Brazilian savanna forest : conservation, medicinal reservoir and bioprospecting

This study aimed to analyse the Brazilian savanna forest from a Legal Reserve (LR) area from a perspective of conservation, reservoir of organic carbon and medicinal biomass for a prospective use of native medicinal plants. An ethnobotanical and ethnopharmacological survey was carried out close to a community settled in the rural area in the south of Tocantins, being selected 9 of the most cited species (caju��- Anacardium othonianum; inharé-Brosimum gaudichaudii; jatobá-Hymenaeae courbaril; jenipapo-Genipa americana, aroeira-Myracrodruon urundeuva; negramina-Siparuna guianensis; barbatimão- Stryphnodendron obovatum; assa peixe-Vernonia brasiliana, embaúba-Cecropia pachystachya). Crude foliar extracts were subjected to a preliminary phytochemical prospection and triage of secondary metabolites with antimicrobial activity of potential interest in health and familiar agriculture. Phenolic compounds, terpenes and flavonoids were detected in the extracts of most species, which suggests the presence of antimicrobial, antioxidant and anti-insect activities. It was evident the need to better know the LR as a reservoir of medicinal biomass in an area under ecological tension where 35% (610ha) of the property is LR and should be protected by law. Therefore, a forest inventory of live woody species was performed using the allometric or indirect method. This identified a rare remnant of Semidecidual Seasonal Forest amidst the largest world savannah, the Cerrado biome. An analysis of the forest average productivity per basal area (m².ha), aerial live biomass (ton.ha-1) and carbon stock was carried out. The forest fragment was considered relatively rich in species and diversity, although showing signs of disturbance and dominance by a few species. Its horizontal structure suggests biotic regeneration conditions. It is an important reservoir of medicinal plants. Of the families (57.5%) presenting medicinal species, 19 from a total of 33 are represented in the area and contain 44% (27) of the total species (61) and 63% (432) of the total individuals catalogued. Medicinal species have ecological importance for the equilibrium of the local flora and represent 80% of the 10 species with higher Importance Value Index (IVI): Tetragastris altissima, Chrysophyllum marginatum, Oenocarpus distichus, Sclerolobium paniculatum, Simarouba versicolor, Alibertia macrophylla, Siparuna guianensis, Maprounea guianensis, Licania parvifolia e Physocalymma scaberrimum. Medicinal productivity was high for this type of phytophysionomy: 183,2 ton. ha-1 of biomass and 91,51 ton. ha-1 of carbon representing 66% of the total biomass and carbon of this Cerrado forest. From this stage S. guianensis (Siparunaceae) was selected for performing bioassays in order to verify its biological activity against microorganisms of health and agricultural relevance. This is a native aromatic medicinal plant recommended as priority for conservation, with local popular medicinal validation and availability of medicinal feedstock (3300 Kg.ha-1), with the foliar fraction giving 38Kg/ha of crude extract and 5L/ha of essential oil. Foliar crude extracts and essential oil were obtained and tested in vitro using a disk diffusion bioassay. Different concentrations of these natural products were tested against gram-positive bacteria (Staphylococcus aureus ATCC 29213), gram-negative bacteria (Escherichia coli ATCC 25922 and ATCC 35218; Pseudomonas aeruginosa ATCC 10145) and fungi (Candida albicans ATCC 6258 e Fusarium oxysporum). The essential oil inhibited the growth of S. aureus in its crude concentration (380μg.mL-1), as well as diluted to half (190μg.mL-1) and a quarter strength (95μg.mL-1). It’s likely that such action is due to sesquiterpenes major components, such as bisabolol and bisabolene (10.35%), measured by gas chromatography (GC-MS, GC-FID). Extracts did not exhibit any antimicrobial activity against the microorganisms tested. The native medicinal plants prospective market is an alternative that favours the conservation of biodiversity while generating benefits for the development of sustainable family productive activities within local ecosystems instead of the current inappropriate uses. This strengthens conservation policies of Legal Reserve in rural settlements and is in agreement with public policy on global warming and climate changes.

