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Arcella Ehrenberg 1832 is one of the most numerous testacean genera. Arcellinids are an aerobic lineage of testate amoeba that live in a wide variety of environments. Probably their ability to survive in such divergent conditions is related to some de- gree of metabolic flexibility. Anaerobic organisms have gained and lost a number of genes related to energetic metabolism. These processes modify classic mitochondria until loss of function and transformation in mitochondrial related organelles (mitosomes and hydrogenosomes). Here I propose that Arcella intermedia adaptation to microarophilic environments is related to the acquisition of new genes. There are two main modes of acquisition of new genes. The traditional view, where duplication is followed by mutations and neo-functionality of the duplicate. Or genes can be acquired from other species (lateral gene transfers). The second process has a major importance in prokaryotic evolution and is probably under considered in eukaryotic evolution. I also propose in this work that genes related to anaerobic metabolism in Arcella are acquired by lateral gene transfer. However, analysis of genomic and transcriptomic data are absent for A.intermedia. Characterizing genome-scale data from eukaryotes is essential for gene discovery and for inferring transitions over the tree of life. The transcriptome dataset from this work provides the first effort of characterization of expressed sequences in A.intermedia. We used single cell from different moments of growth and whole culture RNA extraction in order to increase the diversity of metabolic moments of the cells. Mapped sequences allowed us to identify functional pathways in A.intermedia cells. In general, it seems that metabolic processes are showing up more, followed by signaling and responses to stimuli. We describe functioning of carbohydrate and energy metabolism including even an anaerobic pathway to produce energy. We found ACS-ADP and PFO genes in A.intermedia. We describe amino acid metabolism, with at least 12 amino acids metabolizing pathways described and catabolism mainly related to TCA cycle intermediates. Calcium, Ras GTPases, PI3K-AK and AMPK-mTOR are the main signaling pathways represented in transcriptomes. We described important pathways for amoeba: endocytosis and phagocytosis and it seems to be similar with the ones already described for other amoeba with a dependence on F-actin and small GTPases of Rho subfamily. We couldn\'t find lots of information about programmed cell death in A.intermedia, however cell growth are similar to pathways described for dinoflagellates. We expect that upcoming genomes will finish the description of functioning of those organisms, but we believe our work already is a good starting point. In order to gain a clearer picture of the presence of anaerobic metabolism genes in Amoebozoa, we conducted BLAST searches in Amoebozoa and Arcellinida data bases for the presence/absence of ACS-ADP, PFO and [FeFe]-H2ase. Other Arcellinida species also presented these genes, Difflugia sp., Difflugia compressa and Cyclopyxis lobostoma. Besides these, the already known Mastigamoeba balamuthi, Entamoeba histolytica and Acanthamoeba castelanii. Amoebozoa sequences don\'t form a monophyletic group in any of the three genes. However, Arcellinida sequences always grouped together. As such distinct amoeba groups have those anaerobic metabolism genes, however, most of the Amoebozoa do not. It is more likely to think of lateral transfers occurring independently among these amoeba groups, generating the possibility of occupying a new niche. The main objective of this work was to start generating tools to understand the ability of some testate amoeba to resist environmental harsh conditions. We found lots of interesting questions but the one we focused on this dissertation was (1) the evolution of anaerobic related genes in testate amoeba lineages. The assembled and annotated sequence data will be available as reference sequences, making the work with this group easier. The results can also potentially be applied as biomonitoring markers for the management of water resources. This work will improve the general knowledge on the evolution and function of freshwater organisms. We expect also to make a contribution on the understanding of the impact of lateral gene transfers in Arcellinida diversity / Arcella Ehrenberg 1832 é um dos gêneros de tecamebas mais