Maintenance of a stable arterial blood pressure is a complex physiological phenomenon. In addition to dysfunction of the blood vessels, alterations in homeostasis of circulating signals and humoral factors also contribute significantly to the development of hypertension. Recent evidence indicates that accumulation of the byproducts of cellular respiration, including superoxide anion (O2-) and/or hydrogen peroxide (H2O2), are contributing factors in pathophysiology of hypertension. With respect to the central nervous system, neurons in the rostral ventrolateral medulla (RVLM) play a pivotal role in neural regulation of blood pressure. RVLM neurons not only provide a tonic excitation to maintain the sympathetic vasomotor activity of the blood vessels, they also participate in baroreceptor reflex control of blood pressure. The notion that production of O2- and/or H2O2 in the RVLM participates in central control of blood pressure has recently gained major recognition in the area of hypertension study. Nonetheless, detailed insights into the mechanisms underlying O2- and/or H2O2 promoted hypertension remain to be elucidated. The hypothesis that forms the basis of this study is that enhanced level of O2- and/or H2O2 in the RVLM may be important factors for the manifestation of hypertension in the spontaneously hypertensive rats (SHR), an animal model of human essential hypertension. In comparison to normotensive Wistar-Kyoto (WKY) rats, basal level of O2- in the RVLM region of adult male SHR rats was significantly higher, along with a reduction in the expression of superoxide dismutase 1 (SOD1), SOD2 or catalase. SOD and catalase are enzymes that metabolize cellular O2- or H2O2 respectively. Pharmacologically, microinjection bilaterally into the RVLM of SOD mimetic, Tempol (50 nmol) or a pan SOD/calatase mimetic, FeTMPyP (100 nmol), significantly decreased mean systemic arterial pressure (MSAP) or heart rate (HR) in both SHR and WKY rats. The maximal hypotensive effect produced by Tempol or FeTMPyP was significantly greater in SHR than WKY rats. We also found that in SHR, but not WKY rats, the hypotensive and bradycardiac responses after microinjection bilaterally into the RVLM of FeTMPyP was significantly greater than that by Tempol. In addition, infection of RVLM neurons with adenoviral vector encoding SOD1 (Ad-SOD1), SOD2 (Ad-SOD2) or catalase (Ad-Catalase) gene (5x108 pfu) into the bilateral RVLM resulted in a long-term hypotensive effect in SHR but not WKY rats. The temporal profile of Ad-catalase-promoted hypotension was again longer than that promoted by Ad-SOD1 or Ad-SOD2 alone. At the molecular level, gene transfer of SOD1, SOD2 or catalase into the RVLM region of SHR or WKY rats specifically increased the expression of individual protein, resulting in a reduction in O2- level. Together these results suggest that accumulation of O2- and/or H2O2 in the RVLM is involved in the neural mechanism of hypertension in SHR.

The dissection of etiology of hypertension is a medical imperative. In the central nervous system, rostral ventral lateral medulla (RVLM) plays an essential role in the maintenance of arterial pressure and heart rate through tonic activation of the sympathetic vasomotor activity and regulation of baroreflex response. Oxidative stress of an enhanced cellular content of the reactive oxygen species, in particular the superoxide anion (O2-), has been implicated in hypertension. Superoxide dismutase (SOD) is one of the most important defense enzymes against the oxidative stress through catalysis of O2- into O2 and H2O2. SOD treatment has been demonstrated to decrease arterial pressure. Moreover, in addition to its peripheral vasodilatory effect, nitric oxide (NO) plays an active role in central regulation of arterial pressure and heart rate via modulation of the autonomic system. In the RVLM, both O2- and NO have been demonstrated to be involved in hypertension. Interactions between these two molecules, however, are not understood. The aims of this study are therefore to establish the significance of O2- and NO in the RVLM on blood pressure regulation in hypertension and to examine whether O2- interacts with NO to participate in the pathogenesis of hypertension. To examine their long term effects on mean systemic arterial pressure (MSAP) and heart rate (HR), SOD