Characterisation of ZG16p, a unique mammalian lectin from pancreatic zymogen granules

The mechanisms of secretory granule biogenesis and regulated secretion of digestive enzymes in pancreatic acinar cells are still not well understood. To shed light on these processes, which are of biological and clinical importance (e.g., pancreatitis), a better molecular understanding of the components of the granule membrane, their functions and interactions is required. The application of proteomics has largely contributed to the identification of novel zymogen granule (ZG) proteins but was not yet accompanied by a better characterization of their functions. In this study we aimed at a) isolation and identification of novel membrane-associated ZG proteins; b) characterization of the biochemical properties and function of the secretory lectin ZG16p, a membrane-associated protein; c) exploring the potential of ZG16p as a new tool to label the endolysosomal compartment. First, we have performed a suborganellar proteomics approach by combining protein analysis by 2D-PAGE and identification by mass spectrometry, which has led to the identification of novel peripheral ZGM proteins with proteoglycan-binding properties (e.g., chymase, PpiB). Then, we have unveiled new molecular properties and (multiple) functions of the secretory lectin ZG16p. ZG16p is a unique mammalian lectin with glycan and proteoglycan binding properties. Here, I revealed for the first time that ZG16p is highly protease resistant by developing an enterokinase-digestion assay. In addition I revealed that ZG16p binds to a high molecular weight complex at the ZGM (which is also protease resistant) and forms highly stable dimers. In light of these findings I suggest that ZG16p is a key component of a predicted submembranous granule matrix attached to the luminal side of the ZGM that fulfils important functions during sorting and packaging of zymogens. ZG16p, may act as a linker between the matrix and aggregated zymogens due to dimer formation. Furthermore, ZG16p protease resistance might be of higher importance after secretion since it is known that ZG16p binds to pathogenic fungi in the gut. I have further investigated the role of ZG16p binding motifs in its targeting to ZG in AR42J cells, a pancreatic model system. Point mutations of the glycan and the proteoglycan binding motifs did not inhibit the targeting of ZG16p to ZG in AR42J cells. I have also demonstrated that when ZG16p is present in the cytoplasm it interacts with and modulates the endo-lysosomal compartment. Since it is known that impaired autophagy due to lysosomal malfunction is involved in the course of pancreatitis, a potential role of ZG16p in pancreatitis is discussed.

Structural characterization and biological activity of polysaccharides from infusions used in the popular tradition