numerosos, perten- cente aos Arcellinida. Estas são uma linhagem aeróbia de amebas tecadas que vivem em uma grande variedade de ambientes. Provavelmente, sua capacidade de sobreviver em condições tão divergentes está relacionada a algum grau de flexibilidade metabólica. Os organismos anaeróbicos ganharam e perderam vários genes relacionados ao metabolismo energético. Este processo modifica mitocôndrias clássicas até a perda da função e transformação em organelas relacionadas (mitossomos e hidrogenossomos). Aqui proponho que a adaptação de Arcella intermedia a ambientes microaerófilos está relacionada à aquisição de novos genes. Existem dois modos principais de aquisição de genes. Na visão tradicional, a duplicação gênica é responsável por gerar diversidade, seguida por mutações e neofuncionalidade da duplicata. Alternativamente, os genes podem ser adquiridos de outras espécies (transferências laterais de genes). O segundo processo tem uma grande importância evolutiva e é ainda pouco considerado na evolução eucariótica. Por isso, também proponho neste trabalho que genes relacionados ao metabolismo anaeróbico em Arcella sejam adquiridos por transferência lateral de genes. Entretanto, a análise de dados genômicos e transcriptômicos é inexistente para A.intermedia. A caracterização de dados em escala genômica de eucariotos é essencial para a descoberta de genes e para a inferência transições sobre a árvore da vida. O conjunto de dados de transcriptoma deste trabalho fornece um primeiro esforço de caracterização de sequências expressas em A. intermedia. Utilizamos extrações de célula-única em diferentes momentos de crescimento e extração de RNA de cultura inteira, a fim de aumentar a diversidade de momentos metabólicos das células. Sequências mapeadas permitiram identificar vias funcionais em células de A. intermedia. Em geral, parece que genes relacionados a processos metabo?licos são os que aparecem mais frequentemente, seguidos dos de sinalização e respostas a estímulos. Nós descrevemos a função do metabolismo de carboidratos e energia, incluindo uma via anaeróbica. Encontramos em A.intermedia os genes ACS-ADP e PFO. Descrevemos o metabolismo de aminoácidos, com pelo menos 12 vias metabólicas de aminoácidos descritas e catabolismo relacionado a intermediários do ciclo de TCA. Cálcio, Ras GTPases, PI3K-AK e AMPK-mTOR são as principais vias de sinalização representadas nos transcriptomas. Descrevemos importantes vias para amebas, que são endocitose e fagocitose. Parecem ser vias semelhantes àquelas já descritas para outras amebas, com dependência de F-actina e pequenas GTPases da subfamília Rho. Não conseguimos encontrar muitas informações sobre a morte celular programada em A. intermedia, mas o crescimento celular é semelhante com as vias descritas para os dinoflagelados. Esperamos que os próximos genomas terminem a descrição da função desses organismos, mas acreditamos que nosso trabalho já é um bom ponto de partida. A fim de obter uma visão mais clara da presença de genes de metabolismo anaeróbico em Amoebozoa, realizamos buscas no BLAST em bancos de dados de Amoebozoa e Arcellinida, para a presença/ausência de ACS-ADP, PFO e [FeFe] -H2ase. Outras espécies de Arcellinida também apresentaram estes genes, Difflugia sp, Difflugia compressa e Cyclopyxis lobostoma. Além destes, os já conhecidos Mastigamoeba balamuthi, Entamoeba histolytica e Acanthamoeba castelanii. Sequências de amebozoários não formam um grupo monofilético em nenhum dos três genes. No entanto, as sequencias de Arcellinida sempre se agrupam. Como são grupos de Amoebozoa de tal maneira distintos que possuem estes genes de metabolismo anaeróbico, e sendo que a maioria não possui, é mais provável que sejam transferências laterais independentes entre esses grupos de ameba, gerando a possibilidade de ocupar um novo nicho. O objetivo principal deste trabalho foi gerar ferramentas para entender a capacidade de algumas amebas tecadas em resistir a condições adversas do meio ambiente. Encontramos muitas questões interessantes, mas a que teve nosso foco nesta dissertação foi (1) a evolução de genes relacionados ao metabolismo anaeró?bio em linhagens de amebas tecadas. Os dados da sequência reunidos e anotados estarão disponíveis como sequências de referência, facilitando o trabalho com esse grupo. Os resultados também podem ser aplicados aos marcadores de biomonitoramento para o gerenciamento dos recursos hídricos. Este trabalho irá melhorar o conhecimento geral sobre a evolução e função de organismos de água doce. Esperamos tambem contribuir para a compreensão do impacto das transferências laterais na diversidade de Arcellinida

