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The purpose of this study was to investigate the effects of electrical and chemical stimulation of the sub-lobule IX-b of the cerebellar uvula on the cardiorespiratory responses evoked on stimulation of the carotid chemoreceptors in the anaesthetised and paralysed rabbit. Recordings of arterial blood pressure, ECG, heart rate and phrenic nerve activity were evaluated. Sub-lobule IX-b was activated electrically (1 ms, 20 μA, 100 Hz, 4-s train) and stimulated chemically with sodium glutamate microinjections (2 mM, pH=7.4±0.1) using bipolar concentric or multibarrelled microelectrodes. Carotid body chemoreflexes were evoked by the retrograde injection of sodium cyanide (0.1%, 0.1 ml) into the blood supply of the carotid body through a cannula inserted in the external carotid artery. The interaction of uvula stimulation and the carotid chemoreceptor reflex was studied by following the chemoreceptor stimulus with a stimulus to the uvula. Electrical and chemical stimulation of the uvula evoked a characteristic bradycardia and a depressor response together with a decrease in phrenic nerve activity. Carotid body stimulation evoked a decrease in heart rate accompanied by an increase of both arterial blood pressure and phrenic nerve activity. However, simultaneous delivery of the two stimuli resulted in a depressor response together with a decrease of phrenic nerve activity and an increased bradycardia. These data suggest that stimulation of the cerebellar uvula exerts an inhibitory control of the chemoreflex efficacy as far as respiratory activity and blood pressure is concerned.

Utilizando uma metodologia não-obstrusiva, testámos a hipótese de que a associação entre estímulos visuais não salientes e a pertença grupal dos indivíduos pode interferir na escolha de uma equipa de trabalho sem que estes se apercebam dessa influência. As participantes (66 estudantes dos cursos de Psicologia e de Serviço Social) foram acolhidas por uma Psicóloga vs. Trabalhadora Social após o que deveriam escolher entre duas equipas, fisicamente semelhante vs. diferente da pessoa que as recebeu, para a continuação do mesmo estudo. Os resultados mostraram que as participantes que foram acolhidas pelo membro do exogrupo escolheram mais a equipa diferente do que as participantes acolhidas pelo membro do endogrupo sendo este efeito mediado pela identificação ao endogrupo. Os resultados são discutidos à luz de investigação anterior utilizando medidas implícitas de discriminação social. ABSTRACT: Using a non-obstrusive measure, we tested the hypothesis that the association between non-salient visual stimuli and group membership can affect the choice of a working team beyond participants’ awareness of that influence. Participants (66 Psychology and Social Service students) were received by a Psychologist vs. Social Worker. They then had to choose between two teams, physically similar to vs. different from the person who had just received them, to work with in the second phase. Results show that most participants who had been received by an out-group member chose the different team more than did participants who had been received by an in-group member. This effect was mediated by in-group identification. Results are discussed based on previous research using implicit measures of social discrimination. info:eu-repo/semantics/publishedVersion

The effect on bladder function and arterial blood pressure of adenosine-5'-triphosphate (ATP) and its synthetic analogue, alpha,beta-methylene ATP (alpha,beta-meATP) applied by microinjection to brainstem areas was assessed in the anaesthetised, paralysed and artificially ventilated female rat. Recordings of bladder pressure, changes in the pelvic nerve activity, arterial blood pressure and heart rate were evaluated. The purinergic drugs were microinjected into two brainstem areas the periaqueductal grey matter (PAG) and the area of the Barrington nucleus/locus coeruleus (LC) - only after electrical stimulation (50 Hz, 1 ms, 30-50 microA; n(PAG) = 17; n(LC) = 18) and the microinjection of glutamate (2 mM, pH 7.4+/-0.1; n(PAG) = 16; n(LC) = 16) had shown increases of bladder pressure and/or rate of bladder contractions and/or pelvic nerve activity at specific sites. Electrical and glutamate activation of PAG evoked an increase of arterial blood pressure. Microinjections of ATP (20 mM, pH 7.4+/-0.1; n(PAG) = 11; n(LC) = 11) and alpha,beta-meATP (2 mM, pH 7.4+/-0.1; n(PAG) = 10; n(LC) = 9) both evoked consistent increases of bladder pressure and/or pelvic nerve activity. Stimulation with ATP elicited a biphasic change of arterial blood pressure characterised by an increase followed by a decrease which was accompanied by a rise of heart rate. Microinjection of alpha,beta-meATP into PAG did not elicit a consistent response: a decrease of arterial blood pressure was evoked in five rats, while in two other rats an increase occurred. Electrical stimulation and glutamate activation of Barrington's nucleus/LC evoked an increase of arterial blood pressure, but a decrease was observed after microinjection of both ATP and alpha,beta-meATP. At some sites (n = 8) the effect of alpha,beta-meATP after a pre-injection at the same site of the P2 purino receptor antagonist, suramin (20 mM, pH 7.4+/-0.1) was smaller than the control. At three sites within PAG and two within LC located more medially to sites where an excitatory response had been observed, electrical stimulation evoked a small decrease or no change in bladder pressure. Following the stimulus, a rise in bladder pressure was preceded by an increase of pelvic nerve activity. A similar effect of glutamate was observed in one case. These data suggest that activation of P2 purine receptors in both PAG and Barrington's nucleus/LC is implicated in the neuronal mechanisms that generate patterns of activity in the parasympathetic innervation of the bladder and that purines also act at this level to modify sympathetic outflow to the cardiovascular system.

