject for ecological research, since they spread over a main biogeographic transition between the Eurosiberian and the Mediterranean regions. Despite the awareness of lichen contribution to grey dune stability and protection, there are no recent published studies focusing on their lichen flora. Therefore, the overarching goal of this research was to evaluate the patterns of lichen diversity along biogeographic - and coastal dynamics - related gradients in northern Portugal. A catalogue with 17 species is presented, and includes a new species for Portugal, Cladonia rei Schaer. Patterns of lichen diversity were analysed through Mann-Whitney tests and Analysis of Similarity (ANOSIM), which showed distinct signals when considering different diversity components and scales of analysis. These results have strong implications for conservation and monitoring strategies of this type of ecosystem.Las dunas costeras del norte de Portugal son dinámicas con perturbaciones naturales y humanas. Sin embargo, constituyen un tema interesante para la investigación ecológica, ya que se distribuyen a través de la transición biogeográfica entre las regiones Eurosiberiana y Mediterránea. A pesar de la contribución de los líquenes por la estabilidad y protección de las dunas-grises, no hay estudios recientes sobre su flora liquénica. Lo objetivo de esta investigación es evaluar los patrones de diversidad liquénica a lo largo de gradientes biogeográficos y de dinámica costera del norte de Portugal. Se presenta un catálogo con 17 especies, con una nueva especie para Portugal, Cladonia rei Schaer. Los patrones de diversidad fueron analizados a través de análisis de Mann-Whitney y de similitud, y muestran resultados distintos al considerar los diferentes componentes de la diversidad y escalas de análisis. Los resultados tienen importantes implicaciones para las estrategias de conservación y monitoreo de este tipo de ecosistema

Os testes de provocação conjuntival na avaliação da eficácia da imunoterapia alergénio-específica rush na dermatite atópica canina

