Previous publications from this laboratory demonstrated that administration of leukemia inhibitory factor (LIF) (125 µg/kg) to young, male Sprague-Dawley rats at 6, 24, and 48 h after middle cerebral artery occlusion (MCAO) reduced infract volume, improved sensimotor skills, and alleviated damage to white matter at 72 h after the injury. In vitro studies using cultured oligodendrocytes (OLs) showed that LIF (200 ng/ml) also protects against 24 h of oxygen-glucose deprivation through activation of Akt signaling and upregulation of the antioxidant enzymes peroxiredoxin IV and metallothionein III. Other groups have demonstrated that LIF reduces neurodegeneration in animal models of disease, but the neuroprotective mechanisms of LIF during permanent ischemia have not yet been examined. The overall hypothesis to be tested in this project is whether LIF exerts similar protective mechanisms against neurons during ischemia through increased antioxidant enzyme expression in neurons. In the first set of experiments, superoxide dismutase (SOD) activity was significantly increased in the ipsilateral hemisphere of LIF-treated rats compared to rats that received PBS treatment at 72 h after MCAO. Western blot and immunohistochemical analysis revealed that SOD3 was upregulated in brain tissue and induced specifically in cortical neurons tissue at this time point. Neurons that expressed high levels of SOD3 at 72 h after MCAO also showed high levels of phosphorylated Akt (Ser473). LIF (200 ng/ml) reduced necrotic and apoptotic cell death against 24 h of OGD as measured by lactate dehydrogenase (LDH) release and caspase-3 activation. Quantitative real-time PCR analysis showed that LIF treatment upregulated SOD3 gene expression in vitro during OGD. Treatment with 10 µM Akt Inhibitor IV and transfection with SOD3 siRNA counteracted the neuroprotective effects of LIF in vitro, showing that upregulation of SOD3 and activation of Akt signaling are necessary for LIF-mediated neuroprotection. Several transcription factors that regulated Akt-inducible genes were previously identified by this lab, including myeloid zinc finger-1 (MZF-1) and specificity protein-1 (Sp1). The goal of the second set of experiments was to determine whether LIF exerted protective actions through MZF-1 and Sp1. According to analysis with Genomatix, MZF-1 and Sp1 have multiple binding sites in the promoter for the rat SOD3 gene. Western blot analysis showed that there was a trend towards increased MZF-1 protein expression in the brains of LIF-treated rats that approached significance. Immunohistochemical analysis and quantitative real-time PCR showed a significant in vitro upregulation in MZF-1 expression among LIF-treated neurons compared to PBS-treated neurons. Sp1 gene expression was not changed by LIF treatment, but there was a trend towards increased protein expression. In addition, there was a significant correlation between Sp1 and MZF-1 among brain samples from LIF-treated rats but not PBS-treated or sham rats at 72 h after MCAO. Immunohistochemical analysis revealed that Sp1 and MZF-1 co-localized with neuronal nuclei and SOD3 at 72 h after MCAO. Neurons that were transfected with MZF-1 or Sp1 siRNA following isolation did not show a significant decrease in LDH release after 24 h OGD that was observed among neurons transfected with scrambled siRNA. These data demonstrate that Sp1 and MZF-1 are involved with the neuroprotective signaling of LIF under ischemia. This laboratory has demonstrated that LIF activates transcription of protective genes and increases the activity of transcription factors through modulation of intracellular signaling. However, the upstream signaling mechanisms of LIF during ischemia had not previously been investigated. Previous investigators found that the LIF-specific subunit of the heterodimeric LIF receptor (LIFR) is induced by CNS injury. Western blot analysis was used to determine whether LIFR was induced in the brain and the spleen, which plays a role in the peripheral immune response, after MCAO. According to these results, LIF treatment significantly upregulates LIF in the brain compared to PBS treatment or sham injury at 72 h after MCAO. Genomatix analysis of the LIFR promoter region revealed a binding site for Sp1, which is one of the transcription factors responsible for neuroprotection by LIF. At this same time point, splenic LIFR expression is significantly reduced after MCAO compared to sham injury. LIF treatment did not significantly increase LIFR expression, but did significantly increase spleen size compared to PBS treatment at 72 h after MCAO. Although there was a trend towards increased LIFR expression in the spleen from 24 h to 72 h after MCAO, this increase was not statistically significant. However, there was a significant positive correlation between spleen weight and LIFR expression among rats euthanized 24-72 h after MCAO/sham injury. In addition, there was a significant negative correlation between LIFR expression in the brain and the spleen weight, thus showing that LIFR is upregulated following the splenic response. According to findings from other groups, JAK1 