Novas metodologias para a identificação de adulterações de produtos cárneos com carne de cavalo

O consumo de carne de cavalo depende, principalmente, dos hábitos alimentares das populações que habitam em algumas regiões. Regra geral, este animal é mais consumido nos países onde é produzido [1]. Ao longo dos tempos, o seu consumo tem variado amplamente de acordo com as dife-renças culturais e económicas da sociedade. Este animal fornece uma fonte, relativamente barata, de proteína ani-mal em países onde os cavalos são extensivamente utiliza-dos como animais de trabalho e transporte humano [2]. A textura da carne de cavalo é relativamente firma, e o seu sabor adocicado é-lhe atribuído pelo seu elevado teor em glicogénio, em comparação com outras espécies [1]. Esta carne contém um elevado teor em água, proteínas, ferro e vitaminas, que são solúveis em água. O seu menor teor em lípidos e elevado em fibras musculares e vitaminas insolú-veis em água torna-a diferente, em comparação com a carne bovina e suína [3]. Muitas vezes a ingestão de carne de ca-valo esteve associada a épocas de escassez de alimentos. Contudo, o consumo deste animal é relativamente popular nos países nórdicos e asiáticos, bem como na Itália [4]. Nos países membros da UE, o consumo médio por habitante é de apenas 0,4 kg por ano, mas devido a uma produção insu-ficiente a importação abrange 66,7% das necessidades do mercado [3].Os autores agradecem o apoio financeiro da Fundação para a Ciência e a Tecnologia (FCT) através do projeto PEst-C/EQB/LA0006/2013.info:eu-repo/semantics/publishedVersio

Semi-automated 18F-FDG PET segmentation methods for tumor volume determination in Non-Hodgkin lymphoma patients:a literature review, implementation and multi-threshold evaluation

In the treatment of Non-Hodgkin lymphoma (NHL), multiple therapeutic options are available. Improving outcome predictions are essential to optimize treatment. The metabolic active tumor volume (MATV) has shown to be a prognostic factor in NHL. It is usually retrieved using semi-automated thresholding methods based on standardized uptake values (SUV), calculated from 18F-Fluorodeoxyglucose Positron Emission Tomography (18F-FDG PET) images. However, there is currently no consensus method for NHL. The aim of this study was to review literature on different segmentation methods used, and to evaluate selected methods by using an in house created software tool. A software tool, MUltiple SUV Threshold (MUST)-segmenter was developed where tumor locations are identified by placing seed-points on the PET images, followed by subsequent region growing. Based on a literature review, 9 SUV thresholding methods were selected and MATVs were extracted. The MUST-segmenter was utilized in a cohort of 68 patients with NHL. Differences in MATVs were assessed with paired t-tests, and correlations and distributions figures. High variability and significant differences between the MATVs based on different segmentation methods (p < 0.05) were observed in the NHL patients. Median MATVs ranged from 35 to 211 cc. No consensus for determining MATV is available based on the literature. Using the MUST-segmenter with 9 selected SUV thresholding methods, we demonstrated a large and significant variation in MATVs. Identifying the most optimal segmentation method for patients with NHL is essential to further improve predictions of toxicity, response, and treatment outcomes, which can be facilitated by the MUST-segmenter

Desenvolvimento de metodologias de biologia molecular para a detecão de carne de cavalo (Equus caballus) em produtos cárneos