Dissertação de Mestrado em Etologia A importância da experiência precoce no desenvolvimento das preferências sociais constitui um tema central no estudo do comportamento animal. No entanto, apesar dos extensos estudos realizados, o nosso conhecimento dos detalhes do processo de vinculação (ou attachment) continua bastante incompleto em muitas espécies animais, entre as quais o cão. Com este estudo pretendeu-se contribuir para aprofundar o conhecimento de aspectos quantitativos e qualitativos do processo de vinculação no cão de gado - cães utilizados para proteger o gado dos ataques dos predadores e que não exibem comportamentos predatórios para com os animais domésticos que devem proteger - focando em particular a hipótese da neotenia como um mecanismo de diferenciação rácica. Para tal, foram utilizados cachorros pertencentes a duas raças caninas nacionais - Rafeiro do Alentejo e Cão da Serra de Aires -, que foram mantidos em situações sociais semelhantes até ao fim do período sensível de vinculação, sendo depois colocados em contacto físico directo com um parceiro social (cão ou ovino) da mesma idade, durante um período de tempo igual ao da primeira fase. Após esse período, todos os indivíduos foram sujeitos a um conjunto de testes comportamentais (de preferência social e de isolamento social) por forma a se verificar a existência de um vínculo social com os novos parceiros sociais. Para alguns dos animais envolvidos no estudo, foi igualmente realizada uma segunda experiência de reversão das preferências sociais. Estes testes permitiram verificar a existência de diferenças mais relevantes entre as duas raças de cães que entre as duas espécies estudadas e uma elevada variabilidade individual na raça Rafeiro do Alentejo. Foi confirmada a possibilidade de alterar a vinculação social inicial após um período prolongado de contacto físico directo com um novo parceiro social, a um nível intraespecífico para os cães (mas apenas para a raça Cão da Serra de Aires) e interespecífico para os ovinos. Para os ovinos foi confirmada a reversão da socialização para a espécie inicial de vinculação, num período de tempo muito curto (12 dias), que poderá ter sido facilitada pela socialização intraespecífica inicial. No Rafeiro do Alentejo parece verificar-se um desenvolvimento retardado dos comportamentos que impedem a socialização, permitindo a manutenção da curiosidade social (característica de animais muito jovens), e promovendo, assim, o estabelecimento de novos vínculos sociais. Esta interpretação dos dados parece indicar um grau acentuado de neotenia comportamental nesta raça, apoiando a hipótese da neotenia para a explicação da variação fenotípica no cão, baseada em alterações heterocrónicas durante a filogenia que conduzem ao retardamento do desenvolvimento e à retenção de características ancestrais juvenis no indivíduo adulto de uma espécie. Ao contrário do verificado noutros estudos, não se verificou uma correlação entre o número total e o tipo de vocalizações e o grau de actividade dos cachorros Rafeiro do Alentejo durante o isolamento social. Verificou-se, sim, uma elevada variabilidade interindividual na emissão de vocalizações de perturbação. Os resultados obtidos para os ovinos parecem apoiar o modelo de exclusão competitiva, proposto por Bateson (1981) para explicar o controlo da sensibilidade a estímulos externos durante os períodos sensíveis de vinculação, em detrimento do modelo do relógio interno. Segundo aquele modelo, o desenvolvimento comportamental é influenciado pela acumulação de experiências e não está dependente da regulação por um relógio fisiológico interno. No que diz respeito aos cães, não foi possível tirar conclusões válidas quanto aos modelos existentes, sendo proposto um modelo alternativo semelhante ao da validação funcional proposto por Hale (1969). Toma-se, no entanto, necessária a replicação das experiências com a mesma e outras raças de cães de gado, em virtude da reduzida dimensão da amostra, bem como considerar a realização de outro tipo de testes comportamentais, integrando aspectos neuroendócrinos e fisiológicos, que poderão traduzir de forma mais fiável a existência de vinculação social nos cães de gado.

Recently, an association between atrial fibrillation (AF) and a local increase in sarcoplasmic calcium release was suggested as a mechanism able to generate afterdepolarizations and promote arrhythmias recurrence. The adenosine A2A receptors (A2A) are expressed in the atria, modulate the frequency of spontaneous calcium release, and may be involved in the remodeling process during AF. Aim: To investigate the role of atrial adenosine receptors by analyzing A1 and A2A receptor protein levels through changes in their transcriptional control during atrial high-rate. Methods: The high-right atrium of anesthetized, artificially ventilated rats (n=10; age 12 weeks) was paced epicardially for 2 hours (50Hz). Animals were sacrificed and separate samples of the atria collected for A1 and A2A mRNA Real Time PCR quantification. The housekeeping gene 18SrRNA was used as internal control for the quantification of receptors mRNA. A sham group was used as control (matching number, age and sex). CT values were calculated for each sample, and converted into relative quantities with the ΔCTmethod (Eq.1: ΔCT=CTAdora-CT18SrRNA; Eq.2: ΔΔCT=ΔCTcontrols- ΔCTStimulation). The effects of high-rate pacing were evaluated by one way analysis of variance (significance if P