The purpose of this study was to investigate whether 3 days of dietary nitrate supplementation has positive effects on exercise performance and energy metabolism at rest and during strenuous knee exercise, and exercise recovery. The experimental protocol was a double blind, placebo-controlled, two-period, and within-subjects design. Fourteen healthy nonsmoking males (22.71 ± 0.72yr, 72.93 ± 2.35kg, and 47.67 ± 1.56 ml/kg/min VO2max) participated in the study. Two supplements (nitrate lozenge; NO-L or placebo; PLA) were orally administrated randomly at 48 and 24 hours before each trial day and again 40 minutes before each exercise trial. Total work, peak torque, and respiratory gases were automatically collected during repeated knee extensions/flexions (isokinetic concentric contractions with dominant leg; 4 sets of 28 repetitions at 180°/sec with 30 sec rest intervals) and recovery (6 contractions of 1 repetition maximum with 120 vii sec between contractions). For these results, peak torque, rate of fatigue, work efficiency, and rate of recovery were calculated. Blood specimens were collected at rest before and after the treatment, post exercise, and end of recovery to track the changes in blood glucose and lactate concentrations. There was a significant inverse correlation for total work during knee extension exercise and oxygen consumption (PLA: r = -.560 and NO-L: r = -.546; p < .01, respectively). During the exercise, RER was significantly higher for PLA compared with NO-L (PLA: 1.42 ± 0.02 vs. NO-L: 1.38 ± 0.02: p = .03). Work within each set and total work performed were higher for NO-L, but these differences were not significant. However, NO-L enhanced exercise efficiency by 3.3% when compared with PLA (PLA: 2497.5 ± 134.56 ft-lb/L/min vs. NO-L: 2578.7 ± 132.24 ftlb/L/min; p = .05). Extensor peak torque recovery rate slope was not significant, but meaningfully faster for NO-L (PLA: 2.39 ± 0.52% vs. NO-L: 3.06 ± 0.54%; p = .09) and significant interaction effect (p = .02) was found during recovery contractions, especially from contractions 2 to 3 (p = .03). It is also worth noting that differences in time to peak torque during knee extension exercise for NO-L and PLA approached significances (PLA: 0.213 ± 0.01 sec vs. NO-L: 0.200 ± 0.01 sec; p = .08). The results suggest that 3 days of dietary nitrate supplementation improves rate of exercise recovery and enhances work efficiency during vigorous resistance exercise. / text

Salinity and oxygen availability have long been recognised as important factors influencing animal physiology and therefore species distribution. The maintenance of appropriate cellular ion levels is critical for many essential physiological processes, but at the same time is energetically expensive. Since hypoxia is likely to impose aerobic limitations for ATP generation, the maintenance of salt and water homeostasis could be at risk during hypoxia. The amphidromous inanga (Galaxias maculatus) is well known for its salinity tolerance and its life cycle that involves several salinity related migrations. During these migrations inanga also frequently encounters hypoxic waters, and therefore must maintain energy homeostasis when aerobic metabolism may be compromised. The present study has investigated behavioural, physiological, biochemical and molecular mechanisms by which inanga tolerate changes in salinity and hypoxia. After 14 days of acclimation to salinities ranging from freshwater to 43‰, inanga showed physiological acclimation. This was evident by no changes in metabolic rates or energy expenditures through this salinity range. Energy balance seemed to be tightly and efficiently controlled by changes in the proportion of protein and lipids used as energy substrate. No mortalities and only minor changes in plasma osmolality also indicated salinity acclimation. The remarkable osmoregulatory capacity of inanga was also evidenced after a seawater challenge. The osmotic balance of inanga was only disrupted during the first 24 hours after the challenge, evidenced by an increase in plasma osmolality and plasma Na+, and a decrease in muscle water content. These physiological changes were correlated with changes at the molecular level. Different isoforms of the catalytic subunit of the Na+,K+-ATPase (NKA) were isolated, partially sequenced