Contribuição para a melhoria da qualidade nutricional do sorgo

Este trabalho teve como principal objectivo sugerir uma forma de processamento de sorgo adaptável à escala industrial que, sendo culturalmente aceite e sensível aos hábitos alimentares, aos factores sociais e às limitações económicas e tecnológicas das populações Africanas de consumo, pudesse dar origem a um produto alimentar seguro e enriquecido em termos nutricionais. Numa primeira fase, efectuou-se um estudo comparativo entre os efeitos promovidos por diferentes formas de processamento: cozimento em água, aquecimento em banho-maria, pipocagem, germinação, fermentação e altapressão. Foram ainda determinadas as condições óptimas de aplicação do processo germinativo e da tecnologia de alta-pressão. Verificou-se que a fermentação, a germinação e a alta-pressão permitem uma melhoria significativa da digestibilidade proteica da farinha de sorgo. A pipocagem conduziu a uma redução da extractibilidade das proteínas não promovendo, contudo, alterações na sua digestibilidade. Comparativamente ao cozimento em água, que promove uma diminuição acentuada na digestibilidade proteica, o cozimento em banho-maria promove uma diminuição ténue e significativamente inferior. Deste estudo comparativo, foi possível concluir que a extractibilidade das proteínas não está correlacionada com a sua digestibilidade e que a água exerce um papel fundamental na diminuição da digestibilidade proteica com o aquecimento. Numa segunda fase, pretendeu-se desenvolver um processo fermentativo que conduzisse a um produto de sorgo com características nutricionais incrementadas. Para tal, foram testadas daiferentes espécies de bactérias lácticas isoladas e conjugadas entre si. Foi ainda testada a adição de malte de sorgo previamente à fermentação e a adição de leveduras ao inóculo. Com este estudo, foi possível concluir que a fermentação do sorgo com um inóculo constituído por culturas puras de Lactobacillus brevis, Lactobacillus fermentum e Streptococcus thermophilus permite a obtenção de uma preparação alimentar com características nutricionais melhoradas no que respeita ao balanço em aminoácidos essenciais, à digestibilidade da proteína e do amido e à viscosidade do produto final.

Embryonic and larval ecology and biochemistry of offshore decapods

Understanding the biology of offshore species is hardened by the difficulties of sampling in the deep-sea environment. Additionally, due to the vastness of the open ocean, knowledge of early life histories of pelagic larvae is still relatively scarce. In decapod species with bentho-pelagic lifestyle, the transition from life in the seafloor to the water column not only is associated with drastic morphological metamorphosis, but also with changes in behavior and feeding ecology. The purpose of the present thesis was to investigate physiological, biochemical and behavioral adaptation occurring during early development of such species. The Norway lobster, Nephrops norvegicus, and the crab Monodaeus couchi were used as a model as these two species are encountered off the NE Atlantic shelf at depth greater than 300 m. Chapter 1 introduces the challenges faced by both adult and larvae inhabiting such remote habitats, including the effect of food availability on development and oceanographic processes on dispersal and recruitment. The thesis follows early life histories, starting with within-brood variability in the fatty acid (FA) profile displayed by developing N. norvegicus embryos. There were no differences in the FA composition of embryos sampled from both sides of the brooding chamber in most females. However, all females exhibited significant differences in the FA profiles of embryos sampled from different pleopods. Potential causes for the variations recorded may be differential female investment during oocyte production or shifts in FA catabolism during the incubation period promoted by embryo’s location within the brooding chamber. Next, feeding rates and digestive enzymes activity of the early stage larvae was investigated in N. norvegicus. Both stages were able to maximize food intake when larvae were scarce and showed increased feeding rate following periods of starvation. Amylase activity indicated that carbohydrates are not the primary energy reserve and that feeding may be required soon after hatching to trigger amylase activity. Protease activity indicated that protein reserves are catabolized under starvation. These results indicate that larvae may maximize prey ingestion in the presence of plankton patches with higher food abundance and minimize the deleterious effects induced by previous periods of intermittent starvation or unsuitable prey densities/types. Additionally, changes in enzymatic activity may allow newly hatched N. norvegicus larvae to metabolize protein reserves to overcome short-term starvation. Vertical migration behavior and the influence of oceanographic properties were studied next. All zoeal stages of M. couchi displayed reverse diel vertical migration. Abundance of early stages was correlated with chlorophyll a levels. An ontogenic shift in vertical distribution explained the results; earlier zoeal stages remain in the food-rich upper water column while later stages migrate to the bottom for settlement. This vertical migration behavior is likely to affect horizontal distribution of larvae. Indeed, global current patterns will result in low inter-annual variations in decapod larvae recruitment, but short term variations such as upwelling events will cause deviation from the expected dispersal pattern. Throughout development, from the embryo to metamorphosis into benthic juvenile, offshore decapods face many challenges. For the developing individual survivorship will depend heavily on food availability but also on the reserves passed on by the mother. Even though vertical migration behavior can allow the larvae to take advantage of depth varying currents for transport, the effect of general circulation pattern will superimpose local current and influence feeding conditions and affect dispersal and recruitment.

