Background: Oxidative stress is supposed to be implicated in the pathogenesis of several diseases which are connected with increased formation of reactive oxygen and nitrogen species (RONS). Oxidative stress could play an important role in the pathogenesis of inflammation and sepsis, acute and chronic pancreatitis or in the development of cancer. Organisms are protected against RONS from antioxidant system that is composed of antioxidant enzymes and non-enzymatic antioxidants. To the most important antioxidant enzymes belong superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, glutathione reductase and paraoxonase (PON). The aim of this Doctoral Thesis was to investigate the behaviour of three of these antioxidant enzymes - CuZnSOD, CAT and PON1 in different pathophysiological states. Materials and methods: The activities of CuZnSOD, CAT and PON1 were measured in six different pathophysiological states. Forty patients with metabolic syndrome (MetS), 35 women with depressive disorder (DD), 30 septic patients (SP), 50 patients with pancreatic cancer (PC), 50 patients with chronic pancreatitis (CP) and 13 patients with acute pancreatitis (AP) were included in different studies together with sex- and age-matched healthy controls (CON). Patients with AP and SP were observed in the course...

A dor pediátrica tem sido o foco de estudo de muitos pesquisadores nas últimas décadas devido à constatação de que neonatos apresentam menor limiar para estímulos nocivos e inócuos em comparação aos adultos. Em decorrência disto, o uso de analgésicos tem sido frequente em ambiente hospitalar. A morfina é um dos analgésicos opióides mais utilizados para sedação e analgesia nestes pacientes. Este opióide apresenta maior potência analgésica no período neonatal, sendo o receptor μ mais expresso em neurônios medulares com pico de densidade em P7 e diminuindo até o P21, atingindo então níveis de adulto. Nosso grupo mostrou que ratos neonatos submetidos ao tratamento repetido com morfina não apresentam tolerância. Porém, permanecem maior tempo em analgesia ao final do tratamento do que no 1º dia. Além disto, após dois dias do término do tratamento os animais apresentaram alterações na atividade e expressão gênica da NTPDase 1 (enzima que hidrolisa ATP até adenosina) em medula espinal e córtex cerebral, sugerindo modulação nos níveis extracelulares de nucleotídeos, o que pode levar alterações na resposta nociceptiva. Além do sistema purinérgico outro importante sistema no processamento da resposta nociceptiva é o glutamatérgico. Desde o período neonatal o glutamato é o responsável pelos estímulos nociceptivos em medula espinal, e transportadores no terminal pré-sináptico ou glia são responsáveis pela captação do glutamato liberado, controlando seus níveis. A exposição à morfina também leva a mudanças comportamentais, conhecidas por sensibilização comportamental, que são dependentes, em parte, da ativação do receptor dopaminérgico D2 no sistema límbico. Além disso, esta exposição pode alterar os níveis de BDNF em estruturas relacionadas à nocicepção. Levando em consideração a carência de estudos focados nos efeitos da exposição repetida a opióides em neonatos, o objetivo deste estudo foi verificar os efeitos a curto (P16), médio (P30) e longo prazo (P60) do tratamento com 5 μg de morfina, uma vez ao dia, do P8 ao P14, sobre comportamento nociceptivo; captação de glutamato em medula espinal; comportamentos exploratórios e do tipo ansioso, avaliando o envolvimento do receptor D2; níveis de BDNF e TNF-α, e estresse oxidativo em hipocampo; atividades de nucleotidases solúveis em soro; e além disso avaliar a ação antinociceptiva da melatonina nas respostas nociceptivas alteradas. Os animais que receberam morfina demonstraram em P30 e P60: aumento da resposta nociceptiva que foi revertida por antagonista do receptor NMDA; diminuição da captação de glutamato em medula espinal; aumento dos níveis de BDNF em hipocampo, e diminuição da atividade da SOD no P60; alterações nas atividades das nucleotidases solúveis; aumento do comportamento exploratório no P16 e P30; efeito antinociceptivo da melatonina na hiperalgesia. Estes dados demonstram a necessidade de pesquisas que sejam focadas nos efeitos do tratamento com morfina no período neonatal ao longo da vida, bem como buscar alternativas terapêuticas que possam reverter possíveis alterações. / The study of pediatric pain has been the focus of many researchers in recent decades due to the fact that neonates have a lower threshold for innocuous and noxious stimuli compared to adults. As a result, the use of analgesics has been frequent in hospitals. Morphine is an opioid analgesic commonly used for sedation in these patients. This opioid presents greater analgesic potency in the neonatal period in which the μ opioid receptor is over expressed in neurons of the spinal cord, with peak binding on day 7 and decreasing until day 21, reaching adult levels then. Our research group has shown that neonate rats subjected to repeated treatment with morphine not present tolerance. However, they remain with more time of analgesia at the end of treatment than on first day. Moreover, two days after the end of treatment the animals showed changes in activity and gene expression of NTPDase 1 (enzyme that hydrolyzes ATP to adenosine) in spinal cord and cerebral cortex, suggesting modulation of nucleotides extracellular levels which can alters nociceptive response. Besides the purinergic system another important system associated with nociceptive pathways is glutamatergic. Since the neonatal period glutamate is responsible for nociceptive stimulus in spinal cord, being its level controlled by transporters in the presynaptic terminal or glia responsible for the uptake of glutamate released. Importantly, studies have shown that morphine administration in adult animals can lead to behavioral changes, known as behavioral sensitization, dependent of activation of the limbic dopamine system, with the D2 dopamine receptor is associated with these changes. Moreover, such exposure may lead to altered levels of BDNF on the nociception related structures. Considering the lack of studies focused on the effects of repeated exposure to opioids in neonates, the objective of this study was to assess the short-(P16), medium (P30) and long term (P60) effects of treatment with 5 μg of morphine, once a day, from 8 to 14 days old, on nociceptive behavioral responses; glutamate uptake in the spinal cord; exploratory and type anxiously behavioral responses assessing the involvement of the dopamine receptor D2; levels of BDNF and TNF-α, and oxidative stress in the hippocampus; activities of serum soluble nucleotidases, and furthermore evaluate the antinociceptive action of melatonin on nociceptive responses changed. The results obtained with this study show that in P30 and P60 the animals that had received morphine: increase of the nociceptive response that was reversed by NMDA receptor antagonist, decreased glutamate uptake in spinal cord; increasing levels of BDNF in hippocampus, and decreased activity of the antioxidant enzyme SOD in P60; changes in nucleotide hydrolysis; increased exploratory behavior in P16 and P30; antinociceptive effect of melatonin on nociceptive responses increased. These data demonstrate the need for further research that are focused on the effects of morphine treatment in the neonatal period lifelong and seek alternative therapies that can reverse any changes.

