A deficiência de sulfito oxidase é uma doença autossômica recessiva que afeta o metabolismo da metionina e cisteína. Os indivíduos afetados comumente apresentam, no período neonatal, convulsões refratárias, retardo mental e desordens do movimento cuja fisiopatologia é desconhecida. Os distúrbios no desenvolvimento e o dano cerebral podem ocorrer como resultado do acúmulo tecidual de sulfito no cérebro. Os objetivos deste estudo foram verificar os efeitos in vitro e in vivo do sulfito sobre alguns parâmetros de estresse oxidativo (avaliação de lipoperoxidação e capacidade antioxidante tecidual) e sobre a atividade da Na+, K+-ATPase em córtex cerebral, estriado e hipocampo de ratos. Primeiramente, verificamos o efeito in vitro do sulfito sobre o estresse oxidativo e a Na+, K+-ATPase em cérebro de ratos de 10 e 60 dias. Posteriormente, nos estudos in vivo, investigamos o efeito da administração intracerebroventricular de sulfito sobre os parâmetros estudados in vitro. Os estudos in vitro demonstraram uma ação direta do sulfito (500μM) na indução de estresse oxidativo verificada pela redução na atividade da catalase e aumento da peroxidação lipídica, enquanto que nos estudos in vivo o sulfito não alterou a atividade das enzimas antioxidantes, TRAP ou TBARS. Tanto nos estudos in vitro como in vivo, o sulfito mostrou-se incapaz de alterar a atividade da Na+,K+-ATPase. Nossos resultados, em conjunto, não excluem o potencial efeito neurotóxico do sulfito na fisiopatologia da doença. O conhecimento dos níveis deste composto no cérebro pode evidenciar além da condição de estresse oxidativo, o comprometimento de outras vias metabólicas importantes no funcionamento cerebral e podem apontar estratégias terapêuticas na prevenção dos efeitos neurológicos da deficiência de sulfito oxidase. / The sulfite oxidase deficiency is a rare autosomal recessive disorder affecting the metabolism of methionine and cysteine. Affected individuals commonly present in the neonatal period intractable seizures, mental retardation and movement disorder which the physiopathology is unknown. The disturbed development and damage to the brain might occur as a result of tissue accumulation of sulfite in the cerebro. The objectives of this study was to investigate the in vitro and in vivo effects of sulfite on some parameters of oxidative stress (lipoperoxidation and antioxidant capacity) and on Na+, K+-ATPase activity in cerebral cortex, striatum and hippocampus from rats. Firstly, we verified the in vitro effects of sulfite on oxidative stress and Na+, K+- ATPase in brains from 10 and 60 days old rats. In the subsequent events, in the in vitro studies, we investigated the effect of intracerebroventricular injection of sulfite on the same parameters studied in vitro. The in vitro studies showed a direct action of sulfite (500 μM) in the induction of oxidative stress through the decrease of catalase activity and increase of peroxidation lipid, while the in vivo studies didn’t alter the antioxidants enzyme activity, TRAP or TBARS. Both in vitro and in vivo studies, showed that sulfite was incapable to disturb the Na+,K+-ATPase activity. Our results, together, don’t exclude the potencial neurotoxic effect of sulfite in the physiopathology of disease. The kwonledge of levels from this compound in the brain can show over there the oxidative stress, the compromise of others metabolic patways important to the brain function and can to lead to strategies therapeutics in the prevention of neurologic effects on sulfite oxidase deficiency.

