Evoluzione di micotossine regolate ed emergenti attraverso i processi alimentari: dal campo alla digestione

Nel presente lavoro di tesi sono stati studiati diversi aspetti riguardanti micotossine regolate ed emergenti al fine di fornire informazioni circa la loro mitigazione dal campo al prodotto finito, focalizzando l’attenzione sul potenziale impatto del processo tecnologico. Successivamente, il loro ruolo tossicologico, ancora in discussione, è stato indagato attraverso l’applicazione di modelli gastrointestinali. In particolare, lo studio è stato rivolto a DON, micotossina più comune nel grano, DON-3-Glc, che rappresenta la sua principale forma modificata, e alla micotossina ENN B, maggiore rappresentante tra il gruppo delle cosiddette micotossine “emergenti”. Questo studio è stato quindi suddiviso in tre sezioni. La prima è stata rivolta allo sviluppo di esperimenti in serra effettuati su diversi genotipi di grano duro al fine di comprendere meglio il meccanismo di detossificazione dal DON e la resistenza delle diverse varietà ad una delle malattie più gravi che colpiscono questa specie (fusariosi della spiga). La seconda sezione ha riguardato lo studio del reale impatto di alcune filiere di produzione strategiche sul contenuto finale in micotossine. Inoltre, una strategia di mitigazione è stata messa a punto tenendo conto del possibile sviluppo di altri contaminanti legati al processo stesso e ottenendo un prodotto finito adeguato per il consumatore. Nell'ultima sezione di questo lavoro sono invece state effettuate delle indagini circa i possibili risvolti tossicologici di questi composti, valutando il loro destino durante la digestione umana.

Novel surface-tethered estrogen polymeric platforms in cardiovascular regenerative medicine