Privacy in next generation networks

In the modern society, communications and digital transactions are becoming the norm rather than the exception. As we allow networked computing devices into our every-day actions, we build a digital lifestyle where networks and devices enrich our interactions. However, as we move our information towards a connected digital environment, privacy becomes extremely important as most of our personal information can be found in the network. This is especially relevant as we design and adopt next generation networks that provide ubiquitous access to services and content, increasing the impact and pervasiveness of existing networks. The environments that provide widespread connectivity and services usually rely on network protocols that have few privacy considerations, compromising user privacy. The presented work focuses on the network aspects of privacy, considering how network protocols threaten user privacy, especially on next generation networks scenarios. We target the identifiers that are present in each network protocol and support its designed function. By studying how the network identifiers can compromise user privacy, we explore how these threats can stem from the identifier itself and from relationships established between several protocol identifiers. Following the study focused on identifiers, we show that privacy in the network can be explored along two dimensions: a vertical dimension that establishes privacy relationships across several layers and protocols, reaching the user, and a horizontal dimension that highlights the threats exposed by individual protocols, usually confined to a single layer. With these concepts, we outline an integrated perspective on privacy in the network, embracing both vertical and horizontal interactions of privacy. This approach enables the discussion of several mechanisms to address privacy threats on individual layers, leading to architectural instantiations focused on user privacy. We also show how the different dimensions of privacy can provide insight into the relationships that exist in a layered network stack, providing a potential path towards designing and implementing future privacy-aware network architectures.