has been shown to associate with the heterodimeric LIF receptor (LIFR/gp130) and directly activate PI3K/Akt signaling. To test whether JAK1 contributes neuroprotection during ischemia, cultured neurons were treated with several concentrations (2.5-50 nM) of GLPG0634, a JAK1-specific inhibitor prior to 24 h of OGD. With the exception of the 2.5 nM concentration, all concentrations of GLPG0634 significantly decreased LDH release compared to DMSO treatment, with the 5 nM concentration having the most potent effect on reducing cytotoxicity. However, the 5 nM concentration had no significant did not significantly reduce LDH release compared to DMSO treatment under 24 h of normoxic conditions. These results indicate that JAK1 activity is primarily detrimental to neurons during ischemia. Although it is possible that LIF signaling activates JAK1, it is unlikely that JAK1 is responsible for LIF-mediated neuroprotection during ischemia. The results of these experiments allowed us to determine several molecular mechanisms for LIF-mediated neuroprotection. LIF, which binds to its heterodimeric receptor, activates Akt signaling during ischemia. The transcription factors Sp1 and MZF-1, which are located downstream of Akt, bind to the promoter of the SOD3 gene. In addition, Sp1 also regulates the LIFR gene. SOD3 upregulation increases total SOD activity, which decreases apoptotic and necrotic cell death during apoptosis. Due to its ability to promote antioxidant expression and survival signaling in multiple neural cell types, LIF shows promise as a novel treatment for permanent focal cerebral ischemia.

The research presented in this thesis focuses on the molecular design of an oxidation-sensitive nanocarrier and its enzyme conjugate with a view of their application in the field of biomaterials. I have polarised our attention on a specific class of polymers, the polysulfides, for their environmental responsiveness (towards oxidising substances, a condition often associated with inflammatory reactions), interesting physico-chemical properties, ease of the preparation and multiple possibilities for further modifications and bioconjugations, which are perfectly suitable for the development as systems for drug delivery applications. In this work we firstly have focused on the synthesis of amphiphilic poly(propylene sulfide)-poly(ethylene glycol) (PPS-PEG) block copolymers by employing vinyl sulfone as the functional group to link the blocks and modify the end of the PEG. This study was followed by an investigation of the macromolecular interchange and payload exchange of the formed polymeric micelles to understand the 'co-formulation' events, employing fluorophores (dansyl groups) and quenchers (dabsyl groups) either as terminal groups in macroamphiphiles or as encapsulated hydrophobic payloads. In another part of the work, I have developed a micellar system with which simultaneously to two of the most important ROS: superoxide and hydrogen peroxide, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity towards superoxide with that of hydrophobic thioethers towards hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then 'mopped-up' by the polysulfides. This mode of operation is functionally analogous to the SOD/catalase combination, with the advantage of being based on a single and more stable system.

Aging and disease processes may be due to deleterious and irreversible changes produced by free radical reactions. The enzyme copper-zinc superoxide dismutase (CuZn SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1) performs a protective function by scavenging superoxide radicals. In order to determine whether additional SOD activity affects longevity and oxygen metabolism in Drosophila, our approach was to clone the Sod gene and introduce additional copies of the gene back into the genome via P element mediated transformation. The effects of increased SOD activity on Drosophila life span and oxygen free radical metabolism were investigated. The CuZn SOD cDNA and gene were cloned from Drosophila melanogaster. The sequence of the Sod cDNA and gene revealed an additional C-terminal triplet coding for valine not found in the mature SOD protein. The nucleotide sequence of the coding region has 56% and 57% identity when compared to the corresponding human and rat Sod genes, respectively. A probe of the cloned gene hybridizes to position 68A4-9 on Drosophila polytene chromosomes. In wild-type Drosophila the Sod cDNA hybridizes to a 0.7-0.8 kb transcript which is greatly diminished in a SOD 'null' mutant that produces only 3.5% of the SOD protein. A 1.8 kb EcoRI gene fragment containing the Sod gene was cloned into the P vector pUChsneo and microinjected into Drosophila embryos. Five transformed lines, each of which contain an additional copy of the Sod gene at different chromosomal sites were constructed. The chromosomal positions of the transposed Sod sequence were determined by in situ hybridization of the Sod gene to salivary gland polytene chromosomes. Analysis of RNA from the transformed flies revealed that the transposed Sod gene was expressed. The range of SOD activity