Title of master thesis Effect of ski-mountaineering equipment on energy expenditure of skiing. Work objectives The aim of this study was to determine the effect of weight ski equipment energy performance when walking on snow. Methods The study included 6 skialpinists a mean age of 30 ± 5.5 years. Energy intensity was measured on the basis of breathe oxygen (VO2) and exhaled (VCO2). The measurements were used 3 types of ski-hiking equipment (light - medium x 3160 g - 7754 g x heavy - 9600 g). Each test first completed section in a time of 6 minutes flat terrain (0ř), after which he immediately set out to climb the 360 m long route at an angle (20ř). These two sections (0ř and 20ř) graduated in random order for each type of equipment and at a constant speed of 1 m·s-1 . Results The results show that energy intensity decreases significantly when using ultralight ski equipment. We found that the climb at an angle of 20ř is the difference in oxygen consumption (VO2) between moderate and light equipment 9.6% between heavy and light equipment 17.7% and medium-heavy and heavy equipment 7,4%. The average VO2 test with light equipment was 44.4 ± 3.7 ml·kg-1 ·min-1 , with moderate equipment 48.7 ± 5.6 ml·kg- 1 ·min-1 and heavy equipment 52.2 ± 4.8 ml·kg-1 ·min-1 . At 0ř inclination and moderate use of heavy...

La nitrate réductase périplasmique de Rhodobacter sphaeroides catalyse la réduction du nitrate en nitrite. C'est une métalloenzyme qui comprend un cofacteur à molybdène, un centre fer - soufre et deux hèmes.La réactivité du cofacteur à molybdène reste mal comprise pour plusieurs raisons. Entre autres : l'hétérogénéité des signatures RPE Mo(V), état semi-réduit du site actif, et l'existence d'états inactifs de l'enzyme selon les conditions.Pour comprendre la réactivité et la pertinence catalytique des principales espèces Mo(V), nous avons entrepris une caractérisation des processus d'activation et d'inactivation par électrochimie sur film de protéines, et une étude de leur structure par spectroscopies RPE et HYSCORE.Nos observations cinétiques suggèrent que l'activation irréversible de l'enzyme implique un réarrangement d'une des ptérines du cofacteur à Mo.Ceci est mis en évidence par la modification des couplages magnétiques intercentres du fait de l'activation, et par des modifications de structure au delà de la première sphère de coordination du Mo.Enfin, l'étude de l'inactivation réversible de l'enzyme par électrochimie montre l'implication des différents états redox du site actif dans le mécanisme d'inhibition, et donne les conditions nécessaires au piégeage de formes Mo(V) actives. / Rhodobacter sphaeroides periplasmic nitrate reductase catalyses the reduction of nitrate into nitrite. It is a metalloenzyme containing a molybdenum cofactor, an iron - sulfur cluster, and two haems.The reactivity of the molybdenum cofactor remains elusive for many reasons. Among others : the heterogeneity of the EPR signatures of Mo(V), the semi-reduced state of the active site, and the existence of inactive states of the enzyme, depending on conditions.In order to understand the reactivity and the catalytic relevance of the major Mo(V) species, we have undertaken a characterisation of the activation and inactivation processes by protein-film-electrochemistry, and a study of their structure by EPR and HYSCORE spectroscopies.Our kinetic observations suggest that the irreversible activation of the enzyme involves a rearrangement of one of the pterins of the Mo cofactor.This is evidenced by the modification of intercentre magnetic couplings due to the activation, and by structural modifications beyond the first coordination sphere of Mo.Finally, the study of enzyme reversible inactivation by electrochemistry shows the involvement of the different redox states of the active site in the inhibition mechanism, and yields the necessary conditions to trapping active Mo(V) forms.