No description available.

Orientador: Marcos Ferreira Minicucci / Coorientador: Ana Lúcia Gut / Resumo: O estresse oxidativo é reconhecido como característica fundamental da sepse e pode ser uma via comum para o desenvolvimento da lesão renal aguda (LRA). Alguns biomarcadores para a detecção precoce da LRA tem sido estudados, no entanto, apesar da importância no combate ao estresse oxidativo, a atividade da superóxido dismutase 1 (SOD1) ainda não foi avaliada na LRA em pacientes críticos. Nosso objetivo foi avaliar a atividade eritrocitária da SOD1 como preditora de LRA em pacientes com choque séptico. Este é um estudo prospectivo observacional que avaliou 175 pacientes consecutivos, maiores de 18 anos e com choque séptico na admissão na Unidade de Terapia Intensiva (UTI). Foram excluídos 43 pacientes. Assim, 132 pacientes foram incluídos no estudo. Nas primeiras 24 horas da admissão dos pacientes, foram coletados os dados demográficos e amostras de sangue para determinação da atividade eritrocitária da SOD1 e da concentração do malondialdeído (MDA). Todos os pacientes foram acompanhados durante a internação na UTI e o desenvolvimento da LRA foi avaliado. Além disso, também foram avaliados 17 indivíduos controle. A média de idade dos pacientes com choque séptico foi 63,2 ± 15,7 anos, 53% eram homens e a mediana do tempo de internação na UTI foi de 8 (4- 16) dias. Aproximadamente 51% dos pacientes desenvolveram LRA durante a internação na UTI. A mediana da atividade eritrocitária da SOD1 foi 2,92 (2,19- 3,92) U/mgHb. Quando comparado com os indivíduos controle, os pacientes com ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Oxidative stress is recognized as a fundamental characteristic of sepsis and may be a common pathway for the development of acute kidney injury (AKI). Some biomarkers for early detection of AKI have been studied, however, despite the importance in combating oxidative stress, the activity of superoxide dismutase 1 (SOD1) has not been evaluated in AKI in critically ill patients. Our objective was to evaluate erythrocyte SOD1 activity as predictor of AKI in patients with septic shock. This is a prospective observational study that evaluated 175 consecutive patients over the age of 18 years with septic shock upon Intensive Care Unit (ICU) admission. Forty-three patients were excluded. Thus, 132 patients were enrolled in the study. In the first 24 hours of the patients' enrollment, demographic information was recorded and blood samples were taken to determine the erythrocyte SOD1 activity and the concentration of malondialdehyde (MDA). All patients were followed throughout the ICU stay, and the development of AKI was evaluated. In addition, we also evaluated 17 control subjects. The mean age of patients with septic shock was 63.2 ± 15.7 years; 53% were male, and the median ICU stay was 8 (4-16) days. Approximately 51% of patients developed AKI during the ICU stay. The median erythrocyte SOD1 activity was 2.92 (2.19-3.92) U/mg Hb. When compared to control subjects, septic shock patients had a higher serum MDA concentration [shock group: 1.4 (0.8-2.2) µmol/L vs control group: 0.8 (0.5-1.1) µmol/L; p: 0.003] and lower erythrocyte SOD1 activity [shock group: 2.9 (2.2-3.9) U/mgHb vs control group: 4.4 (3.3-4.7) U/mgHb; p: 0.001]. In univariate analysis, erythrocyte SOD1 activity was lower in patients who developed AKI. The ROC curve analysis revealed that lower erythrocyte SOD1 activity was associated with AKI development at the cutoff of < 3.32 U/mg Hb. In the logistic regression... (Complete abstract click electronic access below) / Doutor

