Considerações sobre o papel da química bioinorgânica na saúde

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Metabolically active, non-nitrogen fixing,Trichodesmiumin UK coastal waters during winter

Trichodesmium, a colonial cyanobacterium typically associated with tropical waters, was observed between January and April 2014 in the western English Channel. Sequencing of the heterocyst differentiation (hetR) and 16S rRNA genes placed this community within the Clade IV Trichodesmium, an understudied clade previously found only in low numbers in warmer waters. Nitrogen fixation was not detected although measurable rates of nitrate uptake and carbon fixation were observed. Trichodesmium RuBisCO transcript abundance relative to gene abundance suggests the potential for viable and potentially active Trichodesmium carbon fixation. Observations of Trichodesmium when coupled with a numerical advection model indicate that Trichodesmium communities can remain viable for >3.5 months at temperatures lower than previously expected. The results suggest that Clade IV Trichodesmium occupies a different niche to other Trichodesmium species, and is a cold- or low-light-adapted variant.

Fonction de l'AmtB dans la régulation de la nitrogénase chez Rhodobacter capsulatus

La fixation de l’azote diatomique est un processus très important à la vie, vu sa nécessité dans la biosynthèse de plusieurs molécules de base; acides aminés, acides nucléiques, etc. La réduction de l’azote en ammoniaque est catalysée par la nitrogénase, une enzyme consommatrice de beaucoup d’énergie étant donné qu’elle nécessite 20 à 30 moles d’ATP pour la réduction d’une mole d’azote. De ce fait une régulation rigoureuse est exigée afin de minimiser le gaspillage d’énergie. Plusieurs systèmes de contrôle sont connus, aussi bien au niveau post-traductionnel que traductionnel. Chez la bactérie photosynthétique pourpre non-sulfureuse R. capsulatus, la régulation de l’activité de la nitrogénase nécessite une panoplie de protéines dont la protéine membranaire AmtB, qui est impliquée dans le transport et la perception d’ammonium, et les protéines PII qui jouent plusieurs rôles clés dans la régulation de l’assimilation d’azote. Suite à l’ajout de l’ammonium dans le milieu, une inhibition réversible de l’activité de la nitrogénase est déclenchée via un mécanisme d’ADP-ribosylation de la nitrogénase. La séquestration de GlnK (une protéine PII) par l’AmtB permet à DraT, une ADP-ribosyltransférase, d’ajouter un groupement ADP-ribose sur la protéine-Fe de la nitrogénase l’empêchant ainsi de former un complexe avec la protéine-MoFe. Donc, le transfert d’électrons est bloqué, engendrant ainsi l’inhibition de l’activité de la nitrogénase qui dure aussi long que la concentration d’azote fixé reste élevé, phénomène appelé le « Switch-off/Switch-on » de la nitrogénase. Dans ce mémoire, pour mieux comprendre ce phénomène de régulation, des mutations ponctuelles au niveau de certains résidus conservés de la protéine AmtB, dont D338, G367, H193 et W237, étaient générées par mutagénèse dirigée, afin d’examiner d’avantage leur rôle dans le transport d’ammonium, la formation du complexe AmtB-GlnK, ainsi que dans le « Switch-off » et l’ADP-ribosylation. Les résultats permettent de conclure l’importance et la nécessité de certains résidus telle que le G367 dans la régulation de la nitrogénase et le transport d’ammonium, contrairement au résidu D338 qui ne semble pas être impliqué directement dans la régulation de l’activité de la nitrogénase. Ces résultats suggèrent d’autres hypothèses sur les rôles des acides aminés spécifiques d’AmtB dans ses fonctions comme transporteur et senseur d’ammonium.

Studies on the isolation and characterization of flavones and triterpenoids from a few plants

In the present scenario, there is an increasing demand for natural products in food industry, pharmaceuticals, cosmetics and agricultural sectors. In this context phytochemical study to identify newer chemicals has got great relevance. Phytochemical studies have become more reliable and encouraging with the development of modern analytical techniques.In the present work the leaves of Piper colubrinum (Piperaceae), aerial parts of Mussaenda fiondosa (Rubiaceae) and Humboldtia vahliana (Leguminosae) and the pericarp of fruits of Artocarpus heterophyllus (Moraceae) were investigated for their secondary metabolites. The major compounds isolated belong to the groups of flavonoids and triterpenoids.Naturally occurring flavonoids have been used widely in chemotaxonomic studies of plants. Flavones and flavonols constitute a group of biosynthetically related natural products. No universal function has been established for flavones and flavonols in plants. However, many functions in individual plants have been demonstrated. These include protection of plants from ultraviolet light, insects and pests; pollinator attractants; antioxidants; plant hormone controllers; enzyme inhibitors and allelopathic agents. Flavonoids are attracting the attention of medical scientists in recent years because of their anticarcinogenic, antiallergic and antiinflammatory properties. The recent discovery that flavonoids are involved in the process of nitrogen fixation in plants also opens the way for agricultural application of these constituents.Triterpenoids are another class of compounds that are ubiquitous in plants. Some triterpenoids present in the latex and resins of plants are believed to be involved in chemical defence against pathogens and herbivores. Triterpenoids possess various biological properties including anti-inflammatory, antifeedant, pesticidal, fungitoxic and antimicrobial activities. Triterpenoids with cytotoxic activity and inhibitory effect on seed germination are also known.

