In advanced stages, cardiac disease causes millions of deaths each year. Superoxide anions (O₂^.-) and their derivative peroxynitrite (ONOO^-) contribute to cardiac disease pathogenesis, yet strategies to reduces these reactive oxygen species through antioxidants in large scale clinical trials have largely been unsuccessful. Better understanding of pathways regulating enzymatic sources of O₂^.- like NADPH oxidases, uncoupled nitric oxide synthases (NOSs) and mitochondrial oxidases are required to regulate myocardial oxidative stress in patients with advanced stages of cardiac disease. Hydrogen sulfide (H2S) is a gaseous signalling molecule generated by transsulfuration pathway enzymes cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MST). H₂S regulates oxidative stress in animal models and shows promise for cardiovascular therapeutic strategy. This thesis investigates whether H₂S/CSE biology is related to human myocardial redox state in a cohort of individuals with advanced cardiac disease (Oxford Heart, Fat, Vessels Cohort; Ox-HVF). Individuals with varying levels of myocardial oxidative stress and function were extensively phenotyped for H₂S biology. Individuals with high myocardial oxidative stress from NADPH oxidases and NOSs were found to have high expression of myocardial CSE. To examine first the positive association with NADPH oxidase activity, CSE expression was examined after myocardial oxidative injury and CSE was found to be redox-sensitive. Furthermore, direct effects of two exogenous H₂S donors (NaHS and GYY4137) demonstrated a direct regulation of O₂^.- from NOSs in myocardium from individuals with advanced cardiac disease, further supporting H₂S's direct role in the regulation of NOS biology. Finally, identification of a SNP in CSE further demonstrated CSE's causal role in the regulation of O2.- generation from mitochondrial oxidases. Taken together, we demonstrate for the first time that H₂S and CSE biology are linked to human myocardial redox state and have a causal role in redox regulation in the human heart. These findings suggest H₂S/CSE biology are important endogenous regulators of myocardial redox state in humans and continued exploration of these pathways may develop novel therapeutic strategies against myocardial oxidative stress in cardiac disease.

The ATP sensitive potassium channel (KATP) in mouse colonic smooth muscle cell is a complex containing a pore forming subunit (Kir6.1) and a sulfonyl urea receptor subunit (SUR2B). These channels are responsible for maintaining the cellular excitability of the smooth muscle cell which in turn regulates the motility patterns in the colon. We used whole-cell voltage-clamp techniques to study the alterations in these channels in smooth muscle cells in experimental model of colitis (colonic inflammation). Colitis was induced in BALB/C mice following an intracolonic administration of trinitrobenzene sulfonic acid (TNBS). KATP currents were measured at Vh -60 mV in high K+ external solution. The dose-response to levcromakalim (LEVC), a KATP channel opener, was significantly shifted to the left in the inflamed smooth muscle cells. Both the affinity and maximal currents induced by LEV were enhanced in inflammation. The EC50 in control was 6259 nM (n=10) and 422 nM (n=8) in inflamed colon while the maximal currents were 9.9 ± 0.71 pA/pF (60 μM) in control and 39.7 ± 8.8 pA/pF (3 μM) following inflammation. Similar to LEVC, KATP currents activated by sodium hydrogen sulfide (NaHS) (10-1000 μM) were significantly greater in inflamed compared to controls. In control cells, pretreatment with 100 µM NaHS shifted the EC50 for LEV-induced currents from 2838 nM (n=6) to 154 nM (n=8). These data suggest that NaHS can act as an allosteric modulator for LEV-induced KATP currents. Decreased colonic motility may result from enhanced KATP activation by increased release of H2S in colitis.