Na sociedade actual, em que a beleza e juventude são sinónimos de sucesso, são cada vez mais fre- quentes as queixas “estéticas” centradas nas imperfeições da pele e do corpo. Os Dermatologistas são muitas vezes procurados para avaliação e tratamento dessas queixas. A Perturbação Dismórfica Corporal (PDC), inicialmente de- nominada “dismorfofobia”, é uma patologia psiquiátrica frequente e incapacitante, caracterizada pela preocupação excessiva com um determinado defeito físico, real ou inexistente, causando angústia e disfunção nos planos social, afectivo e laboral. A ansiedade associada ao “defeito” estético compele os indivíduos com PDC a comportamentos do tipo compulsivo com o objectivo de o camuflar, evidenciando-se comportamentos como a compra compulsiva de lenços e chapéus, utilização de produtos de maquilhagem, manipulação da pele (skin picking), verificação ao espelho e procura de intervenções correctivas em consultas de Dermatologia, Cirurgia Plástica e Medicina Dentária (“doctor shop”). Os doentes com PDC, não reconhecendo o defeito como mínimo ou inexistente, procuram trata- mentos estéticos para uma patologia de natureza psiquiátrica. A prevalência da PDC na população geral é de 1 a 2% e na população de doentes dermatológicos é de 2,9 a 29,4%. Considerando a alta prevalência de PDC naquela população, e tendo em conta que os tratamentos estéticos raramente melhoram os sintomas, a sensibilização e for- mação dos Dermatologistas para a investigação sistemática, diagnóstico e orientação para consulta de Psiquiatria torna-se fundamental.PALAVRAS-CHAVE – Perturbação Dismórfica Corporal; Imagem Corporal; Cirurgia Plástica.

Primary and secondary autonomic nervous system (ANS) disorders often have a severe adverse effect on the quality of life of patients. Diagnostics for ANS disorders are under represented, despite their common occurrence. Precise history taking is of key importance for ANS evaluation: it may help to rule out differential diagnoses and provide important clues to the underlying ANS disorder. In fact, in conjunction with additional bedside tests, it can achieve a clear diagnosis. The analysis of heart rate variability and the results of the standardised tests that make up the Ewing battery are important means of evaluating the parasympathetic and sympathetic nervous system. In addition, sudomotor testing can be used to evaluate cholinergic sympathetic function, and the spontaneous baroreceptor reflex can be assessed using new computerised techniques. These tests provide valuable information on cardiovascular autonomic control. This paper presents a structured review of current standard techniques for diagnosing ANS disorders.

s of the European Congress on Biotechnology, 14, Barcelona, Spain, 2009

O Centro de Crise tem como objectivo prestar apoio psicológico, social, jurídico e médico a mulheres vítimas de violação e/ou espancamento, de forma a permitir a sua reabilitação tanto psicológica como social, proporcionando a reconstrução das suas vidas. Visa-se paralelamente conseguir um processo de sensibilização e informação que facilite uma evolução das mentalidades e permita a denúncia destas formas de violência sobre a mulher. ABSTRACT: The Crisis Center has the main goal of providing psychological, social, legal and medica1 support to victims of rape and/or battering, therefore allowing their psychological and social rehabilitation, as well as the reconstruction of their lives. It’s also an important goal to achieve a process of information and conciousness that promotes an evolution of mentalities and allows public accusation of these forms of violence against women.

Background Escherichia coli has been the organism of choice for the production of many recombinant proteins with high therapeutic value. However, while the research on molecular biology has allowed the development of very strong promoters, there are still some phenomena associated with this process that hamper the full use of those technologies like the so-called stringent response, caused by an imbalance in intracellular amino acid uptake and the endogenous amino acid synthesis rates. It occurs due to high demands of certain amino acids for recombinant protein production [1], causing several cellular responses, from the inhibition of the synthesis of rRNA to the induction of proteases. The main consequence of this response is a decrease in process productivity due to the lower specific growth and production rates observed.

In metabolic engineering it is difficult to identify which set of genetic manipulations will result in a microbial strain that achieves a desired production goal, due to the complexity of the metabolic and regulatory cellular networks and to the lack of appropriate modeling and optimization tools. In this work, Evolutionary Algorithms (EAs) are proposed for the optimization of the set of gene deletions to apply to a microorganism, in order to maximize a given objective function. Each mutant strain is evaluated by resorting to the simulation of its phenotype using the Flux-Balance Analysis approach, together with the premise that microorganisms have maximized their growth along natural evolution. A new set based representation is used in the EAs, using variable size chromosomes, allowing for the automatic discovery of the optimal number of gene deletions. This approach was compared with a traditional binary-based Genetic Algorithm. Two case studies are presented considering the production of succinic and lactic acid as the target, with the bacterium E. coli. The variable size EAs, outperformed the other approaches tested, allowing to reach good results regarding the production of the desired compounds, and additionally presenting low variability among the several runs., Fundação para a Ciência e a Tecnologia (FCT); FEDER

In this work, Evolutionary Algorithms (EAs) are used to achieve optimal feedforward control in a recombinant bacterial fed-batch fermentation process, that aims at producing a bio-pharmaceutical product. Three different aspects are the target of the optimization procedure: the feeding trajectory (the amount of substrate introduced in a bioreactor per time unit), the duration of the fermentation and the initial conditions of the process. A novel EA with variable size chromosomes and using real-valued representations is proposed that is capable of simultaneously optimizing the aforementioned aspects. Outstanding productivity levels were achieved and the results are validated by practice.

In this paper a detailed mathematical model is presented for the fermentative production of fructo-oligosaccharides with Aspergillus sp. The model accounts for hydrolysis and transfructolization reactions, as well as biomass formation and it contains 27 parameters that were determined from experimental data using a System Biology toolbox with the Simulated Annealing method for curve fitting. Several additional experiments were performed in bioreactors where the time variation of 7 state variables (Sucrose, Glucose, Fructose, 1-Kestose, Nystose, 1-fructosyl nystose and Biomass) was measured. Experimental data were compared with results from simulations using the estimated parameters and it was verified that the model can predict the FOS production profile. The good agreement between simulated and experimental data was verified by calculating the relative percentage deviation modulus, which was lower than 10% for all cases except one. The derived and validated model can be used for process optimization, for example for indicating which fed-batch strategy could be used to improve the production of FOS while minimizing glucose concentration.