Alternative plant protein sources for pigs and chickens in the tropics – nutritional value and constraints: a review

In the tropics, a large number of smallholder farms contribute significantly to food security by raising pigs and poultry for domestic consumption and for sale on local markets. The high cost and, sometimes, the lack of availability of commercial protein supplements is one of the main limitations to efficient animal production by smallholders. Locally-grown forages and grain legumes offer ecological benefits such as nitrogen fixation, soil improvement, and erosion control which contribute to improve cropping efficiency. Besides these agronomical assets, they can be used as animal feeds in mixed farming systems. In this paper we review options to include locally-grown forages and grain legumes as alternative protein sources in the diets of pigs and poultry in order to reduce farmers’ dependence on externally-purchased protein concentrates. The potential nutritive value of a wide range of forages and grain legumes is presented and discussed. The influence of dietary fibre and plant secondary metabolites contents and their antinutritive consequences on feed intake, digestive processes and animal performances are considered according to the varying composition in those compounds of the different plant species and cultivars covered in this review. Finally, methods to overcome the antinutritive attributes of the plant secondary metabolites using heat, chemical or biological treatment are reviewed regarding their efficiency and their suitability in low input farming systems.

Iron Uptake and Homeostasis in Microorganisms

Preface. Iron is considered to be a minor element employed, in a variety of forms, by nearly all living organisms. In some cases, it is utilised in large quantities, for instance for the formation of magnetosomes within magnetotactic bacteria or during use of iron as a respiratory donor or acceptor by iron oxidising or reducing bacteria. However, in most cases the role of iron is restricted to its use as a cofactor or prosthetic group assisting the biological activity of many different types of protein. The key metabolic processes that are dependent on iron as a cofactor are numerous; they include respiration, light harvesting, nitrogen fixation, the Krebs cycle, redox stress resistance, amino acid synthesis and oxygen transport. Indeed, it is clear that Life in its current form would be impossible in the absence of iron. One of the main reasons for the reliance of Life upon this metal is the ability of iron to exist in multiple redox states, in particular the relatively stable ferrous (Fe2+) and ferric (Fe3+) forms. The availability of these stable oxidation states allows iron to engage in redox reactions over a wide range of midpoint potentials, depending on the coordination environment, making it an extremely adaptable mediator of electron exchange processes. Iron is also one of the most common elements within the Earth’s crust (5% abundance) and thus is considered to have been readily available when Life evolved on our early, anaerobic planet. However, as oxygen accumulated (the ‘Great oxidation event’) within the atmosphere some 2.4 billion years ago, and as the oceans became less acidic, the iron within primordial oceans was converted from its soluble reduced form to its weakly-soluble oxidised ferric form, which precipitated (~1.8 billion years ago) to form the ‘banded iron formations’ (BIFs) observed today in Precambrian sedimentary rocks around the world. These BIFs provide a geological record marking a transition point away from the ancient anaerobic world towards modern aerobic Earth. They also indicate a period over which the bio-availability of iron shifted from abundance to limitation, a condition that extends to the modern day. Thus, it is considered likely that the vast majority of extant organisms face the common problem of securing sufficient iron from their environment – a problem that Life on Earth has had to cope with for some 2 billion years. This struggle for iron is exemplified by the competition for this metal amongst co-habiting microorganisms who resort to stealing (pirating) each others iron supplies! The reliance of micro-organisms upon iron can be disadvantageous to them, and to our innate immune system it represents a chink in the microbial armour, offering an opportunity that can be exploited to ward off pathogenic invaders. In order to infect body tissues and cause disease, pathogens must secure all their iron from the host. To fight such infections, the host specifically withdraws available iron through the action of various iron depleting processes (e.g. the release of lactoferrin and lipocalin-2) – this represents an important strategy in our defence against disease. However, pathogens are frequently able to deploy iron acquisition systems that target host iron sources such as transferrin, lactoferrin and hemoproteins, and thus counteract the iron-withdrawal approaches of the host. Inactivation of such host-targeting iron-uptake systems often attenuates the pathogenicity of the invading microbe, illustrating the