The use of plants with medicinal purposes is an ancient practice still very common in developing regions, and is rapidly spreading in industrialized countries. This fact is evidenced by the large number of ethnobotanical studies found in the literature referring that these plants are often used as decoctions and infusions. In most studies the reported biological activities are attributed to the presence of phenolic compounds, due to their antioxidant properties, and to polysaccharides, with its anti-tumoral properties. In “Trás-os-Montes” region, some of the most popular infusions used by the popular medicine are prepared with the dried leaves of Fraxinus angustifolia, the dried shoots of Mentha suaveolens, and the dried inflorescences of Pterospartum tridentatum. However, there are no studies about the polysaccharides present in these infusions. Thus, through the structural characterization of the polysaccharides present in the infusions of F. angustifolia, M. suaveolens, and P. tridentatum, the present PhD thesis intends to evaluate the possible relation between polysaccharides and the immunostimulatory activity that these infusions might present. In a preliminary phase, infusions of F. angustifolia were prepared according to the popular tradition, and it was observed that the obtained water soluble material contained approximately 85% of material non-retained in C18 cartridges, with hydrophilic characteristics, with the remaining 15% comprising retained-material with hydrophobic characteristics. It was also shown that the infusions only contained between 2 and 4% of high molecular weight material (HMWM), which comprised approximately 30% of carbohydrate material. Sugar and methylation analysis of the HMWM suggested the presence of pectic polysaccharides, together with type II arabinogalactans, mannans, and xyloglucans. However, the amount of material obtained is to low for the fractionation, and structural analysis of the polysaccharides present. The 4 h decoction, divided in two periods of 2 h, with water renewal, allowed to increase the HMWM yield, relatively to the infusions traditional infusions. It was also observed that the decoction also allowed to increase the HMWM proportion of carbohydrate material, due to an increase in the proportion of uronic acid present, although the neutral sugar residues seemed to be detected in similar proportions. Therefore, in all the experiments subsequently performed, the HMWM used was obtained through the decoction of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences. x After the fractionation, through ethanol precipitation, and anion exchange chromatography, of the polysaccharides from the HMWM obtained by the decoction of the vegetable material of the distinct studied plants, it was observed the presence of high proportions of pectic polysaccharides, containing type I arabinogalactans, together with minor proportions of type II arabinogalactans, mannans, and xyloglucans. The presence of pectic polysaccharides in the extracts from F. angustifolia was also evidenced through endo-polygalacturonase treatment, and ESI-MS and ESI-MS/MS experiments. The detection of linked pentose and uronic acid residues, also seemed to suggest the presence of xylogalacturonan domains in the pectic polysaccharides from F. angustifolia. The extracts from F. angustifolia dried leaves also contained type II arabinogalactans that exhibited a higher structural diversity than those detected in the M. suaveolens, and P. tridentatum extracts, particularly in the substitution degree of the galactan backbone, and in the extension of the (1→5)-Araf side chains. Moreover, for all the plants studied, it was also observed that the type II arabinogalactans, extracted during the 2nd 2h of the extraction process, exhibited a substitution degree of the galactan backbone higher than those extracted during the 1st 2h. The extracts from P. tridentatum dried inflorescences contained higher proportions of mannans, and also of xyloglucans, both presenting a substitution degree higher than those, which were detected in lower proportion in the extracts of F. angustifolia and M. suaveolens. Through ESI-MS and ESI-MS/MS it was possible to evidence that the mannans present in the extracts of P. tridentatum presented acetyl groups on the O-2 of the mannosyl residues. It was also evidenced that the P. tridentatum mannans were more extensively acetylated than the mannans detected in the coffee infusion, LBG, and other non-conventional mannan sources. Moreover, it was detected the presence of oligosaccharides comprising hexose residues linked to non acetylated pentose residues, suggesting the possible presence of arabinose residues in the mannans from P. tridentatum extracts. The immunostimulatory activity of three fractions isolated from the extracts of F. angustifolia, M. suaveolens, and P. tridentatum, was tested and an increase in the NO production by macrophages, without compromising their cellular viability, was observed. The type I, and type II arabinogalactans detected in the extracts from F. angustifolia, and M. suaveolens seem to have contributed for the observed immunostimulatory activity. For the fraction from P. tridentatum, the mannans acetylation, and the presence of type I, and type II arabinogalactans seemed to contribute for the macrophage immunostimulatory activity observed. The possible presence of storage xyloglucans from the inflorescences seeds, also seems to have contributed for the immunostimulatory activity registered when the macrophages were stimulated with higher extract concentrations. The results obtained allow to conclude that the extracts of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences contained high proportions of pectic polysaccharides, exhibiting type I arabinogalactans, together with other polysaccharides, such as type II arabinogalactans, mannans, and xyloglucans. This polysaccharide mixture seems to have contributed to the immunostimulatory activity of fractions isolated from the extracts of the studied plants. Therefore, as the same type of polysaccharides seem to be present in the decoctions and in the infusions, it seems possible that the polysaccharides might contribute for the therapeutic properties frequently associated by the popular tradition to the infusions of these plants.