A mecanismos fisiológicos é frequentemente usada para explicar fenômenos importantes da evolução (p.e. endotermia) e ecologia (p.e. limitações fisiológicas para ocupação de determinado microhabitat) dos vertebrados. Apesar de ser uma área promissora para fornecer explicações e mecanismo que tornam hipóteses evolutivas e ecológicas mais robustas, como já é amplamente praticado no estudo dos vertebrados, ela é raramente usada nas hipóteses elencadas para explicar a megadiversidade das aranhas de teia. Por outro lado, as leis da fisiologia são baseadas fortemente em dados de vertebrados que consistem em menos de 1% da biodiversidade existente no planeta. As aranhas são o sétimo maior táxon em número de espécies e são um bom modelo para testar a validade das regras gerais propostas para os vertebrados. Com insto em mente, o presente trabalho investigou três tipos de taxa metabólica - Metabolismo Padrão, Metabolismo Espontâneo Máximo e Metabolismo Diário - em aranhas de teia. Todas estas taxas foram obtidas através de respirometria intermitente Em um primeiro experimento, usando como modelo Loxosceles, um gênero de aranhas especialmente homogêneo em sua morfologia, testou-se o efeito de sedentarismo e intensidade de construção da teia nas taxas metabólicas. Em um segundo experimento, testou-se como as taxas metabólicas se apresentariam frente a grande diversidade das aranhas do clado Orbiculariae. Os resultados mostram que: as diferenças comportamentais analisadas não afetam significativamente as taxas metabólicas; as taxas metabólicas não possuem sinal filogenético; e o melhor modelo é o que ajusta uma relação alometrica diferente para cada gênero em detrimento de um coeficiente exponencial da massa único para toda a amostra. As diferenças comportamentais de Loxosceles provavelmente apresentam-se na distribuição de gasto energético ao longo do dia partindo de um mesmo montante energético. Os dados de diversidade de Orbiculariae provavelmente precisam ser investigados futuramente sob o aspecto das diferenças morfológicas subjacente à diversidade do clado. / The physiological mechanisms are often applied to explain important evolutionary adaptations (e.g. endothermy) and ecology (e.g. physiological limitations to occupy a particular microhabitat) of vertebrates. Despite the fact that it improves vertebrates ecological and evolutionary hypotheses with possible processes and mechanisms, it is scarcely used to understand the megadiversity of web spiders. On the other hand, the physiological rules are based mainly on vertebrates data, an animal group that represents less than 1% of the world biodiversity. Spiders group is the seventh most diverse taxon in terms of species number on the world and are a good model to test the validity of the general rules proposed for vertebrates. Accordingly, this study investigated three types of metabolic rate - Standard Metabolism, Maximum Spontaneous Metabolism, Daily Metabolism - on web spiders. All these rates were obtained by intermittent respirometry. In the first experiment, the Loxosceles model was chosen, a genus of spider with a especially homogeneous morphology, with whom we tested the differences on metabolic rates of spiders with different sedentarity and web building expenditure. In a second experiment we tested how metabolic rates would vary under the high diversity of Orbiculariae spiders clade. The results show that: behavioral differences do not affect significantly the metabolic rates examined, metabolic rates do not have any significant phylogenetic signal and the best model is the one that adjusts a separate allometric relation for each genus at the expense of an unique allometric model for the whole sample. The behavioral differences of Loxosceles probably could be explained by the distribution of energy expenditure throughout the day under the same amount of energy. The diversity of data of Orbiculariae clade probably need to be investigated in the future under the aspect of morphological differences underlying the diversity of the clade