Hydrogen sulfide (H2S) is receiving increasing interest, as much as nitric oxide (NO) and carbon monoxide have received previously, to understand its physiological functions as it meets all the criteria to define as a third gasotransmitter. Endogenous synthesis from L-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) and the function of H2S as an inhibitor of smooth muscle contraction in gastrointestinal tract are known. However, the loci of generation and action of H2S, and the mechanism of inhibition of contraction are unknown. Hence, my aims in the present study are to: i) identify the expression of enzymes in smooth muscle, ii) determine the effects of endogenously released and exogenously applied H2S on smooth muscle function; and iii) identify the targets and mechanism involved in mediating the effects of H2S using isolated smooth muscle cells from rabbit colon. I have identified the expression of CSE, but not CBS, in smooth muscle and demonstrated that L-cysteine (an activator of CSE) and NaHS (H2S donor): 1) inhibited carbachol-induced contraction in muscle strips and isolated muscle cells that was independent of KATP channels, a known S-sulfhydration target of H2S; 2) induced S-sulfhydration of small G protein, RhoA leading to inhibition of RhoA and Rho kinase activities, a key pathway in the sustained smooth muscle contraction; and 3) inhibited PDE5 activity leading to augmentation NO-induced cGMP formation and muscle relaxation. Sodium nitroprusside (an NO donor) induced an increase in H2S production via PKG-dependent phosphorylation and activation of CSE. We conclude that smooth muscle cells selectively express CSE, and endogenous generation of H2S via activation of CSE inhibits muscle contraction and augments muscle relaxation. Inhibition of contraction is mediated via S-sulfhydration of RhoA and suppression of RhoA/Rho kinase pathway. Augmentation of relaxation is mediated via inhibition of PDE5 activity and stimulation of cGMP/PKG pathway, which in addition initiates generation of H2S via PKG-mediated phosphorylation and activation of CSE. The findings are important in providing the underlying mechanisms involved in the regulation of smooth muscle function by H2S and could offer insights for the development of therapeutic agents that may act on smooth muscle in the gut to treat motility disorders.

Ce travail présente une étude de procédé de traitement des siloxanes et de l’H2S appliqué à la purification des biogaz. Une approche bibliographique montre l’intérêt de développer de nouveaux procédés de traitement à faible coût pouvant s’intégrer facilement dans une filière complète de purification des biogaz. Une partie de l’étude est consacrée aux possibilités de traitement par transfert gaz-liquide dans des huiles. Cette technique est comparée à un procédé plus classique de traitement par adsorption sur charbon actif. Les résultats ont montré que ces 2 techniques étaient complémentaires, l’absorption dans une huile utilisée pour le traitement de fortes concentrations et l’adsorption sur charbon actif en traitement definition. Une autre partie de l’étude a été consacrée au traitement de l’H2S. Les niveaux d’abattement requis étant de plus en plus bas, l’approche choisie est celle d’un traitement definition en complément des traitements principaux classiques. Le procédé étudié est celui d’un système par physi-adsorption sur charbon actif en tissu pre-humidifié. Celui-ci est régénéré in situ par chauffage électrique direct et mise en dépression du réacteur. L’étude des conditions opératoires a permis d’établir des paramètres de régénération et des conditions de traitement permettant de réaliser des cycles pérenne. L’intérêt réside ici dans les conditions douces de température et de pression utilisées pour la régénération. / This work presents a study of the siloxanes and hydrogen sulfide separation process applied to the biogas treatment. A bibliography review shows the interest in the development of new technologies of low cost, to integrate them into an overall process of biogas up-grading. One part of this study is focused on the possible separation process by gas-liquid transfer into oils. This technology is compared with a more classical treatment process by adsorption into activated carbons. The results showed that both technologies are complementary, the absorption into oils used primarily to the abatement of high concentrations and the adsorption into the activated carbon as a finishing process. A second part of this study is focused on the hydrogen sulfide treatment. The requirements of abatement are very low, thus the approach is focused on a finishing process to complement the more classical methods. Thus a system by physic adsorption into pre-humidified activated carbon cloth was studied. The filter was regenerated in situ by direct electric heating under vacuum pressure. The study of the operating conditions allowed establishing the regeneration parameters and the process sustainability. The interest here is focused on the soft conditions of temperature and vacuum used to achieve the regeneration.