L’estradiol (E2) est une hormone femelle qui joue un rôle essentiel, à la fois dans la régulation et dans la détermination de certaines conditions physiologiques in vivo, telle que la différenciation et la prolifération cellulaire. Lorsque l’E2 est donné en supplément, par exemple dans le cas de thérapie hormonale, deux effets sont observés, un effet génomique et un effet non-génomique, de par son interaction avec les récepteurs à œstrogène du noyau ou de la membrane cellulaire, respectivement. L’effet non-génomique est plus difficile à étudier biologiquement parce que l’effet se produit sur une échelle de temps extrêmement courte et à cause de la nature hydrophobe de l’E2 qui réduit sa biodisponibilité et donc son accessibilité aux cellules cibles. C’est pourquoi il est nécessaire de développer des systèmes d’administration de l’E2 qui permettent de n’étudier que l’effet non-génomique de l’œstrogène. Une des stratégies employée consiste à greffer l’E2 à des macromolécules hydrophiles, comme de l’albumine de sérum bovin (BSA) ou des dendrimères de type poly(amido)amine, permettant de maintenir l’interaction de l’E2 avec les récepteurs d’œstrogène de la membrane cellulaire et d’éviter la pénétration de l’E2 dans le noyau des cellules. Toutefois, ces systèmes macromolécules-E2 sont critiquables car ils sont peu stables et l’E2 peut se retrouver sous forme libre, ce qui affecte sa localisation cellulaire. L’objectif de cette thèse est donc de développer de nouvelles plateformes fonctionnalisées avec de l’E2 en utilisant les approches de synthèses ascendantes et descendantes. Le but de ces plateformes est de permettre d’étudier le mécanisme de l’effet non-génomique de l’E2, ainsi que d’explorer des applications potentielles dans le domaine biomédical. L’approche ascendante est basée sur un ligand d’E2 activé, l’acide 17,α-éthinylestradiol-benzoïque, attaché de façon covalente à un polymère de chitosan avec des substitutions de phosphorylcholine (CH-PC-E2). L’estradiol est sous forme de pro-drogue attachée au polymère qui s’auto-assembler pour former un film. L’effet biologique de la composition chimique du film de chitosan-phosphorylcholine a été étudié sur des cellules endothéliales. Les films de compositions chimiques différentes ont préalablement été caractérisés de façon physicochimique. La topographie de la surface, la charge de surface, ainsi que la rhéologie des différents films contenant 15, 25, ou 40% molaires de phosphorylcholine, ont été étudiés par microscopie à force atomique (AFM), potentiel zêta, résonance plasmonique de surface et par microbalance à cristal de quartz avec dissipation (QCM-D). Les résultats de QCM-D ont montré que plus la part molaire en phosphorylcholine est grande moins il y a de fibrinogène qui s’adsorbe sur le film de CH-PC. Des cellules humaines de veine ombilicale (HUVECs) cultivées sur des films de CH-PC25 et de CH-PC40 forment des amas cellulaire appelés sphéroïdes au bout de 4 jours, alors que ce n’est pas le cas lorsque ces cellules sont cultivées sur des films de CH-PC15. L’attachement de l’estradiol au polymère a été caractérisé par plusieurs techniques, telles que la résonance magnétique nucléaire de proton (1H NMR), la spectroscopie infrarouge avec transformée de Fourier à réfraction totale atténuée (FTIR-ATR) et la spectroscopie UV-visible. La nature hydrogel des films (sa capacité à retenir l’eau) ainsi que l’interaction des films avec des récepteurs à E2, ont été étudiés par la QCM-D. Des études d’imagerie cellulaires utilisant du diacétate de diaminofluoresceine-FM ont révélé que les films hydrogels de CH-PC-E2 stimulent la production d’oxyde nitrique par les cellules endothéliales, qui joue un rôle protecteur pour le système cardiovasculaire. L’ensemble de ces études met en valeur les rôles différents et les applications potentielles qu’ont les films de type CH-PC-E2 et CH-PC dans le cadre de la médecine cardiovasculaire régénérative. L’approche descendante est basée sur l’attachement de façon covalente d’E2 sur des ilots d’or de 2 μm disposés en rangées et espacés par 12 μm sur un substrat en verre. Les ilots ont été préparés par photolithographie. La surface du verre a quant à elle été modifiée à l’aide d’un tripeptide cyclique, le cRGD, favorisant l’adhésion cellulaire. L’attachement d’E2 sur les surfaces d’or a été suivi et confirmé par les techniques de SPR et de QCM-D. Des études d’ELISA ont montré une augmentation significative du niveau de phosphorylation de la kinase ERK (marqueur important de l’effet non-génomique) après 1 heure d’exposition des cellules endothéliales aux motifs alternant l’E2 et le cRGD. Par contre lorsque des cellules cancéreuses sont déposées sur les surfaces présentant des motifs d’E2, ces cellules ne croissent pas, ce qui suggère que l’E2 n’exerce pas d’effet génomique. Les résultats de l’approche descendante montrent le potentiel des surfaces présentant des motifs d’E2 pour l’étude des effets non-génomiques de l’E2 dans un modèle in vitro.

Novel surface-tethered estrogen polymeric platforms in cardiovascular regenerative medicine