Dissertação de Mestrado Integrado em Medicina Veterinária, Ciências Veterinárias O cancro da mama é o tipo de neoplasia mais frequentemente diagnosticada na cadela e na mulher e alvo de intensa pesquisa. A utilização de animais de laboratório é, sem dúvida, uma mais-valia para o seu estudo. Os objetivos deste trabalho foram: induzir quimicamente tumores da mama em ratos fêmea da estirpe Sprague-Dawley com 50 dias de idade; acompanhar a evolução dos tumores mamários; avaliar, ultrassonograficamente, o aspeto e vascularização dos tumores, utilizando vários modos de ultrassonografia: modos B, Power Doppler, B Flow e Contraste; determinar as dimensões e volume dos tumores mamários e associar a vascularização ultrassonográfica com a classificação histológica dos tumores. Neste estudo foram utilizados 25 ratos fêmea da estirpe Sprague-Dawley adquiridos à empresa Harlan. Os animais foram aleatoriamente divididos em dois grupos: Grupo I (n=15) e Grupo II (n=10). Aos animais do Grupo I foi administrado o agente carcinogénico N-metil-N-nitrosureia (MNU) por via intraperitoneal aos 50 dias de idade. Os animais, a água e comida foram pesados semanalmente. Os animais foram acompanhados diariamente para avaliação do seu estado geral e foram palpados semanalmente para identificação e registo da localização dos tumores mamários. Os tumores foram monitorizados semanalmente por ultrassonografia, através da utilização dos modos B, Power Doppler e B Flow. No final do protocolo experimental, os tumores foram avaliados pelo modo Contraste e pelos modos de ultrassonografia anteriormente descritos e medidos por caliper e ultrassonografia. O volume tumoral foi calculado de acordo com fórmulas previamente publicadas e por duas fórmulas propostas por nós. O primeiro tumor apareceu dez semanas após a administração da MNU. No final do protocolo experimental foram identificados 28 tumores mamários. O número de tumores da cadeia mamária direita foi sempre superior ao número de tumores da cadeia mamária esquerda. No par três das glândulas mamárias inguinais não se desenvolveu qualquer tumor ao longo do protocolo experimental. A diferença do número de pixéis coloridos detetados pelos modos Power Doppler e B Flow foi estatisticamente significativa (p

Despite some progress over the last years, the development of optimized yeast strains for the production of chemicals and other materials is still a costly and time-consuming process. The availability of suitable chassis cells, pre-optimized for the overproduction of different compounds, can contribute to reduce this cost/time burden. Among several eukaryotic organisms that can be exploited in industrial biotechnology, Saccharomyces cerevisiae is uniquely positioned as a well-established, robust and scalable industrial production platform for a large portfolio of products, due to its high tolerance to harsh industrial conditions and available knowledge. Following this rationale, we herein propose a conceptual framework for the design of platform S. cerevisiae strains towards enhanced production of families of industrially relevant compounds, through the identification of gene deletions to improve the production of the desired targets. The framework includes two main stages: i) Optimization stage, where an OptGene-based [1, 2] multi-objective metaheuristic approach is applied for all targets in a given family and ii) Chassis analysis, where the occurrence of each gene in each of the targeted metabolites solution set is accounted for. Candidate genes for deletion are sorted based on a computed score that favors genes occurring for multiple targets. Top scoring genes are then selected for chassis generation. Afterwards, all the solutions are organized in groups with common chassis. Case studies include the production of metabolites that have found widespread applications in the food, chemical and pharmaceutical industries, namely C4-dicarboxylic acids derived from the TCA cycle (succinate, fumarate and L-malate) and a set of heterologous products derived from the shikimate pathway. Several chassis strains containing a common backbone of non-intuitive gene deletions to the selected targeted metabolites, along with additional genetic targets to guarantee the desired phenotype, were generated and characterized in terms of their biological feasibility. Candidate solutions were ranked based on computed metrics resembling productivity and number of associated gene knockouts. Although experimental validation is still lacking, there is support in the literature for some of the proposed candidate strategies. This work establishes a proof-of-concept showing that it is possible to generate pre-optimized platform strains for enhanced production of different metabolites derived from the same metabolic pathways. Furthermore, we foresee that this model-driven modular design concept may constitute an important step towards realizing the full potential for economical and sustainable production of other families of industrially relevant chemicals., (undefined)

The arterial baroreceptor reflex is an important determinant of autonomic control of cardiovascular system for homeostasis. The present work proposes a wavelet analysis for monitoring baroreflex behavior using wavelets analysis of short time rats R-R and systolic arterial pressure signals (SAP). The instantaneous power of decomposed low-frequency (LF) and high-frequency components was used for a time-variant spectral analysis. Our results show that fast events and changes in autonomic modulation were detected with high temporal resolution. The methods applied yielded the same values of baroreceptor sensitivity as the baroreflex gain by phenylphrine testing. The results indicate that wavelet analysis can be used as a non-invasive method for dynamic assessment of baroreflex response and may be a useful tool in exploring fast autonomic control of the cardiovascular system.