Doctor of Philosophy / Department of Biology / Michael Tobler / Extremophiles are organisms with the ability to survive in environments characterized by strong physicochemical stressors lethal to most other organisms, providing excellent models to further our understanding of life's capacities and limitations to deal with far-from-average conditions. I studied how physiological processes varied among fish residing in starkly different environmental conditions to understand how organisms cope with extreme environments and disentangle the roles of short-term plastic responses and evolved population differences in shaping physiological responses. I used the Poecilia mexicana model, a series of extremophile fish populations that has colonized toxic hydrogen sulfide (H₂S) rich springs and caves, to address three major objectives: (1) I investigated the energetic consequences of life in extreme environments and tested whether predicted reductions in organismal energy demands evolved repeatedly along replicated environmental gradients. (2) I characterized variation in gene expression among populations and organs to test for interactive effects between different stressors and identify potential physiological mechanisms underlying adaptation to H₂S and cave environments. (3) I conducted common garden and H₂S-exposure experiments to test how evolutionary change and plasticity interact to shape variation in gene expression observed in nature. To address these objectives, I measured variation in metabolic physiology and quantified variation in physiological processes through genome-wide gene expression analyses. I found that adaptation to extreme environments directly impacts energy metabolism, with fish living in extreme environments consistently expending less energy overall. Reductions in energy demand have evolved in convergence and were primarily mediated through a life history shift (reduction in body mass). The quantification of gene expression across divergent habitats and organs revealed organ-specific physiological responses in H₂S-rich and cave habitats. Gene expression variation in the relevant genes was primarily shaped by evolutionary change in gene regulation, and ancestral plastic responses play a minor role in causing the observed expression differences between replicated sulfidic and nonsulfidic populations in nature. Overall, my research has implications for understanding the capacities and constraints that shape life in extreme environments and aids in our understanding of modifications in physiological pathways mediating adaptation to elevated H₂S and perpetual darkness.

Generation of hydrogen sulfide (H2S) is a common phenomenon from wastewater collection, transport, and treatment processes. Impacts of H2S emissions from wastewater include corrosion and reduction in the service life of wastewater infrastructure, odor nuisance in the community, and health impacts on wastewater operations and maintenance personnel (Neilsen, et al. WEFTEC 2006). Conventional odor control studies performed by municipalities to design their individual odor/corrosion control strategies largely depend on establishing a dilution to detection threshold (D/T) ratio and ascertaining the recognition threshold (R/T) for air samples collected from the study area. These conventional odor studies based on grab samples using R/T and D/T technique using a few days of data have a number of limitations and potentially lead to inaccurate conclusions. However, H2S emission studies using continuous air monitoring is expensive and time consuming. The objective of this research is to understand the feasibility of utilizing emission factors as a tool to predict hydrogen sulfide emissions from headworks of four different Jefferson Parish, Louisiana wastewater treatment plants (WWTP). Proposed model(s) developed for predicting H2S emission factors that depend on wastewater parameters should be convenient for the municipalities to use as the data required is monitored routinely. Use of H2S emission models should assist rapid identification of H2S emission hot spots, optimize H2S control strategies, predict potential health risks, prevent community odor nuisance, and ascertain infrastructure corrosion. This dissertation attempts to; i) develop a research methodology, ii) identify instruments required, iii) generate emission factor ranges and compare their sensitivity to wastewater parameters, iv) generate preliminary empirical emission models based on flow treated, population serviced and area served by a treatment plant for each sampling location and v) provide a roadmap for future research opportunities to refine the models generated as part of this dissertation. Key words: emission model, emission factor, emission ranges, hydrogen sulfide, odor control, air quality, wastewater treatment.