and identified in inanga. Phylogenetic analysis grouped inanga isoforms (α-1a, α-1b, α-1c) with their respective homologues from salmonids. Patterns of mRNA expression were also similar to salmonids, with α-1a being downregulated and α-1b being up-regulated following seawater challenge. Previous to this study, NKA isoform switching was reported to occur only in salmonids and cichlids. The presence of NKA subunits that change with environmnetal salinity in inanga indicates that this isoform switching phenomenon is much more widespread among teleost lineages than previously thought. Aiming to elucidate the hypoxia tolerance of inanga, oxygen consumption rate as a function of decreasing external PO2 was evaluated. At no point did inanga regulate oxygen consumption, suggesting that this species is an oxyconformer. This is the first robust demonstration of the existence of oxyconforming in fish. Evaluation of the scaling relationship between oxygen consumption and fish size in normoxia, showed that the exponent of this relationship fell within the range previously reported for fish. However, in hypoxic conditions the scaling relationship was less clear suggesting different size-related mechanisms for tolerating hypoxia. Analysis of the aerobic and anaerobic metabolism of small and large fish, showed that smaller inanga were able to sustain aerobic metabolism for longer than larger inanga, which instead relied on anaerobic metabolism for extending their survival. This knowledge is likely to be of value for the conservation of this iconic fish species, by incorporating these size related differences in hypoxia tolerance in streams management. In light of the unusual oxyconforming response of inanga, a study examining the behavioural responses of this species to declining dissolved oxygen was performed. Inanga did not display a behaviour that might reduce energy expenditure during oxygen limitation; instead swimming activity and speed were elevated relative to normoxia. As hypoxia deepened inanga leaped out of the water, emersing themselves on a floating platform. Once emersed, fish exhibited an enhanced oxygen consumption rate compared to fish that remained in hypoxic water. Although this emersion behaviour was hypothesised to be of physiological advantage, both aquatic hypoxia and emersion resulted in similar physiological and biochemical consequences in inanga. While in hypoxic water oxygen availability seemed to be the limiting factor, in air failure of the circulatory system was hypothesised to be the cause of a similar metabolic signature to that found in aquatic hypoxia. Overall, inanga seemed to be not particularly well adapted to tolerate aquatic hypoxia. In light of the increasing likelihood of anthropogenic-induced hypoxia in inanga habitats, this is likely to have negative consequences for the future of inanga populations in the wild. Although this study provides the mechanisms behind the exceptional salinity tolerance of inanga, its susceptibility to hypoxia is likely to impose further constraints for the osmoregulatory processes that guarantee inanga survival during life cycle migrations. The results of the present study are relevant for understanding and managing the fishery of this economically- and culturally important fish species.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Harvesting the apple at the correct maturity stage for long-term storage is becoming more and more difficult, especially due to the lack of manual labor. Thereby, the development of storage techniques that allows the storage of fruit harvested outside the correct maturity is necessary. Thus, on the present dissertation we aimed to evaluate a new dynamic controlled atmosphere storage technique based on the lower oxygen limit (LOL) monitoring by the respiratory quotient (DCA-RQ) and compare it with controlled atmosphere (CA) and the combination of CA with 1-methylcyclopropene application (CA + 1-MCP) on the physical, chemical quality and volatile profile of Galaxy apple harvested in three maturity stages (unripe, ripe and overripe). Two chapters composed the dissertation: the first is directed to evaluate the metabolism, physical and chemical quality and the second to evaluate the metabolism and volatile profile. Fruit of the three maturity stages stored under DCA-RQ showed lower ACC oxidase enzyme activity, internal ethylene, ethylene production and respiration rate as compared to fruit stored under CA. The storage under DCA-RQ 1.3 