Mitochondrial plasticity in pathophysiological conditions

Both skeletal and cardiac muscles daily burn tremendous amounts of ATP to meet the energy requirements for contraction. So, it is not surprising that the maintenance of mitochondrial morphology, number, distribution and functionality in striated muscle are important for muscle homeostasis. In these tissues mitochondria present the added dimension of two populations, the intermyofibrillar (IMF) and the subsarcolemmal (SS) mitochondria, being IMF the most abundant one. In the present thesis, the molecular mechanisms harboured in mitochondria of striated muscles were studied using animal models, to better comprehend the role of mitochondrial plasticity in several pathophysiological conditions such as aging, diabetes mellitus and bladder cancer. The comparative analysis of IMF and SS populations isolated from heart evidenced a higher respiratory chain activity of mitochondria interspersed in the contractile apparatus. The higher susceptible of SS respiratory chain complexes subunits to carbonylation, but not to nitration, seems to justify the lower respiratory chain activity observed in this mitochondrial population. Our results showed that in heart from aged mice there is an accumulation of dysfunctional mitochondria. The age-related decrease of oxidative phosphorylation activity seems to be justified, at least partially, by the increased proneness of mitochondrial proteins as OXPHOS subunits and MnSOD to oxidative modifications. Moreover, a sedentary lifestyle seems to worsen the functional consequences of aging in heart by increasing mitochondrial proteins susceptibility to nitration. In skeletal muscle from rats with type 1 diabetes mellitus induced by streptozotocin administration, we verified the accumulation of dysfunctional mitochondria due, at least in part, to the impairment of PQC system. Indeed, the decreased activity of AAA proteases was accompanied by the accumulation of oxidatively modified mitochondrial proteins with impact in respiratory chain activity. The diminishing of mitochondria activity also underlies cancer-induced muscle wasting. Indeed, using a rat model of chemically induced urothelial carcinoma we verified that the loss of gastrocnemius mass was related to mitochondrial dysfunction due to, at least partially, the down-regulation of PQC system involving the mitochondrial proteases paraplegin and Lon. PQC impairment resulted in the accumulation of oxidatively modified mitochondrial proteins. In overall, regardless the pathophysiological stimuli that promote mitochondrial alterations, there are similarities in the pattern of disease-related mitochondrial plasticity. The diminished capacity for ATP production in striated muscle seems to be due to increased oxidative damage of mitochondrial proteins, namely subunits of respiratory chain complexes, metabolic proteins and MnSOD. Our data highlighted, for the first time, the impact of mitochondrial PQC system impairment in the accumulation of oxidized proteins, exacerbating the dysfunction of this organelle in striated muscle in several pathophysiological conditions.

Nanosensor for assessing the risk of a cardiovascular disease

The incidence of cardiovascular diseases (CVD) has been increasing according to the European and global statistics. Thus, the development of new analytical devices, such as biosensors for assessing the risk of CVD could become a valuable approach for the improvement of healthcare service. In latest years, the nanotechnology has provided new materials with improved electronic properties which have an important contribution in the transduction mechanism of biosensors. Thus, in this thesis, biosensors based on field effect transistors with single-walled carbon nanotubes (NTFET) were developed for the detection of C-reactive protein (CRP) in clinical samples, that is, blood serum and saliva from a group of control patients and a group of CVD risk patients. CRP is an acute-phase protein, which is commonly known as the best validated biomarker for the assessment of CVD, the single-walled carbon nanotubes (SWCNT) were applied as transduction components, and the immunoreaction (interaction between the CRP antigen and the antibodies specific to CRP) was used as the mechanism of molecular recognition for the label-free detection of CRP. After the microfabrication of field effect transistors (FET), the screening of the most important variables for the dispersion of SWCNT, the assemblage of NTFET, and their application on standard solutions of CRP, it was found that NTFET respond accurately to CRP both in saliva and in serum samples, since similar CRP levels were found with the NTFET and the traditional methodology (ELISA technique). On the other hand, a strong correlation between salivary and serum CRP was found with NTFET, which means that saliva could be used, based on non-invasive sampling, as an alternative fluid to blood serum. It was also shown that NTFET could discriminate control patients from CVD risk patients, allowing the determination of a cut-off value for salivary CRP of 1900 ng L-1, which corresponds to the well established cut-off of 3 mg L-1 for CRP in serum, constituting an important finding for the possible establishment of a new range of CRP levels based on saliva. According to the data provided from the volunteer patients regarding their lipoprotein profile and lifestyle factors, it was concluded that the control and the CVD risk patients could be separated taking into account the various risk factors established in literature as strong contributors for developing a CVD, such as triglycerides, serum CRP, total cholesterol, LDL cholesterol, body mass index, Framingham risk score, hypertension, dyslipidemia, and diabetes mellitus. Thus, this work could provide an additional contribution to the understanding of the association of biomarkers levels in serum and saliva samples, and above all, cost-effective, rapid, label-free, and disposable NTFET were developed, based on a noninvasive sampling, for the assessment of CVD risk, thus constituting a potential point-of-care technology.