Hydrogen Sulfide is a corrosive, odorous, and hazardous gas that can cause issues for wastewater collection systems and treatment plants. When evaluating hydrogen sulfide concentrations it is important to consider the source, odor causing mechanism, and its level of toxicity. In this study, continuous hydrogen sulfide monitoring was performed for 8 locations within the New Orleans East Bank Wastewater Treatment Plant for a span of 5 months. Hot spots within the plant are located and data analysis is performed based on daily and hourly averages of hydrogen sulfide concentrations. The data was configured to show peak hydrogen sulfide concentrations at specific time intervals throughout the day.

A manutenção da osmolalidade e do volume dos líquidos extra- (LEC) e intracelular (LIC) são fatores de extrema importância à conservação da vida. Na desidratação (por ingestão insuficiente ou perda excessiva de água), sistemas de controle autonômicos e endócrinos são acionados frente à ativação de barorreceptores (periféricos) e osmorreceptores (centrais e periféricos), visando estimular a sede e inibir o apetite ao sódio, além de reduzir a diurese, promover a natriurese e contrair a musculatura lisa dos vasos. Especificamente para o sistema de controle endócrino é evidente que tais respostas se dão pela secreção dos hormônios neuroipofisários vasopressina (AVP) e ocitocina (OT), e pelo aumento da concentração sanguínea de angiotensina II (ANGII). As secreções de AVP e OT são controladas por sistemas subjacentes, representados por neurotransmissores e também por moduladores gasosos, como óxido nítrico (NO) e monóxido de carbono (CO) (Calapai e cols., 1992; Ventura e cols., 2002 e 2008; Gomes e cols., 2004 e 2010; Reis e cols., 2010). Apesar da existência de modelos que investigassem a modulação do H2S sobre respostas hipocampais, modulação da frequência cardíaca e pressão arterial, e temperatura corporal (Abe e Kimura, 1996; Dawe e cols., 2008; Liu e cols., 2011; Ren e cols., 2011; Kwiatkoski e cols., 2012), nenhuma abordagem sobre a interação do H2S com a secreção de AVP e OT havia sido realizada. No presente estudo, portanto, objetivou-se avaliar, após a privação hídrica de 24 horas (estímulo potente à sede e secreção de AVP e OT), os efeitos do H2S sobre a resposta hipotalâmica. Para tanto, sulfeto de sódio (Na2S 65 mM, doador inorgânico), aminooxiacetato (AOA 50 ?M, inibidor da enzima cistationina ?-sintase) ou veículo (NaCl 0,9%) foram administrados intracerebroventricularmente (icv) em ratos adultos privados de água ou hidratados. Observou-se inicialmente que, em animais apenas submetidos à desidratação (vs. hidratados), houve um aumento da atividade das enzimas hipotalâmicas 8 geradoras de H2S; e, além disso, a administração aguda de Na2S a estes animais potencializou a secreção de AVP, OT e CORT (corticosterona), sem alterar a concentração plasmática de ANGII, ANP e prolactina (hormônios medidos após 5 min. da administração icv da droga ou veículo). O H2S exógeno foi também capaz de reduzir o conteúdo de NO hipotalâmico (medido indiretamente pelo metabólito nitrato/nitrito após 5 min.). O grande aumento na secreção plasmática de AVP e OT provavelmente foi o responsável pela maior concentração plasmática de CORT, uma vez que estes hormônios são secretagogos do hormônio adrenocorticotrófico (ACTH) adenoipofisário. Enquanto o aumento de OT pode ter sido o responsável pela redução da ingestão de água entre os animais privados. Inversamente, a administração de AOA (vs. veículo) aumentou a ingestão de água entre os animais desidratados. Desta forma, estes resultados sugerem que o gás H2S pode ser um importante agente modulador da resposta de sede e secreção neuroipofisária durante a privação hídrica; atuando ou diretamente sobre canais e receptores neuronais ou indiretamente através dos sistemas do NO e CO. / Osmolality and volume maintenance of extra- (ECF) and intracellular fluids (ICF) are factors of extreme importance to life preservation. During dehydration (from insufficient intake or excessive loss of