and/or NO was over-expressed by microinjection of the adenoviral vectors encoding the endothelial NO synthase (AdeNOS) and/or mitochondrial SOD (AdSOD2) into RVLM of the normotensive Wistar-Kyoto (WKY) rats or the spontaneously hypertensive rats (SHR). I found that microinjection of AdeNOS in the RVLM of SHR or WKY rats significantly decreased MSAP or HR that lasted for around 10 days postinjection. The hypotensive effect of AdeNOS was significantly greater in SHR than WKY rats. The AdeNOS-promoted hypotension in SHR, but not WKY rats, was followed by a rebound hypertension, detected in 28 days after the gene transfer. In the AdeSOD2-treated animals, I found a significant decrease in the MSAP in SHR, but not WKY rats, that lasted for about 7 days postinjection. On the other hand, no change in HR was detected in either SHR or WKY rats after the AdSOD2 gene transfer into the RVLM. In animals that received co-microinjection into the bilateral RVLM of AdeNOS and AdSOD2, there was a further prolonged decrease in MSAP or HR in SHR. The rebound hypertension observed in the AdeNOS-treated SHR was reversed to hypotension in the AdeNOS+AdSOD2-treated SHR. There was no difference in the hypotensive or bradycardiac effects in WKY rats that received the AdeNOS+AdSOD2 or AdeNOS gene transfer. Together these results suggest that (1) NO in RVLM plays an important role in central regulation of arterial pressure and heart rate under both normotensive and hypertensive conditions. A greater reduction in MSAP in the AdeNOS-treated SHR further indicates a reduced action of NO at the RVLM in the pathogenesis of hypertension. (2) An excessive oxidative stress of a reduced function of SOD2 in RVLM may be an important factor in neural mechanism of hypertension in SHR. The same mechanism, at the same time, may underlie the rebound hypertensive observed in the AdeNOS-treated SHR. (3) The excessive oxidative stress in the RVLM contributes to hypertension by at least two mechanisms. One is to cause oxidative injury in the RVLM and the other is to interact with NO to decrease already insufficient activity of NO in central cardiovascular regulation.

Peroxisome proliferator activated receptors (PPAR) are members of the nuclear receptor family that act as transcription factors to regulate target gene expression. In addition to their well-known effects in regulation of glucose homeostasis and lipid metabolism, PPAR activators have recently been shown to exert antihypertensive effects, although the underlying mechanism is not clear. Our laboratory has previously demonstrated that oxidative stress of an augmented tissue level of superoxide anion (£R2¡E−) in the rostral ventrolateral medulla (RVLM), where promotor neurons for generation of sympathetic vasomotor outflow reside, contributes to neural mechanism of hypertension. I therefore propose to test in my thesis the hypothesis that protection against oxidative stress after activation of the PPARs in the RVLM may contribute to the antihypertensive effect of these transcription factors. Experiments were performed in the spontaneously hypertensive rats (SHR) or normotensive Wistar-Kyoto (WKY) rats under anesthesia or conscious condition. Compared to WKY rats, microinjection bilaterally into the RVLM of a synthetic activator of PPAR£^, rosiglitazone (1 nmol), evoked significantly greater decreased in mean systemic arterial pressure (MSAP) and heart rate (HR) in SHR. These cardiovascular suppressive effects of rosiglitazone were accompanied by greater decrease in tissue level of O2 - and upregulation of the antioxidant uncoupling proteins (UCPs) in the RVLM of SHR. Rosiglitazone also caused a significant greater increase in PPAR£^ expression in the nuclear extracts from RVLM of SHR than WKY rats. All these cellular events induced by rosiglitazone were antagonized by co-administration into the RVLM of the PPAR£^ inhibitor, GW9662 (5 nmol). This PPAR£^ inhibitor also significantly reversed the cardiovascular depressive effects of rosiglitazone. Together these results suggest that PPAR£^ in the RVLM may participate in central cardiovascular regulation by promoting hypotension and bradycardia via amelioration of O2- production and upregulation of antioxidant UCPs. Moreover, a downregulation of the PPAR£^ in the RVLM may contribute to neural mechanism of hypertension.