for the five transformed lines was 131% to 170% of the value of wild-type. There was good correlation between the amount of Sod mRNA and the level of SOD activity in the transformed lines. Increased SOD levels in the transformed lines did not confer greater resistance to paraquat-generated superoxide radicals, nor increase their lifespan. The SOD 'null' mutant with 3.5% of the wild-type SOD activity was hypersensitive to paraquat when compared to wild-type, whereas the heterozygous SOD deficiency Df(3L)1xd⁹/TM3SbSer with 50% of the wild-type SOD activity was not. Mutants lacking SOD are dramatically impaired in oxygen metabolism and a few percent of wild-type activity appears to provide significant protection against superoxide, while 50% of the wild-type levels confers essentially the same resistance as wild-type. Despite the observation that the SOD activities found in a wide range of animals correlates directly with their longevity, Drosophila melanogaster appears to be well protected against the toxic effects of oxygen by its native levels of SOD. / Arts, Faculty of / Philosophy, Department of / Graduate

Superoxide dismutase is an enzyme which scavenges superoxide radicals and is thought to be a longevity determinant, as there exists a positive correlation between superoxide dismutase concentration and maximum life span potential. The cytosolic CuZn superoxide dismutase in D. melanogaster has been purified and sequenced, but the gene has not been cloned. However, when it is available the CuZn SOD gene may be reintroduced into the Drosophila genome via the P-element transformation system so its effects on the life span potential of Drosophila may be studied. This study describes attempts to clone the CuZn SOD gene from D. melanogaster using two mixed sequence oligonucleotide probes. The SI probe corresponds to amino acids 43-48 of the protein sequence and contains 128 different oligonucleotide sequences representing all possible codon combinations predicted from the amino acid sequence. The GT3 probe is targeted to amino acids 90-95 of the protein. In this probe, deoxyguanosine was placed in positions where all four nucleotides may occur to decrease probe heterogeneity. The probes were used to screen D. melanogaster Canton-S and Oregon-R genomic lambda libraries. Three positive clones isolated from the Canton-S library had identical nucleotide sequence in the GT3 probe binding region, and sequencing of the probe binding site revealed that one member of the GT3 probe had formed a 15 bp duplex with the phage DNA. Screening of the Oregon-R library produced four clones which hybridized with both GT3 and S1 probes. When these phage DNA were hybridized to polytene chromosomes by in situ hybridization, none mapped to 68AB on the third chromosome, the location of the CuZn SOD gene. These results suggest that modification of the classical strategy used in this study is necessary, and implications on probe design are discussed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Orientador: Felix Guillermo Reyes Reyes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-18T15:46:40Z (GMT). No. of bitstreams: 1 Maciel_RaquelApratto_M.pdf: 938949 bytes, checksum: 248a32d15aed7abd02f0ac036eb29872 (MD5) Previous issue date: 2011 / Resumo: As doenças cardiovasculares ocupam o primeiro lugar em causas de morte no mundo e, segundo pesquisas, estas podem estar associadas ao estresse oxidativo - desequilíbrio entre produção de radicais livres e defesas antioxidantes. O tratamento e prevenção não farmacológico, dessas doenças, preconizado pela Organização Mundial da Saúde (OMS), considerando os hábitos alimentares, consiste em aumentar o consumo de frutas, hortaliças, cereais integrais e grãos, devido as suas propriedades antioxidantes, assim como aos efeitos fisiológicos das fibras alimentares também presentes nessas fontes alimentares. Por outro lado, estudos mostram que algumas substâncias xenobióticas podem estar presentes nos alimentos, como por exemplo, os inseticidas piretróides, aumentando o risco do desenvolvimento ou agravamento de alterações cardiovasculares e, ao mesmo tempo, desestruturando as defesas antioxidantes e produzindo radicais livres nocivos às células, colocando em risco a saúde humana. É inquestionável a necessidade do uso de agrotóxicos na produção agrícola, em programas de saúde pública, na saúde animal, bem como na silvicultura. O não cumprimento da legislação em vigor tem sido uma realidade preocupante, pois coloca em risco a saúde dos trabalhadores que manipulam essas substâncias, as pessoas que consomem os alimentos e o ecossistema. Assim, pesquisas que dimensionem os efeitos adversos que os agrotóxicos podem causar na saúde humana, contribuem com o cumprimento da legislação específica e, até mesmo, na atualização da mesma. Este trabalho teve por objetivo avaliar a exposição aguda, por via oral, de deltametrina, agrotóxico da classe dos piretróides, na excitação e condução cardíaca e no estresse oxidativo em ratos Wistar machos adultos, através de parâmetros eletrocardiográficos e da atividade de enzimas antioxidantes nos tecidos cardíacos e hepáticos. O