importance of ‘the battle for iron’ in the infection process. The role of iron sequestration systems in facilitating microbial infections has been a major driving force in research aimed at unravelling the complexities of microbial iron transport processes. But also, the intricacy of such systems offers a challenge that stimulates the curiosity. One such challenge is to understand how balanced levels of free iron within the cytosol are achieved in a way that avoids toxicity whilst providing sufficient levels for metabolic purposes – this is a requirement that all organisms have to meet. Although the systems involved in achieving this balance can be highly variable amongst different microorganisms, the overall strategy is common. On a coarse level, the homeostatic control of cellular iron is maintained through strict control of the uptake, storage and utilisation of available iron, and is co-ordinated by integrated iron-regulatory networks. However, much yet remains to be discovered concerning the fine details of these different iron regulatory processes. As already indicated, perhaps the most difficult task in maintaining iron homeostasis is simply the procurement of sufficient iron from external sources. The importance of this problem is demonstrated by the plethora of distinct iron transporters often found within a single bacterium, each targeting different forms (complex or redox state) of iron or a different environmental condition. Thus, microbes devote considerable cellular resource to securing iron from their surroundings, reflecting how successful acquisition of iron can be crucial in the competition for survival. The aim of this book is provide the reader with an overview of iron transport processes within a range of microorganisms and to provide an indication of how microbial iron levels are controlled. This aim is promoted through the inclusion of expert reviews on several well studied examples that illustrate the current state of play concerning our comprehension of how iron is translocated into the bacterial (or fungal) cell and how iron homeostasis is controlled within microbes. The first two chapters (1-2) consider the general properties of microbial iron-chelating compounds (known as ‘siderophores’), and the mechanisms used by bacteria to acquire haem and utilise it as an iron source. The following twelve chapters (3-14) focus on specific types of microorganism that are of key interest, covering both an array of pathogens for humans, animals and plants (e.g. species of Bordetella, Shigella, , Erwinia, Vibrio, Aeromonas, Francisella, Campylobacter and Staphylococci, and EHEC) as well as a number of prominent non-pathogens (e.g. the rhizobia, E. coli K-12, Bacteroides spp., cyanobacteria, Bacillus spp. and yeasts). The chapters relay the common themes in microbial iron uptake approaches (e.g. the use of siderophores, TonB-dependent transporters, and ABC transport systems), but also highlight many distinctions (such as use of different types iron regulator and the impact of the presence/absence of a cell wall) in the strategies employed. We hope that those both within and outside the field will find this book useful, stimulating and interesting. We intend that it will provide a source for reference that will assist relevant researchers and provide an entry point for those initiating their studies within this subject. Finally, it is important that we acknowledge and thank wholeheartedly the many contributors who have provided the 14 excellent chapters from which this book is composed. Without their considerable efforts, this book, and the understanding that it relays, would not have been possible. Simon C Andrews and Pierre Cornelis

Comparative genomics suggests that an ancestral polyploidy event leads to enhanced root nodule symbiosis in the Papilionoideae

Root nodule symbiosis (RNS) is one of the most efficient biological systems for nitrogen fixation and it occurs in 90% of genera in the Papilionoideae, the largest subfamily of legumes. Most papilionoid species show evidence of a polyploidy event occurred approximately 58 million years ago. Although polyploidy is considered to be an important evolutionary force in plants, the role of this papilionoid polyploidy event, especially its association with RNS, is not understood. In this study, we explored this role using an integrated comparative genomic approach and conducted gene expression comparisons and gene ontology enrichment analyses. The results show the following: (1) approximately a quarter of the papilionoid-polyploidy-derived duplicate genes are retained; (2) there is a striking divergence in the level of expression of gene duplicate pairs derived from the polyploidy event; and (3) the retained duplicates are frequently involved in the processes crucial for RNS establishment, such as symbiotic signalling, nodule organogenesis, rhizobial infection and nutrient exchange and transport. Thus, we conclude that the papilionoid polyploidy event might have further refined RNS and induced a more robust and enhanced symbiotic system. This conclusion partly explains the widespread occurrence of the Papilionoideae.

Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida

Taxonomic characterization was performed on the putative N-2-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of Sao Sebastiao (Sao Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n = 76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n = 7), V. alginolyticus (n = 8), V. campbellii (n = 3), and V parahaemolyticus (n = 1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N, might explain why they are so abundant in the mucus of different coral species. (C) 2008 Published by Elsevier GmbH.

Application Of Real-Time Control Strategy To Improve Nitrogen Removal In Wastewater Treatment

Biological nitrogen removal is an important task in the wastewater treatment. However, the actual removal of total nitrogen (TN) in the wastewater treatment plant (WWTP) is often unsatisfactory due to several causes, one of which is the insufficient availability of carbon source. One possible approach to improve the nitrogen removal therefore is addition of external carbon source, while the amount of which is directly related to operation cost of a WWTP. It is obviously necessary to determine the accurate amount of addition of external carbon source according to the demand depending on the influent wastewater quality. This study focused on the real-time control of external carbon source addition based on the on-line monitoring of influent wastewater quality. The relationship between the influent wastewater quality (specifically the concentration of COD and ammonia) and the demand of carbon source was investigated through experiments on a pilot-scale A/O reactor (1m3) at the Nanjing WWTP, China. The minimum doses of carbon source addition at different situations of influent wastewater quality were determined to ensure the effluent wastewater quality meets the discharge standard. The obtained relationship is expected to be applied in the full-scale WWTPs. .

Inoculação com Azospirillum brasilense, fontes e doses de nitrogênio na cultura do milho em duas épocas de semeadura

Pós-graduação em Agronomia - FEIS

Consórcio de milho e Urochloa ruziziensis e inoculação com Azospirillum brasilense e seu efeito residual associado à adubação nitrogenada em feijoeiro de inverno

Pós-graduação em Agronomia - FEIS

Epicoccum nigrum P16, a Sugarcane Endophyte, Produces Antifungal Compounds and Induces Root Growth

Background: Sugarcane is one of the most important crops in Brazil, mainly because of its use in biofuel production. Recent studies have sought to determine the role of sugarcane endophytic microbial diversity in microorganism-plant interactions, and their biotechnological potential. Epicoccum nigrum is an important sugarcane endophytic fungus that has been associated with the biological control of phytopathogens, and the production of secondary metabolites. In spite of several studies carried out to define the better conditions to use E. nigrum in different crops, little is known about the establishment of an endophytic interaction, and its potential effects on plant physiology. Methodology/Principal Findings: We report an approach based on inoculation followed by re-isolation, molecular monitoring, microscopic analysis, plant growth responses to fungal colonization, and antimicrobial activity tests to study the basic aspects of the E. nigrum endophytic interaction with sugarcane, and the effects of colonization on plant physiology. The results indicate that E. nigrum was capable of increasing the root system biomass and producing compounds that inhibit the in vitro growth of sugarcane pathogens Fusarium verticillioides, Colletotrichum falcatum, Ceratocystis paradoxa, and Xanthomomas albilineans. In addition, E. nigrum preferentially colonizes the sugarcane surface and, occasionally, the endophytic environment. Conclusions/Significance: Our work demonstrates that E. nigrum has great potential for sugarcane crop application because it is capable of increasing the root system biomass and controlling pathogens. The study of the basic aspects of the interaction of E. nigrum with sugarcane demonstrated the facultative endophytism of E. nigrum and its preference for the phylloplane environment, which should be considered in future studies of biocontrol using this species. In addition, this work contributes to the knowledge of the interaction of this ubiquitous endophyte with the host plant, and also to a better use of microbial endophytes in agriculture.

Responses to rapid warming at Termination 1a at Gerzensee (Central Europe): Primary succession, albedo, soils, lake development, and ecological interactions

The transition from the Oldest Dryas to the Bølling around 14,685 cal yr BP was a period of extremely rapid climatic warming. From a single core of lake marl taken at Gerzensee (Switzerland) we studied the transition in stable isotopes of oxygen and carbon on bulk sediment and charophyte remains, as well as on monospecific samples of ostracods, after Pisidium a; in addition pollen, chironomids, and Cladocera were analyzed. The δ18O record serves as an estimate of mean air temperature, and by correlation to the one from NGRIP in Greenland it provides a timescale. The timing of responses: The statistically significant zone boundaries of the biostratigraphies are telescoped at the rapid increase of about 3‰ in δ18O at the onset of Bølling. Biotic responses may have occurred within sampling resolution (8 to 16 years), although younger zone boundaries are less synchronous. Gradual and longer-lasting responses include complex processes such as primary or secular succession. During the late-glacial interstadial of Bølling and Allerød, two stronger and two weaker cool phases were found. Biological processes involved in the responses occurred on levels of individuals (e.g. pollen productivity), of populations (increases or decreases, immigration, or extinction), and on the ecosystem level (species interactions such as facilitation or competition). Abiotic and biotic interactions include pedogenesis, nitrogen-fixation, nutrient cycling, catchment hydrology, water chemistry of the lake and albedo (controlled by the transition from tundra to forest). For the Swiss Plateau this major change in vegetation induced a change in the mammal fauna, which in turn led to changes in the tool-making by Paleolithic people.