nÃo hà / No mundo, existem cerca de 3.000 espÃcies de serpentes das quais 10 a 14% sÃo peÃonhentas. Dentre os paÃses sul-americanos, o Brasil à o que apresenta maior nÃmero de acidenÂtes/ano com cerca de 20.000 acidentes ofÃdicos por ano. De acordo com o MinistÃrio da SaÃde, as serpentes do gÃnero Bothrops sÃo as principais envolvidas nos acidentes ofÃdicos no paÃs e a insuficiÃncia renal aguda (IRA) à uma complicaÃÃo grave dos envenenamentos produzidos por estas serpentes. Tendo em vista que a fraÃÃo L-aminoÃcido oxidase (LAAO) constitui grande parte da composiÃÃo total do veneno de serpentes, em algumas serpentes chegando a constituir mais de 30% do total de proteÃnas do veneno, neste trabalho, foram investigados os efeitos renais da fraÃÃo L-aminoÃcido oxidase isolada do veneno da serpente Bothrops marajoensis (LAAOBM) em sistema de perfusÃo de rim isolado e em cultura de cÃlulas tubulares renais da linhagem MDCK (Madin-Darby Canine Kidney). Para perfusÃo de rim isolado foram utilizados ratos Wistar pesando entre 250 e 300g, cujos rins foram excisados cirurgicamente e perfundidos com soluÃÃo de Krebs-Hanseleit contendo 6%p/v de albumina bovina previamente dialisada. Foram investigados os efeitos da LAAOBM (10 Âg/mL; n=4) sobre a PressÃo de PerfusÃo (PP), ResistÃncia Vascular Renal (RVR), Fluxo UrinÃrio (FU), Ritmo de FiltraÃÃo Glomerular (RFG), Percentual de Transporte Tubular Proximal de SÃdio (%pTNa+),Percentual de Transporte Tubular de SÃdio (%TNa+), de PotÃssio (%TK+) e de Cloreto (%TCl-). As cÃlulas MDCK foram cultivadas em meio de cultura RPMI 1640 suplementado com 10% v/v de Soro Bovino Fetal e entÃo incubadas com a LAAOBM nas concentraÃÃes de 50; 25; 12,5; 6,25; 3,125 e 1,652 Âg/mL. ApÃs 24 horas de incubaÃÃo, foram realizados os ensaios de viabilidade e proliferaÃÃo celular utilizando-se o mÃtodo do MTT. A LAAOBM promoveu uma reduÃÃo da pressÃo de perfusÃo aos 90 minutos de experimento e esta reduÃÃo foi ainda discretamente maior aos 120 minutos. Observou-se tambÃm queda da resistÃncia vascular renal aos 120 minutos. Houve uma queda abrupta e acentuada do fluxo urinÃrio aos 90 minutos que, apesar da tendÃncia à recuperaÃÃo observada aos 120 minutos, ainda mostrou-se bastante reduzido quando comparado ao grupo controle. A infusÃo da LAAOBM tambÃm promoveu uma reduÃÃo do ritmo de filtraÃÃo glomerular aos 90 minutos quando comparada ao grupo controle e este parÃmetro manteve-se ainda no mesmo patamar de reduÃÃo aos 120 minutos. A LAAOBM reduziu gradativamente o percentual de transporte tubular de sÃdio aos 90 e 120 minutos e o percentual de transporte tubular de cloreto nos tempos de 60, 90 e 120 minutos. A anÃlise histolÃgica dos rins perfundidos com LAAOBM mostrou a presenÃa de alteraÃÃes morfolÃgicas significativas, como acÃmulo de proteÃnas nos espaÃos tubulares e glomerulares. Na cultura de cÃlulas MDCK a LAAOBM promoveu uma reduÃÃo da viabilidade celular a partir da concentraÃÃo de 3,25Âg/mL atà a concentraÃÃo de 50Âg/mL, com valor da CI50 de 2,43Âg/mL. Foram observadas tambÃm, atravÃs de microscÃpio Ãptico invertido, alteraÃÃes morfolÃgicas destas cÃlulas, tais vacuolizaÃÃo citoplasmÃtica, alteraÃÃo do estado de confluÃncia e desprendimento das mesmas do substrato de cultura. Estes resultados demonstram que a LAAOBM alterou todos os parÃmetros vasculares e renais avaliados na perfusÃo de rim isolado e possui aÃÃo citotÃxica sobre as cÃlulas MDCK apÃs 24 horas de incubaÃÃo. / There are about 3.000 species of snakes worldwide, but only 10 to 14% are venomous. Among South American countries, Brazil is the one with the largest number of accidents, with approximately 20.000 snakebites each year. According to the Department of Health of Brazil, the genus Bothrops are the main involved in snakebites in the country. Acute renal failure (ARF) is a serious complication of snake poisoning. The fraction L-amino acid oxidase (LAAO) constitutes a main part of the total composition of snake venoms. In some cases this amount can reach 30% of total venom proteins. The renal effects of fraction L-amino acid oxidase isolated from the venom of Bothrops marajoensis (LAAOBM) was investigated in this study. Isolated perfused rat kidney and the cultured renal tubular cells line MDCK (Madin-Darby Canine Kidney) were used here. For the