Zielstellung dieser Studie war es zu überprüfen, ob durch Fütterung einer mit Vitamin E und Selen angereicherten Mineralstoffmischung in der Transitphase eine Beeinflussung des antioxidativen Status mit Reaktionen GPX [Glutathionperoxidase], SOD [Superoxiddismutase], TEAC [Trolox equivalent antioxidative capacity] und ACW [nichtenzymatische wasserlösliche Antioxidantien] sowie des Stoffwechsels erreicht werden kann und ob damit die Häufigkeit der in der Frühlaktation typischen Erkrankungen sinkt. Zur Beantwortung dieser Fragestellung wurden in einem Milchviehbestand mit 1400 Kühen und Färsen zwei Gruppen von je 26 Tieren zu Beginn der Transitfütterung zusammengestellt. Die Versuchsgruppe erhielt drei Wochen ante partum bis drei Wochen post partum eine Mineralstoffmischung mit einem Vitamin E- Gehalt von 300 mg/kg TM (= 447 IU /kg TM) und einem Selengehalt von 0,5 mg/ kg TM, die Kontrollgruppe die stallübliche Mineralstoffmischung mit 0,3 mg Selen/kg TM ohne zusätzliche Vitamin E Ergänzung. Jedem Tier wurde drei Wochen ante partum, 2 bis 4 Tage post partum und 3 Wochen post partum zur klinisch- chemischen Kontrolle Blut entnommen.Zur Bestimmung des antioxidativen Status wurden die GPX, SOD, TEAC und ACW untersucht. Zur Bewertung des peripartalen Stoffwechsels wurden die Parameter des Energie-, Fett- und Leberstoffwechsels (BHB [ß-0H-Butyrat], Cholesterol, AST [Aspartat-Amino-Transferase], GLDH [Glutamat- Dehydrogenase]), des Eiweißstoffwechsels (Albumin, TP [Gesamt-Eiweiß]), sowie des Mineralstoffwechsels (Ca [Calcium], Pi [anorganisches Phosphat] und der CK [Creatinkinase] bestimmt und mit den Kühen der Kontrollgruppe verglichen. Im Blutbild wurden die Erythrozytenzahl, die Leukozytenzahl, die Erythrozytenindices (MCH, MCHC, MCV), Hämatokrit, Hämoglobin und Thrombozytenzahlen verglichen. Die Häufigkeit des Auftretens der klinischen Krankheitsbilder Mastitis, Gebärparese, Retentio secundinarum, Klauenerkrankungen und puerperale Septikämie und die Produktionsdaten Milchleistung nach 100 Tagen, Milchleistung nach 305 Tagen und Zwischenkalbezeit wurden nach Ende der Untersuchungen statistisch ausgewertet. Eine direkte Beeinflussung des SOD und der GPX ist möglich. Durch die Gabe der mit Vitamin E und Selen angereicherten Mineralstoffmischung konnte in der Versuchsgruppe ein Anstieg der GPX-Aktivität und eine Plateaubildung erreicht werden. Die SOD-Aktivitäten lagen in der Versuchsgruppe drei Wochen post partum signifikant höher als in der Kontrollgruppe. Eine bessere Adaptation an den oxidativen Stress im peripartalen Zeitraum kann durch eine mit Vitamin E und Selen angereicherte Mineralstoffmischung erreicht werden. Die Inzidenz der Mastitiserkrankungen in der Frühlaktation wurde signifikant gesenkt.Die Inzidenz der Mastitiserkrankungen in der Frühlaktation wurde signifikant gesenkt. Signifikante Unterschiede ergaben sich auch in der Aktivität der GLDH. In der Versuchsgruppe wurden 3 Wochen post partum deutlich niedrigere GLDH- Aktivität gemessen als in der Kontrollgruppe, woraus auf einen besseren Leberzellschutz in der kritischen biologischen Phase der Milchkuh zu schließen ist. Hinsichtlich der Häufigkeit des Auftretens weiterer klinischer Erkrankungen im peripartalen Zeitraum konnte jedoch keine Verbesserung erzielt werden. Ebenso haben sich die Produktionsparameter Milchleistung und Zwischenkalbezeit nicht verbessert.