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In recent years a remarkable effort has been made in the development of new sensors and process analytical technology. However, it is still difficult to find reliable and low cost commercial sensors for on-line measurements of important variables. Therefore, considerable attention has been focused on the development of on-line software sensors. Nevertheless, the application of those algorithms to complex biological processes is still very incipient. In this work two different state estimators have been studied regarding their applicability to the recombinant Escherichia coli fed-batch fermentation. Both algorithms showed the ability to estimate on-line biomass and acetate concentrations. However, the extended Kalman observer exhibited a better convergence in spite of being less flexible regarding the combination of the measured and estimated variables., PRODEP ; Fundação para a Ciência e a Tecnologia (FCT)

Although important contributions have been made in recent years within the field of bioprocess model development and validation, in many cases the utility of even relatively good models for process optimization with current state-of-the-art algorithms (mostly offline approaches) is quite low. The main cause for this is that open-loop fermentations do not compensate for the differences observed between model predictions and real variables, whose consequences can lead to quite undesirable consequences. In this work, the performance of two different algorithms belonging to the main groups of Evolutionary Algorithms (EA) and Differential Evolution (DE) is compared in the task of online optimisation of fed-batch fermentation processes. The proposed approach enables to obtain results close to the ones predicted initially by the mathematical models of the process, deals well with the noise in state variables and exhibits properties of graceful degradation. When comparing the optimization algorithms, the DE seems the best alternative, but its superiority seems to decrease when noisier settings are considered., This work was supported by the Portuguese Foundation for Science and Technology under project POSC/EIA/59899/2004, partially funded by FEDER.

The Systems Biology approach has been replacing the reductionist view that dominated biology research in the last decades. Present biochemical knowledge and genomic databases allowed the development of metabolic models for several organisms, which, however, are still incomplete. The availability of the genome sequence of H. pylori has allowed the construction of a genome-scale metabolic model for this organism. The purposes of this work were to study the growth of H. pylori in a chemically defined medium, to compare the experimental data obtained with the simulated data supplied by the model and analyse the composition of the in silico media used. Cultures were grown at 37ºC under microaerophilic conditions in Ham´s F-12 medium supplemented with fetal bovine serum. Optical density and the counting of CFU/mL were performed for assessing the growth. OptFlux, a software platform for metabolic engineering, which includes several tools such as flux balance analysis (FBA) was employed for simulate the behavior of wild type H. pylori under the conditions used in vivo. The simultaneous use of both approaches allows to correct the in silico model, and on the other hand, to rationally adjust the medium components present in F-12. For instance pimelate, that has been considered to be essential in the latest metabolic model, is lacking in F-12 and is likely to be redundant in the model. Our future work is not only to improve the genome-scale metabolic model, but also, identify potential targets for design more effective drugs for the inactivation of H. pylori.