Os éteres de bifenilas polibromados (PBDEs) são largamente usados como retardadores de chama e têm sido detectados no sangue humano, tecido adiposo e leite materno devido às suas propriedades físico-químicas e bioacumuladoras e à sua elevada persistência no meio ambiente. Muitos estudos têm relatado toxicidade hepática relacionada à exposição aos PBDEs. Como efeitos citotóxicos são frequentemente associados à disfunção mitocondrial,no presente estudo, investigamos a toxicidade do BDE-47 em mitocôndrias isoladas de fígado de rato. Nos seguintes parâmetros: potencial de membrana mitocondrial, consumo de oxigênio, interação com a membrana mitocondrial, liberação de cálcio, inchamento mitocondrial e os níveis de ATP da suspensão mitocondrial com a finalidade de observar a capacidade do composto em interferir com a bioenergética da organela. Avaliou-se ainda a formação de espécies reativas de oxigênio (ROS), dos níveis de GSH/GSSG, níveis mitocondriais de grupos sulfidrila de proteínas e níveis mitocondriais de NAD(P)H com o intuito de analisar o estado redox mitocondrial. Os congêneres BDE-47 e BDE-99 foram avaliados em concentrações que variaram entre 0,1 e 50 ?mol/L. Os dois congêneres de PBDEsapresentaram toxicidade mitocondrial afetando todos os parâmetros que avaliam a bioenergética mitocondrial culminandoum uma depleção do ATP. Por outro lado, nos ensaios de avaliação do estresse oxidativo os PBDEs estudados não demonstraram qualquer efeito, no entanto, afetaram a cadeia respiratória mitocondrial, o que normalmente reflete em um acúmulo de espécies reativas de oxigênio. Estes resultados são explicados pelo mecanismo de ação destes compostos como retardante de chama, pois durante a combustão os retardantes de chama bromados realizam a neutralização de radicais livres (OHo e Ho). Conclui-se assim que os PBDEs podem levar à disfunção bioenergética e assim induzir a morte celular pela diminuição do ATP. Observou-se ainda que BDE-47 apresentou maior toxicidade do que o BDE-99 sobre os parâmetros estudados. / Polybrominated biphenyls ethers (PBDE) , widely used as flame retardants, have been detected in human blood, adipose tissue and breast milk due to their physicochemical and high bioaccumulative properties and environmental persistence. Many studies have reported liver toxicity related to exposure to PBDEs. As cytotoxic effects are often associated with mitochondrial dysfunction,in the present study, we investigated the toxicity of the BDE-47 and BDE-99 on isolated rat liver mitochondria. Assessing mitochondrial membrane potential, oxygen consumption, interaction with the mitochondrial membrane, calcium release, mitochondrial swelling and ATP levels of the mitochondrial suspension in order to observe the ability of the compounds to interfere with the bioenergetic organelle, and the formation of reactive oxygen species (ROS), GSH/GSSG levels, mitochondrial sulfhydryl groups and levels of mitochondrial NAD(P)H in order to analyze mitochondrial redox state. The congeners BDE-47 and BDE -99 were evaluated at concentrations ranging between 0.1 and 50 ?mol/L. Both PBDEs congeners showed mitochondrial toxicity affecting all parameters that assess mitochondrial bioenergetics and culminating with ATP depletion. On the other hand, no effect was observed in tests assessing oxidative stress, although they were able to inhibit the mitochondrial respiratory chain, which is usually reflected in an accumulation of reactive oxygen species. These results are explained by the mechanism of action of this compounds as flame retardants, because during the combustion brominated flame retardants perform the neutralization of free radicals (OHo and Ho). It follows that PBDEs can lead to mitochondrial dysfunction and thus induce bioenergetic cell death. It was also observed that The BDE-47 showed higher toxicity than BDE-99 in the studied model

The rapid emergence of multidrug resistant bacterial strains represents a major global healthcare issue. Amongst five known classes of membrane transporters, which play a huge role in multidrug efflux, primary-active ATP-binding cassette (ABC) transporters are ATP powered whilst secondary-active transporters utilize electrochemical ion gradients to drive substrate transport. Mechanistic insights into transport by these proteins can help with the design and development of novel therapeutic agents against multidrug resistance, and can increase our understanding of the physiological functions of these transporters. Although available crystal structures illustrate a common alternate access model for transport by ABC transporters, the mechanisms by which metabolic energy is coupled to the transport cycle is still elusive. This thesis presents a series of functional studies using whole cells as well as artificial phospholipid membranes to study the energetics of transport, and the influence of membrane phospholipids on substrate transport by the homodimeric Escherichia coli lipid A/multidrug ABC exporter MsbA. Current alternating access models for ABC exporters involve cycling between conformations with inward- and outward-facing substrate-binding sites in membrane domains (MDs) in response to engagement and hydrolysis of ATP at the nucleotide-binding domains (NBDs). Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. In this thesis, analogous substrate transport reactions are also studied for two other ABC exporters, the MsbA homologue LmrA and the human multidrug transporter ABCG2. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. It also raises questions about the role of NBDs in the transport process. Comparisons of drug transport and resistance in cells expressing MsbA-MD (truncated MsbA lacking the NBD) and full length MsbA (MsbA-WT) demonstrate increased transport efficiency of MsbA-WT compared to MsbA-MD. In addition, growth studies using E. coli WD2 cells, which are conditionally defective in MsbA’s essential activity in lipid A transport, show that lipid A transport can be restored by the expression of MsbA-WT but not MsbA-MD or ATP-hydrolysis impaired Walker A mutant (MsbA- ΔK382). Lastly, we also present biochemical experiments with proteoliposomes with a defined phospholipid composition, which suggest that cardiolipin is essential for the transport activity of MsbA. These techniques open the way to further explore lipid-proteins interactions and examine the physiological role(s) of MsbA. In conclusion, this thesis produces new insights in the mechanisms of transport and energy coupling in ABC exporters.