water), autonomic and endocrine control systems are triggered mainly by (peripheral) baroreceptors and (central and peripheral) osmoreceptors to stimulate water intake and inhibit sodium appetite, in addition to restraining diuresis, promoting natriuresis and contracting smooth muscle of vessels. Specifically for endocrine control system, it is evident that such responses take place by neurohypophysis secretion of vasopressin (AVP) and oxytocin (OT), and by increase of plasma angiotensin II (ANGII) levels. Both AVP and OT secretion are controlled by underlying systems, which are represented by neurotransmitters and also by gaseous modulators like nitric oxide (NO) and carbon monoxide (CO) (Calapai et al., 1992; Ventura et al., 2002 and 2008; Gomes et al., 2004 and 2010; Reis et al., 2010). Despite the existence of models which investigated H2S modulation on hippocampus responses, heart frequency and blood pressure, and body temperature (Abe and Kimura, 1996; Dawe et al, 2008; Liu et al, 2011; Ren et al, 2011; Kwiatkoski et al, 2012), no approach of H2S interaction with AVP and OT secretion had been conducted. In this study, we thus aimed to evaluate the effects of H2S on hypothalamus response after a 24-hour water deprivation (a powerful stimulus for thirst and AVP/OT secretion). To that, 65 mM sodium sulfide (Na2S, inorganic donor), 50 ?M aminooxyacetate (AOA, cystathionine ?-synthase inhibitor) or vehicle (NaCl 0,9%) were administered intracerebroventricularly (icv) to water-deprived or euhydrated adult rats. It has been initially observed that hypothalamus H2S-generating enzymes activity of dehydrated-only animals was increased (vs. euhydrated); and, further, acute Na2S administration to dehydrated animals potentiated AVP, OT and CORT 10 (corticosterone) secretion without modifying ANGII, ANP and prolactin plasma levels (measured after 5 min. of drug or vehicle icv administration). Exogenous H2S could reduce hypothalamus NO content as well (indirectly measured by metabolite nitrate/nitrite after 5 min.). The great increase of AVP and OT secretion was probably responsible for higher levels of corticosterone, since these hormones are secretagogues of anterior pituitary adrenocorticotropic hormone (ACTH). While OT rise might have been the responsible for reduced water intake among deprived animals. On the other hand, AOA central injection (vs. vehicle) augmented water intake among water-deprived rats. These results therefore suggest that the gas H2S may be an important modulating agent of thirst and posterior pituitary secretion response during water deprivation; acting either directly on neuron channels and receptors or indirectly by NO and CO systems.

O objetivo deste estudo foi realizar ensaios de adsorção dinâmica, em coluna de leito fixo sem etapa regenerativa, para tal finalidade utilizou-se como adsorventes o carvão ativado comercial e material adsorvente à base de lodo biológico, para o tratamento de sulfeto de hidrogênio em corrente gasosa e obtenção das capacidades de adsorção. O lodo foi obtido de forma bruta em estação de tratamento de efluentes de abatedouro de aves, e tratado por processos térmicos e químicos em laboratório, para a geração de adsorvente. A concentração de H2S alimentada foi de 500 ppm na coluna, presente em duas misturas gasosas, uma com N2 e H2S, e outra mistura simulando o biogás contendo CO2, CH4 e H2S. As concentrações de H2S foram monitoradas por meio de um analisador de gás conectado a saída da coluna. Usou-se a técnica de interpretação das curvas de efluência para a determinação da capacidade de adsorção dos adsorventes estudados. Os resultados obtidos nesse trabalho indicaram que o aparato experimental usado não se mostrou viável para a adsorção com adsorvente à base de lodo. No entanto foi possível aumentar sua área superficial por meio dos tratamentos submetidos, passando de 0,005 g/m² na condição de lodo seco, para 86,4 g/m² depois de tratado. O lodo tratado com ácido sulfúrico mostrou maior capacidade de adsorção para a vazão de 4,0 L/min, apresentando remoção média de 0,21 