Bioethanol production from a sub-product of pulping industry

A vida da sociedade atual é dependente dos recursos fósseis, tanto a nível de energia como de materiais. No entanto, tem-se verificado uma redução das reservas destes recursos, ao mesmo tempo que as necessidades da sociedade continuam a aumentar, tornando cada vez mais necessárias, a produção de biocombustíveis e produtos químicos. Atualmente o etanol é produzido industrialmente a partir da cana-de-açúcar e milho, matérias-primas usadas na alimentação humana e animal. Este fato desencadeou o aumento de preços dos alimentos em todo o mundo e, como consequência, provocou uma série de distúrbios sociais. Os subprodutos industriais, recursos independentes das cadeias alimentares, têm-se posicionado como fonte de matérias-primas potenciais para bioprocessamento. Neste sentido, surgem os subprodutos gerados em grande quantidade pela indústria papeleira. Os licores de cozimento da madeira ao sulfito ácido (SSLs) são uma matériaprima promissora, uma vez que durante este processo os polissacarídeos da madeira são hidrolisados originando açúcares fermentáveis. A composição dos SSLs varia consoante o tipo de madeira usada no processo de cozimento (de árvores resinosas, folhosas ou a mistura de ambas). O bioprocessamento do SSL proveniente de folhosas (HSSL) é uma metodologia ainda pouco explorada. O HSSL contém elevadas concentrações de açúcares (35-45 g.L-1), na sua maioria pentoses. A fermentação destes açúcares a bioetanol é ainda um desafio, uma vez que nem todos os microrganismos são capazes de fermentar as pentoses a etanol. De entre as leveduras capazes de fermentar naturalmente as pentoses, destaca-se a Scheffersomyces stipitis, que apresenta uma elevada eficiência de fermentação. No entanto, o HSSL contém também compostos conhecidos por inibirem o crescimento de microrganismos, dificultando assim o seu bioprocessamento. Neste sentido, o principal objetivo deste trabalho foi a produção de bioetanol pela levedura S. stipitis a partir de HSSL, resultante do cozimento ao sulfito ácido da madeira de Eucalyptus globulus. Para alcançar este objetivo, estudaram-se duas estratégias de operação diferentes. Em primeiro lugar estudou-se a bio-desintoxicação do HSSL com o fungo filamentoso Paecilomyces variotii, conhecido por crescer em resíduos industriais. Estudaram-se duas tecnologias fermentativas diferentes para a biodesintoxicação do HSSL: um reator descontínuo e um reator descontínuo sequencial (SBR). A remoção biológica de inibidores do HSSL foi mais eficaz quando se usou o SBR. P. variotii assimilou alguns inibidores microbianos como o ácido acético, o ácido gálico e o pirogalol, entre outros. Após esta desintoxicação, o HSSL foi submetido à fermentação com S. stipitis, na qual foi atingida a concentração máxima de etanol de 2.36 g.L-1 com um rendimento de 0.17 g.g-1. P. variotti, além de desintoxicar o HSSL, também é útil na produção de proteína microbiana (SCP) para a alimentação animal pois, a sua biomassa é rica em proteína. O estudo da produção de SCP por P. variotii foi efetuado num SBR com HSSL sem suplementos e suplementado com sais. A melhor produção de biomassa foi obtida no HSSL sem adição de sais, tendo-se obtido um teor de proteína elevado (82,8%), com uma baixa concentração de DNA (1,1%). A proteína continha 6 aminoácidos essenciais, mostrando potencial para o uso desta SCP na alimentação animal e, eventualmente, em nutrição humana. Assim, a indústria papeleira poderá integrar a produção de bioetanol após a produção SCP e melhorar a sustentabilidade da indústria de pastas. A segunda estratégia consistiu em adaptar a levedura S. stipitis ao HSSL de modo a que esta levedura conseguisse crescer e fermentar o HSSL sem remoção de inibidores. Operou-se um reator contínuo (CSTR) com concentrações crescentes de HSSL, entre 20 % e 60 % (v/v) durante 382 gerações em HSSL, com uma taxa de diluição de 0.20 h-1. A população adaptada, recolhida no final do CSTR (POP), apresentou uma melhoria na fermentação do HSSL (60 %), quando comparada com a estirpe original (PAR). Após esta adaptação, a concentração máxima de etanol obtida foi de 6.93 g.L-1, com um rendimento de 0.26 g.g-1. POP possuía também a capacidade de metabolizar, possivelmente por ativação de vias oxidativas, compostos derivados da lenhina e taninos dissolvidos no HSSL, conhecidos inibidores microbianos. Por fim, verificou-se também que a pré-cultura da levedura em 60 % de HSSL fez com que a estirpe PAR melhorasse o processo fermentativo em HSSL, em comparação com o ensaio sem pré-cultura em HSSL. No entanto, no caso da estirpe POP, o seu metabolismo foi redirecionado para a metabolização dos inibidores sendo que a produção de etanol decresceu.