No description available.

Graduation date: 1994

Mangroves and seagrass beds serve as essential fish habitat for many economically- and ecologically-valuable species. Depending on their location, these shallow-water habitats are often characterized by substantial fluctuation in salinity levels, which can represent a source of osmoregulatory stress for associated organisms. In South Florida, one of the most important fish species that utilizes these habitats is the gray snapper (Lutjanus griseus). Although this species constitutes a significant portion of the region?s total recreational fishery harvest, the effects of salinity on its distribution, physiology and behavior remain poorly understood. The main goal of this thesis was then to investigate the ecophysiological basis of habitat selection by the gray snapper. Specific objectives include to: (1) examine patterns of distribution and abundance across gradients in environmental salinity; (2) measure physiological status and responses to controlled salinity challenges and; (3) conduct behavioral trials to examine for salinity preferenda (if any). To begin investigating if salinity could be a primary factor structuring the gray snapper assemblages, I examined empirical data collected from Biscayne Bay to test the null hypothesis that gray snapper abundances were evenly distributed along the full salinity range at which samples have been collected. Using the delta approach, three abundance metrics (frequency of occurrence, concentration and delta density) were used as an index for the distribution and abundance of this species. Results indicated that abundance patterns for the smaller gray snapper were consistent with a strategy of reducing osmoregulatory costs by selecting intermediate salinities. However, corresponding abundance patterns for subadult gray snapper were inconsistent with this strategy of minimizing energetic costs, suggesting that this life stage may be indifferent to the range of salinities at which they were observed. These patterns helped developed further hypotheses regarding the ecophysiology of juvenile and subadult gray snapper, the latter of which was then tested via laboratory experiments. Subsequently, I challenged fish in the laboratory with six different salinity treatments (0, 5, 30, 50, 60 and 70ppt, including control) for 192 consecutive hours and collected blood samples at different time points. Results indicated that physiological stress to salinity changes is unlikely to occur at a salinity range of 5 to 50 ppt. At salinities of 0 and 60 ppt transient significant changes in plasma osmolality and/or blood haematocrit were observed, but were corrected after an initial adjustment period of approximately 96 hours. At the highest salinity treatment (70 ppt), a constant osmolality could not be maintained, resulting in death for all fish within 48 hours of exposure. Overall, these findings demonstrate the strong euryhalinity and extraordinary tolerance of this species to both extreme hypo- and hypersaline environments. Finally, I investigated the salinity preference and effects on swimming behavior of the gray snapper in an automated salinity choice shuttlebox via 48-hr trials. In general, gray snapper tested displayed either one of two distinctively different salinity preferences. Half of gray snappers displayed a salinity preference in the range of 9-15 ppt, whereas the other half displayed a salinity preference in the range of 19-23 ppt. Recorded swimming speeds in all fish tested reflected a significant, but weak negative linear relationship with salinity during both time periods of the day (light and dark); however, gray snapper were usually most active during the dark period across all salinities. Overall, these findings reveal that gray snapper prefer slightly hyperosmotic salinities that may minimize the physiological costs of osmoregulation compared to extreme salinities.