Tese de Doutoramento em Ciências Veterinárias - Clínica O cancro é um importante problema de saúde pública a nível mundial. De acordo com a Organização Mundial de Saúde, no ano de 2012, esta doença afetou 14 milhões de pessoas em todo o mundo. O cancro da mama é o mais frequente na mulher, tendo sido responsável por mais de meio milhão de mortes no ano de 2012. Este tipo de cancro é também o mais frequente nas cadelas não castradas e o terceiro mais frequente na gata. O desenvolvimento de cancro da mama encontra-se associado a diversos fatores de risco, como a idade, o sexo, a raça, fatores reprodutivos, a exposição aos estrogénios, a massa corporal, mutações genéticas e o estilo de vida. Existem diversas opções médicas para o tratamento do cancro da mama, como a cirurgia, a radioterapia, a quimioterapia, a terapia hormonal e a imunoterapia. Contudo, a possível ineficácia, a imprevisibilidade da resposta do cancro à terapia instituída e os efeitos secundários associados à terapia constituem uma permanente preocupação no tratamento do cancro. Desta forma, é essencial investir na pesquisa de novas abordagens terapêuticas que possam melhorar o prognóstico e a qualidade de vida dos doentes oncológicos. O modelo de cancro da mama quimicamente induzido no rato pela administração do agente carcinogénico N-metil-N-nitrosureia (MNU) é internacionalmente reconhecido para o estudo da carcinogénese mamária. Este modelo permite estudar diversos aspetos da carcinogénese mamária, como a patogenia, e as alterações genéticas e moleculares, bem como avaliar novas abordagens preventivas e terapêuticas. Com esta tese pretendemos contribuir para a monitorização e tratamento do cancro da mama utilizando este modelo animal. Neste sentido, o crescimento e a vascularização das neoplasias foram monitorizados de forma não invasiva, recorrendo a métodos de diagnóstico por imagem, e foram avaliadas duas abordagens terapêuticas: o efeito do estilo de vida (prática de exercício físico) e a ação do fármaco anti-histamínico H1 de segunda geração e estabilizador dos mastócitos - cetotifeno. Neste trabalho foram realizados dois protocolos experimentais, um para avaliar o efeito da prática de exercício físico moderado durante 35 semanas e outro para avaliar o efeito do cetotifeno na progressão e vascularização do cancro da mama. No primeiro protocolo, 50 ratos do sexo feminino da estirpe Sprague-Dawley foram aleatoriamente divididos em quatro grupos experimentais: MNU sedentário (n=15), MNU exercitado (n=15), controlo sedentário (n=10) e controlo exercitado (n=10). Aos 50 dias de idade, os animais dos grupos MNU receberam uma injeção intraperitoneal do agente carcinogénico MNU. Após a administração, os animais foram exercitados num tapete rolante a uma velocidade de 20 m/min, 60 min/dia, 5 dias/semana, durante 35 semanas. No segundo protocolo experimental, 34 ratos do sexo feminino da estirpe Sprague-Dawley foram aleatoriamente divididos em cinco grupos experimentais: MNU (n=10), MNU + cetotifeno-1 (n=10), MNU + cetotifeno-2 (n=10), cetotifeno (n=2) e controlo (n=2). O desenvolvimento de cancro da mama foi induzido nos animais dos grupos MNU através da administração do agente carcinogénico MNU de acordo com as condições descritas no primeiro protocolo experimental. Os animais do grupo MNU + cetotifeno-1 receberam o fármaco anti-histamínico e inibidor da desgranulação dos mastócitos cetotifeno por via oral após a administração da MNU, durante 18 semanas. Os animais do grupo MNU + cetotifeno-2 apenas receberam o cetotifeno após o desenvolvimento da primeira neoplasia mamária. Os restantes grupos foram utilizados como controlos. Em ambos os protocolos experimentais, o crescimento das neoplasias e a vascularização foram monitorizados recorrendo a métodos não invasivos (utilização do paquímetro, ultrassonografia e termografia). No final dos ensaios, os animais sobreviventes foram sacrificados e foi realizada uma necrópsia completa. De acordo com os nossos resultados, as neoplasias da mama induzidas pela MNU em ratos do sexo feminino crescem como uma superfície circular com pouca profundidade, assemelhando-se à forma de um esferoide oblato. A ultrassonografia é o método recomendado para a determinação do volume das neoplasias in vivo, enquanto o deslocamento de água e a determinação do volume com base na massa das neoplasias constituem as metodologias mais adequadas para determinar o volume após o sacrifício dos animais ou a excisão cirúrgica das neoplasias. A ultrassonografia e a termografia são métodos de imagem recomendados para a avaliação da vascularização das neoplasias. A análise das neoplasias por microscopia eletrónica revelou semelhanças entre as neoplasias induzidas pela MNU no rato e o cancro da mama da mulher. A prática de exercício físico moderado durante 35 semanas inibiu a carcinogénese mamária, reduzindo a inflamação, o número total de neoplasias, o número de neoplasias por animal e a sua malignidade, e aumentou o período de latência. Adicionalmente, as neoplasias mamárias dos animas exercitados exibiram maior imunoexpressão dos recetores de estrogénios α, sugerindo uma maior diferenciação destas neoplasias e melhor resposta à terapia hormonal. Apesar de apresentarem menor malignidade, as neoplasias dos animais exercitados exibiram maior vascularização quando comparadas com as neoplasias dos animais sedentários. Foi também observado que o cancro e a prática de exercício físico induzem algumas alterações ao nível do músculo esquelético. Nos animais com neoplasias (grupos MNU) foi observada uma menor ecogenicidade do músculo gastrocnémio sugestiva de uma menor infiltração de tecido adiposo, e níveis séricos de miostatina mais elevados provavelmente devido ao efeito catabólico do processo neoplásico no tecido muscular. O músculo gastrocnémio dos animais exercitados (MNU e controlo) exibiu menor ecogenicidade quando comparada com a ecogenicidade do músculo dos animais dos respetivos grupos sedentários devido à diminuição da infiltração de tecido adiposo nos grupos exercitados. A administração do cetotifeno inibiu a desgranulação dos mastócitos. O principal efeito positivo desta inibição na carcinogénese mamária foi a redução da proliferação das neoplasias quando a desgranulação dos mastócitos foi inibida antes do desenvolvimento das mesmas. De acordo com os nossos resultados, a prática de exercício físico moderado durante um longo período de tempo e a administração do cetotifeno são recomendadas para a prevenção e tratamento do cancro da mama. Cancer is a major public health problem around the globe. According to the World Health Organization, 14 million of people were affected by this disease in the year 2012. Breast cancer is the most frequent cancer among women, being responsible for more than a half a million of deaths in 2012. It is also the most frequent cancer in intact female dogs and the third most frequent in female cats. Breast cancer development is intimately associated with several risk factors, namely age, sex, race, reproductive factors, estrogens exposure, body weight, genetic mutations and lifestyle. Several medical options are available for breast cancer treatment, such as surgery, radiotherapy, chemotherapy, hormone therapy and immunotherapy. However, the possible ineffectiveness, the distinct response of cancer to the therapies and the devastating effects of some of these therapies for patients are the major concerns in cancer treatment. So, it is crucial to search for new or at least adjuvant therapies that may improve the lifespan and quality of life of oncologic patients. The rat model of chemically-induced mammary cancer by the administration of the carcinogen N-methyl-N-nitrosourea (MNU) is internationally recognized for the study of mammary carcinogenesis. It allows to better understand several aspects of mammary carcinogenesis, namely pathogenesis, genetic and molecular basis, as well as to evaluate new preventive and therapeutic approaches. With this thesis, we intend to contribute to the monitoring and treatment of mammary cancer using this animal model. For this purpose, mammary tumor growth and vascularization were non-invasively evaluated by imaging modalities, and two distinct therapeutic approaches were tested: we evaluated the role of lifestyle (practice of physical exercise) and the effects of the second-generation H1-antihistamine and mast cell stabilizer drug - ketotifen. Two distinct experimental protocols were performed: one to evaluate the effects of long-term moderate exercise training and another to evaluate the effects of ketotifen in the progression and vascularization of mammary cancer. In the first experiment, 50 female Sprague-Dawley rats were randomly divided into four experimental groups: MNU sedentary (n=15), MNU exercised (n=15), control sedentary (n=10) and control exercised (n=10). At 50 days of age, animals from MNU groups received a single intraperitoneal injection of the carcinogen agent MNU. Then, animals were exercised on a treadmill running at a velocity of 20 m/min, 60 min/day, five days a week, for 35 weeks. In the second experiment, 34 female Sprague-Dawley rats were randomly divided into five experimental groups: MNU (n=10), MNU + ketotifen-1 (n=10), MNU + ketotifen-2 (n=10), ketotifen (n=2) and control (n=2). Mammary tumor development was induced in animals from MNU groups by the administration of MNU according to the conditions described above. Animals from MNU + ketotifen-1 group were orally administered with ketotifen immediately after MNU administration for 18 weeks, while each animal from MNU + ketotifen-2 group only received ketotifen after the development of the first mammary tumor. The remaining groups were used as controls. Mammary tumor growth and vascularization were non-invasively monitored throughout both experiments, using caliper, ultrasound and thermography. At the end of the experiments, all survived animals were humanely sacrificed and submitted to a complete necropsy. According to our results, MNU-induced mammary tumors in female rats grow as a circular surface with small depth, resembling the shape of an oblate spheroid. Ultrasonography is the recommended way to determine tumor volume in vivo, while water displacement or tumor weight are the best ways to assess tumor volume after animal sacrifice or tumor excision. Ultrasound and thermographic techniques are applicable to provide information on tumor vascularization. Electron microscopy analysis revealed similar features between MNU-induced mammary tumors in rats and women mammary tumors. Lifelong moderate exercise training inhibited the carcinogenic response, by reducing inflammation, multiplicity, burden and malignancy, and increasing the cancer latency. Additionally, mammary tumors from exercised animals exhibited higher immunoexpression of estrogen receptors α, which is an indicator of tumor differentiation and better response to hormone therapy. Surprisingly, despite low malignancy, mammary tumors from exercised animals were more vascularized when compared with mammary tumors from sedentary animals. It was also observed that cancer and the practice of exercise training promoted some changes in the skeletal muscle. A lower echogenicity of gastrocnemius muscle suggestive of lower fat infiltration and higher myostatin serum levels were observed in animals with cancer (MNU-exposed groups) probably due to cancer-associated catabolic effects on body tissues. Exercised groups (MNU and control) also exhibited lower echogenicity when compared with respective sedentary groups due to the reduction of muscular fat. Ketotifen inhibited mast cell degranulation. The main positive effect of this inhibition on mammary carcinogenesis was the reduction of mammary tumor proliferation when the mast cell degranulation was inhibited before tumor development. According to our results, the practice of moderate exercise training for a long period of time and the administration of ketotifen are recommended for mammary cancer prevention and treatment. Fundação para a Ciência e Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior (Portugal)