plano de trabalho consistiu na administração aguda, por gavagem, de deltametrina (DMT), com os animais distribuídos em 4 grupos, com 10 animais,pesando em média 200 ± 10g cada, assim constituídos: 1) Grupo controle (5 mL de óleo de milho); 2) Grupo DMT10 [10% do valor da DL50 da deltametrina (3,2 mg/5mL de óleo de milho)]; 3) Grupo DMT25 [25% do valor da DL50 da deltametrina (8 mg/5mL de óleo de milho)]; 4) Grupo DMT100 [100% do valor da DL50 de deltametrina (32mg/mL de óleo de milho)]. No início do ensaio, foi introduzida uma sonda de gavagem nos ratos. Logo após, os animais foram submetidos à anestesia para realização do eletrocardiograma (ECG). Após 5 min de início dos registros do ECG, a DMT foi administrada através de gavagem, conforme o planejamento experimental de cada grupo, registrando-se o ECG por mais 30 min. Após, fez-se o sacrifício dos animais por aprofundamento da anestesia. Foram retirados o fígado e o coração para mensurar a atividade das enzimas: catalase, superóxido dismutase, fosfatase alcalina, glutationa S-transferase e quantificação da glutationa reduzida e do produto da peroxidação lipídica malondialdeído. A deltametrina alterou a condutividade elétrica do coração. O eletrocardiograma mostrou redução significativa da frequência cardíaca e aumento do intervalo RR, indicando bradicardia. Também foi constatado aumento da duração do complexo QRS e redução da amplitude da onda R, o que sugere alterações na excitação ventricular. A amplitude P não foi significativamente diferente, mostrando que a excitação e a condução elétrica atrial não foram prejudicadas. Da mesma forma, os intervalos QT e QTc não foram alterados significativamente, embora o grupo DMT100 para o intervalo QT tenha se mostrado elevado, descartando-se assim, o risco de morte súbita. O segmento ST apresentou infradesnivelamento progressivo, embora não significativo. A onda T mostrou-se positiva e sem alterações significativas, não podendo assim sugerir possibilidade de isquemia miocárdica induzida por deltametrina. As análises bioquímicas mostraram que a deltametrina provocou estresse oxidativo tanto no fígado como no coração. A lipoperoxidação ocorreu nos hepatócitos e não parece ter ocorrido nos miócitos. No fígado, o estresse oxidativo foi confirmado pelo aumento significativo da atividade das enzimas glutationa S-transferase e fosfatase alcalina, aumento da concentração da glutationa reduzida, e significativa redução da atividade das enzimas superóxido dismutase e catalase. Já no coração, o estresse oxidativo foi evidenciado pelo significativo aumento da atividade da catalase e redução significativa da superóxido dismutase e glutationa S-transferase, assim como, pelo aumento da concentração da enzima glutationa reduzida. Em conclusão, este trabalho constatou que a deltametrina, administrada por via oral, diferentes dosagens, em ratos Wistar, causou aumento do estresse oxidativo e este pode ter ocasionado alterações na condução e excitação cardíaca verificadas no presente estudo / Abstract: Cardiovascular diseases are in the first rank among causes of death in the world and, according to researches, they can be associated with oxidative stressimbalance between free radicals productions and antioxidant defenses. The treatment and non-pharmacological prevention of these diseases, advocated by the World Health Organization (WHO), consist in to increase consumption of fruits, vegetables, whole grains and beans, because of their antioxidant properties and the physiological effects of dietary fiber also present on those foods. On the other hand, studies have shown that some xenobiotic substances may be present in food, such as pyrethroid insecticides, increasing the risk of development or aggravation of cardiovascular alterations and, at the same time, destabilizing the antioxidant defenses and producing free radicals which are harmful to cells, endangering human health. There is no doubt about the necessity of the use of pesticides in agricultural production, in public health programs, animal health, as well as in forestry. The non-compliance with the legislation in force has been a troubling reality because it puts in risk the health of workers who handle those substances, the people who consume foods and the ecosystem. Researches that evaluate the harmful effects that pesticides can cause to the human health, contribute to the fulfilling of the specific legislation and, even, in the update of it. This work aims to evaluate the acute oral exposure of deltamethrin, a pyrethroid pesticide, on the excitation system of the heart and on the oxidative stress in adult male Wistar rats, by means of electrocardiographic parameters, and antioxidant enzymes in liver and heart tissues, respectively. The work consisted on the acute administration, by gavagem, of deltamethrin (DMT), to animals distributed in 4 groups, containing 10 animals each, as follows: 1) Control Group (5 mL of corn oil); 2) DMT10 Group [10% of the LD50 value of deltamethrin (3,2mg/5mL corn oil)]; 3) DMT25 Group [25% of the LD50 value of deltamethrin (8 mg/5mL corn oil)]; 4) DMT100 Group [100% of the LD50 value of deltamethrin (32mg/mL corn oil)]. At the beginning of the assay, it was introduced in the rats a probe of gavagem. Soon after, the animals were subjected to anesthesia for conduction of the electrocardiogram (EGC). After 5 minutes of the beginning of the ECG record, the deltamethrin was administered via gavagem, accordingly to the experimental planning of each group, and the ECG recorded for additional 30 minutes. Upon completion of the ECG recording, the rats were killed by deepening of anesthesia. The liver and heart were withdrawn to measure the activity of the enzymes: catalase, superoxide dismutase, glutathione transferase, alkaline phosphatase, as well as of the quantification of the malondialdehyde biomarker and reduced glutathione. Deltamethrin was capable of changing the electrical conductivity of heart. The electrocardiogram showed significant reduction of the heart rate and increase of the RR interval, indicating bradycardia. It was also found to increase of the duration QRS complex and reduction of the R wave amplitude, which means that there were alterations in ventricular excitation. The P amplitude was not significantly different, showing that arousal and atrial electrical conduction was not impaired. Similarly, the QT and QTc intervals were not altered significantly, although has been shown high of the group DMT100, discarding the risk of sudden death. The ST segment depression showed a progressive, though not significant. The T wave was positive and no significant changes and therefore can not suggest the possibility of myocardial ischemia induced by deltamethrin. Biochemical analysis showed that deltamethrin causes oxidative stress in both liver and heart. Lipid peroxidation occurred in hepatocytes and not observed in myocytes and this is confirmed by checking the increased activity of the alkaline phosphatase, glutathione s-transferase and reduced activity of the superoxide dismutase and catalase and increased of concentration of the reduced glutathione in the liver. In the heart, oxidative stress has been shown by increased activity of the catalase, increased concentration of the glutathione reduced and reduced activity of the superoxide dismutase and glutathione s-transferase. In conclusion, this study found that deltamethrin administered orally at different doses in rats, caused increased oxidative stress and this may have caused changes in cardiac excitation conduction and verified / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos

Vitamin C is used in the diets in order to improve growth, resistance to stress and disease, as well as the survival of fish. It can also be effective in the conservation of fish during processing and storage, inhibiting the degradation of lipids by oxidation. This study evaluated the stocking density and the effects of supplementation of vitamin C in the final quality of the fillet tilapia (Oreochromis nilotius, Linneaus, 1875). We used one hundred and twenty fingerlings for each trial, the study of fish stocking density had an initial weight of 4.0 g and evaluation of supplementation with vitamin C, 11.0 g. Both studies had a randomized design, comprising four treatments of different stocking densities (50, 75, 100 and 125 fish/m3) with five replications. Already working with vitamin C (ascorbic acid 2 - sulfate acid - protected form) was carried out in four treatments with six replicates, characterized by four levels of supplementation of vitamin C in the diets (250, 500 and 750 mg/kg diet) and control group (zero mg/kg diet) with two different stocking densities (50 and 100 fish/m3). The ration given to the first study contained 36% crude protein and 3100 kcal/DE/kg feed and the second was 28% crude protein and 3400 kcal/DE/kg. After ninety days, the fish were slaughtered and evaluated the growth performance parameters for the study of stocking density, chemical composition, cholesterol content, fatty acid profile, quality of lipid fraction in both studies. The liver of fish from the second stage of labor was frozen at - 80°C for lipid peroxidation analysis and determination of enzyme activity of oxidative stress (catalase and superoxide dismutase). It follows that for the study of stoking significant difference ρ <0.05 between the final average weight and total weight gain between the different densities tested, the highest weight was found in the group density of 75 fish/m3. The body composition analysis, differences were observed ρ <0.05 between the densities on moisture, total lipid content, protein, calories and cholesterol. The ratio polyunsaturated / saturated was higher in density of 11.76 for 100 fish/m3, since the density of 50 fish/m3 presented a sum of 6.85 for the levels of EPA + DHA. In assessing the nutritional quality of lipids, the densities studied showed the contents of n-6/n-3, hypocholesterolemic/hypercholesterolemic index of polyunsaturated fatty acids of atherogenicity and index of thrombogenicity as favorable for consumption. Fish reared at a density of 50 fish/m3 can be seen as a good food from a nutritional standpoint, they presented a good level of fatty acids, especially omega-3 fatty