The hallmark feature of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), is the accumulation of cytoplasmic inclusions of key disease-linked proteins. Two of these proteins, TDP-43 and SOD1, represent a significant proportion of sporadic and familial ALS cases, respectively. The population of potentially aggregation-prone partially-folded states on the folding free-energy landscape may serve as a common mechanism for ALS pathogenesis. A detailed biophysical understanding of the folding and misfolding energy landscapes of TDP-43 and SOD1 can provide critical insights into the design of novel therapeutics to delay onset and progression in ALS. Equilibrium unfolding studies on the RNA recognition motif (RRM) domains of TDP-43 revealed the population of a stable RRM intermediate in RRM2, with residual structure localized to the N-terminal half of the domain. Other RRM domains from FUS/TLS and hnRNP A1 similarly populate RRM intermediates, suggesting a possible connection with disease. Mutations, which enhance the population of the RRM2 intermediate, could serve as tools for deciphering the functional and misfolding roles of this partially-folded state in disease models, leading to the development of new biomarkers to track ALS progression. ALS mutations in SOD1 have been shown to destabilize the stable homodimer to result in increased populations of the monomeric and unfolded forms of SOD1. Mechanistic insights into the misfolding of SOD1 demonstrated that the unfolded state is a key species in the initiation and propagation of aggregation, suggesting that limiting these populations may provide therapeutic benefit to ALS patients. An in vitro time-resolved Förster Resonance Energy Transfer assay to screen small molecules that stabilize the native state of SOD1 has identified several lead compounds, providing a pathway to new therapeutics to treat ALS.

Masters of Science / In this study, we observed that phosphate (P) deficiency stunted plant growth and produced plants with poor morphological characteristics (yellow and small leaves). Furthermore, we treated plants with 0.8 mM (control) and 0.02 mM P (deficient) in addition to 6 μM methylglyoxal (MG) and we observed that the plants treated with MG had a higher germination, and better morphological characteristics (the leaves were more dark green and bigger in size) compared to the P deficient plants. However, we also observed that the P deficient plants treated with MG had low levels of both O2- and H2O2 and this could be a possible reason for the improved growth and morphological characteristics. In contrast, the P deficient plants not treated with MG had high levels of O2- and H2O2 which could be the possible reason for the observed cell death. We also performed biochemical assays including superoxide dismutase, ascorbate peroxidase, malondialdehyde content, ascorbic acid content, catalase, and most of the assays showed high levels of reactive oxygen species (ROS) and low levels of antioxidant activities in plants not treated with MG while high levels of antioxidant activities and low levels of ROS were observed in plants treated with exogenous MG. Since nitric oxide (NO) is also known to be a signalling molecule, we did a NO assay and observed that NO content increased under low exogenous doses of MG. From our findings we came to a hypothesis that MG modulates P deficiency stress in P. vulgaris through NO signalling or it might be that NO and MG work in tandem to modulate signalling pathways under P deficiency. Finally, we looked at the nutrient profile and the results showed that while there was a poor nutrient profile generally under P deficiency, there was an improvement in nutrient profile when MG was administered at low doses.