Dissertação de Mestrado Integrado em Medicina VeterináriaA atopia é uma doença frequente no cão, mas pouco se sabe acerca do envolvimento ocular, desconhecendo-se a prevalência e o impacto da conjuntivite alérgica (CA) na dermatite atópica canina (DAc). O teste de provocação conjuntival (TPC) é um modelo útil ao seu estudo, sendo utilizado na avaliação da eficácia de várias formas de tratamento, incluindo a imunoterapia alergénio-específica (ITAE). Através da realização de TPC em pacientes atópicos com sensibilização a Dermatophagoides farinae (Df) pretendeu-se avaliar a contribuição deste alergénio nas manifestações oculares e a eficácia da imunoterapia alergénio específica rush (ITAEr) na remissão dos sinais oculares e cutâneos. Foram incluídos no estudo 15 pacientes (n=15) com diagnóstico de DAc e sinais compatíveis com CA. Numa primeira fase todos os animais foram submetidos a TPC para Df e avaliados quanto ao prurido e quanto à gravidade das lesões dermatológicas através do Canine Atopic Dermatitis Extend and Severity Index (CADESI-03). O TPC foi realizado com doses crescentes do alergénio (0.08, 0.4 e 2 BU/mL) até ao aparecimento de um score positivo (≥6), dado pelo somatório das classificações de hiperémia conjuntival, quemose, epífora e prurido ocular avaliadas de 0 a 3 consoante a sua gravidade (ausente, suave, moderado ou grave, respectivamente). Ao prurido foi permitido um grau adicional de 4 (muito grave). O alergénio foi instilado no olho direito (olho teste) e o olho esquerdo serviu de controlo. O teste foi considerado inválido sempre que o olho controlo apresentou uma pontuação ≥6. Na segunda fase do estudo, 2 meses após o início da ITAE rush (ITAEr), os pacientes foram sujeitos a novo TPC e os seus CADESI-03 e escala de prurido reavaliados. Os animais não fizeram qualquer tipo de tratamento, que não a ITAEr, durante o período que decorreu entre fases. As classificações finais relativas aos TPC, prurido e CADESI-03 foram posteriormente comparadas pelo teste de Wilcoxon para amostras emparelhadas (p <0.005). Após a ITAEr, os scores oculares finais foram mais baixos em 87% dos pacientes e 3 animais (20%) apresentaram um TPC negativo. Os valores do CADESI-03 e TPC alteraram-se de 122.80 ± 55.11 e 6.73 ± 0.76 para 97.67 ± 43.95 (p <0.005) e 5.40 ± 1.12 (p <0.002), respectivamente. O prurido decresceu de 8.80 ± 0.86 para 5.70 ± 2.09 (p <0.001). O TPC permitiu estabelecer uma nítida relação entre a afecção conjuntival e a exposição a alergénios para os quais se sabe existir sensibilização, concluindo-se que pode ser útil na avaliação da eficácia da ITAE. A ITAEr pode beneficiar cães atópicos com CA associada, uma vez que a maioria dos animais demonstrou uma resolução parcial ou total sintomatologia ocular e cutânea.ABSTRACT - EFFECTIVENESS OF RUSH IMMUNOTHERAPY IN ATOPIC DOGS AS ASSESSED BY CONJUCTIVAL PROVOCATION TESTS - Canine atopic dermatitis (cAD) is frequent in dogs, but little is known about eye involvement and the true prevalence and impact of canine allergic conjunctivitis (AC) is still unknown. The conjunctival provocation test (CPT) is used in human to study the ocular response to allergenic stimuli and to evaluate anti-allergic therapy, including specific immunotherapy. The aims of this study are to evaluate the clinical use of the CPT in atopic dogs as a mean of testing the efficacy of rush specific immunotherapy (rSIT) and to understand if this form of treatment is of benefit in canine atopic patients that suffer from AC. 15 dogs with cAD known to be sensitized to Dermatophagoides farinae (Df) that had history of allergic ocular findings and scored positive on the CPT on initial presentation were enrolled in the study. Pruritus was assessed and dermatological sings were graded using Canine Atopic Dermatitis Extend and Severity Index (CADESI-03). Patients were challenged with increasing doses of specific allergen (0.08, 0.4 e 2 BU/mL) until a positive score was reached (≥6). Concerning CPT allergic ocular findings, four criteria (conjunctival hyperemia, epifora, chemosis and pruritus) were scored on a three or four–point scale, separately for each eye. Patients were tested in the right eye before they began rSIT and two months later. The left eye served as control. No other treatment protocol was established for these patients apart from the vaccine administration. In the end CPT, CADESI-03 and pruritus initial and final scores were compared using Wilcoxon test for paired samples (p <0.005). After rSIT, 87% of the patients scored lower on the CPT and 3 animals (20%) showed no response. CADESI-03 and CPT scores decreased from 122.80 ± 55.11 and 6.73 ± 0.76 to 97.67 ± 43.95 (p <0.005) and 5.40 ± 1.12 (p <0.002), respectively. Mean pruritus was significantly lower (8.80 ± 0.86 to 5.70 ± 2.09, p <0.001). With CPT a link can be made between conjunctival pathology and exposure to allergens for which the patient is known to be sensitized. We have demonstrated that CPT can be used to monitor the clinical response of patients to rSIT and that atopic patients with ocular signs benefit from this specific treatment, since the majority showed partial or complete resolution of allergic ocular signs. It is therefore considered that rSIT is of benefit in canine atopic patients that suffer from AC

