Physical, Metabolic, and Energetic Investigations of Methane-Metabolizing Microbial Communities

Understanding the roles of microorganisms in environmental settings by linking phylogenetic identity to metabolic function is a key challenge in delineating their broad-scale impact and functional diversity throughout the biosphere. This work addresses and extends such questions in the context of marine methane seeps, which represent globally relevant conduits for an important greenhouse gas. Through the application and development of a range of culture-independent tools, novel habitats for methanotrophic microbial communities were identified, established settings were characterized in new ways, and potential past conditions amenable to methane-based metabolism were proposed. Biomass abundance and metabolic activity measures – both catabolic and anabolic – demonstrated that authigenic carbonates associated with seep environments retain methanotrophic activity, not only within high-flow seep settings but also in adjacent locations exhibiting no visual evidence of chemosynthetic communities. Across this newly extended habitat, microbial diversity surveys revealed archaeal assemblages that were shaped primarily by seepage activity level and bacterial assemblages influenced more substantially by physical substrate type. In order to reliably measure methane consumption rates in these and other methanotrophic settings, a novel method was developed that traces deuterium atoms from the methane substrate into aqueous medium and uses empirically established scaling factors linked to radiotracer rate techniques to arrive at absolute methane consumption values. Stable isotope probing metaproteomic investigations exposed an array of functional diversity both within and beyond methane oxidation- and sulfate reduction-linked metabolisms, identifying components of each proposed enzyme in both pathways. A core set of commonly occurring unannotated protein products was identified as promising targets for future biochemical investigation. Physicochemical and energetic principles governing anaerobic methane oxidation were incorporated into a reaction transport model that was applied to putative settings on ancient Mars. Many conditions enabled exergonic model reactions, marking the metabolism and its attendant biomarkers as potentially promising targets for future astrobiological investigations. This set of inter-related investigations targeting methane metabolism extends the known and potential habitat of methanotrophic microbial communities and provides a more detailed understanding of their activity and functional diversity.

Part I. Regulation of development in the cellular slime mold Dictyostelium discoideum. Part II. Polysomes and RNA synthesis during early development of the surf clam Spisula solidissima

Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively.

The cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: ρ = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23°C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot_1/2 (pure) 0.28) and the single-copy DNA 70% (Cot_1/2(pure) 70). The complexity of the nuclear genome is 30 x 10⁹ daltons and that of the mitochondrial DNA is 35-40 x 10⁶ daltons (Cot_1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a ρ = 1.682.

RNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.

Part II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-³H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-³H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.

RNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.

In the early cleavage stages the light polysomes are mostly affected by actinomycin.

The E2F2 Transcription Factor Sustains Hepatic Glycerophospholipid Homeostasis in Mice

Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified "lipid metabolism regulation" as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2(+/+)) and E2F2 deficient (E2F2(-/-)) mice to investigate the in vivo role of E2F2 in the composition of liver lipids and fatty acids in two metabolically different contexts: quiescence and 48-h post-PH, when cellular proliferation and anabolic demands are maximal. We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2(+/+) and E2F2(-/-) mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2(-/-) mice resembles the phenotype of proliferating E2F2(+/+) liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance.

Resveratrol: Anti-Obesity Mechanisms of Action

Resveratrol is a non-flavonoid polyphenol which belongs to the stilbenes group and is produced naturally in several plants in response to injury or fungal attack. Resveratrol has been recently reported as preventing obesity. The present review aims to compile the evidence concerning the potential mechanisms of action which underlie the anti-obesity effects of resveratrol, obtained either in cultured cells lines and animal models. Published studies demonstrate that resveratrol has an anti-adipogenic effect. A good consensus concerning the involvement of a down-regulation of C/EBPa and PPAR. in this effect has been reached. Also, in vitro studies have demonstrated that resveratrol can increase apoptosis in mature adipocytes. Furthermore, different metabolic pathways involved in triacylglycerol metabolism in white adipose tissue have been shown to be targets for resveratrol. Both the inhibition of de novo lipogenesis and adipose tissue fatty acid uptake mediated by lipoprotein lipase play a role in explaining the reduction in body fat which resveratrol induces. As far as lipolysis is concerned, although this compound per se seems to be unable to induce lipolysis, it increases lipid mobilization stimulated by beta-adrenergic agents. The increase in brown adipose tissue thermogenesis, and consequently the associated energy dissipation, can contribute to explaining the body-fat lowering effect of resveratrol. In addition to its effects on adipose tissue, resveratrol can also acts on other organs and tissues. Thus, it increases mitochondriogenesis and consequently fatty acid oxidation in skeletal muscle and liver. This effect can also contribute to the body-fat lowering effect of this molecule.

