Dynamics and kinetics of the O2-evolution step in Photosystem II: a time-resolved polarography and X-ray spectroscopy study

Photosystem II is a large oxidoreductase protein complex with a crucial role in oxygenic photosynthesis where it makes use of its unique catalytic ability to facilitate water oxidation. Apart from this fundamental importance, the research on this protein has attracted extra attention over the last couple decades as PSII also serves as a paragon for the development of synthetic catalysts for solar fuel production. Consequently, great advancements have been made, with important contributions from crystallography, spectroscopy, and computational methods, so that the structure and reactive cycle of PSII is now much better understood. Although some questions remain open, like the identities of the substrate water molecules, the type of mechanism involved in the formation of the O-O bond, the proton dynamics around the reaction center of PSII, the role of structural and molecular reorganizations and the impact of special features, like red-shifted pigments in the catalytic cycle. To tackle the first question, and indirectly the second, a temperature dependent study of the interaction of ammonia, a substrate water analogue that binds directly to the Mn-cluster of PSII, was performed. From it, three possible binding spots directly to or in the close vicinity of the Mn-cluster were suggested, one of which might be a substrate water binding site. Further O2 polarography measurements of several PSII cores with unique point mutations of H-bond active residues offered a broader perspective on the protonation dynamics away from the OEC specially identing the broad channel as the most probable H+ pathway in S3→S0, with E65 as a key participant in this process. As most dynamics of the S-state cycle freeze out below 200 K, an investigation of dark-adapted PSII by means of a temperature-dependent EXAFS study identified a breakpoint in the Debye-Waller factor at this exact temperature point, revealing the importance of vibrational mobility and molecular reorganizations in the S-state transitions, which may relate to the much slower τ-value than theoretically predicted for the ET from OEC → YZ●+. Lastly, the study of PSII containing red-shifted chlorophylls showed for FRL C. thermalis a significant slowdown of the O2 release and an increase in a ΔG‡ 40 mev higher than the average value found for common PSIIs, further supporting that the reaction center of PSII from FRL C. thermalis have at least one Chl replaced by a red-shifted one.Photosystem II ist ein Schlüsselenzym der oxygenen Photosynthese, das als Oxidoreduktase die Wasserspaltung ermöglicht. Neben dieser Eigenschaft, hat das Enzym in den letzten Jahrzehnten zudem weitere Aufmerksamkeit als Vorlage für die Entwicklung synthetischer Katalysatoren für die Gewinnung von Brennstoffen mittels Sonnenenergie erhalten. Durch die Nutzung verschiedener Methoden wie Röntgenkristallographie, Spektroskopie oder Computersimulationen konnten grundlegende Erkenntnisse über dessen Funktion und Struktur gewonnen werden, wobei einige Fragen aber weiterhin offenblieben. So ist weiterhin unklar, welche Wassermoleküle als Substrat dienen, wie die O-O-Bindung im Detail erfolgt, welche Protonierungsdynamiken im Reaktionszentrum ablaufen und wie sich strukturelle und molekulare Umorientierungen im Proteinkomplex , wie z.B. die Einlagerung rot-verschobener Pigmente auf den katalytischen Zyklus auswirken. Um die ersten beiden Fragen zu adressieren, wurden im Rahmen dieser Arbeit, temperaturabhängige und zeitaufgelöste Sauerstoffmessungen mit Ammonium, einem Substratwasseranalogon, das direkt an den PSII-Mangan-Cluster bindet, durchgeführt. Diese zeigen drei Bindungsmöglichkeiten in der unmittelbaren Nähe des Mn-Clusters, mit einer als mögliche Substratwasserbindungsstelle. Weitere Sauerstoffpolarografie-Experimente mit PSII-Varianten bei denen Wasserstoffbrücken-bildende Aminosäuren ausgetauscht wurden, ermöglichten die Untersuchung der Protonierunsgdynamiken um den OEC (oxygen evolving center) und unterstützen die Hypothese, dass der breite Kanal mit E65 als Hauptprotonenweg im S3→S0 Übergang fungiert. Durch temperaturabhängige EXAFS-Studien an dunkel-adaptiertem PSII konnte ein Umschlagpunkt des Debye-Waller-Faktors bei 200 K bestimmt werden, der auch dem Temperaturpunkt entspricht, an dem der S-Zyklus zum Erliegen kommt. Diese Ergebnisse unterstützen, dass Vibrationsmobilität und molekularer Umorientierung während der S-Zyklus-Übergänge eine entscheidende Rolle spielen und in Verbindung mit dem im Vergleich zur Theorie deutlich verlangsamten τ-Wert beim Elektrontransfer vom OEC zu YZ●+ stehen könnten. Abschließende Untersuchungen an einer PSII-Variante mit rot-verschobenen Chlorophyllmolekülen, zeigte eine deutliche Verlangsamung der Sauerstofffreisetzung und eine Erhöhung in ΔG‡ um 40 meV im Vergleich zu gewöhnlichen Chlorophyll a-PSII-Varianten. Diese Unterschiede stützen die Hypothese, dass mindestens ein Chlorophyllmolekül im Reaktionszentrum durch ein rot-verschobenes Chlorophyll ersetzt wurde

