Chemical composition and phytoplankton characteristics of water samples obtained during POLARSTERN cruise ANT-XXIV/3

The fixation of dissolved inorganic carbon (DIC) by marine phytoplankton provides an important feedback mechanism on concentrations of CO2 in the atmosphere. As a consequence it is important to determine whether oceanic primary productivity is susceptible to changing atmospheric CO2 levels Among numerous other factors, the acquisition of DIC by microalgae particularly in the polar seas is projected to have a significant effect on future phytoplanktonic production and hence atmospheric CO2 concentrations. Using the isotopic disequilibrium technique the contribution of different carbon species (CO2 and bicarbonate) to the overall DIC uptake and the extent to which external Carbonic Anhydrase (eCA) plays a role in facilitating DIC uptake was estimated. Simultaneous uptake of CO2 and HCO3- was observed in all cases, but the proportions in which different DIC species contributed to carbon assimilation varied considerably between stations. Bicarbonate as well as CO2 could be the major DIC source for local phytoplankton assemblages. There was a positive correlation between the contribution of CO2 to total DIC uptake and ambient concentration of CO2 in seawater suggesting that Southern Ocean microalgae could increase the proportion of CO2 uptake under future high atmospheric CO2 levels. Results will be discussed in view of metabolic costs related to DIC acquisition of Southern Ocean phytoplankton.

Analysis of Alpha CA Genes in Early Vertebrates and ; High-Invertebrates

Carbonic anhydrases are enzymes that are ubiquitously found in all organisms that are engaged in catalyzing the hydration of carbon dioxide to form bicarbonate and proton and vice versa. They are crucial in the process of respiration, bone resorption, pH regulation, ion transport, and photosynthesis in plants. Out of the five classes of carbonic anhydrase α, β, γ, δ, ζ this study focused in the α carbonic anhydrases. This class of CAs constitute of 16 subfamilies in mammals that include 3 non-active enzymes known as Carbonic Anhydrase Related Proteins. The inactiveness of these enzymes is due to the loss of one or more Histidine residues in the active site. This thesis was conducted based on the aim of studying evolutionary analysis of carbonic anhydrase sequences from organisms spanning from the Cambrian age. It was carried out in two phases. The first phase was the sequence collection, which involved many biological sequence databases as a source. The scope of this segment included sequence alignments and analysis of the sequence manually and in an automated form incorporating few analysis tools. The second Phase was phylogenetic analysis and exploring the subcellular location of the proteins, which was key for the evolutionary analysis. Through the medium of the methods conducted with respect to the phases mentioned above, it was possible to accomplish the desired result. Certain thought-provoking sequences were come across and analyzed thoroughly. Whereas, Phylogenetics showed interesting results to bolster previous findings and new findings as well which lay bedrock for future intensified studies.

Produção e extração da enzima anidrase carbônica e de ficobiliproteínas a partir de microalgas