Infection mechanism of Diplodia corticola

Diplodia corticola is regarded as the most virulent fungus involved in cork oak decline, being able to infect not only Quercus species (mainly Q. suber and Q. ilex), but also grapevines (Vitis vinifera) and eucalypts (Eucalyptus sp.). This endophytic fungus is also a pathogen whose virulence usually manifests with the onset of plant stress. Considering that the infection normally culminates in host death, there is a growing ecologic and socio-economic concern about D. corticola propagation. The molecular mechanisms of infection are hitherto largely unknown. Accordingly, the aim of this study was to unveil potential virulence effectors implicated in D. corticola infection. This knowledge is fundamental to outline the molecular framework that permits the fungal invasion and proliferation in plant hosts, causing disease. Since the effectors deployed are mostly proteins, we adopted a proteomic approach. We performed in planta pathogenicity tests to select two D. corticola strains with distinct virulence degrees for our studies. Like other filamentous fungi D. corticola secretes protein at low concentrations in vitro in the presence of high levels of polysaccharides, two characteristics that hamper the fungal secretome analysis. Therefore, we first compared several methods of extracellular protein extraction to assess their performance and compatibility with 1D and 2D electrophoretic separation. TCA-Acetone and TCA-phenol protein precipitation were the most efficient methods and the former was adopted for further studies. The proteins were extracted and separated by 2D-PAGE, proteins were digested with trypsin and the resulting peptides were further analysed by MS/MS. Their identification was performed by de novo sequencing and/or MASCOT search. We were able to identify 80 extracellular and 162 intracellular proteins, a milestone for the Botryosphaeriaceae family that contains only one member with the proteome characterized. We also performed an extensive comparative 2D gel analysis to highlight the differentially expressed proteins during the host mimicry. Moreover, we compared the protein profiles of the two strains with different degrees of virulence. In short, we characterized for the first time the secretome and proteome of D. corticola. The obtained results contribute to the elucidation of some aspects of the biology of the fungus. The avirulent strain contains an assortment of proteins that facilitate the adaptation to diverse substrates and the identified proteins suggest that the fungus degrades the host tissues through Fenton reactions. On the other hand, the virulent strain seems to have adapted its secretome to the host characteristics. Furthermore, the results indicate that this strain metabolizes aminobutyric acid, a molecule that might be the triggering factor of the transition from a latent to a pathogenic state. Lastly, the secretome includes potential pathogenicity effectors, such as deuterolysin (peptidase M35) and cerato-platanin, proteins that might play an active role in the phytopathogenic lifestyle of the fungus. Overall, our results suggest that D. corticola has a hemibiotrophic lifestyle, switching from a biotrophic to a necrotrophic interaction after plant physiologic disturbances.This understanding is essential for further development of effective plant protection measures.