resulted in similar quality keeping as compared to CA + 1-MCP. Lower mealiness incidence and flesh breakdown were observed in DCA-RQ and CA + 1-MCP stored fruit. Higher flesh firmness was verified in DCA-RQ 1.3, if fruit were harvested unripe, higher in CA + 1-MCP, DCA-RQ 1.3 and DCA-RQ 1.5 in ripe harvested fruit and higher in CA + 1-MCP overripe harvested apple. The storage under DCA-RQ 1.5 resulted in acetaldehyde, ethanol and ethyl acetate accumulation, but at a concentration that did not increase the electrolyte leakage and physiological disorders related to fermentation, like flesh breakdown. Oxygen lowering down to extremely low partial pressure, by the storage under DCA-RQ 1.5, resulted in fruit with higher volatile compounds concentration that composed the aroma, regardless the maturity stage, as compared to fruit stored under CA. Additionally, the volatile compounds increase was not only observed for ethyl esters, but also in characteristic Galaxy apple esters, like butyl acetate, hexyl acetate and 2-methylbutyl acetate. With the 1-MCP application was observed a reduction in the aroma production as compared to CA and not allowed the increment of esters concentration with the maturity advance, resulting in fruit with similar ester concentration on the three maturity stages. Taken in account the physical, chemical quality and the volatile compounds, the best storage conditions for all maturity stages follow this order: DCA-RQ 1.5 > DCA-RQ 1.3 > CA + 1-MCP > CA. / A colheita de maçãs no estádio de maturação correto para longos períodos de armazenamento é uma tarefa cada vez mais complicada, especialmente em função da falta de mão-de-obra. Em função disso, o desenvolvimento de técnicas de armazenamento que possibilitem o armazenamento de frutas colhidas fora do estádio de maturação adequado é necessário. Nesse sentido, na presente dissertação, buscou-se avaliar uma nova técnica de armazenamento em atmosfera controlada dinâmica baseada no monitoramento do limite mínimo de O2 (LMO) pelo quociente respiratório (ACD-QR) e comparar com a da atmosfera controlada convencional (AC) e a combinação de AC com aplicação de 1-metilciclopropeno (AC + 1-MCP) sobre a qualidade física, química e o perfil volátil de maçãs Galaxy colhidas em três estádios de maturação (verde, madura e sobremadura). A dissertação é composta de dois capítulos (artigos): o primeiro direcionado à avaliação do metabolismo e da qualidade física e química e o segundo ao metabolismo e à composição volátil. Frutos dos três estádios de maturação armazenados ACD-QR apresentaram menor atividade da enzima ACC oxidase, etileno interno, produção de etileno e respiração em comparação aos frutos armazenados em AC. O armazenamento em ACD-QR 1,3 resultou em manutenção da qualidade similar ao armazenamento em AC + 1-MCP, independentemente do estádio de maturação. Menor incidência de polpa farinácea e degenerescência de polpa foi verificada em frutos armazenados em ACD-QR e AC + 1-MCP. A firmeza de polpa foi maior em frutos armazenados em ACD-QR 1,3, quando colhidos verdes, maior em AC + 1-MCP, ACD-QR 1,3 e ACD-QR 1,5 em frutos maduros e maior em AC + 1-MCP em frutos colhidos sobremaduros. O armazenamento em ACD-QR 1,5 resultou em acúmulo de acetaldeído, etanol e acetato de etila, porém numa concentração que não causa aumento no extravasamento de eletrólitos e distúrbios fisiológicos relacionados à fermentação, como a degenerescência de polpa. A redução do O2 a pressões parciais extremamente baixas, pelo armazenamento em ACD-QR 1,5, resultou em frutos com maior concentração de compostos voláteis que compõe o aroma, independentemente do estádio de maturação, em comparação aos frutos armazenados em AC. Adicionalmente, esse incremento na produção de compostos voláteis não ocorreu apenas em forma de acetatos de etila, mas também na forma de ésteres característicos de maçãs Galaxy , como o acetato de butila, acetato de hexila e acetato de 2-metilbutila. Com a aplicação de 1-MCP houve redução na produção de aroma em comparação a AC sem uso do produto, também não houve incremento na produção de ésteres com o avanço da maturação nos frutos tratados com 1-MCP, resultando em frutos com concentração de ésteres similar entre os três estádios de maturação. Levando em consideração a qualidade física, química e a produção de compostos voláteis, as melhores condições de armazenamento para todos os estádios de maturação segue a seguinte ordem: ACD-QR 1,5 > ACD-QR 1,3 > AC + 1-MCP > AC.