A identificação de espécies em alimentos é cada vez mais importante para avaliar a sua autenticidade, principalmente nos produtos cárneos. O consumidor tem direito a uma escolha informada, uma vez que esta pode ser um reflexo de um estilo de vida, práticas religiosas ou problemas de saúde. Assim, uma rotulagem correta e verdadeira é fundamental para manter o consumidor informado sobre a identidade e qualidade dos produtos alimentares que está a adquirir. Recentemente, surgiu um escândalo de dimensões internacionais, relacionado com a adição de carne de cavalo não declarada em alimentos processados. Contudo, a presença desta espécie não representa um risco para a saúde do consumidor, a não ser que a carne de cavalo contenha fenilbutazona. Esta droga não é permitida para o consumo humano, uma vez que pode causar doenças sanguíneas, como a anemia aplástica. Animais que forem medicados com esta droga não podem ser utilizados para consumo, uma vez que não é possível definir um limite máximo de resíduo para este medicamento veterinário. Todavia, a presença não declarada desta espécie representa uma fraude alimentar. Assim, este trabalho teve como objetivo o desenvolvimento de técnicas de PCR específicas e sensíveis para a identificação de carne de cavalo em produtos alimentares. Inicialmente foi desenvolvida a técnica de PCR qualitativa, onde foram estabelecidos limites de deteção (LOD) absolutos e relativos. A sensibilidade obtida para o LOD absoluto foi de 1pg e 10 pg para ADN de cavalo cruo e autoclavado, respetivamente. Relativamente ao LOD relativo também se alcançaram limites distintos para misturas binárias cruas e processadas, de 0,001% e 0,01%, respetivamente. Esta técnica foi aplicada a um conjunto de 77 amostras adquiridas antes e depois do “escândalo da carne de cavalo”, sendo que 2 revelaram a presença da espécie. Posteriormente, de forma a confirmar e quantificar a espécie de cavalo, recorreu-se à técnica de PCR em tempo real com o corante EvaGreen e análise de melting. Atingiu-se um limite de deteção absoluto de 0,1 pg de ADN de cavalo cruo e autoclavado, e um LOD relativo de 0,0001% de carne de cavalo em misturas cruas e processadas. Por fim, foi construído e validado o modelo normalizado de quantificação com base no método ΔCt, tendo-se passado à sua aplicação nas amostras que amplificaram positivamente a espécie de cavalo na PCR qualitativa. O método permitiu confirmar a presença de ADN de cavalo, no entanto os valores baixos encontrados indicaram que a presença de cavalo deverá ser resultado de contaminação cruzada e não de possível fraude. Pode-se concluir que neste trabalho foram desenvolvidas ferramentas úteis e eficazes para a deteção de adulterações em produtos cárneos com carne de cavalo.Nowadays, identification of species in food is of major importance to evaluate food authenticity, with special emphasis on meat products. The consumer has the right of an informed choice, which may be a reflection of lifestyles, religious practices or health problems. Therefore, a correct and truthful labelling is crucial to inform the consumer about the identity and quality of food products they are buying. Recently, associated with the undeclared addition of horse meat to processed foods, an international scandal emerged. The presence of this species does not pose a health risk to the consumer, unless the horse meat contains phenylbutazone. This drug is not allowed for human consumption since it can cause blood diseases such as aplastic anaemia. Animals that are treated with this drug cannot be used for consumption since it is not possible to set a maximum residue limit for this veterinary medicine. However, the presence of non-declared horse-species is a food fraud. This work aimed at developing sensitive and specific PCR-based methods for the identification of horse meat in food products. Initially qualitative PCR assay was developed, where absolute and relative limits of detection (LOD) were established. The sensitivity obtained for the absolute LOD was 1 pg of raw horse DNA and 10 pg of processed horse DNA. The relative sensitivity was achieved by means of raw and processed binary reference mixtures, showing LOD of 0.001 % and 0.1%, respectively. This technique has been successfully applied to a set of 77 samples, prior and after the “horse meat scandal”, which showed the presence of two positive samples to horse DNA. Subsequently, in order to confirm and quantify the presence of horse, we resorted to the technique of real-time PCR with EvaGreen dye and melting curve analysis. The technique reached a absolute LOD of 0.1 pg in raw and autoclaved horse DNA and a relative LOD 0.0001% of horse meat in raw and processed mixtures. Finally, the standard model for quantification was built based on ΔCt method, successfully validated and applied to the positive samples in qualitative PCR. However, this model can be only applied in one sample. Quantification revealed the presence of the specie at very low amount, which suggests the possibility of cross-contamination. The method allowed confirming the presence of horse DNA, though the low values indicate that its presence should have been the result of cross-contamination and not an eventual food fraud. This work allowed concluding that useful and effective tools were developed for the adulteration detection in meat products with horse meat