mg H2S/g lodo. O carvão ativado comercial se apresentou propício para a remoção de H2S nas duas correntes gasosas, apresentando uma capacidade de adsorção média de 2,70 mg H2S/g carvão para a mistura gasosa simulando biogás e 1,26 mg H2S/g carvão para a corrente gasosa formado por nitrogênio e sulfeto de hidrogênio. / The objective of this study was to perform dynamic adsorption tests in fixed-bed column without regenerative stage, for this purpose it was used as adsorbents commercial activated carbon and biological sludge-based adsorbent material, for the treatment of hydrogen sulfide in the gas stream and obtaining the adsorption capacities. The sludge was obtained in raw form in treatment effluent plant from slaughterhouse poultry, and treated by thermal and chemical processes in the laboratory, to generate adsorbent. The concentration of H2S fed was 500 ppm in the column, present in two gas mixtures, one with N2 and H2S, and other mixing simulating the biogas containing CO2, CH4 and H2S. The H2S concentration was monitored by a gas analyzer connected to the outlet of the column. It was used breakthrough curves to determine the adsorption capacity of the adsorbents studied. The results of this work indicated that the experimental apparatus used was not suitable for adsorption with sludge-based adsorbent. However it was possible to increase the surface area through of undergoing treatments, from 0.005 g/m² on dry sludge condition to 86.4 g/m² after treated. The treated sludge with sulfuric acid showed a higher adsorption capacity for the flow rate of 4.0 L/min, with an average removal of H2S 0.21 mg/g sludge. The commercial activated carbon proved itself suitable for the removal of H2S in the two gas streams, with an average adsorption capacity of 2.70 mg H2S/g carbon for the gaseous mixture simulating biogas and 1.26 mg/g carbon for the gaseous stream consisting of nitrogen and hydrogen sulfide.

A dor e o prurido são sensações sensoriais que auxiliam na defesa do organismo. Apesar das ações anti-nociceptivas do novo gasotransmissor endógeno H2S, nada se sabe sobre seu efeito no prurido. Avaliou-se aqui o efeito de doadores de H2S (Na2S e reagente de Lawesson - LR) no prurido e inflamação induzidos pela injeção i.d. de histamina e composto 48/80 (C48/80) na pele de camundongos. Verificou-se a produção do H2S e a expressão de suas enzimas (CBS e CSE) na pele. A injeção i.d. de histamina ou C48/80 aumentou significativamente a frequência de coceira e induziu extravasamento plasmático e influxo de leucócitos na pele dos animais em relação ao veículo. A co-injeção do Na2S ou LR reduziu (P<0.05) o extravasamento plasmático evocado por ambos agentes, mas somente o prurido induzido por histamina foi inibido. Conclui-se que a suplementação exógena de H2S inibe o extravasamento plasmático induzido por histamina e C48/80, mas somente inibiu o prurido evocado por histamina. As enzimas CSE e CBS estão expressas constitutivamente na pele e sintetizam o H2S local. / Pain and pruritus are sensory sensations that help in defending the organism. Despite the anti-nociceptive actions of the new gasotransmitter H2S, little is known about its effect on pruritus. We evaluated the effect of H2S donors (Na2S and Lawesson\'s reagent - LR) in itching and inflammation induced by i.d. injection of histamine and compound 48/80 (C48/80) in mouse dorsal skin. We also investigated the production of H2S and expression of its enzymes (CBS and CSE) in the mouse skin. The i.d. injection of histamine or C48/80 significantly increased the frequency of itching and induced plasma leakage and leukocyte influx into the skin in relation to vehicle. Co-injection of Na2S or LR decreased (P<0.05) plasma extravasation evoked by both agents, but only the itching induced by histamine was inhibited. In conclusion, the exogenous supplementation of H2S inhibits plasma extravasation induced by histamine and C48/80, but only inhibited histamine-induced pruritus. CSE and CBS are constitutively expressed in the skin and are responsible for local H2S synthesis.