L’estradiol (E2) est une hormone femelle qui joue un rôle essentiel, à la fois dans la régulation et dans la détermination de certaines conditions physiologiques in vivo, telle que la différenciation et la prolifération cellulaire. Lorsque l’E2 est donné en supplément, par exemple dans le cas de thérapie hormonale, deux effets sont observés, un effet génomique et un effet non-génomique, de par son interaction avec les récepteurs à œstrogène du noyau ou de la membrane cellulaire, respectivement. L’effet non-génomique est plus difficile à étudier biologiquement parce que l’effet se produit sur une échelle de temps extrêmement courte et à cause de la nature hydrophobe de l’E2 qui réduit sa biodisponibilité et donc son accessibilité aux cellules cibles. C’est pourquoi il est nécessaire de développer des systèmes d’administration de l’E2 qui permettent de n’étudier que l’effet non-génomique de l’œstrogène. Une des stratégies employée consiste à greffer l’E2 à des macromolécules hydrophiles, comme de l’albumine de sérum bovin (BSA) ou des dendrimères de type poly(amido)amine, permettant de maintenir l’interaction de l’E2 avec les récepteurs d’œstrogène de la membrane cellulaire et d’éviter la pénétration de l’E2 dans le noyau des cellules. Toutefois, ces systèmes macromolécules-E2 sont critiquables car ils sont peu stables et l’E2 peut se retrouver sous forme libre, ce qui affecte sa localisation cellulaire. L’objectif de cette thèse est donc de développer de nouvelles plateformes fonctionnalisées avec de l’E2 en utilisant les approches de synthèses ascendantes et descendantes. Le but de ces plateformes est de permettre d’étudier le mécanisme de l’effet non-génomique de l’E2, ainsi que d’explorer des applications potentielles dans le domaine biomédical. L’approche ascendante est basée sur un ligand d’E2 activé, l’acide 17,α-éthinylestradiol-benzoïque, attaché de façon covalente à un polymère de chitosan avec des substitutions de phosphorylcholine (CH-PC-E2). L’estradiol est sous forme de pro-drogue attachée au polymère qui s’auto-assembler pour former un film. L’effet biologique de la composition chimique du film de chitosan-phosphorylcholine a été étudié sur des cellules endothéliales. Les films de compositions chimiques différentes ont préalablement été caractérisés de façon physicochimique. La topographie de la surface, la charge de surface, ainsi que la rhéologie des différents films contenant 15, 25, ou 40% molaires de phosphorylcholine, ont été étudiés par microscopie à force atomique (AFM), potentiel zêta, résonance plasmonique de surface et par microbalance à cristal de quartz avec dissipation (QCM-D). Les résultats de QCM-D ont montré que plus la part molaire en phosphorylcholine est grande moins il y a de fibrinogène qui s’adsorbe sur le film de CH-PC. Des cellules humaines de veine ombilicale (HUVECs) cultivées sur des films de CH-PC25 et de CH-PC40 forment des amas cellulaire appelés sphéroïdes au bout de 4 jours, alors que ce n’est pas le cas lorsque ces cellules sont cultivées sur des films de CH-PC15. L’attachement de l’estradiol au polymère a été caractérisé par plusieurs techniques, telles que la résonance magnétique nucléaire de proton (1H NMR), la spectroscopie infrarouge avec transformée de Fourier à réfraction totale atténuée (FTIR-ATR) et la spectroscopie UV-visible. La nature hydrogel des films (sa capacité à retenir l’eau) ainsi que l’interaction des films avec des récepteurs à E2, ont été étudiés par la QCM-D. Des études d’imagerie cellulaires utilisant du diacétate de diaminofluoresceine-FM ont révélé que les films hydrogels de CH-PC-E2 stimulent la production d’oxyde nitrique par les cellules endothéliales, qui joue un rôle protecteur pour le système cardiovasculaire. L’ensemble de ces études met en valeur les rôles différents et les applications potentielles qu’ont les films de type CH-PC-E2 et CH-PC dans le cadre de la médecine cardiovasculaire régénérative. L’approche descendante est basée sur l’attachement de façon covalente d’E2 sur des ilots d’or de 2 μm disposés en rangées et espacés par 12 μm sur un substrat en verre. Les ilots ont été préparés par photolithographie. La surface du verre a quant à elle été modifiée à l’aide d’un tripeptide cyclique, le cRGD, favorisant l’adhésion cellulaire. L’attachement d’E2 sur les surfaces d’or a été suivi et confirmé par les techniques de SPR et de QCM-D. Des études d’ELISA ont montré une augmentation significative du niveau de phosphorylation de la kinase ERK (marqueur important de l’effet non-génomique) après 1 heure d’exposition des cellules endothéliales aux motifs alternant l’E2 et le cRGD. Par contre lorsque des cellules cancéreuses sont déposées sur les surfaces présentant des motifs d’E2, ces cellules ne croissent pas, ce qui suggère que l’E2 n’exerce pas d’effet génomique. Les résultats de l’approche descendante montrent le potentiel des surfaces présentant des motifs d’E2 pour l’étude des effets non-génomiques de l’E2 dans un modèle in vitro.

Determination of multi-mycotoxin occurrence in maize based porridges from selected regions of Tanzania by liquid chromatography tandem mass spectrometry (LC-MS/MS), a longitudinal study