Target and Non-Target Approaches for Food Authenticity and Traceability

Over the last few years, the subject of food authenticity and food fraud has received increasing attention from consumers and other stakeholders, such as government agencies and policymakers, control labs, producers, industry, and the research community. Among the different approaches aiming to identify, tackle, and/or deter fraudulent practices in the agri-food sector, the development of new, fast, and accurate methodologies to evaluate food authenticity is of major importance. This book, entitled “Target and Non-Target Approaches for Food Authenticity and Traceability”, gathers original research and review papers focusing on the development and application of both targeted and non-targeted methodologies applied to verify food authenticity and traceability. The contributions regard different foods, among which some are frequently considered as the most prone to adulteration, such as olive oil, honey, meat, and fish. This book is intended for readers aiming to enrich their knowledge through reading contemporary and multidisciplinary papers on the topic of food authentication

Despiste de fraude alimentar em processados de carne por métodos biomoleculares

Mestrado em Biologia Molecular e CelularA identificação de espécies, em alimentos para consumo humano é cada vez mais importante para avaliar a sua autenticidade. Uma rotulagem correta, verdadeira e precisa é essencial para manter o consumidor informado sobre a identidade e qualidade dos produtos alimentares que pretende adquirir, especialmente nos produtos processados, onde a diferenciação das espécies utilizadas é extremamente difícil, tornando-os alvos fáceis de adulterações. A crescente demanda por transparência na indústria alimentar e a aplicação de uma correta rotulagem, proporcionaram uma força motriz para o desenvolvimento de metodologias analíticas adequadas à identificação de espécies de carne. Assim, o recurso a técnicas de biologia molecular, em especial a Reação em Cadeia da Polimerase (PCR), tem-se mostrado como uma alternativa específica, rápida, sensível e adequada para a identificação de espécies em produtos alimentares. Neste estudo, inicialmente procedeu-se à extração de ADN de diferentes espécies de carne fresca (amostras de referência) adquiridas no talho, Bos taurus (vaca), Sus scrofa (porco), Eqqus caballus (cavalo) e Ovis aries (ovelha). De seguida, as espécies em estudo foram identificadas com recurso a um protocolo de amplificação multiplex em tempo real. Posteriormente avaliou-se a autenticidade de 38 amostras comerciais de produtos cárneos, que apenas continham carne de vaca na sua rotulagem, de forma a averiguar a veracidade e rigor da rotulagem em relação ao conteúdo do produto. De todas as amostras comerciais analisadas, nenhuma revelou a presença de qualquer outra das espécies em estudo, para além da mencionada no rótulo de cada uma, pelo que não foi necessário proceder-se à posterior quantificação da mesma. Assim, através da utilização de metodologias de biologia molecular, podemos concluir que a rotulagem está em conformidade com o conteúdo do produto alimentar.In food the identification of species for human consumption is increasingly important to evaluate it authenticity. A correct truthful and accurate labeling is essential to keep the consumer informed about the identity and quality of the food products that he intends to acquire, especially in processed products, where species differentiation is extremely difficult, making them easy targets for adulteration. A growing demand of the transparency on food industry and an application of a correct labeling, have provided a driving force for the development of analytical methodologies suitable for the identification of meat species. Thus, the use of molecular biology techniques, in particular Polymerase Chain Reaction (PCR), is a specific, fast, sensitive and adequate alternative for the identification of species in food products. In this study, DNA from different fresh meat species (reference samples) acquired in the butcher's shop, Bos taurus (cow), Sus scrofa (pig), Eqqus caballus (horse) and Ovis aries (sheep) were initially extracted. Next, the species under study were identified using a real-time multiplex amplification protocol. Subsequently, the authenticity of 38 commercial samples of meat products containing only beef in their labeling was assessed to ascertain the veracity and accuracy of the labeling in relation to the product content. Of all the commercial samples analyzed, none revealed the presence of any of the other species under study, in addition to the one mentioned in the label of each one, reason why it was not necessary to be quantified later. Thus, through the use of molecular biology methodologies, we can conclude that the labeling is in conformity with the content of the food product

Challenges and opportunities: adjustment to life post transplant for adults with cystic fibrosis and the impact on their professional support needs.