acid and its value in hypocholesterolemic/hypercholesterolemic. Regarding the second stage of labor was observed difference ρ<0.05 between the levels of vitamin C supplementation levels in total lipid content, crude protein, calories and cholesterol. The ratio polyunsaturated/saturated was higher in the supplementation level of 250 mg of vitamin C (1.26), since the level of 500 mg of vitamin C showed a sum of 6.48 for the levels of EPA + DHA. In assessing the nutritional quality of lipids, the levels of vitamin C study showed rates of n-6/n-3, hypocholesterolemic / hypercholesterolemic index of atherogenicity and index of thrombogenicity for subsequent food consumption. Fish reared with diets supplemented with 750 mg of vitamin C in the density of 50 fish/m3 can be seen as a good food from a nutritional standpoint, they presented a good level of fatty acids, especially omega-3 fatty acid and its value in hypocholesterolemic/hypercholesterolemic. In the study of redox balance results showed that there was an increase in lipid peroxidation, suggesting that there may have been damage to the liver of animals and the antioxidant defenses were not sufficient to prevent oxidative stress. Density of 50 fish/m3, the dietary supplementation with vitamin C can be pro-oxidant. Since the density 100 fish/m3, vitamin C supplementation may be an antioxidant. The catalase activity of the enzyme showed low activity, suggesting an impairment in hepatic redox balance for this enzyme in animals receiving diets containing different levels of vitamin C. Since superoxide dismutase activity of the enzyme showed high activity, suggesting a redox balance in protecting liver for this enzyme in animals receiving diets containing different levels of vitamin C. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A vitamina C é usada nas dietas com a finalidade de melhorar o crescimento, a resistência ao estresse e às doenças, assim como a sobrevivência de peixes. Também pode ser eficiente na conservação do pescado durante o processamento e estocagem, inibindo a degradação dos lipídeos pela oxidação. O presente trabalho avaliou a densidade de estocagem e os efeitos da suplementação de vitamina C na qualidade final do filé de Tilápia-do-Nilo (Oreochromis nilotius, Linneaus, 1875). Foram utilizados cento e vinte alevinos para cada estudo, sendo que os peixes do estudo da densidade de estocagem tinham peso inicial de 4,0 g e os da avaliação com a suplementação de vitamina C de 11,0 g. O delineamento inteiramente casualizado foi utilizado em ambos os estudos perfazendo quatro tratamentos de diferentes densidades de estocagem (50, 75, 100 e 125 peixes/m3) com cinco repetições. Já o trabalho com a vitamina C (ácido 2 - sulfato ascórbico - forma protegida) foi realizado em quatro tratamentos com seis repetições, caracterizado pela suplementação de quatro níveis de vitamina C nas rações (250, 500 e 750 mg/kg de ração) e o grupo controle (zero mg/kg de ração) com duas diferentes densidades de estocagem (50 e 100 peixes/m3). A ração fornecida para o primeiro estudo continha 36% de proteína bruta e 3100 kcal/energia digestiva/kg de ração e a segunda tinha 28% de proteína bruta e 3400 kcal/energia digestiva/kg. Após noventa dias, os peixes foram abatidos e foram avaliados os parâmetros de desempenho zootécnico para o estudo de densidade de estocagem, composição centesimal, teor de colesterol, perfil de ácidos graxos, qualidade da fração lipídica para ambos os estudos. O fígado dos peixes da segunda etapa do trabalho foi congelado a – 80ºC para análises de peroxidação lipídica e determinação da atividade das enzimas do estresse oxidativo (catalase e superóxido dismutase). Conclui-se que para o estudo de densidade de estocagem houve diferença significativa ρ<0,05 entre o peso médio final e no ganho de peso total entre as diferentes densidades populacionais testadas; o maior peso foi encontrado no grupo de densidade de 75 peixes/m3. Quanto à análise da composição corporal observou-se diferença ρ<0,05 entre as densidades nos teores de umidade, lipídeos totais, teor de proteína bruta, valor calórico e de colesterol. A relação poliinsaturados/saturados foi mais elevada na densidade de 100 peixes/m3 de 11,76, já a densidade de 50 peixes/m3 apresentou uma somatória de 6,85 para os teores de ácido eicosapentaenóico + ácido docosahexaenóico. Na avaliação da qualidade nutricional dos lipídeos, as densidades estudadas mostraram os índices de n-6/n-3, hipocolesterolêmicos/hipercolesterolêmicos, índice de ácidos graxos poliinsaturados de aterogenicidade e índice de trombogenicidade como favoráveis para consumo alimentar. Os peixes criados na densidade de 50 peixes/m3 podem ser considerados como um bom alimento do ponto de vista nutricional, pois apresentaram um bom teor de ácidos graxos, principalmente de ácido ômega-3 e pelo seu valor em hipocolesterolêmicos/hipercolesterolêmicos. Com relação à segunda etapa do trabalho foi observado diferença ρ<0,05 entre os níveis de suplementação da vitamina C nos teores de lipídeos totais; teor de proteína bruta; valor calórico e de colesterol. A relação poliinsaturados/saturados