isolated perfused rat kidney method, we used Wistar rats weighing between 250 and 300. Their right kidneys were surgically excised and perfused with Krebs-Hanseleit containing 6% w/v bovine albumin previously dialyzed. The effects of LAAOBM (10 mg/mL, n=4) were analyzed on the Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR) Percentage of Sodium Proximal Tubular Transport (%pTNa+), Percentage of Sodium (%TNa+), Potassium (%TK+) and Chloride (%TCl-) Tubular Transport. MDCK cells were cultured in RPMI 1640 medium supplemented with 10% v/v fetal bovine serum and incubated with LAAOBM at concentrations of 50, 25, 12.5, 6.25, 3.125 and 1.652 mg/mL. After 24 hours of incubation, assays were performed on cell proliferation and viability using the MTT method. The LAAOBM promoted a reduction of perfusion pressure at 90 minutes of the experiment and this reduction was even slightly higher at 120 minutes. It was also observed decrease in renal vascular resistance at 120 minutes. There was a sharp and sudden drop in urine flow at 90 minutes, despite the tendency of recovery observed at 120 minutes, still proved to be quite small when compared to the control group. The infusion of LAAOBM also promoted a reduction in glomerular filtration rate at 90 minutes compared to the control group and this parameter still remained at the same level of reduction at 120 minutes. The LAAOBM gradually reduced the percentage of sodium tubular transport at 90 and 120 minutes and the percentage of chloride tubular transport in the periods of 60, 90 and 120 minutes. Histological analysis of kidneys perfused with LAAOBM showed the presence of significant morphological changes such as accumulation of proteins in tubular and glomerular spaces. In the MDCK cell culture LAAOBM promoted a reduction in cell viability from concentrations of 3.25 mg / mL until 50μg/mL, with IC50 value of 2.43 mg/mL. Inverted light microscopy showed morphological changes of these cells, such as vacuolation, alteration of the state of confluence and detachment of the substrate culture. These results demonstrated that LAAOBM changed all the parameters evaluated in renal and vascular perfusion of isolated kidney and had cytotoxic activity on MDCK cells after 24 hours of incubation.

Master of Science / Department of Agronomy / Kassim Al-Khatib / Field experiments were conducted near Manhattan, KS in 2005 and 2006 and Sabetha, KS in 2005 to determine the efficacy of S-metolachlor tank mixed with fomesafen on common waterhemp in soybean. Preemergence treatments included S-metolachlor + fomesafen at 0.91 + 0.22, 1.21 + 0.28, 1.52 + 0.36, and 1.82 + 0.43 kg ha-1 and S-metolachlor + metribuzin at 0.55 + 0.14 kg ha-1. These treatments were applied alone or followed by a postemergence glyphosate application at 0.88 kg ha-1. Ratings were taken 2, 4 and 8 weeks after treatment. The study showed that S-metolachlor + fomesafen gave excellent early season control of common waterhemp at both Sabetha and Manhattan. S-metolachlor + fomesafen at the 1.52+0.36 kg ha-1 rate gave greater weed control than S-metolachlor + metribuzin. A separate study was conducted to determine the competitiveness and fitness of a protox-resistant common waterhemp biotype. Protox-resistant and protox-susceptible biotypes of common waterhemp were grown under noncompetitive and competitive arrangements in the greenhouse. In the noncompetitive study a single plant of both biotypes was planted in 15-cm-diam pots. Photosynthesis, leaf area, and plant biomass were measured 10, 20, 30, and 40 day after transplanting (DATP). In general, photosynthesis rate and plant biomass was similar between biotypes. However, the protox-resistant biotype had higher leaf area then the susceptible biotype at 20, 30, and 40 DATP. Under competitive conditions, a replacement series study, photosynthesis, leaf area, plant height, and plant biomass were measured 7, 14, 21, and 28 DATP. In general protox-resistant and –susceptible common waterhemp values were similar 28 DATP. Relative crowding coefficient values 28 DATP were 0.86, 0.89, 1.09, and 1.13 for photosynthesis, leaf area, plant height, and plant biomass, respectively. Suggesting, protox resistance did not change the ability of common waterhemp to grow normally under competitive conditions.