Lung transplant can improve both quality and quantity of life for a person with Cystic Fibrosis (CF) at end stage respiratory disease. However, life post transplant can be challenging both medically and psychologically due to the need to adjust to a significantly changed health status, as well as understand and manage the side effects and medical complications of transplant. This study questioned, whether from a service perspective, the support needs of adults with CF changes with transplant and how a specialist CF centre should accommodate this. In order to do this, a more detailed understanding of the experiences of a post transplant group was sought utilising qualitative methodology. Eleven participants or sixty-five percent of adults with CF post transplant who attend one of Scotland's largest specialist CF centres participated in semi-structured interviews. Framework analysis was chosen as the method of analysis due to its relevance in a health care setting. A framework was generated consisting of four broad areas of post transplant adjustment: Recovering; Adjusting and realising; Redefining and pursuing and; Managing the issues of post transplant life. Each area has activities and key factors which provide more information about post transplant adjustment life as well as factors that may account for individual differences. In general, and in the absence of medical complications, participants adjusted to transplant with the support of partners, families and local CF and transplant services. They did not indicate the need for dedicated post transplant services in their local CF centre, but found communications between service providers to be inefficient. When faced with medical complications especially rejection, participants reported needing more psychosocial support. Recommendations include an increased awareness of the processes of psychosocial adjustment post transplant for health professionals, psychological intervention at times of crisis and more efficient communication between transplant and local CF services.sub_psunpub1811_ethesesunpu