Microglia Susceptibility to Free Bilirubin Is Age-Dependent

Funding: This work was supported by National Funds (Fundacão para a Ciencia e a Tecnologia—UID/DTP/04138/2015-2019) to iMed.ULisboa. AF had a post-doctoral research position (C2007-FFUL/UBMBE/02/ 2011) and AV a post-doc fellowship (SFRH/BPD/76590/2011), both granted by FCT. CS and ES were Master students from University of Bologna who developed their thesis at Universidade de Lisboa, with fellowships from Erasmus+ Programme. We thank Professor Stefano Girotti from the University of Bologna for establishing such collaborative Program and for the local supervision of the students. The funding organization had no role in data collection and analysis, decision to publish, or preparation of the manuscripIncreased concentrations of unconjugated bilirubin (UCB), namely its free fraction (Bf), in neonatal life may cause transient or definitive injury to neurons and glial cells. We demonstrated that UCB damages neurons and glial cells by compromising oligodendrocyte maturation and myelination, and by activating astrocytes and microglia. Immature neurons and astrocytes showed to be especially vulnerable. However, whether microglia susceptibility to UCB is also age-related was never investigated. We developed a microglia culture model in which cells at 2 days in vitro (2DIV) revealed to behave as the neonatal microglia (amoeboid/reactive cells), in contrast with those at 16DIV microglia that performed as aged cells (irresponsive/dormant cells). Here, we aimed to unveil whether UCB-induced toxicity diverged from the young to the long-cultured microglia. Cells were isolated from the cortical brain of 1- to 2-day-old CD1 mice and incubated for 24 h with 50/100 nM Bf levels, which were associated to moderate and severe neonatal hyperbilirubinemia, respectively. These concentrations of Bf induced early apoptosis and amoeboid shape in 2DIV microglia, while caused late apoptosis in 16DIV cells, without altering their morphology. CD11b staining increased in both, but more markedly in 2DIV cells. Likewise, the gene expression of HMGB1, a well-known alarmin, as well as HMGB1 and GLT-1–positive cells, were enhanced as compared to long-maturated microglia. The CX3CR1 reduction in 2DIV microglia was opposed to the 16DIV cells and suggests a preferential Bf-induced sickness response in younger cells. In conformity, increased mitochondrial mass and NO were enhanced in 2DIV cells, but unchanged or reduced, respectively, in the 16DIV microglia. However, 100 nM Bf caused iNOS gene overexpression in 2DIV and 16DIV cells. While only arginase 1/IL-1β gene expression levels increased upon 50/100 nM Bf treatment in long-maturated microglia, MHCII/arginase 1/TNF-α/IL-1β/IL-6 (>10-fold) were upregulated in the 2DIV microglia. Remarkably, enhanced inflammatory-associated microRNAs (miR-155/miR-125b/miR-21/miR-146a) and reduced anti-inflammatory miR-124 were found in young microglia by both Bf concentrations, while remained unchanged (miR/21/miR-125b) or decreased (miR-155/miR-146a/miR-124) in aged cells. Altogether, these findings support the neurodevelopmental susceptibilities to UCB-induced neurotoxicity, the most severe disabilities in premature babies, and the involvement of immune-inflammation neonatal microglia processes in poorer outcomes.publishersversionpublishe

Hierarchic species-area relationships and the management of forest habitat islands in intensive farmland

Habitat loss and fragmentation due to land use changes are major threats to biodiversity in forest ecosystems, and they are expected to have important impacts on many taxa and at various spatial scales. Species richness and area relationships (SARs) have been used to assess species diversity patterns and drivers, and thereby in the establishment of conservation and management strategies. Here we propose a hierarchical approach to achieve deeper insights on SARs in small forest islets in intensive farmland and to address the impacts of decreasing naturalness on such relationships. In the intensive dairy landscapes of Northwest Portugal, where small forest stands (dominated by pines, eucalypts or both) represent semi-natural habitat islands, 50 small forest stands were selected and surveyed for vascular plant diversity. A hierarchical analytical framework was devised to determine species richness and inter- and intra-patch SARs for the whole set of forest patches (general patterns) and for each type of forest (specific patterns). Differences in SARs for distinct groups were also tested by considering subsets of species (native, alien, woody, and herbaceous). Overall, values for species richness were confirmed to be different between forest patches exhibiting different levels of naturalness. Whereas higher values of plant diversity were found in pine stands, higher values for alien species were observed in eucalypt stands. Total area of forest (inter-patch SAR) was found not to have a significant impact on species richness for any of the targeted groups of species. However, significant intra-patch SARs were obtained for all groups of species and forest types. A hierarchical approach was successfully applied to scrutinise SARs along a gradient of forest naturalness in intensively managed landscapes. Dominant canopy tree and management intensity were found to reflect differently on distinct species groups as well as to compensate for increasing stand area, buffering SARs among patches, but not within patches. Thus, the maintenance of small semi-natural patches dominated by pines, under extensive practices of forest management, will promote native plant diversity while at the same time contributing to limit the expansion of problematic alien invasive specie