Note: Portions of this abstract have been previously published in the journal Blood, Basiorka et al. Blood. 2016 Oct 13, and has been reproduced in this manuscript with permission from the publisher. Myelodysplastic syndromes (MDS) are genetically diverse hematopoietic stem cell malignancies that share a common phenotype of cytological dysplasia, ineffective hematopoiesis and aberrant myeloid lineage maturation. Apoptotic cell death potentiated by inflammatory cytokines has been considered a fundamental feature of MDS for over two decades. However, this non-inflammatory form of cell death cannot account for the inflammatory nature of these disorders. We report that a hallmark of lower-risk (LR) MDS is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptosis, a caspase-1-dependent programmed cell death induced by danger-associated molecular pattern (DAMP) signals. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPC) overexpress pyroptosis-related transcripts, inflammasome proteins and manifest activated NLRP3 inflammasome complexes that direct caspase-1 activation, IL-1β and IL-18 maturation and pyroptotic cell death. Using the S100A9 transgenic (S100A9Tg) mouse model that phenocopies human MDS, we demonstrated that forced expression of S100A9 was sufficient to drive pyroptosis in vivo, implicating pyroptosis as the principal mechanism of HSPC cell death in S100A9Tg mice. The lytic cell death releases intraceullar contents that include alarmins and catalytically active ASC specks, which can propagate bystander inflammation. Notably, MDS mesenchymal stromal cells (MSC) and stromal-derived linages were found to predominantly undergo pyroptosis, with marked activation of caspase-1 and NLRP3 inflammasome complexes. These findings may account for the clusters of both HSPC and stromal cell death previously described in the bone marrows of patients with MDS. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPC and bone marrow (BM) plasma. Further, both somatic gene mutations and S100A9-induced signaling activate NADPH oxidase (NOX), generating reactive oxygen species (ROS) that initiate cation influx, cell swelling and β-catenin activation. Accordingly, ROS and active β-catenin were significantly increased in MDS BM mononuclear cells (BM-MNC) and S100A9Tg mice compared to normal controls, as well as in human cell lines harboring gene mutations and in murine models of gene mutation knock-in or gene loss. ROS and β-catenin nuclear translocation were significantly reduced by NLRP3 or NOX inhibition, indicating that S100A9 and somatic gene mutations prime cells to undergo NOX1/4-dependent NLRP3 inflammasome assembly, pyroptosis and β-catenin activation. Together, these data explain the concurrent proliferation and inflammatory cell death characteristic of LR-MDS. Given that loss of a gene-rich area in del(5q) disease results in derepression of innate immune signaling, we hypothesized that this genetic deficit would trigger assembly of the NLRP3 inflammasome complex, akin to the pathobiological mechanism characteristic of non-del(5q) MDS. To this end, we utilized two distinct murine models of del(5q) disease, namely in the context of Rps14 haploinsufficiency and concurrent loss of mDia1 and microRNA (miR)-146a. In both models, pyroptosis was not evident in the HSPC compartment; however, early erythroid progenitors displayed high fractions of pyroptotic cells. This was associated with significant increases in caspase-1 and NLRP3 inflammasome activation, ROS and nuclear localization of β-catenin, which was extinguished by inflammasome or NOX complex inhibition. These data suggest that early activation of the inflammasome drives cell death and prevents terminal maturation of erythroid precursors, accounting for the progressive anemia characteristic of del(5q) disease, whereby hematopoietic defects are primarily restricted to the erythroid compartment. Importantly, these data implicate a similar pathobiological mechanism in del(5q) MDS as is observed in non-del(5q) patients. The identification of the NLRP3 inflammasome as a pathobiological driver of the LR non-del(5q) and del(5q) MDS phenotype allows for novel therapeutic agent development. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppresses pyroptosis, ROS generation and nuclear β-catenin in MDS, and are sufficient to restore effective hematopoiesis. In del(5q) murine models, inhibition of the NLRP3 inflammasome significantly improved erythroid colony forming capacity by a mechanism distinct from that of lenalidomide, highlighting the translational potential for targeting this innate immune complex in this subset of MDS. Thus, alarmins and founder gene mutations in MDS license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention. Furthermore, aggregated clusters of the NLRP3 adaptor protein ASC [apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD)] are referred to as ASC specks. During pyroptosis, ASC specks are released from dying cells and function as DAMP signals that propagate inflammation. In this way, specks are a surrogate marker for NLRP3 inflammasome activation and pyroptotic cell death. Given that pyroptosis is the predominant mechanism of cell death in MDS and ASC specks are readily quantified by flow cytometry, we hypothesized that BM or peripheral blood (PB) plasma-derived ASC specks may be a biologically rational biomarker for the diagnosis of MDS. The percentage of ASC specks were significantly increased in MDS BM plasma compared to normal, healthy donors, which was validated by confocal microscopy. PB plasma-derived ASC specks were significantly greater in LR- versus HR-MDS, consistent with the greater extent of cell death and myeloid-derived suppressor cell (MDSC) expansion in LR disease. As hyperglycemia induces NLRP3 inflammasome activation, plasma glucose levels were measured to adjust for this confounding variable. Subsequently, the percentage of glucose-adjusted, PB plasma-derived ASC specks was measured in a panel of specimens of varied hematologic malignancies. The corrected percentage of ASC specks was significantly increased in MDS compared to normal donors and to each other malignancy investigated, including other myeloid and lymphoid leukemias, myeloproliferative neoplasms and overlap syndromes, like chronic myelomonocytic leukemia (CMML). These data indicate that the glucose-adjusted ASC speck percentage is MDS-specific and may be a valuable diagnostic biomarker. At a cutoff of 0.039, the biomarker minimizes misclassification error and achieves 95% sensitivity and 82% specificity in classifying MDS from normal donors, other hematologic malignancies and T2D. Lastly, the biomarker declined with treatment response to lenalidomide in LR-MDS patients, but not to erythropoietin stimulating agent (ESA) or hypomethylating agent (HMA) therapy. As such, the percentage of ASC specks represents the first biologically rational, diagnostic biomarker for MDS that can be implemented with current diagnostic practices to reduce diagnostic error.