Isolation and characterisation of starch biosynthesis genes from cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz) is a tropical crop grown for its starchy thickened roots, mainly by peasant farmers, in the tropics, for whom it is a staple food. There is an increasing demand for the use of cassava in processed food and feed products, and in the paper and textile industries amongst others. This thesis describes research on the cloning of the genes encoding ADP-glucose pyrophosphorylase small and large subunits (AGPase B and S, respectively) and granule bound starch synthase II (GBSSII). These genes and their products were extensively characterised to determine their role in starch biosynthesis in cassava. Functional verification of the genes was carried out by transforming potato and cassava followed by analysis of the starch produced by the transgenic plants.In Chapter 1 cassava production in the world in general and in Zimbabwe in particular is examined against the backdrop of new cloning and transformation strategies to improve starch quality and quantity. The development of cassava cultivars whose starches have novel physico-chemical properties by genetic modification of the process of starch biosynthesis is examined therein. The main criteria for these new cultivars to emerge are set forth as being: the availability of cloned and characterised starch biosynthesis genes, a universally applicable transformation and regeneration procedure for cassava, transfer to appropriate cassava cultivars, and biosafety analysis of transgenic cassava plants before disbursement to farmers.The cloning of the cassava starch biosynthesis genes encoding granule bound starch synthase II (GBSSII) and the large and small subunits of ADP-glucose pyrophosphorylase (AGPase) is described in Chapters 2 and 3. The cloning of GBSSII reveals that there is indeed a second isoform of this enzyme in cassava as in other plants species. While sharing very little amino acid sequence homology with cassava GBSSI the GBSSII isophorm shares high amino acid sequence homology to other GBSSII genes from pea and potato. Cassava GBSSII seems to be more important in leaf tissue where it is more highly expressed than in tuber tissue where GBSSI predominates. Mapping of GBSSII revealed that this is a single copy gene located on the male derived linkage group T of the cassava mapping population.Cloning of the cassava genes coding for the small (B) and large subunit (S) of AGPase revealed interesting aspects about the cassava enzyme. The cassava AGPase is likely to be heterotetrameric in constitution as had been found in other plant species. Comparison of the cassava AGPase sequences with those of already cloned AGPases revealed that AGPase B is more similar to small subunit genes from other plants than to cassava AGPase S coding for the large subunit (Chapter 3). Segregation analysis of a cassava mapping population revealed that AGPase S is a single copy gene that is localised on the female derived linkage group E of the cassava genetic map. Both genes are expressed in all cassava tissues but AGPase B was shown to have a higher steady state mRNA level than AGPase S especially in leaf and tuber tissue. Post-transcriptional control of small subunit polypeptide levels could be inferred from the discrepancy between AGPase B mRNA and polypeptide levels. The AGPase enzyme activity was much higher in young cassava leaves than older leaves and tubers. Cassava leaf AGPase activity was increased 3 fold by the addition of 3-PGA (3-phospho-glycerate) and inhibited by up to 90% in the presence of inorganic phosphate (Pi). The tuber enzyme was relatively unaffected by 3PGA, but was highly inhibited by Pi.In order to verify the biological role of the AGPase B gene antisense constructs were made of the cassava AGPase B behind a CaMV35S promoter (chapter 3). This was transferred into potato plants by Agrobacterium tumefaciens. While the 224 transgenic antisense AGPase B potato plants did not differ in appearance from normal potato plants, 45 transgenic plants, however, had more numerous and smaller tubers than control plants. Antisense plants with reduced AGPase B mRNA levels had 1.5 to 3 times less starch than tubers from the control plants. The levels of the soluble sugars in the antisense plants increased significantly (up to 10 times more glucose, 6 times the amount of fructose, and 5 times the amount of sucrose) when compared to those found in control plants. These results show that a heterologous gene from cassava can have an antisense effect in potato, but that the number of plants required to find plants exhibiting maximum antisense effect has to be very large. This is probably due to sequence homology differences between the cassava AGPase B and potato AGPase B genes which share only 68% amino acid sequence homology.Chapter 5 describes the further development of an efficient, time and labour saving protocol for transforming cassava based on stringent selection of the luciferase (firefly) marker gene. In addition the first reported transformation of cassava with a gene (AGPase B) other than a marker gene is described. An antisense construct was made for transforming cassava. This consisted of the cassava AGPase B gene which was placed in antisense orientation behind the CaMV35S promoter. This was then coupled to the luciferase gene driven by another CaMV35S promoter. After particle bombardment of cassava FEC transgenic tissue was selected using three different selection regimes: non stringent luciferase selection, stringent luciferase selection and combined chemical (phosphinothrycin) and luciferase selection. Stringent luciferase selection whereby luciferase positive FEC units were precisely pinpointed, isolated and cultured was found to be the most effective and time saving method. It was possible to generate cultures having more than 90% luciferase positive FEC tissue after 12 weeks of stringent LUC selection, compared to 45% and Cassava plants carrying the AGPase B antisense gene had extremely low levels of starch, compared to control plants, as shown by iodine staining of in vitro induced thick stems. In plants exhibiting the highest AGPase B antisense effect, starch formation was limited only to the epidermal layer. These results functionally confirm the identity of cassava AGPase B as well as emphasising the critical role of AGPase in starch formation in cassava.A discussion about the significance and implications of cloning cassava genes and producing transgenic cassava for culture in developing countries is carried out in Chapter 6. While there are clearly many economic and nutritional benefits to producing transgenic cassava, for resource poor farmers, many people in the South are not aware of the biosafety implications of growing transgenic crops. It is further emphasised that discussions and debate should be initiated to make local communities aware of the issues surrounding transgenic crops and their products. In addition it is recommended that some form of international legal framework be set up to ensure that resource poor farmers are not disadvantaged by the patenting of material originating from their communities by individuals and companies in the North. This thesis clearly demonstrates how it will be possible in the near future to produce new cassava cultivars carrying the appropriate genes to affect pronounced changes on tuber productivity and starch quality