Design normal: memória e invisibilidade dos objectos

As últimas décadas têm sido caracterizadas por uma crescente indefinição do design, que refletindo um dia-a-dia cada vez mais complexo, não pode mais ser encarado como actividade fechada no projecto e acabada no produto. O seu universo aumentou e dispersou-se em mercados menores, alargando o seu alcance e sua importância, transformando-o em termo quantitativo e inflacionário. A crescente atenção de que é alvo dramatizou a sua relação com o consumo, o ruído nas prateleiras e a superficialidade da generalização de uma perspectiva do projecto focada nos new media, validando uma abordagem narcisista e de dispensa do diálogo com a indústria, tornaram-no sinónimo de especial. Este trabalho procura confirmar e refletir sobre a existência de um universo de produtos concretos e abordagens que de forma consciente partilham esta noção do contexto actual, em primeiro lugar, sugerindo caminhos alternativos e reacções resultantes da prática e discurso. Em segundo lugar procura analisar se tal reacção pode ser considerada como um movimento. Partindo de uma revisão bibliográfica, e de uma reflexão sobre um universo de produtos do dia-a-dia, procura-se, em terceiro lugar, interpretar e organizar as diversas formas e estratégias de rejeição da noção de design especial. Da procura de abordagens opostas a especial resulta uma ideia base de normal, normal sem ser banal, expressa na exposição Super Normal. Da extensão a um universo maior de produtos, reacções e autores, resulta a ideia de normal como um conceito mais abrangente, mas composto por conceitos menores, mais específicos, mais concretos e fáceis de identificar nos objectos. A organização e interpretação dessas abordagens resulta num triângulo de conceitos que constroem a noção de normal pela procura da evolução por recurso à memória e ao conhecimento, da invisibilidade pela integração e diluição do produto e projecto, e da liberdade como meio de adequação ao contexto actual. O livro resultante reflete uma abordagem definida de uma época, e não todo o panorama. A sistematização do conceito de normal a partir da segmentação em ideias menores, mais concretas e identificáveis nos produtos, procura proporcionar a consulta organizada de reacções ao contexto dominante actual, à ideia de especial, mas mais do que regras, procura fornecer um documento de reflexão a visitar no acto de projectar, seja como um todo, ou em partes.

Evaluation of the matabolic response of osteosarcoma cells to conventional and new anticancer drugs by NMR metabolomics

The main scope of this work was to evaluate the metabolic effects of anticancer agents (three conventional and one new) in osteosarcoma (OS) cells and osteoblasts, by measuring alterations in the metabolic profile of cells by nuclear magnetic resonance (NMR) spectroscopy metabolomics. Chapter 1 gives a theoretical framework of this work, beginning with the main metabolic characteristics that globally describe cancer as well as the families and mechanisms of action of drugs used in chemotherapy. The drugs used nowadays to treat OS are also presented, together with the Palladium(II) complex with spermine, Pd2Spm, potentially active against cancer. Then, the global strategy for cell metabolomics is explained and the state of the art of metabolomic studies that analyze the effect of anticancer agents in cells is presented. In Chapter 2, the fundamentals of the analytical techniques used in this work, namely for biological assays, NMR spectroscopy and multivariate and statistical analysis of the results are described. A detailed description of the experimental procedures adopted throughout this work is given in Chapter 3. The biological and analytical reproducibility of the metabolic profile of MG-63 cells by high resolution magic angle spinning (HRMAS) NMR is evaluated in Chapter 4. The metabolic impact of several factors (cellular integrity, spinning rate, temperature, time and acquisition parameters) on the 1H HRMAS NMR spectral profile and quality is analysed, enabling the definition of the best acquisition parameters for further experiments. The metabolic consequences of increasing number of passages in MG-63 cells as well as the duration of storage are also investigated. Chapter 5 describes the metabolic impact of drugs conventionally used in OS chemotherapy, through NMR metabolomics studies of lysed cells and aqueous extracts analysis. The results show that MG-63 cells treated with cisplatin (cDDP) undergo a strong up-regulation of lipid contents, alterations in phospholipid constituents (choline compounds) and biomarkers of DNA degradation, all associated with cell death by apoptosis. Cells exposed to doxorubicin (DOX) or methotrexate (MTX) showed much slighter metabolic changes, without any relevant alteration in lipid contents. However, metabolic changes associated with altered Krebs cycle, oxidative stress and nucleotides metabolism were detected and were tentatively interpreted at the light of the known mechanisms of action of these drugs. The metabolic impact of the exposure of MG-63 cells and osteoblasts to cDDP and the Pd2Spm complex is described in Chapter 6. Results show that, despite the ability of the two agents to bind DNA, the metabolic consequences that arise from exposure to them are distinct, namely in what concerns to variation in lipid contents (absent for Pd2Spm). Apoptosis detection assays showed that, differently from what was seen for MG-63 cells treated with cDDP, the decreased number of living cells upon exposure to Pd2Spm was not due to cell death by apoptosis or necrosis. Moreover, the latter agent induces more marked alterations in osteoblasts than in cancer cells, while the opposite seemed to occur upon cDDP exposure. Nevertheless, the results from MG-63 cells exposure to combination regimens with cDDP- or Pd2Spm-based cocktails, described in Chapter 7, revealed that, in combination, the two agents induce similar metabolic responses, arising from synergy mechanisms between the tested drugs. Finally, the main conclusions of this thesis are summarized in Chapter 8, and future perspectives in the light of this work are presented.