is food missing in the equation?

publishersversionpublishe

Safety mechanisms for the reliable operation of 3D vehicles

The safety and reliability of unmanned vehicles is a growing concern in our modern society. This work proposes and implements mechanisms to minimize risks in the operation of 3D vehicles. A brief analysis is performed to identify high priority risks and low complexity solutions are proposed in order to avoid or minimize their impact. To cope with critical power failures, an autonomous current monitoring system was studied and implemented after analyzing two different techniques: resistive and magnetic current sensing. Furthermore, a fall detection system capable of detecting rotational and free falls was developed and evaluated. Lastly, an obstacle detection and avoidance system relying on multiple smart sensors was proposed. Several simulation tests were performed for different velocities to obtain processing delays and stopping times and thus, the minimal safe flying distance for the avoidance of obstacles.A segurança na operação fiável de veículos não tripulados é uma preocupação crescente na nossa sociedade moderna. Este trabalho propõe e implementa mecanismos para minimizar os riscos no manuseamento destes veículos. Uma breve análise é realizada para identificar os componentes com maior risco de ocorrerem problemas e soluções de baixa complexidade são propostas a fim de evitar ou minimizar o seu impacto. Para lidar com falhas de energia críticas, um sistema de monitorização de corrente foi estudado e implementado após analisar duas técnicas diferentes: detecção de corrente resistiva e magnética. Além disso, foi desenvolvido e avaliado um sistema de detecção de quedas rotacionais e livres. Por último, foi proposto um sistema de detecção e anti-colisão de obstáculos baseado em múltiplos sensores inteligentes. Diversos testes de simulação foram realizados para obter atrasos de processamento e tempos de travagem. Deste modo foi possível calcular a distância de segurança mínima de travagem face à detecção de um obstáculo

Children exposure to multiple mycotoxins through food consumption: a holistic approach for risk assessment

Food ingestion is considered a major route of human exposure to chemical contaminants, namely mycotoxins. Considering their toxic and carcinogenic effects, mycotoxins exposure assessment assumes particular importance, especially when vulnerable populations as children, are involved. Although there are increasing evidences of mycotoxins co-contamination in food, scarce data are available concerning children exposure to multiple mycotoxins, their bioaccessibility and the potential toxic effects resulting from intestinal exposure. Addressing these considerations, this thesis main objectives were: to characterize Portuguese children (under 3 years old) exposure to multiple mycotoxins through food consumption; to determine mycotoxins bioaccessibility in foods usually consumed by children; to evaluate intestinal toxic effects associated to mycotoxins exposure; and, to characterize the risk associated to the consumption of foods, considering data from exposure assessment, bioaccessibility and intestinal toxicity assays. The present thesis applied for the first time a holistic approach, gathering results obtained through different first-line methodologies, comprising probabilistic tools to estimate mycotoxins daily intake and in vitro assays to determine bioaccessibility and toxic effects. Results revealed a potential health concern for aflatoxins exposure with regard to the high percentiles of children intake. For the first time, a potential synergism was described relatively to the combined intestinal toxic effects of patulin and ochratoxin A, two known enterotoxins. The obtained results reinforce the importance to use a holistic approach to multiple mycotoxins risk assessment, especially for vulnerable populations as children. Particular attention should be dedicated to evaluate the consequences of intestinal exposure to mycotoxins, in particular to their impact on the development of intestinal diseases; RESUMO: EXPOSIÇÃO DE CRIANÇAS A MÚLTIPLAS MICOTOXINAS ATRAVÉS DO CONSUMO DE ALIMENTOS: UMA ABORDAGEM HOLÍSTICA À AVALIAÇÃO DE RISCO A ingestão de alimentos é considerada a principal via de exposição humana a contaminantes químicos, dos quais se destacam as micotoxinas. Em virtude dos seus efeitos tóxicos e carcinogénicos, é fundamental avaliar a exposição a estes compostos, particularmente em populações vulneráveis, como é o caso das crianças. Nos últimos anos, alguns estudos têm dedicado especial atenção à co-contaminação de alimentos por micotoxinas. No entanto, são ainda escassos os dados disponíveis relativos à exposição das crianças a múltiplas micotoxinas assim como a sua bioacessibilidade e potenciais efeitos tóxicos a nível intestinal. Neste contexto, a presente Tese teve como principais objetivos: caracterizar a exposição das crianças portuguesas (idade inferior a 3 anos) a múltiplas micotoxinas através da alimentação; determinar a bioacessibilidade das micotoxinas nos alimentos habitualmente consumidos pelas crianças; avaliar os efeitos tóxicos intestinais associados à exposição simultânea a micotoxinas; e, caracterizar o risco associado ao consumo de alimentos, coligindo resultados da avaliação da exposição, bioacessibilidade e toxicidade intestinal. A presente Tese desenvolveu pela primeira vez uma abordagem holística, reunindo resultados obtidos através de diferentes metodologias, nomeadamente ferramentas probabilísticas para estimar a ingestão diária de micotoxinas e ensaios in vitro para determinar a bioacessibilidade e efeitos tóxicos. Os principais resultados revelaram, pela primeira vez, que a exposição a aflatoxinas pode representar um risco potencial para as crianças que apresentaram os percentis mais elevados de ingestão. Foram também descritos efeitos sinérgicos inéditos decorrentes da exposição intestinal simultânea a duas enterotoxinas: patulina e ocratoxina A. Os resultados obtidos reforçam a importância da implementação de uma abordagem holística para a avaliação de risco de misturas de micotoxinas, em especial quando envolvidas populações vulneráveis como as crianças. As potenciais consequências da exposição intestinal a micotoxinas deverão ainda ser ponderadas, principalmente no que respeita às implicações no desenvolvimento de doenças intestinais