Muito interesse tem sido focado no potencial biotecnológico das microalgas, principalmente devido à identificação de diversas substâncias sintetizadas por estes organismos, dentre elas a anidrase carbônica e as ficobiliproteínas. A anidrase carbônica é uma metaloenzima que catalisa a hidratação reversível do CO2 em bicarbonato com alta eficiência, sendo utilizada para captação de CO2 através de sistemas biológicos. A C-ficocianina e a aloficocianina, corantes naturais, são os dois principais componentes das ficobiliproteínas em cianobactérias e apresentam diversas aplicações dentro da indústria alimentícia, cosmética e farmacêutica. O objetivo principal desta tese foi avaliar a produção e a extração da anidrase carbônica e das ficobiliproteínas a partir de diferentes microalgas. Para isso, primeiramente foi realizado uma investigação da produção da anidrase carbônica pela microalga Dunaliella tertiolecta, onde foi estudada a extração da enzima e sua aplicação em sistemas de captura enzimática de CO2. Posteriormente foi avaliada a produção da enzima ao longo do cultivo de diferentes microalgas marinhas e dulcícolas (Dunaliella tertiolecta, Tetraselmis sueccica, Phaeodactylum tricornutum, Nannochloropsis oculata, Isochysis galbana, Chlorella vulgaris e Scenedesmus obliquus). A produção da enzima e de ficobiliproteínas, também, foi estudada para as cianobactérias Spirulina platensis LEB 52, Spirulina sp. LEB 18 e Synechococcus nidulans. Todos os cultivos foram acompanhados em termos de biomassa e pH. Por último, foi realizado um estudo de extração da enzima de P. tricornutum e extração conjunta da anidrase carbônica e de ficobiliproteínas da cianobactéria S. sp. LEB 18. Os cultivos foram realizados em frascos erlenmeyer contendo os meios Conway (marinhas), BG-11 (dulcícolas) e Zarrouk 20% (cianobactérias). Na avaliação da ruptura celular foram testadas as técnicas de maceração em gral e pistilo, agitação em vórtex com pérolas de vidro, sonicação com pérolas de vidro, homogeneizador ultrassônico, secagem, congelamento e descongelamento e a combinação de tratamentos. Maiores rendimentos de extração da enzima a partir da microalga D. tertiolecta foram obtidos utilizando tratamento ultrassônico, juntamente com baixas concentrações de biomassa úmida (0,1 e 0,2 g/L), e a mesma apresentou potencial para aplicação em processos de captação enzimática do CO2. Durante os cultivos, a microalga C. vulgaris se destacou como maior produtora da enzima anidrase carbônica, atingindo valores de atividade enzimática de 44,0 U/L. As cianobactérias apresentaram valores de atividade entre 41,6 e 45,9 U/L, sendo que a S. sp. LEB 18 foi a que apresentou maiores produções de C-ficocianina e aloficocianina no ponto de máxima atividade volumétrica, 65,9 e 82,2 µg/mL, respectivamente. A enzima extraída da biomassa de S. platensis LEB 52 catalisou a hidratação do CO2 que precipitou na forma de CaCO3. Maiores rendimentos de extração da enzima a partir das microalgas P. tricornutum e S. sp. LEB 18 foram obtidos utilizando homogeneizador ultrassônico, que foram 31,3 U/g e 25,5 U/g, respectivamente. A biomassa de S. sp. LEB 18, também apresentou potencial para a extração de ficobiliproteínas, obtendo- se altas concentrações de C-ficocianina (100,5 mg/g) e aloficocianina (69,9 mg/g). Através dos resultados obtidos, pode-se verificar a potencialidade das microalgas e das cianobactérias para produção da enzima anidrase carbônica e das ficobiliproteínas, biomoléculas de alto valor industrial. Este trabalho apresenta processos eficientes para a extração da enzima e de ficobiliproteínas tanto para escala laboratorial como industrial.

Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex

The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific 'carbonic anhydrase domain' of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe 'life without complex I'.

The conserved and divergent roles of carbonic anhydrases in the filamentous fungi Aspergillus fumigatus and Aspergillus nidulans

P>Carbon dioxide (CO(2)) and its hydration product bicarbonate (HCO(3)-) are essential molecules in various physiological processes of all living organisms. The reversible interconversion between CO(2) and HCO(3)- is in equilibrium. This reaction is slow without catalyst, but can be rapidly facilitated by Zn2+-metalloenzymes named carbonic anhydrases (CAs). To gain an insight into the function of multiple clades of fungal CA, we chose to investigate the filamentous fungi Aspergillus fumigatus and A. nidulans. We identified four and two CAs in A. fumigatus and A. nidulans, respectively, named cafA-D and canA-B. The cafA and cafB genes are constitutively, strongly expressed whereas cafC and cafD genes are weakly expressed but CO(2)-inducible. Heterologous expression of the A. fumigatus cafB, and A. nidulans canA and canB genes completely rescued the high CO(2)-requiring phenotype of a Saccharomyces cerevisiae Delta nce103 mutant. Only the Delta cafA Delta cafB and Delta canB deletion mutants were unable to grow at 0.033% CO(2), of which growth defects can be restored by high CO(2). Defects in the CAs can affect Aspergilli conidiation. Furthermore, A. fumigatus Delta cafA, Delta cafB, Delta cafC, Delta cafD and Delta cafA Delta cafB mutant strains are fully virulent in a low-dose murine infection.

Quantification of ruminal microbiota and production of methane and carbonic dioxide from diets with inclusion of glycerin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The carbonic acid industry

Mode of access: Internet.