Leishmaniasis is a virulent parasitic infection that causes a significant threat to human health worldwide. The existing drugs are becoming less effective due to the ability of Leishmania spp. to alter its metabolism to adapt to harsh environments. Understanding how this parasite manipulates its metabolism inside the host (e.g. sandfly and human) might underpin new ways to prevent the disease and develop effective treatment strategies. Despite significant advances in omics technologies, biochemistry of parasites still lacks the understanding of molecular components that determine the metabolic behavior under varying conditions. Metabolic network modeling might be of interest to identify physiologically relevant nodes in a metabolic network. The present work proposes a metabolic model iSK570 (an extension of the iAC560 model) with additional reactions for the metabolism of lipids, long chain fatty acids and carbohydrates to study the metabolic behavior of this parasite. Gene Inactivity Moderated by Metabolism and Expression (GIMME) algorithm was used to verify the consistency between model flux predictions and gene expression data. Improved flux distributions were obtained, allowing a more accurate understanding of stage-specific metabolism in of promastigotes and amastigotes., This work was supported by the Initial Training Network, GlycoPar, funded by the FP7 Marie Curie Actions of the European Commission (FP7-PEOPLE-2013-ITN-608295). The authors gratefully express appreciation to SilicoLife Lda for providing required infrastructural facilities related to this work. We also thank Bruno Pereira (systems biologist at SilicoLife) and Hugo Giesteira (programmer at SilicoLife) for scientific and technical assistance during various phases of the project., info:eu-repo/semantics/publishedVersion

A system for the on-line monitoring of the major state variables during the production of recombinant proteins with E. coli was developed. Substrate (glucose) and the major by-product (acetate) were measured with a Flow Injection Analysis system, while the carbon dioxide and oxygen transfer rates were calculated from data obtained with exhaust gas analysis. The fermentation culture weight was also continuously assessed with a balance placed below the fermenter, allowing the use of more precise mass-based concentrations. The graphical programming environment LabVIEW was used to acquire and integrate these variables in a supervisor computer, allowing to perform integrated monitoring and control of the process. This framework is illustrated with different experiments for model calibration, state estimation by software sensors and process performance optimisation by exponential feed control., PROTEXPRESS project (Agência de Inovação). Fundação para a Ciência e a Tecnologia (FCT)

An optimal experimental design for yield coefficients estimation in an unstructured growth model of fed-batch fermentation of E. coli is presented. The feed profile is designed by optimisation of a scalar function based on the Fischer Information Matrix. A genetic algorithm is proposed as the optimisation method due to its efficiency and independence on the initial values., Programa de Desenvolvimento Educativo para Portugal (PRODEP)., Fundação para a Ciência e a Tecnologia (FCT) – PRAXISXXI/BD/16961/98.

Evolutionary Algorithms (EAs) have been used to achieve optimal feedforward control in a number of fed-batch fermentation processes. Typically, the optimization purpose is to set the optimal feeding trajectory, being the feeding profile over time given by a piecewise linear function, in order to reduce the number of parameters to the optimization algorithm. In this work, a novel representation scheme for the encoding of the feeding trajectory over time is proposed. Each gene in the variable sized chromosome has two components: a time label and the real value of the variable. The new approach is compared with a traditional real-valued EA, with chromosomes of constant size and fixed discretization steps. Three distinct case studies are presented, taken from previous work from the authors and literature, all considering the optimization of fed-batch fermentation processes. The experimental results show that the proposed approach is capable of results better or at the same level of quality of the best traditional EAs and is able to automatically evolve the best discretization steps for each case, thus simplifying the EA's setup., Fundação para a Ciência e Tecnologia (FCT) - 59899/EIA/POSC/2004.