Burden of disease of dietary exposure to aflatoxins in European countries

publishersversionpublishe

human biomonitoring (HBM) as a tool to support policy and regulatory action to prevent chemicals exposure

publishersversionpublishe

Challenges in risk assessment of multiple mycotoxins in food

Review articleMost fungi are able to produce several mycotoxins simultaneously and, consequently, to contaminate a wide variety of foodstuffs. Therefore, the risk of human co-exposure to multiple mycotoxins is real, raising a growing concern about their potential impact on human health. Besides, government and industry regulations are usually based on individual toxicities, and do not take into account the complex dynamics associated with interactions between co-occurring groups of mycotoxins. The present work assembles, for the first time, the challenges posed by the likelihood of human co-exposure to these toxins and the possibility of interactive effects occurring after absorption, towards knowledge generation to support a more accurate human risk assessment. Regarding hazard assessment, a physiologically-based framework is proposed in order to infer the health effects from exposure to multiple mycotoxins in food, including knowledge on the bioaccessibility, toxicokinetics and toxicodynamics of single and combined toxins. The prioritisation of the most relevant mixtures to be tested under experimental conditions that attempt to mimic human exposure and the use of adequate mathematical approaches to evaluate interactions, particularly concerning the combined genotoxicity, were identified as the main challenges for hazard assessment. Regarding exposure assessment, the need of harmonised food consumption data, availability of multianalyte methods for mycotoxin quantification, management of left-censored data, use of probabilistic models and multibiomarker approaches are highlighted, in order to develop a more precise and realistic exposure assessment. To conclude, further studies on hazard and exposure assessment of multiple mycotoxins, using harmonised methodologies, are crucial towards an improvement of data quality and a more reliable and robust risk characterisation, which is central for risk management and, consequently, to prevent mycotoxins-associated adverse effects. A deep understanding of the nature of interactions between multiple mycotoxins will contribute to draw real conclusions on the health impact of human exposure to mycotoxin mixtures.This research was performed under the MycoMix project ‘Exploring the toxic effects of mixtures of mycotoxins in infant food and potential health impact’ (PTDC/DTP-FTO/0417/2012), through CESAM: UID/AMB/50017/2013 and ToxOmics: UID/BIM/00009/2013, all funded by the Fundação para a Ciência e Tecnologia (FCT), Portugal.info:eu-repo/semantics/publishedVersio

Salmonella e os ovos um perigo real?