This report details the activities and inhibition of metal-substituted human carbonic anhydrase II (M-HCA-II). The traditional activities (hydrolysis of CO2 and para-nitrophenol acetate) in addition to new activities (oxidation of 2-aminophenol, disproportionation of H2O2, and disproportionation of superoxide) were investigated. Values reported for the relative hydrolytic activities of M-HCA-IIs are reported here for the first time, ranging from 47.5 % (plus or minus 0.6) to 86 % (plus or minus 4) for the hydrolysis of CO2 and from 0.299 % (plus or minus 0.012) to 4.72 % (plus or minus 0.015) for the hydrolysis of para-nitrophenol acetate. With respect to new activities, only the oxidation of 2-aminophenol was observed. Turnover was observed for Fe-HCA-II (kcat/KM = 3.6 plus or minus 1.3 mM-1 s-1) and Cu-HCA-II (kcat/KM = 8 plus or minus 2 mM-1 s-1). Inhibition of Zn-, (di-substituted) Cu2-, and Cu/Zn-HCA-II hydrolysis of CO2 and para-nitrophenol acetate by sulfanilamide, coumarin, and ortho-coumaric acid were investigated. Sulfanilamide was shown to inhibit: Zn-HCA-II, Cu2-HCA-II, and Cu/Zn-HCA-II - (with CO2) KM = 8.9 plus or minus 1.1 microM, 11 plus or minus 2 microM, 8.8 plus or minus 1.4 microM and (with p-nitrophenyl acetate) KM = 8.4 plus or minus 1.0 microM, (none), 8.4 plus or minus 1.4 microM, respectively. No inhibition was observed for coumarin or ortho-coumaric acid or its derivatives for any CAs studied.

No description available.

Amyotrophic Lateral Sclerosis (ALS) is an always lethal motor neuron disease with unknown pathogenesis. Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) have limited neuroprotection in some models of motor neuron degeneration. However the direct effect of Hsp90 inhibition on motor neurons is unknown. Here we show that Hsp90 inhibition induced motor neuron death through activation of the P2X7 receptor. Motor neuron death required phosphatase and tensein homolog (PTEN)-mediated inhibition of the PI3K/AKT pathway leading to Fas receptor activation and caspase dependent death. The relevance of Hsp90 for motor neuron survival was investigated in mutant Cu/Zn superoxide dismutase (SOD) transgenic animal models for ALS. Nitrated Hsp90, a posttranslational modification known to induce cell death (Franco, Ye et al. 2013), was present in motor neurons after intracellular release of zinc deficient (Zn, D83S) and the SOD in which copper binding site was genetically ablated (Q) but not after copper deficient (Cu) wild type SOD. Zn deficient and Q mutant SOD induced motor neuron death in a peroxynitrite mediated and copper dependent mechanism. Nitrated Hsp90 was not detected in the spinal cord of transgenic animals for ALS-mutant SOD animal models until disease onset. Increased nitrated Hsp90 concentrations correlated with disease progression. Addition of Zn or Q SOD to nontransgenic brain homogenate treated with peroxynitrite led to an increase level of nitrotyrosine in comparison to wild type controls. However, in the same samples there was a 2 to 10 time increase in Hsp90 nitration as compared to nitrotyrosine. The selective increase is likely due to the binding of Hsp90 to Zn deficient and Q SOD as oppose to wild type SOD. These results suggest that Hsp90 nitration facilitated by mutant SOD may cause motor neuron degeneration in ALS. Targeted inhibition of nitrated Hsp90 may be a novel therapeutic approach for ALS. An alternative therapeutic strategy is to target the production of survival factors by glial cells. Riluzole is the only FDA approved drug for the treatment of ALS and it shows a small but significant increase in patient lifespan. Our results show that acute riluzole treatment stimulated trophic factor production by astrocytes and Schwann cells. However long-term exposure reversed and even inhibited the production of trophic factors, an observation that may explain the modest increase in patient survival in clinical trials. Discontinuous riluzole treatment can maintain elevated trophic factor levels and prevent trophic factor reduction in spinal cords of nontransgenic animals. These results suggest that discontinuous riluzole administration may improve ALS patient survival. In summary, we demonstrated that Hsp90 has an essential function in the regulation of motor neuron survival. We have also shown that Hsp90 was nitrated in the presence of mutant SOD and was present during symptom onset and increases as disease progresses, which may explain the toxic gain of function of mutant SOD. Finally we demonstrate a biphasic effect of riluzole on trophic factor production and propose changes in administration to improve effects in ALS patients.