foi mais elevada no nível de suplementação de 250 mg de vitamina C (1,26), já o nível de 500 mg de vitamina C apresentou um somatório de 6,48 para os teores de ácido eicosapentaenóico + ácido docosahexaenóico. Na avaliação da qualidade nutricional dos lipídeos, os níveis de suplementação de vitamina C estudados mostraram os índices de n-6/n-3, hipocolesterolêmicos/hipercolesterolêmicos, índice de aterogenicidade e índice de trombogenicidade favoráveis quanto ao consumo alimentar. Os peixes criados com ração suplementada com 750 mg de vitamina C na densidade de 50 peixes/m3 podem ser considerados como um bom alimento do ponto de vista nutricional, pois apresentaram um bom teor de ácidos graxos, principalmente de ácido ômega-3 e pelo seu valor em hipocolesterolêmicos/hipercolesterolêmicos. Já no estudo do balanço redox os resultados mostraram que houve aumento na peroxidação lipídica, sugerindo que podem ter existido danos ao fígado dos animais e as defesas antioxidantes não foram suficientes para evitar o estresse oxidativo. Na densidade de 50 peixes/m3, a ração suplementada com vitamina C pode ser pró-oxidante. Já na densidade 100 peixes/m3, a vitamina C suplementada pode ser antioxidante. A atividade da enzima catalase apresentou uma baixa atividade, sugerindo um comprometimento no balanço redox hepático para esta enzima nos animais submetidos à ração com diferentes níveis de suplementação de vitamina C. Já atividade da enzima superóxido dismutase apresentou elevada atividade, sugerindo uma proteção no balanço redox hepático para esta enzima nos animais submetidos à ração com diferentes níveis de suplementação de vitamina C.

No description available.

Superoxide dismutase scavenges oxygen radicals, which have been implicated in ischemia/reperfusion (I/R) injury in the heart. Our experiments were designed to study the effect of a moderate increase of copper/zinc superoxide dismutase (CuZnSOD) on myocardial I/R injury in TgN(SOD1)3Cje transgenic mice. A species of 0.8 kb human CuZnSOD mRNA was expressed, and a 273% increase in CuZnSOD activity was detected in the hearts of transgenic mice with no changes in the activities of other antioxidant enzymes. Furthermore, immunoblot analysis revealed no changes in the levels of HSP-70 or HSP-25 levels. Immunocytochemical study indicated that there was increased labeling of CuZnSOD in the cytosolic fractions of both endothelial cells and smooth muscle cells, but not in the myocytes of the hearts from transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x LVDP, was 48 ± 3% in the transgenic hearts as compared to 30 ± 5% in the nontransgenic hearts (p < .05). The improved cardiac function was accompanied by a significant reduction in lactate dehydrogenase release from the transgenic hearts. Our results demonstrate that overexpression of CuZnSOD in coronary vascular cells renders the heart more resistant to I/R injury.

Sponges are the descendants of the oldest members of the metazoan phylogenetic lineage and their genome contains animal specific genes but lack true tissues, organ systems, and neurons. Thus, the sponge model system can be used to elucidate origin of developmental processes. The PSED (RDGN) network (Pax/Six/Eya/Dac) is important in development of eyes, muscles, and other structures in Bilaterians. Similarly, sponges contain a precursor Pax-Six gene network. The Ephydatia muelleri (Em) PaxB protein binds to a Pax2/5/8 consensus sequence site and two cis-regulatory elements upstream and one intron sequence of EmSix1/2 (Rivera et al., 2013). This study aimed to determine if transcription factor EmSix1/2 binds upstream of EmPaxB using gel shift mobility assays, identify other downstream targets of EmSix1/2 using DNA immunoprecipitation, and to identify the recognition sequence of Six in sponges through sequencing. In conclusion, purified EmSix binds to DNA specific fragments (1 and 3), which may contain enhancer sequences located in the PaxB promoter region.Possible consensus recognition sequence of Six in sponges were also identified. The host immune response has various mechanisms to protect the organism from infections and invasions of microorganisms and cell damaging chemicals. One such mechanism is the elimination of reactive oxygen species aided by superoxide dismutase (SOD). A study showed that anti-neutrophil chemotactic factor antibodies recognize Tritrichomonas foetus SOD (Granger et al., 1997). During first casualties, SOD is released and triggers a host immune response. The parasites could use SOD to counter oxidative attacks by the leukocytes and damaged cells to protect surrounding parasites. We used the parasite Trichomonas vaginalis that causes trichomoniasis to determine if SOD acts a neutrophil (or other leukocyte) chemotactic factor in this parasite and characterize the expression and secretions of epithelial cells when treated with SOD6. HeLa cells treated with SOD6 showed an increase in the expression of IL-8 chemokine relative to TrxR treated cells. Scratch test assays showed that SOD may act as a macrophage chemoattractant as compared to TrxR but further tests are needed to confirm this.