Dans les environnements côtiers, soumis à l’impact des marées noires, les microorganismes jouent un rôle crucial dans le devenir des hydrocarbures pétroliers. Toutefois, de nombreux facteurs influencent les activités microbiennes, notamment par les organismes bioturbateurs qui modifient la pénétration de l’oxygène dans les sédiments. Le travail de la thèse vise à mieux comprendre l’impact d’une pollution pétrolière sur les communautés microbiennes dans des sédiments bio turbés. Il s’agissait de comparer les remaniements structuraux de la communauté microbienne liée à la contamination pétrolière dans des sédiments présentant une faible et une forte activité de bioturbation. Des sédiments marins ont été maintenus en microcosmes durant 9 mois et soumis à quatre conditions : (i) pas de traitement (contrôle), (ii) pétrole, (iii) bioturbation et (iv) pétrole et bioturbation. Les efficacités de dégradation des hydrocarbures pétroliers se sont révélées similaires dans les deux types de sédiments. Par des approches moléculaires, la diversité taxonomique et fonctionnelle des communautés microbiennes totales et métaboliquement actives a été évaluée au cours du temps. Les communautés microbiennes ont subi d’importants remaniements structuraux spécifiques à chaque traitement. Nous suggérons que le fonctionnement global de la communauté est modifié par l’activité bioturbatrice sans pour autant modifier l’activité de dégradation. Ces travaux ont mis en évidence une redondance fonctionnelle de l’activité de biodégradation des hydrocarbures pétroliers des communautés microbiennes. L’isolement de communautés hydrocarbonoclastes a permis de confirmer cette redondance fonctionnelle. / Coastal areas such as mudflats are affected by oil spills. In these environments, microorganisms play a crucial role in the fate of petroleum hydrocarbons. However, many factors influence microbial activities, especially the bioturbating organisms, which altered the oxygen penetration in sediments. The present work attempts to better understand the impact of petroleum contamination on microbial community associated with petroleum contamination in sediments with low and high bioturbation activity. This study is based on microcosm experiments with a device simulating tidal cycles. Marine sediments were maintained for 9 months in microcosms and subjected to four conditions: (i) no treatment (control), (ii) oil, (iii) bioturbation and (iv) oil and bioturbation. Chemical, microbiological and biological analyses were conducted throughout the experiment. The efficiencies of degradation of petroleum hydrocarbons were similar in both sediments. By molecular approaches, we assessed the dynamic of the functional and taxonomic diversity of the total and metabolically active communities during the oil contamination. Microbial communities showed significant structural rearrangements specific for each treatment that resulted in distinct microbial communities in both sediments. Hence, the overall microbial community structure was changed by bioturbating activity without changing the degradation capacity revealing a functional redundancy of the biodegradation capacity of hydrocarbons. This result was further supported by the isolation and characterization of hydro carbonoclastic communities.