Ambiência aérea em alojamento de frangos de corte: poeira e gases

Air quality in animal production environment has been refereed as an interesting point for studies in environmental control systems with the focus both to the animal health which live in total confinement, as to the workers. The objective of this research was to determine the variation on the aerial environmental quality in two types of broiler housing: conventional (Gc) and tunnel type (Gt). The total dust values in both houses offered adequate rearing conditions to the birds; however, regarding the inhale dust in the air was above the limits recommended for humans. Carbon monoxide concentration in the heating phase during the evaluated period was above the 10 ppm maximum recommended, and it was higher during the cold season in Gt house (30 ppm) when compared to the Gc house (18 ppm). Ammonia concentration peaks in the air were above the 20 ppm recommended from the 20th day of production in both houses and in daily average, for a period higher in Gt (4h30) when compared to Gt (2h45). Only traces of nitrate oxide and methane were found while carbonic dioxide gas concentration evaluated during daytime met the limits allowed for both birds and labor.

Chemical and in vitro study of the potential of 3-(aryl)-2-sulfanylpropenoic acids and their Zn(II) complexes as protective agents against cadmium toxicity

The free H(2)xspa ligands [xspa = pspa, Clpspa, tspa or fspa where p = 3-(phenyl), Clp = 3-(2-chlorophenyl), t = 3-(2-thienyl), f = 3-(2-furyl) and spa = 2-sulfanylpropenoato], their Zn(II) complexes of formula [HQ](2)[Zn(xspa)(2)] (HQ=diisopropylammonium) and the Cd(II) equivalents were prepared and characterized by elemental analysis and by IR, Raman and NMR ((1)H, (13)C) spectroscopy. X-Ray studies of the crystal structures of [HQ](2)[Zn(pspa)(2)], [HQ](2)[Zn(Clpspa)2], [HQ](2)[Zn(tspa)(2)] and [HQ](2)[Zn(fspa)(2)] show that the zinc atom is coordinated to two O atoms and two S atoms of the ligands in a distorted tetrahedral ZnO(2)S(2) environment. In the structures of [HQ](2)[Cd(pspa)(2)] and [HQ](2)[Cd(Clpspa)(2)] the cadmium atom is coordinated to three S atoms and two carboxylato O atoms of the ligands in a distorted trigonal bipyramidal environment. The interchange of ligands between Zn( II) and Cd( II) was studied by (113)Cd NMR spectroscopy. The in vitro protective effect of H(2)xspa and their Zn( II) complexes against Cd toxicity was investigated using the human hepatocarcinoma HepG2 cell line and the pig renal proximal tubule LLC-PK1 cell line. The incorporation of Zn( II) was found to be relevant in the case of H(2)pspa, with an increase observed in the cell viability of the LCC-PK1 cells with respect to the value for the free ligand.

Gene expression profile analysis of primary glioblastomas and non-neoplastic brain tissue: identification of potential target genes by oligonucleotide microarray and real-time quantitative PCR

The prognosis of glioblastomas is still extremely poor and the discovery of novel molecular therapeutic targets can be important to optimize treatment strategies. Gene expression analyses comparing normal and neoplastic tissues have been used to identify genes associated with tumorigenesis and potential therapeutic targets. We have used this approach to identify differentially expressed genes between primary glioblastomas and non-neoplastic brain tissues. We selected 20 overexpressed genes related to cell cycle, cellular movement and growth, proliferation and cell-to-cell signaling and analyzed their expression levels by real time quantitative PCR in cDNA obtained from microdissected fresh tumor tissue from 20 patients with primary glioblastomas and from 10 samples of non-neoplastic white matter tissue. The gene expression levels were significantly higher in glioblastomas than in non-neoplastic white matter in 18 out of 20 genes analyzed: P < 0.00001 for CDKN2C, CKS2, EEF1A1, EMP3, PDPN, BNIP2, CA12, CD34, CDC42EP4, PPIE, SNAI2, GDF15 and MMP23b; and NFIA (P: 0.0001), GPS1 (P: 0.0003), LAMA1 (P: 0.002), STIM1 (P: 0.006), and TASP1 (P: 0.01). Five of these genes are located in contiguous loci at 1p31-36 and 2 at 17q24-25 and 8 of them encode surface membrane proteins. PDPN and CD34 protein expression were evaluated by immunohistochemistry and they showed concordance with the PCR results. The present results indicate the presence of 18 overexpressed genes in human primary glioblastomas that may play a significant role in the pathogenesis of these tumors and that deserve further functional investigation as attractive candidates for new therapeutic targets.