Phenolic compounds are a group of organic chemicals present in the wastewaters of many synthetic industrial processes. Due to their extreme toxicity to man and animals, and deleterious impact on the environment, a range of techniques exist for the effective treatment and disposal of these pollutants. Biological degradation using microbial enzymes presents a valuable alternative to conventional wastewater treatment systems. This research was therefore initiated to investigate the polyphenol oxidase enzyme system and the feasibility of its application for effluent treatment and studies in organic solvents. The enzyme system is widely distributed in nature, with Agaricus bisporus (the common mushroom) being the best known producer. Biochemical investigations of the enzyme system were therefore carried out using this extract. A screening programme was initiated to identify microbial polyphenol oxidase producers which could be cultured in liquid media, thereby enabling the production of large quantities of enzyme in fermentation systems. Extensive growth optimization and enzyme induction and optimization studies were carried out on selected cultures. A number of good producers were isolated, namely a bacterial culture designated AECI culture no. 26, Streptomyces antibioticus, Streptomyces glaucescens and a manipulated strain, Streptomyces lividans (pIJ702). Enzyme production by Agaricus bisporus mycelia was optimized in deep-liquid culture; enzyme extracts showed high phenol removal efficiencies. Streptomyces antibioticus, Streptomyces glaucescens, Streptomyces lividans (pIJ702) and AECI culture no. 26 whole cells were also investigated for phenol-removing ability in simulated phenolic effluents. The use of whole cells reduces enzyme inactivation and instability due to the protection of the enzyme system within the cell. All cultures showed improved removal efficiencies in phenolic growth media. These results strongly suggest their use for phenol removal in continuous systems.

Investigations into the cognitive effects of tobacco smoking have generally focused on nicotine and its effect on nicotinic acetylcholine receptors (nAChRs) in the brain. However, it is now known that chronic smokers exhibit robust inhibition of the monoamine oxidase (MAO) enzyme through the actions of non-nicotine components in tobacco smoke. Therefore, the primary aim of this thesis is to elucidate the effects of nicotine and MAO-inhibition on electroencephalographic (EEG) and event-related potential (ERP) measures of cognition. 24 healthy nonsmoking males were administered 75 mg of moclobemide, and chewed 6 mg nicotine gum, in order to simulate the effects of acute smoking. Four experimental conditions included placebo, nicotine, moclobemide, and a combination of nicotine and moclobemide. Early auditory ERPs were used as measures of cognition, such as the auditory P50 sensory gating paired-stimulus paradigm, the acoustic-change-elicited mismatch-negativity (MMN), the novel sound-elicited P3a, and the target sound-elicited P3b. Three minutes of eyes closed EEG were also recorded. Because these ERPs are often identified as biomarkers for schizophrenia, drug effects were also measured after individuals were stratified for low-baseline amplitude of each ERP measure, as a laboratory model of cognitive deficits in schizophrenia. Overall results showed a synergistic improvement in sensory gating via nicotine combined with moclobemide, accompanied by a reduction in theta band power. Nicotine in the absence of moclobemide increased P3b amplitude, accompanied by an increase in alpha2 band power. Moclobemide in the absence of nicotine increased P3a amplitude, accompanied by a decrease in beta2 power. Stratifying participants by placebo amplitude revealed both nicotine and moclobemide exhibited an inverted-U pattern of effect, i.e. showing greater amplitude increases in individuals with the lowest baseline amplitudes. Overall, this thesis demonstrates how these two components of tobacco smoke affect different facets of auditory processing in different ways, with synergistic effects in some paradigms but antagonizing effects in others. Therefore, chronic smokers and schizophrenia patients who seek transient cognitive improvement through smoking may actually experience cognitive detriments overall, possibly contributing to withdrawal symptoms and/or an exacerbation of already-present psychiatric symptoms.

