Development of biosensors for early detection of anastomotic leak and sepsis

Anastomotic leak is a catastrophic surgical complication leading to high morbidity, mortality and cancer recurrence. Currently detection is difficult, with a paucity of available diagnostic tests that have variable sensitivity and specificity. The work described in this thesis evaluated the use of local biomarkers within the anastomotic environment coupled with a biosensor application to assess proof-of-concept feasibility as a point-of-care diagnostic tool for anastomotic leak. Using a small animal model of caecal ligation and puncture to replicate abdominal sepsis, local abdominal biomarkers lactate, TNFα, and E. coli were all found to significantly increase compared to sham control models at 24 and 36 hours. Chronoamperometry and electrochemical impedance spectroscopy (EIS) interrogation of biosensors were then used to detect and quantitate levels of these respective biomarkers in real patient samples, and data compared to that obtained by existing commercial assays to evaluate accuracy. Characterisation of each biosensor utilised cyclic voltammetry, SEM, Midland blotting, SDS-PAGE and dot blotting techniques to optimise the fabrication methodology. The lactate biosensor consisted of a pre-impregnated Prussian Blue carbon electrode with lactate oxidase enzyme immobilised onto the surface via polyethyleneimine. Using chronoamperometry, the lactate biosensor gave significantly similar results to a commercial enzyme-based lactate colorimetric assay in ten abdominal fluid patient samples. E. coli immunosensors were constructed using a polytyramine matrix onto which half polyclonal antibody fragments raised against multiple strains of E. coli were immobilised. EIS was used to measure the charge transfer resistance of the biosensors when incubated with a varying concentration of E. coli, with a limit of detection found to be 104 cells ml-1. EIS of E. coli biosensors in the ten patient samples showed statistically significant equivalent results to those from flow cytometry. Immunosensors to TNFα were constructed using a similar methodology to E. coli, with whole antibody to TNFα immobilised onto a polytyramine electrode surface. Initial EIS results in buffered solution showed good biosensor response to varying concentrations of TNFα, but further studies are required for complete biosensor development

Políticas de Copyright de Publicações Científicas em Repositórios Institucionais: O Caso do INESC TEC

A progressiva transformação das práticas científicas, impulsionada pelo desenvolvimento das novas Tecnologias de Informação e Comunicação (TIC), têm possibilitado aumentar o acesso à informação, caminhando gradualmente para uma abertura do ciclo de pesquisa. Isto permitirá resolver a longo prazo uma adversidade que se tem colocado aos investigadores, que passa pela existência de barreiras que limitam as condições de acesso, sejam estas geográficas ou financeiras. Apesar da produção científica ser dominada, maioritariamente, por grandes editoras comerciais, estando sujeita às regras por estas impostas, o Movimento do Acesso Aberto cuja primeira declaração pública, a Declaração de Budapeste (BOAI), é de 2002, vem propor alterações significativas que beneficiam os autores e os leitores. Este Movimento vem a ganhar importância em Portugal desde 2003, com a constituição do primeiro repositório institucional a nível nacional. Os repositórios institucionais surgiram como uma ferramenta de divulgação da produção científica de uma instituição, com o intuito de permitir abrir aos resultados da investigação, quer antes da publicação e do próprio processo de arbitragem (preprint), quer depois (postprint), e, consequentemente, aumentar a visibilidade do trabalho desenvolvido por um investigador e a respetiva instituição. O estudo apresentado, que passou por uma análise das políticas de copyright das publicações científicas mais relevantes do INESC TEC, permitiu não só perceber que as editoras adotam cada vez mais políticas que possibilitam o auto-arquivo das publicações em repositórios institucionais, como também que existe todo um trabalho de sensibilização a percorrer, não só para os investigadores, como para a instituição e toda a sociedade. A produção de um conjunto de recomendações, que passam pela implementação de uma política institucional que incentive o auto-arquivo das publicações desenvolvidas no âmbito institucional no repositório, serve como mote para uma maior valorização da produção científica do INESC TEC.The progressive transformation of scientific practices, driven by the development of new Information and Communication Technologies (ICT), which made it possible to increase access to information, gradually moving towards an opening of the research cycle. This opening makes it possible to resolve, in the long term, the adversity that has been placed on researchers, which involves the existence of barriers that limit access conditions, whether geographical or financial. Although large commercial publishers predominantly dominate scientific production and subject it to the rules imposed by them, the Open Access movement whose first public declaration, the Budapest Declaration (BOAI), was in 2002, proposes significant changes that benefit the authors and the readers. This Movement has gained importance in Portugal since 2003, with the constitution of the first institutional repository at the national level. Institutional repositories have emerged as a tool for disseminating the scientific production of an institution to open the results of the research, both before publication and the preprint process and postprint, increase the visibility of work done by an investigator and his or her institution. The present study, which underwent an analysis of the copyright policies of INESC TEC most relevant scientific publications, allowed not only to realize that publishers are increasingly adopting policies that make it possible to self-archive publications in institutional repositories, all the work of raising awareness, not only for researchers but also for the institution and the whole society. The production of a set of recommendations, which go through the implementation of an institutional policy that encourages the self-archiving of the publications developed in the institutional scope in the repository, serves as a motto for a greater appreciation of the scientific production of INESC TEC