O interesse socioeducativo (e as dificuldades) de traduzir em português-espanhol

O Projeto que apresentamos insere-se nos estudos do Mestrado em Tradução Especializada dentro da vertente da Saúde e Ciências da Vida; é por isso que, partindo de um texto/suporte que visa promover uma alimentação saudável nas escolas (manual de referência difundido pelos Ministérios da Educação e Ciência e Ministério da Saúde de Portugal), procedemos à tradução (de português para espanhol) de um conteúdo que suscita, nos nossos dias, um grande interesse socioeducativo e cultural. É preciso destacar que o facto de ter nascido e vivido na Venezuela durante doze anos permitiu-me superar com êxito os meus estudos de espanhol na Licenciatura e no referido Mestrado; mas, ao mesmo tempo, tive que me adaptar, enquanto cidadã portuguesa, ao complexo e inacabado processo de ir interiorizando estas duas línguas – português/espanhol – tão próximas e no entanto com sistemas linguísticos bem distintos. Esta evidência encontra a sua maior justificação no grande capítulo dos erros (de forma e de significado) que consegui detetar e analisar graças à ajuda da minha professora orientadora, ao longo deste meu primeiro trabalho académico (texto traduzido para espanhol). Só agora, depois da minha primeira e “ingénua” tradução desse texto/manual, sou capaz de perceber com toda nitidez a dimensão do erro na atividade tradutora, assim como das suas consequências. Além desta importante reflexão, também o facto de elaborar este projeto permitiu-me abordar um aspeto linguístico da língua espanhola que me cativa pessoalmente (no que me diz respeito): o das variantes lexicais do castelhano nos países hispanofalantes. Não duvido em apontar um outro aspeto fundamental: o esforço de ter tido que corrigir minuciosamente e com rigor esta tarefa tradutora (com os seus respetivos comentários tradutológicos) converteu-se numa fonte de múltiplas satisfações pessoais; são aquelas que têm a ver com o cultivo de valores tais como o esforço, o espírito de superação, o exercício da responsabilidade e o (re)conhecimento da honestidade intelectual, entre outros.

Impact of exercise training on cancer-induced cardiac dysfunction

Cachexia is a complex syndrome characterized by severe weight loss frequently observed in cancer patients and associated with poor prognosis. Cancer cachexia is also related to modifications in cardiac muscle structure and metabolism leading to cardiac dysfunction. In order to better understand the cardiac remodeling induced by bladder cancer and the impact of exercise training after diagnosis on its regulation, we used an animal model of bladder cancer induced by exposition to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in the drinking water. Healthy animals and previously BBN exposed animals were submitted to a training program in a treadmill at a speed of 20m/min, 60 min/day, 5 days/week during 13 weeks. At the end of the protocol, animals exposed to BBN presented a significant decrease of body weight, in comparison with control groups, supporting the presence of cancer cachexia. Morphological analysis of the cardiac muscle sections revealed the presence of fibrosis and a significant decrease of cardiomyocyte’s cross-sectional area, suggesting the occurrence of cardiac dysfunction associated with bladder cancer. These modifications were accompanied by heart metabolic remodeling characterized by a decreased fatty acid oxidation given by diminished levels of ETFDH and of complex II subunit  from the respiratory chain. Exercise training promoted an increment of connexin 43, a protein involved in cardioprotection, and of c-kit, a protein present in cardiac stem cells. These results suggest an improved heart regenerative capacity induced by exercise training. In conclusion, endurance training seems an attractive non-pharmacological therapeutic option for the management of cardiac dysfunction in cancer cachexia.