Three experiments were conducted to evaluate the effects of dietary chitin and chitosan on growth, immune responses and resistance of grass prawn Penaeus monodon against white spot syndrome virus (WSSV) and Vibrio infections. In the first experiment, two levels (0.5¡B1 g/100g diet) of chitin and three levels (0.5¡B1¡B5 g/100g diet) of chitosan were evaluated. The results show that weight gain of the shrimp fed on diet containing no chitosan or the lowest level of chitosan (0.5 %) was higher than other groups. In the second experiment, four levels of chitosan (0¡B0.5¡B1¡B5 g/100g diet) were tested. Weight gains of the control (0 %) and 0.5 % chitosan groups were significantly (P<0.05) higher than the 0.1 and 1 % chitosan groups. Shrimp survival rate was not influenced by chitosan inclusion. The test shrimp of the first experiment were evaluated for their immune responses after dietary exposures. The results show that phenoloxidase activity and superoxide dismutase were not significantly different (P>0.05) among treatments. The production of superoxide anion in the 0.5 % chitin group was significantly (P<0.05) lower than the other groups at day 3 and 12. The last experiment evaluated the effectiveness of dietary chitosan against infection of WSSV and Vibrio damsela. Shrimp were fed for 20 days on test diets containing four levels of chitosan (0¡B0.5¡B1¡B5 g/100g diet) and then challenged by injection of WSSV or Vibrio solution. In the WSSV challenge, except at day 7, shrimp survivals were not different among treatments. At day 7, however, the survival rates of the shrimp fed the diet containing 0.1 or 1 % chitosan were significantly (P<0.05) higher than those of the other groups. When challenged with Vibrio damsela, there was no difference in shrimp survival among dietary treatments. The present study shows that dietary chitin and chitosan do not significantly enhance immune responses and disease resistance of juvenile P. monodon. Dietary incorporation of chitin or chitosan negatively affects shrimp growth.

O Brasil possui uma imensa diversidade biológica, na qual muitos compostos bioativos podem ser encontrados e utilizados em beneficio à sociedade. No entanto, processos de degradação do ambiente e introdução de espécies exóticas têm contribuído ao conhecimento e uso limitadosde muitas plantas nativas, sendopequena a quantidade de estudos sobre sua composição química e potencial biológico. A prevenção de doenças crônicas constantemente vem sendo associada à atividade antioxidante de metabólitos secundários dos vegetais, principalmente os fenólicos. Assim, o objetivo deste trabalho foi avaliar a atividade antioxidante e composição fenólica de cinco espécies frutíferas nativas do Brasil (G. brasiliensis, E. leitonii, E. involucrata, E. brasiliensis e E. myrcianthes). Os métodos capacidade de redução do Folin-Ciocalteau, autoxidação do sistema ?-caroteno/ácido linoleico, capacidade de absorção de radicais oxigênio (ORAC), sequestro dos radicais livres DPPH e ânion superóxido foram aplicados na determinação da atividade antioxidante dos extratos etanólicos de folha, casca, semente e polpa das espécies selecionadas. As amostras demonstraram significativa atividade antioxidante e, em alguns casos, superior as frutas comumente consumidas pela população brasileira. Em geral, folhas apresentaram as maiores atividades, mas o destaque foi a semente de E. leitonii que exibiu os melhores resultados em quatro dos cinco métodos utilizados: 120,67 mg AG.g-1 na redução do reagente Folin; 7,08 Gmol Trolox.g-1 no ?-caroteno; EC50 de 0,26 mg.mL-1 e 0,07 mg.mL-1 no sequestro do ânion superóxido e DPPH, respectivamente; 514,75 Gmol Trolox.g-1 no ORAC para o qual a folha de E. involucrata obteve o maior valor (1393,3 Gmol Trolox.g-1). Os extratos das espécies nativas também demonstram efeito antioxidante contra radicais de relevância biológica, como peroxila e superóxido. Por meio de CG-EM e CLAE acoplado a arranjo de fotodiodos, os principais compostos fenólicos encontrados nos extratos vegetais foram catequina, epicatequina e ácido gálico. Este trabalho demonstrou o grande potencial antioxidante das frutíferas nativas brasileiras, evidenciando assim possível efeito positivo em sistemas biológicos. / Brazil has a great biodiversity, in which many bioactive compounds can be found and used to benefit the society. However, environmental degradation processes and introduction of exotic species have contributed to limited use and knowledge of many native plants, reflecting in few studies about chemical composition and biological potential. The prevention of chronic diseases has been constantly associated with the antioxidant activity of plants secondary metabolites, mainly the phenolics. The objective of this study was to evaluate the antioxidant activity and the phenolic composition of five Brazilian native fruitful species (G. brasiliensis, E. leitonii, E. involucrata, E. brasiliensis e E. myrcianthes). The methods of Folin-Ciocalteau reducing capacity, co-oxidation of ?-carotene/linoleic acid system, oxygen radical absorbance capacity (ORAC), DPPH and superoxide free radical scavenging were used to determine the antioxidant activity of ethanolic extracts of leaf, peel, seed and pulp of the selected species. The samples showed significant antioxidant activity and, in some cases, it was superior to fruits commonly consumed by Brazilian population. In general, leaves presented the highest activities, but the seed of E. Leitonii stood out exhibiting the best results in four of the five methods: 120.67 mg GA.g-1 in the Folin reducing; 7.08 Gmol Trolox.g-1 in the ?-carotene; EC50 of 0.26 mg.mL-1 and 0.07 mg.mL-1 in the superoxide and DPPH scavenging, respectively; 514.75 Gmol Trolox.g-1 in the ORAC, for which the E. Involucrata leaf had the highest value (1393.3 GmolTrolox.g-1). The extracts of native species also demonstrate antioxidant effect against radicals of biological relevance, such as peroxyl and superoxide. By GC-MS and HPLC coupled to a photodiode array, the major phenolic compounds found in the plant extracts were catechin, epicatechin and gallic acid. In this study, Brazilian native fruitful presented high antioxidant potential, showing a possible positive effect on biological systems.