As fishery production and habitat quality continues to experience declines, there is a growing need to improve the scientific methodologies used to assess and sustain economically and ecologically important fisheries. This requires a sound understanding of the life histories and population dynamics of each species, and development of a robust framework for population modeling. Realization of the multi-species nature of fisheries has prompted a shift towards ecosystem-based approaches to modeling. To evaluate alternative methods for modeling predator-prey interactions within a physically variable coastal-estuarine ecosystem, a food web of national economic and ecological significance comprised of Atlantic menhaden (Brevoortia tyrannus), striped bass (Morone saxatilis), and bluefish (Pomatomus saltatrix) is considered in this study. Understanding the dynamics of biological communities such as this is challenging and requires the formulation of realistic mathematical models. This should be a stepwise process in which the underlying assumptions, parameter sensitivities, and fundamental behaviors of interacting species dynamics described by relatively "simple" to more "complex" models are delineated and quantified. In this study two alternative multispecies modeling frameworks were utilized to evaluate the dynamic interactions between predator and prey populations, and to understand the influence of fisheries and environmental temperature change on predator-prey and food chain communities. First, relatively "simple," age-independent, predator-prey and food chain models representing generalized, ecological-scale interactions between different trophic groups were developed and analyzed. Sensitivity analyses revealed the relative importance of model parameters and the effect of varying levels of fishing mortality on model dynamics. Overall, the predator-prey and food chain models were shown to be a valuable tool for understanding general patterns in the dynamic behavior of interacting populations. Next, an environment-dependent, age-structured, Atlantic coast spatial dynamic multispecies model was investigated. This more complex model links individual-scale bioenergetic processes controlling growth to ecological-scale rates of natural and predation mortality. Simulations were performed using recent stock assessment estimates of fishery condition and stock sizes to evaluate the nature and magnitude of linkages among menhaden and their key predators, specifically bluefish. This ecosystem model was demonstrated to provide valuable insights into the dynamics of menhaden and bluefish given the underlying dynamics and forcing in the Atlantic States fishery coastal ocean ecosystem. Additionally, the influence of environmental temperature on both modeling frameworks was investigated. This iterative process of model development and analysis advances the current understanding of the species and ecosystem of interest, and ultimately provides an improved basis for multispecies fisheries assessments.

Previous studies on fishes exposed to effluent from the Key Lake uranium mill in northern Saskatchewan have demonstrated elevated lipids in young-of-the-year pike (Esox lucius), deformities in larval pike and decreased survival of fathead minnows (Pimephales promelas). The objectives of this thesis were to evaluate possible factors that could be contributing to altered bioenergetics of juvenile northern pike inhabiting lakes receiving effluent from the Key Lake operation and to examine the effects of effluent exposure on biomarkers of oxidative stress and histopathology of target organs. Although glycogen and triglycerides stores were significantly greater in pike from exposure lakes compared to the reference, triglycerides stores of juvenile pike prey items showed no overall differences among lakes. Measures of parasitism, however, were negatively correlated with pike bioenergetics thereby reflecting a possible energetic cost of parasitism on reference lake fish. The degree of infection by intestinal parasites and gill monogeneans was greatest in reference pike and intermediate in low exposure pike, whereas high exposure pike harboured no parasites. <p>Arsenic, nickel and selenium are elevated in lakes downstream of the Key Lake mill and have been shown to be associated with increased reactive oxygen species (ROS) in biological systems causing oxidative stress. The potential for oxidative stress was assessed in pike liver and kidney using several biomarkers. Overall, the concentrations of total, reduced and oxidized glutathione and the ratio of oxidized to reduced glutathione did not differ significantly among exposure and reference pike. The activity of glutathione peroxidase was greater in high exposure than reference liver whereas, contrary to predictions, lipid peroxidation was greater in reference than exposure pike tissues. <p>Histopathological evaluations revealed greater kidney and gill pathology in reference lake pike, whereas for liver, hepatocyte morphology differed among lakes without any clear signs of pathology. Trace metal analyses of muscle showed that eight elements (arsenic, cobalt, copper, iron, molybdenum, selenium, thallium, uranium) were significantly elevated in exposure pike. These results provide only limited evidence of oxidative stress in exposure pike tissues and no evidence of histopathology despite indications that metals are bioaccumulating in tissue. Overall, the results from this thesis suggest that the health and condition of juvenile northern pike living downstream of the Key Lake uranium mill may not be compromised by effluent exposure.