Diversité des vésicules extracellulaires dans le lait bovin et leurs activités dans les maladies inflammatoires de l'intestin

Les vésicules extracellulaires (VE) sont des « fragments » de cellules activement libérés dans tous les fluides biologiques. Elles transitent dans la circulation corporelle et transmettent leur contenu bioactif à d’autres cellules. Le lait est le fluide qui contient le plus de VE et celles-ci encapsulent plusieurs éléments bioactifs qui ont des effets anticancéreux, anti-inflammatoire et diminuent notamment les symptômes de la polyarthrite rhumatoïde in vivo. Durant ma thèse j’ai exploré la diversité des VE présentes dans le lait bovin commercial et j’ai étudié leurs activités biologiques dans le cadre des maladies inflammatoires chroniques de l’intestin (MICI). Les résultats que j’ai obtenus démontrent l’existence de plusieurs populations de VE dans le lait bovin commercial que j’ai pu discriminer grâce à l’utilisation du citrate de sodium pour leur isolation. J’ai découvert qu’elles étaient capables de survivre lors de la digestion in vitro durant laquelle elles protègent leur contenu bioactif, notamment des microARN. Après avoir décrit en détail les différents microARN et protéines encapsulées dans ces VE, j’ai pu trouver des marqueurs spécifiques pour certains sous-ensembles de VE du lait. J’ai aussi montré le transfert de miR-223 bovin à des cellules humaines in vitro et son activité biologique sur l’expression d’un gène rapporteur. J’ai alors exploré l’activité biologique des VE du lait dans un modèle murin de colite induite par le Dextran sulfate sodium (DSS). La prise orale de VE du lait a diminué les symptômes de la maladie, restauré en partie le microbiote intestinal et rétabli la barrière digestive et les niveaux de mucines. J’ai aussi découvert que différentes populations de VE du lait ont différents effets sur l’inflammation du côlon, notamment en modulant le niveau de certains microARN impliqués dans le développement des MICI. Le lait contient donc différentes VE avec des activités biologiques différentes capables de moduler l’inflammation et le développement de pathologies digestives. L’étude des mécanismes qui sous-tendent leur bioactivité pourrait impacter la prise en charge des maladies inflammatoires, permettrait une amélioration des formulations de lait pour les nouveau-nés et serait d’importance pour la santé publique et le traitement industriel du lait commercial.Extracellular vesicles (EVs) are cellular “fragments” actively released in all biological fluids. They are transported through body circulation and transmit their bioactive content to remote recipient cells. Milk is the biological fluid most enriched in EVs and these encapsulate several bioactive elements with anti-cancer and anti-inflammatory effects and reduce the symptoms of rheumatoid arthritis in vivo. During my thesis, I explored the diversity of EVs present in commercial bovine milk and studied their biological activities in the context of inflammatory bowel diseases (IBD). The results I obtained demonstrate the existence of several EV subsets in commercial bovine milk that I could discriminate using sodium citrate for their isolation. I found these EVs can survive during in-vitro digestion and protect their bioactive content, including microRNAs. After detailing the different microRNAs and proteins encapsulated in these EVs, I found specific markers for certain populations of milk EVs. I also reported the transfer of vesicular bovine miR-223 to human cells in vitro and its biological activity on the expression of a reporter gene. I then explored the biological activities of milk EVs in a mouse model of DSS-induced colitis. The oral intake of milk EVs decreased the symptoms of the disease, restored part of the intestinal microbiota, restored the intestinal barrier and replenished mucin levels. Also, different populations of milk EVs differentially modulated inflammation in the colon, notably by regulating the level of certain IBD-associated microRNAs. Milk therefore contains different EV subsets with different biological activities capable of modulating inflammation and the development of digestive pathologies. Studying the mechanisms underlying their bioactivity could impact the management of inflammatory diseases, improve milk formulations for newborns, and be of importance to public health and industrial milk processing