Dysregulation of the autonomic cardiovascular control is a complication of diseases including diabetes, hypertension, sleep apnea, and aging. A common factor in these conditions is an increase in reactive oxygen species (ROS) in neural, cardiac, and endothelial tissues. Cu/Zn superoxide dismutase (SOD1) is an intracellular anti-oxidant enzyme that catalyzes dismutation of the superoxide anion (O2.-) to hydrogen peroxide (H2O2). Expression and function of this enzyme are diminished in pathologies that impair cardiovascular autonomic control. This study employed mice overexpressing a transgene for human SOD1 (hSOD1) to determine if its overexpression would alter autonomic regulation of BP, HR, and BRS in healthy animals, and if this animal line (C57B6SJL-Tg (SOD1)2 Gur/J) could be used in future studies to determine if hSOD1 overexpression can preserve cardiac autonomic function in disease models. To accomplish this aim, using anesthetized SOD1 and C57 (control) mice, we recorded HR, and aortic depressor nerve (ADN) activity changes in response to pharmacologically-induced BP changes in order to measure baroreflex and baroreceptor sensitivity, respectively. In order to identify any alterations in central, efferent, and cardiac components of the baroreflex arc, we electrically stimulated the left ADN and left cervical vagus and compared the reductions in BP and HR between the C57 and SOD1 mice. Time- and frequency-domain analysis of heart rate variability (HRV) was performed using pulse pressure recordings prior to pharmacologic or surgical procedures. We found that hSOD1 overexpression in the SOD1 mouse line, in comparison to C57 controls did not significantly affect resting HR (C57: 558 ± 8 vs. SOD1:553 ± 13 beats-per-minute) or blood pressure (C57: 88.8 ± 2.9 vs.SOD1: 85.8 ± 2.1 mmHg). hSOD1 overexpression did not affect the decrease in average mean arterial pressure (MAP) following injection of sodium nitroprusside (SNP) (C57: 38.7 ± 1.4 vs. SOD1: 39.5 ± 1.3 mmHg) or increase in average MAP (C57: 135.8 ± 3.1 vs. SOD1: 136.6 ± 3.5 mmHg) following injection of phenylephrine (PE). BRS, as measured by the averaged regression lines for ΔHR/ΔMAP for the SNP-induced tachycardic baroreflex (C57: 0.57 ± 0.06 bpm/mmHg, SOD1: 0.61 ± 0.08 bpm/mmHg)) and the PE-induced bradycardic baroreflex (C57: -2.9 ± 0.57 bmp/mmHg, SOD1: -4.3 ± 0.84 bpm/mmHg) are not significantly different between C57 and SOD1. Baroreceptor activation showed a significant increase in gain (C57: 5.4 ± 0.3 vs. SOD1: 7.4 ± 0.5 %/mmHg, P < 0.01) in the SOD1 transgenic mice. Heart rate depression in response to electrical stimulation of the left ADN and cervical vagus was comparable between C57 and SOD1, though MAP reduction in response to ADN stimulation is slightly, but significantly increased at 50 Hz in SOD1 animals. Time- domain analysis of HRV did not reveal any significant difference in beat-to-beat variability between SOD1 and C57 (SDNN: C57: 2.78 ± 0.20, SOD1: 2.89 ± 0.27), although frequency-domain analysis uncovered a significant reduction in the low-frequency power component of the HRV power spectral distribution (C57: 1.19 ± 0.11, SOD1: 0.35 ± 0.06, P < 0.001). This study shows that although hSOD1 overexpression does not affect overall baroreflex function, it does potentiate baroreceptor sensitivity and brain stem control of arterial pressure, and reduces low-frequency beat-to-beat variations in HR, without affecting total HRV.