Development of an in vitro model for the early stages of cardiovascular disease is a current necessity. Cardiovascular disease is the leading cause of death in the United States and throughout the world. Oxidative stress and reactive oxygen species have been implicated in cardiovascular disease development. An in vitro model of these processes will improve our understanding of cardiovascular disease development and allow for the development of additional treatments. Atherosclerosis is an inflammatory disease and increased levels of H2O2 are associated with inflammation. The model focuses on H2O2-induced oxidative stress under static and shear conditions. Previous studies have documented increased O2.- and increased cytotoxicity in smooth muscle cells exposed to H2O2. Under static culture, endothelial cells exposed to H2O2, exhibited increased O2.- over basal levels via NOS and NAPDH oxidase pathways. Increased O2.- was attenuated by MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. Endothelial cell cytotoxicity was increased with H2O2 exposure. The increase in cytotoxicity was diminished upon exposure to Tiron or L-NAME. Under shear conditions (8.2 dynes/cm2), endothelial cells exposed to H2O2 exhibited increased O2.- compared to control via an L-NAME (specific inhibitor NOS) and Apocynin (NADPH oxidase inhibitor) inhibitable mechanism. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. The increased O2.- was attenuated with MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron (an O2.-scavenger). Endothelial cell attachment under shear with exposure to H2O2 was improved with MnSOD adenoviral-mediated upregulation as observed by decreased loss of the endothelial cell monolayer compared with H2O2 exposed endothelial cells. Endothelial cells exposed to H2O2 exhibit increased O2.-, suggesting that H2O2-induced oxidative stress may be a reasonable model for atherosclerosis. NOS and NADPH oxidase co-inhibition under shear and static culture demonstrated that NOS and NADPH oxidase inhibition is non-additive under static culture, yet additive under shear. Co-inhibition results suggest a complex relationship between the two enzymes that requires additional experimentation to deconvolve.

<i>In vivo</i> and <i>in vitro</i> studies on numerous types of blood vessels obtained from a variety of vascular beds and species have demonstrated that oxygen free radicals (OFRs) can evoke both vasodilation and vasoconstriction. Specific OFRs have been shown to elicit different and often times opposite effects on vascular smooth muscle. Therefore, this thesis attempts to define the vascular actions and mechanism of oxygen free radicals (OFRs) [superoxide anion (O₂^-), hydrogen peroxide (HO₂) and hydroxyl radical (OH)] on isolated rabbit aorta. This thesis will examine the role of OH in Ach- and nitroglycerin (NTG)-induced relaxation of isolated rabbit aorta. Superoxide anions generated by xanthine (X) plus xanthine oxidase (XO) produced concentration-dependent contractions of isolated rabbit aorta. The contractile response to O₂^- was completely abolished in preparations denuded of endothellum or pretreated with superoxide dismutase (SOD), a scavenger of O₂^-. The contractile response was reduced by indomethacin (I), a cyclooxygenase inhibitor. These results suggest that O₂^- mediated by vasoconstrictor arachidonic acid metabolites. Hydrogen peroxide generated by glucose and glucose oxidase produced contraction (low concentrations) and relaxation followed by contraction (high concentrations) in isolated rabbit aorta. The contractile response was abolished in the presence of catalase, a scavenger of H₂O₂ however the relaxant effect was exaggerated. Indomethacin markedly reduced the H₂O₂-induced contraction. Relaxation was completely prevented by de-endothelialization or pretreatment with N^G-monomethyl-L-arginine (LNMMA), an inhibitor of