Residents of certain areas of Tanzania are exposed to mycotoxins through the consumption of contaminated maize based foods. In this study, 101 maize based porridge samples were collected from villages of Nyabula, Kikelelwa and Kigwa located in different agro-ecological zones of Tanzania. The samples were collected at three time points (time point 1, during maize harvest; time point 2, 6 months after harvest; time point 3, 12 months after harvest) over a 1-year period. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to detect and quantify 9 mycotoxins: aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), fumonisin B1 (FB1), fumonisin B2 (FB2), deoxynivalenol (DON), ochratoxin A (OTA) and zearaleneone (ZEN) in the samples following a QuEChERS extraction method. Eighty two percent of samples were co-contaminated with more than one group of mycotoxins. Fumonisins (FB1 + FB2) had the highest percentage occurrence in all 101 samples (100%) whereas OTA had the lowest (5%). For all three villages the mean concentration of FB1 was lowest in samples taken from time point 2. Conversely, In Kigwa village there was a distinct trend that AFB1 mean concentration was highest in samples taken from time point 2. DON concentration did not differ greatly between time points but the percentage occurrence varied between villages, most notably in Kigwa where 0% of samples tested positive. ZEN occurrence and mean concentration was highest in Kikelelwa. The results suggest that mycotoxin contamination in maize can vary based on season and agro-ecological zones. The high occurrence of multiple mycotoxins found in maize porridge, a common weaning food in Tanzania, presents a potential increase in the risk of exposure and significant health implications in children.

Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients

Aflatoxins are a group of carcinogenic compounds produced by Aspergillus fungi that can grow on different agricultural crops. Both acute and chronic exposure to these mycotoxins can cause serious illness. Due to the high occurrence of aflatoxins in crops worldwide fast and cost-effective analytical methods are required for the identification of contaminated agricultural commodities before they are processed into final products and placed on the market. In order to provide new tools for aflatoxin screening two prototype fast ELISA methods: one for the detection of aflatoxin B₁ and the other for total aflatoxins were developed. Seven monoclonal antibodies with unique high sensitivity and at the same time good cross-reactivity profiles were produced. The monoclonal antibodies were characterized and two antibodies showing IC₅₀ of 0.037 ng/mL and 0.031 ng/mL for aflatoxin B₁ were applied in simple and fast direct competitive ELISA tests. The methods were validated for peanut matrix as this crop is one of the most affected by aflatoxin contamination. The detection capabilities of aflatoxin B₁ and total aflatoxins ELISAs were 0.4 μg/kg and 0.3 μg/kg for aflatoxin B₁, respectively, which are one of the lowest reported values. Total aflatoxins ELISA was also validated for the detection of aflatoxins B₂, G₁ and G₂. The application of the developed tests was demonstrated by screening 32 peanut samples collected from the UK retailers. Total aflatoxins ELISA was further applied to analyse naturally contaminated maize porridge and distiller's dried grain with solubles samples and the results were correlated with these obtained by UHPLC-MS/MS method.

Sex steroid feedback regulation of pituitary gonadotropins during early secondary oocyte growth in coho salmon (Oncorhynchus kisutch) – a potential target of endocrine disruption.

Thesis (Ph.D.)--University of Washington, 2016-06

Assessment of toxigenic fungi in poultry feed

Introduction - Feed supplies the necessary nutrients for the growth of healthy animals, which are a part of the human diet. The presence of toxigenic fungi in animal feed such as Aspergillus spp. may contribute to 1) the loss of nutritional value of feedstuff, since fungi will assimilate the most readily available nutrients present in the feed, and 2) the development of mycotoxicoses and chronic conditions, which can raise economic issues due to animal disease and contamination of animal derived products. Aim of the study - The goal of this work was to evaluate the incidence of Aspergilli, particularly from the Circumdati, Flavi and Fumigati sections, through real-time quantitative PCR (qPCR) in 11 feed samples.

Fungi distribution in poultry feed

Feed can easily be contaminated and colonized by fungi that use up the nutrients for their own metabolism and growth, producing secondary metabolites such as mycotoxins that are not eliminated throughout the feed processing. The major problems associated with mycotoxin contaminated animal feed are metabolic disturbances resulting in poor animal productivity. In addition, handling contaminated animal feed can also raise health issues regarding workers exposure to fungi and mycotoxins. The scope of this work was to characterize fungal distribution in 11 poultry feed samples. Twenty grams of feed were suspended in 180 mL of distilled water and homogenized during 20 minutes at 200 rpm. The washed supernatant was plated in malt extract agar (MEA) and dichloran glycerol agar base (DG18) media for morphological identification of the mycobiota present. Using macro- and microscopic analysis of the colonies, fungal contamination was evident in 72.7% of the analyzed poultry feed samples. Fungal load ranged from 0 to 13140 CFU/g, and the most prevalent species/genera were F. graminearum complex (71.1%), Penicillium sp. (11.6%), Cladosporium sp. (8.8%), and Fusarium poae (3.6%). In addition to these species, we also isolated Aspergillus sections Circumdati, Nigri and Aspergilli, and Mucor and Rhizopus genus albeit at a lower abundance. The data obtained showed that, besides high fungal contamination, mycotoxins contamination is probably a reality, particularly in the final product since mycotoxins resist to all the processing operations including thermal treatment. Additionally, data claimed attention for the probable co-exposure to fungi and mycotoxins of the workers in feed industries.