Perigo Salmonella associado ao consumo de ovo

Years of life lost associated with COVID-19 deaths in Portugal: two years of pandemic

Abstract publicado em. Eur J Public Health. 2023 Oct; 33(Suppl 2):ckad160.701. https://doi.org/10.1093/eurpub/ckad160.701Background: The impact of the COVID-19 pandemic on the Portuguese population is still underexplored. Therefore, the characteriza tion of years of life lost (YLL) due to this epidemic may provide relevant data for establishing effective strategies in future epidemics. The aim of this study was to calculate YLL associated with COVID-19 deaths in Portugal between 2020 and 2022. Methods: This was an observational, cross-sectional study. Data on the average resident population and life expectancy at birth by age group and sex were obtained from Statistics Portugal (INE) and GBD 2019 standard life-table, respectively. Data on COVID-19 deaths were extracted from the Directorate-General of Health’s (DGS) reports. YLL was calculated as the number of COVID-19 deaths multiplied by standard life expectancy at the age of death, globally, by sex and age group. YLL was calculated by 100,000 population and age-standardized. Results: Between 2020 and 2022, 3413013 cases of COVID-19 were reported in Portugal, of which 21342 (0.6%) eventually died of the disease. The number of deaths by sex was similar globally or by age group. Globally, the YLLs estimated for COVID-19 in Portugal were 309383,8. YLLs were higher for males than for females for most age groups except of age groups 10-19 yrs (76.56 vs 153.11) and > 80 yrs (58710.88 vs 68491.44). YLLs age-standardized also showed a steady increase as we progressed in the age group [from 3.25 (0-9 yrs) to 885.54 (>80 yrs)], in which from the 50-59 yrs age group onwards we observed a steeper slope of increase in YLLs. Conclusions: COVID-19 has had a major impact on mortality rates in Portugal, with this impact being greater in the older population, especially in people aged over 70 years. Also, males presented higher YLLs than females, with this difference increasing substantially as age increases. These insights can be useful in terms of public health as the disease progresses to an endemic phase.Key messages: - COVID-19 has had a major impact on mortality rates in Portugal; - Calculating YLLs associated with COVID-19 in Portugal provides relevant data for establishing effective strategies in future epidemics.info:eu-repo/semantics/publishedVersio

Patulin bioaccessibility in fruit juices: what happens after ingestion?

As micotoxinas são contaminantes identificados com frequência em diversos géneros alimentícios incluindo fruta e produtos derivados. A exposição a estes metabolitos secundários produzidos por fungos pode provocar efeitos tóxicos para a saúde humana e animal. A patulina é uma micotoxina que ocorre sobretudo em maçãs e alimentos à base de maçã, incluindo os que são consumidos por crianças, tais como purés ou sumos de fruta. A ingestão de alimentos contaminados é considerada a principal via de exposição à patulina pelo que se considera relevante estudar a influência do processo digestivo na potencial toxicidade desta micotoxina. Para o efeito, neste estudo foram determinados os valores de bioacessibilidade da patulina nas fases oral (O), gástrica (G) e intestinal (I) quando presente em sumos de fruta à base de maçã, contaminados artificialmente, aplicando-se, pela primeira vez, o método harmonizado de digestão in vitro. Os valores de bioacessibilidade obtidos (O: 77,8%; G: 79,3%; I: 21,6%) sugerem uma redução dos teores de patulina que ocorre maioritariamente durante a fase intestinal, revelando também a existência de elevada bioacessibilidade nas fases oral e gástrica (superior a 75%). Os resultados indicam a necessidade de um estudo mais detalhado sobre os efeitos da patulina na fase gástrica atendendo ao potencial impacto na saúde humana, em especial em populações mais vulneráveis como as crianças.Mycotoxins are contaminants usually found in foodstuf fs including fruit and products thereof. These secondary metabolites produced by fungi can cause serious human and animal health problems. Patulin is a mycotoxin that occurs primarily in apples and products thereof, including those consumed by children, such as apple purées or fruit juices. The ingestion of contaminated food is the main route of exposure to patulin therefore, it is impor tant to clarify the influence of the digestive process on the potential toxicity of this mycotoxin. The patulin’s bioaccessibility values in the oral (O), gastric (G) and intestinal (I) phases were determined in this study, considering ar tificially contaminated apple-based juices and applying, for the first time, the harmonized in vitro digestion method. The bioaccessibility values obtained (O: 77.8%, G: 79.3%, I: 21.6%) suggest that the reduction of patulin occurs mostly during the intestinal phase, also revealing the existence of high bioaccessibility during the oral and gastric phase (above 75%). These results point the need for fur ther studies on the potential toxic ef fects of patulin at gastric phase par ticularly for vulnerable population groups as children.info:eu-repo/semantics/publishedVersio