Diagnóstico Energético e proposta de melhorias numa unidade de Produção de Peças de Borracha

O consumo de energia de forma irracional acarreta desvantagens a nível económico para o consumidor e problemas ambientais para toda a sociedade, como a escassez de recursos naturais e o aumento da poluição. Neste contexto, a otimização energética na indústria, e em particular no setor das borrachas, é indispensável de forma a utilizar racionalmente a energia e assim contribuir para a viabilidade das empresas. Este trabalho, efetuado na Flexocol - Fábrica de Artefactos de Borracha, Lda., teve como principal objetivo efetuar um levantamento energético à unidade fabril e propor alternativas que permitissem a redução do consumo de energia elétrica. Foi ainda realizado um estudo sobre a possibilidade de substituir o n-hexano, solvente utilizado na limpeza dos moldes, por um solvente mais adequado. O levantamento energético efetuado permitiu identificar o consumo das utilidades existentes na Flexocol. Esta empresa consome gasóleo e energia elétrica, sendo esta última, a forma de energia mais consumida correspondendo a 96%. O consumo global de energia é cerca 151 tep anuais, inferior a 500 tep/ano, ou seja é considerada uma empresa não consumidora intensiva de energia. Com base neste levantamento determinou-se os indicadores de consumo específico de energia e da intensidade carbónica, 2,73 tep/ ton e 1684,5 kg CO2/tep. A análise do consumo de energia elétrica dos diferentes equipamentos permitiu verificar que o setor que mais consome energia elétrica é a Vulcanização com 45,8%, seguido do setor da Mistura e Serralharia com 27,5% e 26,7%, respetivamente. O sistema de iluminação nos vários setores foi também alvo de estudo e permitiu identificar a Vulcanização como o setor com mais consumo e o da Mistura como o que menos consome. O estudo das variáveis anteriormente referidas permitiu apresentar algumas propostas de melhoria. Uma das propostas analisada foi implementação de condensadores no quadro parcial de forma a diminuir a energia reativa. Com esta medida prevê-se uma poupança de 5631 €/ano e um retorno de investimento de 0,045 anos. Foi também analisada relativamente à iluminação a possibilidade de instalação de balastros eletrónicos que conduziria a uma poupança na energia elétrica de cerca 7072 kWh/ano, mas com um retorno de investimento desfavorável. Por último estudou-se o solvente alternativo ao n-hexano. A acetona foi o solvente proposto uma vez que tem as propriedades indicadas para o fim a que se destina.

Focus on: II – physiological principles of acid-base balance: an integrative perspective

Normal human metabolism leads to the daily production of large amounts of volatile and non-volatile acids. The maintenance of the pH within physiological limits is a demanding task in which several mechanisms are involved. The most immediate answer comes from several physiological buffers that quickly neutralize pH deviations caused by the addition of strong acids or bases to the body. Bicarbonate/carbonic acid is the most important buffer pair of the extracellular milieu, but is chemically inefficient and depends on the continuous activity of the lung and kidney. Other physiological buffers have higher efficacy and are very important in the intracellular environment and renal tubules. The capacity of the various chemical buffers is kept by operating in an open system and by several controlling mechanisms. The lung is responsible for the elimination of the carbon dioxide (CO2) produced in the body. In metabolic disorders, respiratory adjustment of the elimination of CO2 prolongs the effect of the bicarbonate/carbonic acid buffer, but this process consumes bicarbonate. The kidney contributes to acid-base balance through several mechanisms: 1) controls the reabsorption of filtered bicarbonate; 2) regenerates bicarbonate consumed in buffer reactions; 3) eliminates non-volatile acids. Renal elimination of acid and bicarbonate regeneration is only possible due to the existence of several urinary buffers and to the ability of the kidneys to produce ammonia