nitric oxide synthetase. These results suggest that H₂O₂ in large concentrations produces a biphasic response, relaxation followed by contraction. Relaxation is endothelium dependent and is mediated by endothelium-derived relaxing factor (EDRF), nitric oxide (NO). The contractile response is endothelium independent and is mediated by vasoconstrictor arachidonic acid metabolites of smooth muscle. Hydroxyl radicals generated by dihydroxyfumarate (DHF), ferric chloride (FeCl₃) and adenosine diphosphate (ADP) (DHF/FeCl₃-ADP) produced concentration dependent relaxations of NE-precontracted rabbit aorta. Mannitol (Ml) completely inhibited OH-induced relaxation. Relaxation was markedly reduced in aortic rings mechanically denuded of endothelium. The relaxant effect was reduced by an inhibitor of NO synthesis (LNMMA), by an inhibitor of guanylate cyclase (methylene blue), by an inhibitor of cyclooxygenase (indomethacin) and by an inhibitor of an ATP-sensitive K⁺ channel blocker (glyburide). These results indicate that OH produces relaxation that is endothelium-dependent and partially mediated by an endothelium-derived relaxing factor (NO), vasodilatory arachidonic acid metabolites and an ATP-sensitive K⁺ channel. We hypothesized that Ach-induced vascular relaxation is mediated by OH derived from the interaction of NO and O₂^-. To test this hypothesis we investigated the effect of Ach and NTG on NE-precontracted isolated rabbit aortic preparations in the absence or presence of scavengers of O₂^- (SOD), and OH (dimethylthiourea (DMTU) or mannitol or Garlicin). The OFR scavengers (SOD, dimethylthiourea, mannitol, garlicin and histidine) alone or the combination of SOD and DMTU markedly reduced Ach- or NTG-induced relaxation. However, the combination of histidine, (a ¹O₂ scavenger) SOD and DMTU completely abolished Ach-induced relaxation.

In the interest of identifying natural substances from marine invertebrates collected off the waters of Taiwan, we have searched the bioactive metabolites from the organic extracts of gorgonian corals Rumphella antipathies and Echinomuricea sp. This study had led to the isolation of thirty compounds (1¡V30), including nine new caryophyllane-related metabolites, rumphellaones A (1), B (2) and C (3), rumphelloic acids A (4) B (5) and C (6), rumphellolides J (7), K (8) and L (9), five new clovane-related metabolites, rumphellclovanes A (12), B (13), C (14), D (15) and E (16), two new disesquiterpenoid dimers, rumphelladimers A (24) and B (25), eight new natural products, (8R,9R)-isocaryolane-8,9-diol (10), 4£],8£]-epoxycaryophyllan-5-ol (11), 9£\-hydroxyclovan-2-one (17), 2£]-hydroxyclovan-9-one (18), clovan-2,9-dione (19), 2£]-acetoxyclovan-9£\-ol (20), 9£\-acetoxyclovan-2£]-ol (21) and 2£],9£]-dihydroxyclovane (22), along with a known compound, clovan-2£],9£\-diol (23) from Rumphella antipathies. In addition, three new labdane-, halimane-, and clerodane-related metabolites, echinolids A (26), B (27) and C (28), a new sesquiterpenoid natural product, (7S,10R)-(+)-10,11-epoxycurcuphenol (29), along with a known compound, (+)-curcuphenol (30) were also found in Echinomuricea sp. The structures of metabolites 1¡V30 were established by spectroscopic methods and by comparison of the spectral data with those of related known compounds. The absolute configurations of clovane-type compounds were determined using a modified Mosher¡¦s method for 23. The biosyntheses of compounds 1¡V5 and 12 were proposed. In the biological activity experiments, compounds 5 and 19 displayed significant inhibitory effects on superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB. Compounds 24 and 27 displayed significant inhibitory effects on elastase release by human neutrophils. Compound 27 was found to exhibit inhibition against the growth of DLD-1 (human colon adenocarcinoma) and Lovo (human colorectal adenocarcinoma) tumor cells.

Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia Sepsis is a complex pathophysiologic state resulting from an exaggerated whole-body inflammatory response to infection or injury. Metabolic disturbances, abnormal regulation of blood flow and diminished utilization of oxygen at the cellular level may account for tissue damage and lead to multiple organ failure and death. As the primary site of cellular energy generation is the mitochondrion, it presents itself as an important target for the septic cascade. In this regard, the notion that bioenergetic failure due to mitochondrial dysfunction contributes to organ failure during sepsis has received attention. We established the low frequency fluctuations in the systemic arterial pressure signals are related to the sympathetic neurogenic vasomotor tone, and reflect the functional integrity of the brain stem. Their origin is subsequently traced to the premotor sympathetic neurons at the rostral ventrolateral medulla (RVLM), whose neuronal activity is intimately related to the ¡§life-and-death¡¨ process. Based on a rat model of experimental endotoxemia that provides continuous information on changes in neuronal activity in the RVLM, the present study was undertaken to evaluate whether changes in mitochondrial respiratory functions are associated with death arising from sepsis. We also evaluated the efficacy of a new water-soluble coenzyme Q10 (CoQ10, ubiquinone) formula in the protection against fatality during endotoxemia by microinjection into bilateral RVLM. Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia in the Rat We investigated the functional changes in mitochondrial respiratory chain at the RVLM in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. Experiments were carried out in adult male Sprague-Dawley rats that were maintained under propofol anesthesia. Intravenous administration of E. coli lipopolysaccharide (LPS; 30 mg/kg) induced progressive hypotension, with death ensued within 4 hours. The sequence of cardiovascular events during this LPS-induced endotoxemia can be divided into a reduction (Phase I), followed by an augmentation (Phase II; ¡§pro-life¡¨ phase) and a secondary decrease (Phase III; ¡§pro-death¡¨ phase) in the power density of the vasomotor components (0-0.8 Hz) of systemic arterial pressure (SAP) signals. Enzyme assay revealed significant decrease of the activity of NADH cytochrome c reductase (Complex I+III) and cytochrome c oxidase (Complex IV) in the RVLM during all 3 phases of endotoxemia. On the other hand, the activity of succinate cytochrome c reductase (Complex II+III) remained unaltered. Neuroprotective Effects of Coenzyme Q10 at Rostral ventrolateral Medulla Against Fatality During Experimental Endotoxemia in the Rat CoQ10 is a highly mobile electron carrier in the mitochondrial respiratory chain that also acts as an antioxidant. We evaluated the neuroprotective efficacy of CoQ10 against fatality in an experimental model of endotoxemia, using a novel water-soluble formulation of this quinone derivative. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of E. coli LPS (30 mg/kg) induced experimental endotoxemia. Pretreatment by microinjection bilaterally of CoQ10 (1 or 2 mg) into RVLM significantly diminished mortality, prolonged survival time, and reduced the slope or magnitude of the LPS-induced hypotension. CoQ10 pretreatment also significantly prolonged the duration of Phase II endotoxemia and augmented the total power density of the vasomotor components of SAP signals in Phase II endotoxemia. The increase in superoxide anion production induced by LPS at the RVLM during Phases II and III endotoxemia was also significantly blunted. Conclusion The present study revealed that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM is closely associated with fatal endotoxemia. CoQ10 provides neuroprotection against fatality during endotoxemia by acting on the RVLM. We further found that a reduction in superoxide anion produced during endotoxemia at the RVLM may be one of the mechanisms that underlie the elicited neuroprotection of CoQ10. These findings therefore open a new direction for future development of therapeutic strategy in this critical, complicated and highly fatal condition known as sepsis.