Prevalence of Aspergillus section Fumigati in Portuguese slaughterhouses: a fungal and mycotoxin concern

Introduction - Within the Aspergillus genus, Aspergillus fumigatus species is one of the most ubiquitous saprophytic fungi and is considered the species with higher clinical relevance. The fungi belonging to the Fumigati section are the most common cause of invasive aspergillosis and a major source of infection related mortality in immunocompromised patients. One of the most abundant metabolites produced by Aspergillus fumigatus is the metabolite gliotoxin, which exhibits a diverse array of biologic effects on the immune system. Further, environments contaminated with A. fumigatus may be the cause or enhance respiratory problems in the workers of those specific settings. These species produce specific allergens and mycotoxins that could cause respiratory disorders. Aim of the study - The aim of the present work was to determine the prevalence of Aspergillus section Fumigati by cultural and molecular methods in poultry; swine and bovine; and large animal (bovine and horses) slaughterhouses.

Toxigenic fungi in coffee samples: a menace to public health

Introduction - Mycotoxin contamination was reported to occur in some food and commodities, such as coffee, particularly due to the presence of toxigenic fungi such as Aspergillus, Penicillium and Fusarium spp. Aspergilli are known to produce high levels of mycotoxins, such as ochratoxin and aflatoxin. Aspergillus ochraceus has been proposed as the major cause of ochratoxin A contamination in coffee beans. Aim of the study - The aim of this work was to evaluate the prevalence of Aspergillus sections Circumdati, Flavi and Fumigati in 28 green coffee samples to be used by Portuguese coffee industry, from Coffea arabica (Arabica coffee) and Coffea canephora (Robusta coffee) species from different origins.

Importance of size-selective particle measuring for assessing occupational exposures: a case study “from field to fork”

Sampling the total air concentration of particulate matter (PM) only provides a basic estimate of exposure that normally not allows correlating with the observed health effects. Therefore is of great importance to recognize the particles size distribution and, particularly, the exposure to fine particles (≤ 2.5 μm). This particles dimension corresponds to the respirable fraction, the one that can implicate local and systemic effects due to particle deposition and clearance from the lungs and transport within the organism. This study intended to describe occupational exposure to PM2.5 in three units related with swine production and consumption, namely: feed production, swine production and swine slaughterhouse. A size-selective particle measuring in five to six workplaces of each unit was performed. Measurements of PM were done using a portable direct-reading hand-held equipment (Lighthouse, model 3016 IAQ). Data showed slaughterhouse unit with higher values, with values ranging from 0.030 to 0.142 mg/m3 (0.073 + 0.043), being the cutting room the workplace with higher values. In feed production unit, values were between 0.026 and 0.033 mg/m3 (0.028 + 0.003) with the warehouse of pharmacy products as the workplace with higher values. Finally, in swine unit values ranged from 0.006 to 0.048 mg/m3 (0.023 + 0.017) with the batteries area presenting the higher values. PM can be rich in fungi and bacteria and their metabolites, such as endotoxins and mycotoxins. Previous publications already showed high contamination in these occupational settings and particles can have an important role in exposure since can easily act as carrier of these agents. Data acquired allow not only a better prediction of particle penetration into respiratory regions of the respiratory tract, but also a better estimation of PM health effects. Moreover, data permit to identify the workplaces where investment should be made to prevent and reduce exposure.

Fungal contamination in coffee samples: a public health concern

Introduction - Fungi are natural coffee contaminants and under certain environmental conditions have the potential to produce toxins. Many studies revealed that the important toxigenic fungal genera (Aspergillus and Penicillium) are natural coffee contaminants, and are present from the field to storage. Aspergilli from the Circumdati and Nigri sections are known to produce high levels of ochratoxin A, a mycotoxin known as nephrotoxic for animals and humans. This work aimed to evaluate fungal distribution and also the prevalence of Aspergillus sections Fumigati, Flavi, Nigri and Circumdati from Coffea arabica (Arabica coffee) and Coffea canephora (Robusta coffee) green samples.