Vegetation responses to rapid warming and to minor climatic fluctuations during the Late-Glacial Interstadial (GI-1) at Gerzensee (Switzerland)

High-resolution pollen analyses made on the same samples on which the ratios of oxygen isotopes were measured that provided the time scale and a temperature proxy after correlation to NorthGRIP. (1) A primary succession: The vegetation responded to the rapid rise of temperatures around 14,685 yr BP, with a primary succession on a decadal to centennial time scale. The succession between ca 15,600 and 13,000 yr BP included: (1.1.) The replacement of shrub-tundra by woodland of Juniperus and tree birch (around 14,665 yr BP) (1.2.) The response of Juniperus pollen to the shift in oxygen isotopes in less than 20 yr, (1.3.) A sequence of population increases of Hippophaë rhamnoides (ca 14,600 yr BP), Salix spp. (ca 14,600 yr BP), Betula trees (ca.14,480 yr BP), Populus cf. tremula (ca. 14,300 yr BP), and Pinus cf. sylvestris (ca. 13,830 yr BP). (2) Biological processes: Plants responded to the rapid increase of summer temperatures on all organisational levels: (2.1) Individuals may have produced more pollen (e.g. Juniperus); (2.2) Populations increased or decreased (e.g. Juniperus, Betula, later Pinus), and (2.3) Populations changed their biogeographical range and may show migrational lags. (2.4) Plant communities changed in their composition because the species pools changed through immigration and (local) extinction. Some plant communities may have been without modern analogue.These mechanisms require increasing amounts of time. (2.5) Processes on the level of ecosystems, with species interactions, may involve various time scales. Besides competition and facilitation, nitrogen fixation is discussed. (3) The minor fluctuations of temperature during the Late-Glacial Interstadial, which are recorded in δ18O, resulted in only very minor changes in pollen during the Aegelsee Oscillation (Older Dryas biozone, GI-1d) and the Gerzensee Oscillation (GI-1b). (4) Biodiversity: The afforestation at the onset of Bølling coincided with a gradual increase of taxonomic diversity up to the time of the major Pinus expansion.

Biotic and abiotic parameters and cynobacteria abundance during an experimental set-up with varying pCO2 conditions

Heterocystous cyanobacteria of the genus Nodularia form extensive blooms in the Baltic Sea and contribute substantially to the total annual primary production. Moreover, they dispense a large fraction of new nitrogen to the ecosystem when inorganic nitrogen concentration in summer is low. Thus, it is of ecological importance to know how Nodularia will react to future environmental changes, in particular to increasing carbon dioxide (CO2) concentrations and what consequences there might arise for cycling of organic matter in the Baltic Sea. Here, we determined carbon (C) and dinitrogen (N2) fixation rates, growth, elemental stoichiometry of particulate organic matter and nitrogen turnover in batch cultures of the heterocystous cyanobacterium Nodularia spumigena under low (median 315 µatm), mid (median 353 µatm), and high (median 548 µatm) CO2 concentrations. Our results demonstrate an overall stimulating effect of rising pCO2 on C and N2 fixation, as well as on cell growth. An increase in pCO2 during incubation days 0 to 9 resulted in an elevation in growth rate by 84 ± 38% (low vs. high pCO2) and 40 ± 25% (mid vs. high pCO2), as well as in N2 fixation by 93 ± 35% and 38 ± 1%, respectively. C uptake rates showed high standard deviations within treatments and in between sampling days. Nevertheless, C fixation in the high pCO2 treatment was elevated compared to the other two treatments by 97% (high vs. low) and 44% (high vs. mid) at day 0 and day 3, but this effect diminished afterwards. Additionally, elevation in carbon to nitrogen and nitrogen to phosphorus ratios of the particulate biomass formed (POC : POP and PON : POP) was observed at high pCO2. Our findings suggest that rising pCO2 stimulates the growth of heterocystous diazotrophic cyanobacteria, in a similar way as reported for the non-heterocystous diazotroph Trichodesmium. Implications for biogeochemical cycling and food web dynamics, as well as ecological and socio-economical aspects in the Baltic Sea are discussed.