Submitted by LUANA CHIQUETTO PARACATÚ null (luanachiquetto@hotmail.com) on 2016-07-14T18:15:23Z No. of bitstreams: 1 TESE - Luana Chiquetto.pdf: 2823701 bytes, checksum: 584996ba0cd6c6754d03400de9c6de73 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-18T20:01:01Z (GMT) No. of bitstreams: 1 paracatu_lc_dr_arafcf.pdf: 2823701 bytes, checksum: 584996ba0cd6c6754d03400de9c6de73 (MD5) / Made available in DSpace on 2016-07-18T20:01:01Z (GMT). No. of bitstreams: 1 paracatu_lc_dr_arafcf.pdf: 2823701 bytes, checksum: 584996ba0cd6c6754d03400de9c6de73 (MD5) Previous issue date: 2016-06-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Inúmeras patologias têm a sua gênese e/ou progressão relacionadas à produção desregulada de intermediários oxidantes. O complexo multienzimático NADPH oxidase é um dos componentes de maior relevância neste contexto, pois é uma das principais fontes de ânion superóxido no organismo animal. Sendo expresso em inúmeros tecidos, incluindo leucócitos e células do tecido endotelial, o desenvolvimento de inibidores eficientes deste complexo enzimático poderá significar uma nova terapêutica para o tratamento de doenças inflamatórias crônicas. Com objetivo de explorar a relação entre a estrutura molecular, as propriedades químicas e atividades biológicas, utilizamos o éster fenetílico do ácido cafeico (CAPE) como inibidor do complexo enzimático NADPH oxidase e comparamos sua eficácia com o seu precursor ácido cafeico e os derivados, éster fenetílico do ácido cinâmico e o ácido clorogênico, correlacionando-os a respeito a sua hidrofobicidade, propriedades redox e inibição do complexo NADPH oxidase em leucócitos ativados. A hipótese seria de que um aumento da hidrofobicidade provocado pela esterificação do ácido cafeico poderia facilitar o seu acesso à membrana celular e assim alterar seu efeito como possível inibidor de NADPH Oxidase. Os resultados, em ensaios in vitro, mostraram que as alterações na hidrofobicidade não provocaram alterações significativas no potencial de oxidação e potencial antioxidantes dos compostos testados. Quando testados em leucócitos ativados (modelos ex vivo), a esterificação provocou uma melhora significativa na capacidade de inibição do complexo NADPH oxidase. Este potente efeito se propagou às EROs decorrentes de ânion superóxido e produzidas por leucócitos, como peróxido de hidrogênio e ácido hipocloroso, entretanto, sem alterar a capacidade fagocítica dos leucócitos. Os resultados deste estudo mostram que nos ensaios celulares o CAPE foi o composto mais potente em relação ao seu precursor ácido e ácido clorogênico, sendo significativamente mais efetivo na inibição da produção das EROs. Da mesma forma, CAPE foi o inibidor mais eficaz da expressão de TNF-α e IL-10 por Staphylococcus aureus células estimuladas. Em conclusão, a presença do grupo catecol e a maior hidrofobicidade, do CAPE, foram essenciais para os efeitos biológicos, confirmando nossa hipótese. Considerando-se o envolvimento da NADPH-oxidases na génese e progressão de doenças inflamatórias, CAPE deve ser considerada como uma droga anti-inflamatória promissora. / Several diseases have their genesis and / or progression related to unregulated production of oxidants intermediates. The multienzymatic complex NADPH oxidase is one of the most important components in this context because it is a major source of superoxide anion in animal organisms. It is expressed in numerous tissues, including leukocytes and endothelial tissue cells. Developing effective inhibitors of this enzyme complex may indicate a new therapy for the treatment of chronic inflammatory diseases. Several studies have described numerous anti-inflammatory properties attributed to caffeic acid phenethyl ester (CAPE), an active component found in propolis. In order to explore the relationship between the molecular structure, chemical properties and biological activities, we used CAPE to inhibit the enzyme complex NADPH oxidase and compare its efficacy with the its precursor caffeic acid and derivatives, phenethyl ester of cinnamic acid and chlorogenic acid, correlating them with regard to hydrophobicity, redox properties and inhibition of NADPH oxidase complex on activated leukocytes. The hypothesis was that an increase of hydrophobicity caused by the esterification of caffeic acid could facilitate access to cell membranes and thereby alter its effect as a possible inhibitor of NADPH oxidase. The results (in vitro), showed that the changes in hydrophobicity did not provoke significant changes in the oxidation potential and antiradical potency of the tested compounds. But when tested in activated leukocytes (ex vivo), the esterification caused a significant improvement in the ability to inhibit the NADPH oxidase complex. This potent inhibition effect resulted also in the blockage of production of hypochlorous acid, however, without altering the phagocytic ability of leukocytes. The results of this study show that in cellular assays, CAPE was the most potent compound in comparison to caffeic acid and chlorogenic acid, significantly more effective in inhibiting the production of ROS. Likewise, CAPE was the most effective inhibitor of expression of TNF-α and IL-10 in Staphylococcus aureus stimulated cells. In conclusion, the presence of the catechol moiety and the higher hydrophobicity of CAPE were essential for the biological effects. Considering the involvement of NADPH oxidases in the genesis and progression of inflammatory diseases, CAPE should be considered as a promising anti-inflammatory drug.