Efeitos da suplementação dietética com cálcio sobre a plasticidade ontogenética decorrente do desmame precoce ou da exposição materna a nicotina na lactação

O cálcio tem se mostrado útil na regulação do metabolismo energético, favorecendo a perda de peso. Visto que tanto o desmame precoce como a exposição materna à nicotina na lactação são fatores condicionantes para o desenvolvimento de obesidade, hiperleptinemia e resistência à insulina, além de outras alterações endócrinas na idade adulta, decidimos avaliar os possíveis efeitos da suplementação dietética com cálcio sobre as disfunções apresentadas pelos seguintes modelos experimentais: 1) Desmame precoce (DP): ratas lactantes foram envolvidas com atadura para interromper o acesso da prole ao leite nos 3 últimos dias da lactação. As proles das ratas controles tiveram livre acesso ao leite materno durante todo o período da lactação (21 dias). 2) Exposição materna à nicotina (N): Dois dias após o nascimento, ratas lactantes receberam implantes de minibombas osmóticas contendo solução de nicotina (6 mg/kg/dia, 14 dias) ou salina (C), nas mesmas condições. Aos 120 dias de idade, as proles de ambos os modelos de obesidade experimental receberam dieta padrão ou dieta suplementada com cálcio (10g de carbonato de cálcio/kg de ração). O sacrifício ocorreu aos 180 dias de idade. Os dados foram considerados significativos quando p<0,05. Corroborando dados anteriores do nosso grupo, as proles de ambos os modelos de programação (N e DP) apresentaram maior gordura corporal total e visceral, hiperleptinemia, resistência hipotalâmica a leptina e distúrbios na homeostase glicêmica. Além disto, verificamos que as proles N e DP também exibiram aumento dos níveis séricos de 25-hidroxivitamina D3. Todos essas alterações endócrino-metabólicas foram corrigidas pelo tratamento com suplementação com cálcio. Além disso, a prole DP aos 180 dias mostrou hiperfagia e hipertrigliceridemia, que também foram normalizados pela suplementação dietética com cálcio. A prole N apresentou hipotireoidismo, maior conteúdo de catecolaminas e maior expressão de tirosina hidroxilase (TH). A terapia com cálcio reverteu a disfunção adrenal, embora não tenha sido eficaz para normalizar a hipofunção tireoideana. Assim, a suplementação dietética com cálcio normalizou a maioria dos parâmetros da síndrome metabólica observadas nos dois modelos de plasticidade ontogenética. É possível que a redução da adiposidade central induzida pela terapia com cálcio, por si, tenha sido o principal mecanismo que resultou na melhora dos parâmetros estudados. Uma vez que a suplentação de cálcio reverteu as concentrações séricas de 25-hidroxivitamina D3 dos animais obesos, é possível que o efeito anti-obesidade do cálcio também ocorra via ação do calcitriol sobre o adipócito.

Physiological Determinants of Invasive Success Linking Distribution Patterns to Metabolic Physiology in Native and Invasive Blue Mussels (genus Mytilus)

In the early 20th century, a blue mussel species from the Mediterranean invaded the California coast and subsequently out-competed the native species south of Monterey Bay. Like other invasive species, Mytilus galloprovincialis has physiological traits that make it successful in habitats formerly occupied by the native M. trossulus, namely its adaptation to warm sea surface temperatures. This study looks at the current genotype distributions and enzymatic activities of field-acclimatized mussels within the hybrid zone where the species co-occur as well as mussels that have been acclimated for four weeks to different temperature and salinity conditions. In the field-acclimatized and laboratory-acclimated mussels, the native species exhibited significantly higher enzyme rates, which may reflect an evolutionary adaptation to compensate to low habitat temperatures. Indeed, the results of the laboratory acclimation indicate that these differences are genetically based. Whether an acclimation capacity exists may require even longer-term acclimation to different temperatures. Current findings suggest that the further spread of the invasive species is likely to be governed in large measure by the potentially counteracting effects of rising temperatures, which would favor the northerly spread of M. galloprovincialis, and increased winter precipitation, which would favor the persistence of M. trossulus. However, the success of M. galloprovincialis during acclimation to ‘dilute’ salinity (25 ppt) suggests that the invasive species can tolerate a greater salinity range than previously thought. Thus, further investigation is needed to build a comprehensive predictive model of the movement of M. galloprovincialis and the hybrid zone along the California coast.

European Market Infrastructure Regulation

El trabajo trata de dar unas nociones básicas de la regulación EMIR y de la descripción de la implantación de la solución que toma la empresa en la que trabajé en prácticas. Y lo he realizado en castellano

Standard and routine metabolic rates of juvenile sandbar sharks (Carcharhinus plumbeus), including the effects of body mass and acute temperature change*

Standard and routine metabolic rates (SMRs and RMRs, respectively) of juvenile sandbar sharks (Carcharhinus plumbeus) were measured over a range of body sizes (n=34) and temperatures normally associated with western Atlantic coastal nursery areas. The mean SMR Q10 (increase in metabolic rate with temperature) was 2.9 ±0.2. Heart rate decreased with increasing body mass but increased with temperature at a Q10 of 1.8−2.2. Self-paired measures of SMR and RMR were obtained for 15 individuals. Routine metabolic rate averaged 1.8 ±0.1 times the SMR and was not correlated with body mass. Assuming the maximum metabolic rate of sandbar sharks is 1.8−2.75 times the SMR (as is observed in other elasmobranch species), sandbar sharks are using between 34% and 100% of their metabolic scope just to sustain their routine continuous activity. This limitation may help to explain their slow individual and population growth rates, as well as the slow recoveries from overfishing of many shark stocks worl