Assessment of genotoxicity of aflatoxin M1 and B1 contaminated milks after in vitro human digestion

Introduction - Milk is considered a complete food from the nutritional point of view. Milk can be exposed to various types of contamination, such as mycotoxins. These metabolites are naturally occurring toxic compounds produced by fungi. Several studies on milk samples have reported the presence of aflatoxin B1 (AFB1) and M1 (AFM1), due to the high incidence in samples intended for human consumption, carcinogenicity proven AFB1 and resistance of the contaminants to the process of digestion, making those available for intestinal absorption. Considering these aspects, the objective of this study was to evaluate the genotoxicity of milk samples contaminated by AFB1 and AFM1 before and after the action of lactic acid bacteria using Caco-2 intestinal human cells.

Endocrine disruptors mixtures: the real scenario of human exposure

Endocrine disrupting chemicals (EDCs) are exogenous agents that have the ability to interfere with/or mimic estrogenic hormones and, therefore can simultaneously and differentially trigger specific signaling pathways responsible for the nature and magnitude of biological responses in diverse cell types. Human exposure to EDCs, particularly at low-doses, is ubiquitous, persistent and occurs in complex mixtures. These compounds can bioaccumulate in lipid compartments of tissues forming a mixed “body burden” of contaminants of different origins. Although the independent action of chemicals has been considered the main principle in EDCs mixture toxicity, several effects cannot be predicted when analyzing single compounds individually. Based in a revision of the literature, focused in studies that evaluated EDCs mixtures, we hypothesize the scenario of a pregnant woman environmentally exposed to three different EDCs as a potential real scenario of human exposure supported by data describing where exposure to these compounds occur.

Bibliotecas na saúde… e a saúde nas bibliotecas?

Em arquivos e bibliotecas a presença de fungos é considerada nefasta pelas suas implicações na conservação e leitura de documentos históricos e pela sua associação a problemas de saúde sentidos pelos funcionários e utentes que frequentam estes locais. De acordo com alguns autores, os problemas de saúde mais reportados por funcionários em Bibliotecas e Arquivos são dermatite, rinite, alergias e asma. Embora revestida de inegável importância, existem poucos estudos internacionais sobre a temática e, em Portugal, a contaminação fúngica em ambiente arquivístico e em bibliotecas é ainda muito pouco conhecida. O estudo realizado em quatro Arquivos Portugueses teve como objectivo conhecer a contaminação fúngica, contribuindo para a análise da qualidade do ar interior desses espaços e sua comparação com estudos internacionais. Para isso foram recolhidas amostras de ar e de superfícies e estas foram analisadas por métodos clássicos de cultura e, quando necessário, por métodos de biologia molecular. A avaliação foi feita quantitativa e qualitativamente, considerando os requisitos legais em vigor. No que respeita à análise do ar, o número de unidades formadoras de colónias (UFC)/m3 nunca excedeu as 500 (limite legislado), tendo sido verificada contaminação interior em todos os locais estudados. Comparativamente aos estudos realizados anteriormente em contextos semelhantes foram encontrados níveis elevados de contaminação por leveduras nas amostras de ar analisadas em Arquivos Portugueses. Não foi identificado nenhum fungo patogénico neste estudo, mas em quase todas as amostras estavam presentes fungos potencialmente toxinogénicos. Dentro do grupo dos Aspergillus, o A.versicolor mostrou predominância, tendo este fungo reconhecidas capacidades de emissão de micotoxinas em ambiente de interior. A inclusão de amostras de superfície revelou-se vital para conhecer todo o espectro fúngico existente em cada um dos locais estudados, incluindo a detecção de Stachybotrys chartarum e a do fungo potencialmente queratinofílico, Chrysosporium carmichaelli. Tanto para a saúde como para a conservação, o recente estudo realizado em quatro arquivos permitiu retirar importantes conclusões e reforçar a necessidade de vigilância, sendo também útil para a definição de padrões de qualidade no campo do património cultural.