Background One of the European Union directives indicates that 10% of all fuels must be bio-synthesized by 2020. In this regard, biobutanolnatively produced by clostridial strainsposes as a promising alternative biofuel. One possible approach to overcome the difficulties of the industrial exploration of the native producers is the expression of more suitable pathways in robust microorganisms such as Escherichia coli. The enumeration of novel pathways is a powerful tool, allowing to identify non-obvious combinations of enzymes to produce a target compound. Results This work describes the in silico driven design of E. coli strains able to produce butanol via 2-oxoglutarate by a novel pathway. This butanol pathway was generated by a hypergraph algorithm and selected from an initial set of 105,954 different routes by successively applying different filters, such as stoichiometric feasibility, size and novelty. The implementation of this pathway involved seven catalytic steps and required the insertion of nine heterologous genes from various sources in E. coli distributed in three plasmids. Expressing butanol genes in E. coli K12 and cultivation in High-Density Medium formulation seem to favor butanol accumulation via the 2-oxoglutarate pathway. The maximum butanol titer obtained was 85±1 mg L1 by cultivating the cells in bioreactors. Conclusions In this work, we were able to successfully translate the computational analysis into in vivo applications, designing novel strains of E. coli able to produce n-butanol via an innovative pathway. Our results demonstrate that enumeration algorithms can broad the spectrum of butanol producing pathways. This validation encourages further research to other target compounds., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of a Ph.D. Grant (PD/BD/52366/2013) from MIT Portugal Program and the strategic funding of UID/BIO/04469 unit. Additional support was received by COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte. The authors also thank the Times New Roman project “Dynamics”, Ref. ERA-IB-2/0002/2014, funded by national funds through FCT/MCTES. The genes thl, hbd, crt and adhE1 were kindly provided by Kristala L. Jones Prather from MIT. The authors thank the project DDDeCaF - Bioinformatics Services for Data-Driven Design of Cell Factories and Communities, Ref. H2020-LEIT-BIO-2015-1 686070–1, funded by the European Commission and the Project LISBOA010145 FEDER007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT Fundacao para a Ciencia e a Tecnologia, info:eu-repo/semantics/publishedVersion

BACKGROUND:Rational approaches for Metabolic Engineering (ME) deal with the identification of modifications that improve the microbes' production capabilities of target compounds. One of the major challenges created by strain optimization algorithms used in these ME problems is the interpretation of the changes that lead to a given overproduction. Often, a single gene knockout induces changes in the fluxes of several reactions, as compared with the wild-type, and it is therefore difficult to evaluate the physiological differences of the in silico mutant. This is aggravated by the fact that genome-scale models per se are difficult to visualize, given the high number of reactions and metabolites involved.FINDINGS:We introduce a software tool, the Topological Network Analysis for OptFlux (TNA4OptFlux), a plug-in which adds to the open-source ME platform OptFlux the capability of creating and performing topological analysis over metabolic networks. One of the tool's major advantages is the possibility of using these tools in the analysis and comparison of simulated phenotypes, namely those coming from the results of strain optimization algorithms. We illustrate the capabilities of the tool by using it to aid the interpretation of two E. coli strains designed in OptFlux for the overproduction of succinate and glycine.CONCLUSIONS:Besides adding new functionalities to the OptFlux software tool regarding topological analysis, TNA4OptFlux methods greatly facilitate the interpretation of non-intuitive ME strategies by automating the comparison between perturbed and non-perturbed metabolic networks. The plug-in is available on the web site http://www.optflux.org webcite, together with extensive documentation., This work is funded by ERDF - European Regional Development Fund through the COMPETE Programme (operational programme for competitiveness) and by National Funds through the FCT (Portuguese Foundation for Science and Technology) within projects ref. COMPETE FCOMP-01-0124-FEDER-015079 and PEst-OE/EEI/UI0752/2011. JPP and RP work is funded by PhD grants from the Portuguese FCT (ref. SFRH/BD/41763/ 2007 and SFRH/BD/51111/2010).

[EN] Miniaturized, high-throughput, cost-effective sensing devices are needed to advance lab-on-a-chip technologies for healthcare, security, environmental monitoring, food safety, and research applications. Quartz crystal microbalance with dissipation (QCMD) is a promising technology for the design of such sensing devices, but its applications have been limited, until now, by low throughput and significant costs. In this work, we present the design and characterization of 24-element monolithic QCMD arrays for high-throughput and low-volume sensing applications in liquid. Physical properties such as geometry and roughness, and electrical properties such as resonance frequency, quality factor, spurious mode suppression, and interactions between array elements (crosstalk), are investigated in detail. In particular, we show that the scattering parameter, S 21 , commonly measured experimentally to investigate crosstalk, contains contributions from the parasitic grounding effects associated with the acquisition circuitry. Finite element method simulations do not take grounding effects into account explicitly. However, these effects can be effectively modelled with appropriate equivalent circuit models, providing clear physical interpretation of the different contributions. We show that our array design avoids unwanted interactions between elements and discuss in detail aspects of measuring these interactions that are often-overlooked., The authors would also like to thank Jorge Martínez from the Laboratory of High Frequency Circuits (LCAF) of the Universitat Politècnica de València (UPV) for assistance with profilometry, and Manuel Planes, José Luis Moya, Mercedes Tabernero, Alicia Nuez, and Joaquin Fayos from the Electron Microscopy Services of the UPV for helping with the AFM, and SEM measurements. M. Calero is the recipient of the doctoral fellowship BES-2017-080246 from the Spanish Ministry of Economy, Industry and Competitiveness, Madrid, Spain.