O sulfito e o tiossulfato estão acumulados em tecidos e líquidos biológicos de pacientes afetados pela deficiência da sulfito oxidase (SO), uma enzima mitocondrial que catalisa a oxidação de sulfito derivado do metabolismo de aminoácidos sulfurados. A deficiência da SO é causada pela deficiência isolada da enzima SO ou por uma deficiência na rota de biossíntese de seu cofator molibdênio. Os indivíduos afetados por esta desordem apresentam disfunção neurológica progressiva, convulsões neonatais severas, subluxação do cristalino, hipotonia axial, hipertonicidade periférica e atraso no desenvolvimento, resultando geralmente em morte prematura. Considerando que a fisiopatologia do dano neurológico encontrado em pacientes deficientes para a SO ainda não está esclarecida, o objetivo do presente trabalho foi investigar os efeitos in vitro do sulfito e do tiossulfato sobre parâmetros de metabolismo energético e homeostase redox e mitocondrial em cérebro de ratos jovens. Inicialmente, verificamos que o sulfito inibe a atividade do complexo IV da cadeia respiratória, indicando que este composto prejudica o fluxo de elétrons, enquanto que o tiossulfato não afetou a atividade de nenhum dos complexos da cadeia respiratória em sobrenadantes de córtex cerebral. Também foi verificado que o sulfito e o tiossulfato diminuem a atividade da creatina quinase total (tCK) e de suas isoformas mitocondrial e citosólica, sugerindo que estes compostos prejudicam o tamponamento e a transferência de energia celular no cérebro. Além disso, melatonina, trolox (análogo solúvel do α-tocoferol), glutationa e o inibidor da óxido nítrico sintase Nω-nitro-L-arginina metil éster atenuaram ou preveniram totalmente a inibição da tCK induzida por sulfito e tiossulfato, sugerindo o envolvimento de espécies reativas de oxigênio e nitrogênio nestes efeitos. O sulfito e o tiossulfato também aumentaram a oxidação da 2’,7’-diclorofluorescina e inibiram a atividade da aconitase, enquanto que somente o sulfito aumentou a produção de peróxido de hidrogênio, reforçando o envolvimento de dano oxidativo nos efeitos provocados por estes metabólitos. Contudo, a atividade da enzima Na+,K+-ATPase sináptica não foi alterada pelo sulfito e tiossulfato. Em seguida, observamos que o sulfito dissipa o potencial de membrana mitocondrial na presença de Ca2+, de forma dose-dependente de sulfito e Ca2+ em preparações mitocondriais de cérebro de ratos. O sulfito também induziu inchamento e diminuiu a capacidade de retenção de Ca2+, os níveis de NAD(P)H na matriz e o imunoconteúdo de citocromo c em mitocôndrias quando Ca2+ estava presente no meio. Além disso, as alterações provocadas pelo sulfito foram prevenidas por rutênio vermelho, ciclosporina A e ADP, sugerindo que o sulfito induz transição da permeabilidade mitocondrial (MPT). Também foi verificado que dentre vários inibidores da MPT, incluindo antioxidantes, inibidores da fosfolipase A2 e o regente redutor ditiotreitol, apenas o agente alquilante de tióis N-etilmaleimida foi capaz de prevenir o inchamento mitocondrial causado por sulfito. O sulfito também diminuiu o conteúdo de grupamentos tiol de proteínas de membrana em preparações mitocondriais de cérebro, indicando que este composto age diretamente sobre grupamentos tiol contidos no poro de MPT. Assim, pode-se presumir que o prejuízo no metabolismo energético e na homeostase redox causados pelo sulfito e pelo tiossulfato e que a indução de MPT pelo sulfito podem estar envolvidos na disfunção neurológica observada nos portadores da deficiência da SO. / Sulfite and thiosulfate accumulate in tissues and biological fluids of patients affected by the deficiency of sulfite oxidase (SO), which is a mitochondrial enzyme that catalyzes the oxidation of sulfite derived from the metabolism of sulfur amino acids. SO deficiency is caused by the isolated deficiency of the enzyme SO itself or by a deficiency in the biosynthetic pathway of its molybdenum cofactor. Individuals affected by this disorder present progressive neurological dysfunction, severe neonatal seizures, lens subluxation, axial hypotonia, limb hypertonicity and failure to thrive, resulting often in early childhood death. Considering that the pathophysiology of the neurological damage found in SO deficient patients has not been totally established, the aim of the present work was to investigate the in vitro effect of sulfite and thiosulfate on parameters of energy metabolism, as well as redox and mitochondrial homeostasis in rat brain. First, we verified that sulfite inhibited the activity of complex IV of the respiratory chain in cerebral cortex supernatants, indicating that this compound impairs the electron transfer flow, whereas thiosulfate did not affect any of the activities of the respiratory chain complexes. It was also found that sulfite and thiosulfate markedly decreased the activity of total creatine kinase (tCK) and its mitochondrial and cytosolic isoforms, suggesting that these compounds impair brain cellular energy buffering and transfer. Moreover, melatonin, trolox (soluble analogue of α-tocopherol), glutathione and the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester attenuated or fully prevented the inhibition of tCK induced by sulfite and thiosulfate, suggesting the involvement of reactive oxygen and nitrogen species in these effects. Sulfite and thiosulfate also increased 2’,7’-dichlorofluorescin oxidation and inhibited the activity of aconitase, whereas only sulfite increased hydrogen peroxide production, reinforcing the involvement of oxidative damage in the effects elicited by these metabolites. In contrast, synaptic Na+,K+-ATPase activity was not altered by sulfite and thiosulfate. Next, we observed that sulfite dissipates mitochondria membrane potential in the presence of Ca2+, in a sulfite and Ca2+ dose-dependent manner. Sulfite also induced swelling and decreased Ca2+ retention capacity, matrix NAD(P)H pool and cytochrome c immunocontent in mitochondria when Ca2+ was present in the medium. Furthermore, the alterations elicited by sulfite were prevented by ruthenium red, cyclosporine A and ADP, supporting the involvement of mitochondrial permeability transition (MPT) in these effects. It was also verified that among various MPT inhibitors, including antioxidants, phospholipase A2 inhibitors and the reductant reagent dithiothreitol, only the thiol alkylating agent N-ethylmaleimide was able to prevent the sulfite-elicited mitochondrial swelling. Moreover, sulfite decreased membrane protein thiol group content in brain mitochondria, indicating that this compound acts directly on MPT pore containing thiol groups. Taken together, it may be presumed that the mitochondrial energy and redox homeostasis impairment caused by sulfite and thiosulfate and MPT induced by sulfite may be involved in the neurological dysfunction observed in patients affected by SO deficiency.

>Magister Scientiae - MSc / Alzheimer’s Disease (AD) is a neurodegenerative disease which results from the irreversible loss of neurons in the brain. The disease is characterized by progressive cognitive impairment with recurrent short-term memory loss. AD is the leading cause of dementia and 4th leading cause of death in the elderly. Success in the treatment of AD has been limited, with drugs only treating it at a symptomatic level due to its pathology being complex and poorly understood. However, it is known that the cholinesterase and MAO-B enzymes play an important role in the disease through their association with production of amyloid plaques and oxidative stress respectively, two mechanisms associated with cell death and the symptoms seen in AD.

Les neutrophiles participent à la défense de l'hôte en phagocytant les agents pathogènes et en les détruisant via notamment la production de formes réactives de l'oxygène (FRO). Les FRO sont produites par un complexe multi- protéique :la NADPH oxydase (NOX2). Celle-ci peut s’assembler à la membrane du phagosome lors de la phagocytose mais aussi à la membrane plasmique lors de la stimulation des neutrophiles par des agents bactériens ou des médiateurs de l’inflammation. La NADPH oxydase est une arme à double tranchant; une activation excessive ou inappropriée de la NADPH oxydase génère un stress oxydant, facteur aggravant des nombreuses pathologies. Cette enzyme doit donc être finement régulée. La NADPH oxydase est activée lorsque les sous-unités cytosoliques de NOX2 (p67phox, p47phox, p40phox) et la petite GTPase Rac s’assemblent avec les sous-unités membranaires (p22phox et gp91phox) à la membrane phagosomale ou plasmique. P67phox régule le flux d'électrons qui transite via gp91phox du NADPH à O2.-. Des travaux récents indiquent que les phospholipides anioniques contribueraient à la régulation de la NADPH oxydase. De plus, Les protéines organisatrices p40phox et p47phox possèdent des domaines de liaison à ces phosphoinositides : p40phox peut se lier au phosphatidylinositol 3-phosphate (PI(3)P) et p47phox au phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Nous avons donc voulu comprendre le rôle des ces phospholipides dans la régulation de la NADPH oxydase. Dans un premier temps nous nous sommes intéressés au rôle du PI(3)P, présent au phagosome après la fermeture de celui-ci, dans l’activation de la NADPH oxydase. Nos données indiquent que p40phox fonctionne comme un adaptateur, PI(3)P dépendant, permettant de maintenir p67phox dans le complexe de la NADPH oxydase. Le PI(3)P agit comme un « timer » pour l'activation de la NADPH oxydase au phagosome. Nous avons ensuite voulu examiner le rôle du PI(3,4)P2 dans la régulation de la NADPH oxydase à la membrane plasmique. Ce lipide est formé à la membrane plasmique par phosphorylation du PI(4)P par la PI3K de classe I lors de l’activation des neutrophiles. Nous avons montré que, l'activité PI3K de classe I est nécessaire pour maintenir l’activation, intégrine-dépendante, de la NADPH oxydase à la membrane plasmique. / The NADPH oxidase of the professional phagocyte is essential for the immune system. The phagocyte NADPH oxidase, NOX2, catalyze the reduction of molecular oxygen to superoxide. Superoxide is transformed rapidly into other reactive oxygen species (ROS) which play a critical role in the killing of pathogens in host defense. Indeed neutrophils, the first cells that arrive at the site of infections, engulf pathogens in a process called phagocytosis. The production of reactive oxygen species is then triggered by the NADPH oxidase in the phagosome. The importance of ROS production is demonstrated by the recurrent bacterial and fungal infections that face patients who lack functional NADPH oxidase as in the rare genetic disorder known as the chronic granulomatous disease (CGD). Upon stimulation by bacterial peptide or in some pathological conditions, NADPH oxidase can also be activated at the phagocyte plasma membrane producing ROS in the extracellular medium. So, an excessive or inappropriate NADPH oxidase activation generates oxidative stress involve in chronic inflammation, cardiovascular disease and neurodegenerative disease. The NADPH oxidase activity should be tightly regulated. The activity of the enzyme is the result of the assembly of cytosolic subunits (p47phox, p67phox, p40phox and Rac2) with membranous subunits (gp91phox and p22phox). P67phox regulates the electron flow through gp91phox from NADPH to oxygen leading to the formation of superoxide. Recent data indicate that the anionic phospholipids are important for the NADPH oxidase regulation. Moreover, p40phox and p47phox bear a PX domain that binds respectively phosphatidylinositol3-phosphate (PI3P) and phosphatidylinositol (3,4)-bisphosphate(PI(3,4)P2). Our objective was to decipher the importance of these phosphoinositides on the NADPH oxidase activity. We first examined the role of PI3P, which is present on the cytosolic leaflet of phagosome after its sealing, in NADPH oxidase activation. Our data indicate that p40phox works as a late adaptor controlled by PI3P to maintain p67phox in the NADPH oxidase complex. Thus, PI3P acts as a timer for NADPH oxidase assembly. We then examined the role of PI(3,4)P2 in the activation of the NADPH oxidase assembled at the plasma membrane. PI(3,4)P2 and PI(3,4,5)P3 are formed at the plasma membrane, upon neutrophil activation, by phosphorylation by Class I PI3K of respectively PI4P and PI(4,5)P2. We found that class I PI3K activity is required to maintain the integrin-dependent activation of NADPH oxidase at the plasma membrane.

Thesis (D.Sc.D.)--Boston University, Goldman School of Dental Medicine, 2008 (Dept. of Periodontology and Oral Biology). / Includes bibliographical references: leaves 140-148. / Lysyl oxidases constitute a family of enzymes responsible for the formation of cross links in collagen and elastin. These enzymes have also been linked to pathological fibrosis. The importance of collagen in the structural and mechanical properties of bone led us to investigate the hypothesis that the absence of one or more of these enzymes could lead to a significant bone phenotype. This phenotype could resemble osteoporosis or diabetic bone disease. In addition, we tried to overexpress lysyl oxidase proenzyme in vitro. The ability to produce enough amounts of lysyl oxidase proenzyme and the ability to process it and activate it could facilitate the development of drugs that control its activity in pathological fibrosis. Bones from 12-week old mice (8 males and 8 females) with the compound genotype LOX+/-, LOXLl -/- were analyzed. 5 males of the genotype LOX+/+, LOXLl-/were also analyzed. 16 wild type mice (8 males and 8 females) were used as controls. μCT was used to analyze the trabecular and cortical bone morphology of both left femur and L5 vertebrae (n=5). The femora were subsequently subjected to mechanical testing using the twist failure in torsion. Right femurs (n=5) were used for histology and histromorphometric analysis. Tibia and fibula (n=5) were used for cross-link analysis. Two way factor ANOV A with post-hoc Tukey HSD test was used for statistical analysis. A P value of less than 0.05 was used to declare significance. μCT analysis of the trabecular bone in femur distal ... [TRUNCATED]