The aim of this work was to investigate the problems involved in production and separation of a yeast microsomal enzyme, cytochrome P450. Immobilisation of the microsomal preparation, and utilisation of the immobilised system for the removal of polycyclic aromatic hydrocarbons, particularly the carcinogen benzo(a)pyrene [B(a)P], from aqueous systems was also investigated. High recoveries of the microsomal enzyme were obtained using rapid, low-speed centrifugation (5 minutes, 3000xg) by prior precipitation with a non-ionic polymer, polyethylene glycol (PEG). PEG was found to effectively replace centrifugal acceleration. A semi-empirical mathematical model of the process based on the size distribution of the agglomerates formed and the proportion of the agglomerates sedimented by centrfugation was developed. The effect of PEG was consistent with its increasing the mean effective diameter of protein agglomerates in proportion to PEG concentration to an exponent of 0.619 and with its increasing the spread of the size distribution in proportion to the mean effective agglomerate diameter. Binding spectra studies established that B(a)P binds to the active site of yeast cytochrome P450 and is unaffected by PEG. The B(a)P assay by fluorescence following hexane extraction was also unaffected by PEG. The stability of the microsomal cytochrome P450 preparation at different temperatures was investigated. The enzyme half-life was 66 minutes at 37° and was also unaffected by PEG. The enzyme preparation was satisfactorily immoblilsed by encapsulation in calcium alginate gel and enzyme distribution profiles were determined in sections of alginate stained with coomasie blue by a novel technique using a scanning optical densitometer, Diffusivity coefficients of B(a)P and NADP in alginate gel beads were determined as 7.5 and 2.5 x 10[-10] m[2]/S, respectively by the Tanaka method, fitting solute depletion profiles to Crank's theoretical model. The microsomal enzyme preparation immobilised in alginate beads was used to remove B(a)P from aqueous solution. B(a)P removal was shown to be by a non-specific affinity absorption mechanism and the removal profile was found to correspond well to a theoretical model of a diffusion-reaction system, for an affinity ligand system. The activaiton energy for deactivation of microsomal P450 was measured as 33.56kJ/mole.

The active site of porcine microsomal aminopeptidase was probed by studying the inhibition of the enzyme using derivatives of ethylenediamine and diaminopropionic acid. In addition, some amino acids, substituted hydroxamates and phosphates were also tested. In order to synthesize diaminopropionic acid derivatives, CBZ-amino acid p-nitrophenyl esters were reduced to the corresponding aldehydes by lithium tri-t-butoxy-aluminohydride. Through the Strecker synthesis, the aldehyde intermediates were converted to diaminopropionitrile analogues which were then hydrolysed in acid to the desired products. Unfortunately, these compounds were not potent inhibitors for this enzyme. α-Amino acids were found to be better inhibitors than their β-amino counterparts and the Kᵢ of α-leucine was about 7-fold lower than its B-analogue. The amino group position of the amino acids is therefore important for enzyme recognition. On the other hand, N-alkylation of ethylenediamine was observed to abolish its inhibition potential. Furthermore, another unexpected finding in this work is that N- or 0-methylation of the hydroxamate group hinders the ability of these inhibitors to act as a bidentate zinc ligand. Although some phosphate derivatives that we tested showed poor inhibitory potency, phosphonamidate, a potential transition state analogue, might serve as a powerful inhibitor. In summary, the relationship between the structure and inhibitory potency of some inhibitors was demonstrated. / Thesis / Master of Science (MSc)

The active site of porcine kidney microsomal aminopeptidase was investigated using single, multiple and EDTA inactivation kinetic studies. Good inhibitors invariably contained a zinc-coordinating group such as the mercapto moiety, which proved to be the best ligand for aminopeptidase. Due to the potency of β -mercaptoethylamine, derivatives of this compound were examined for aminopeptidase inhibition. (S)-2-amino-4-methyl-l-pentanethiol (L-leucinthiol) exhibited the largest potency and specificity towards aminopeptidase when compared against carboxypeptidase A and thermolysin, two similar zinc-peptidases. The presence of a zinc-coordination subsite, two hydrophobic pocket subsites and a second amine-binding subsite (distinct from that responsible for substrate recognition) were discerned and the binding modes of amino acid hydroxamates and mercaptoamines compared using Yonetani-Theorell inhibition kinetics. Aminopeptidase does not show virtually any stereoselectivity between L-and D-leucine hydroxamate while greater than a 1,000-fold preference is seen for L-leucinthiol over the D isomer. Also, the amino group of mercaptoamines is crucial to the binding of these inhibitors whereas that of the hydroxamate compounds does not seen to contribute much to their binding. The differences in binding between hydroxamates and mercaptoamines are postulated to be a consequence of the product analogue nature of the former and transition state analogue character of the latter. L-leucine hydroxamate is proposed to bind in a backwards orientation while the D isomer binds in the normal substrate-like position. Similarly, L-leucinthiol is proposed to bind in the same fashion as substrate. Design of future inhibitors should endeavour to: (1) lower the pᵏₐ of the α-amino group, (2) include an extended chain structure capable of binding to additional hydrophobic pockets, (3) incorporate a second amine moiety into the structure to interact with the second amine-binding subsite and (4) replace the mercapto group with a more potent zinc ligand such as the selenol group. / Thesis / Master of Science (MSc)

Induction of hepatic microsomal hydroxylative enzymes is an important aspect of detoxication of fat-soluble toxicants. The magnitude of induction depends on numerous factors, such as the nature and dose of toxicant as well as dietary factors. Research was conducted on (1) endrin tolerance in rats, (2) preliminary comparisons of inductive effects of various organochlorine pesticides in rats to select compounds for further study in guinea pigs, (3) general effects of various dietary factors on induction, and (4) effects of ascorbic acid deficiency on induction of hepatic microsomal hydroxylative enzymes by organochlorine pesticides in guinea pigs. Measurements were made of body weight gain, feed consumption, liver weight, in vivo and in vitroenzyme activities, and body levels of pesticides and vitamins. Tolerance developed in rats fed 25 ppm endrin in the diet. There was severe intoxication during the first week but complete recovery of rate of body weight gain and feed consumption occurred during the second week, in spite of continued ingestion of the endrin-containing diet. Induction of endrin-degrading microsomal enzymes was proposed as the mechanism for tolerance. Pretreatment with the potent inducer, dieldrin, diminished the severity of endrin intoxication. However, pretreatment with another inducer, phenobarbital, afforded less protection in proportion to the extent of microsomal enzyme induction. Organochlorine pesticides, tested for their inductive capacity in rats, in decreasing order of effectiveness then fed as 25 ppm of the diet for 15 days, were heptachlor epoxide, dieldrin, endrin, 1,1-Bis-(p-chlorophenyl)-2,2,2-Trichloroethane (DDT), Ovex, gamma-chlordane, and lindane. Of these, DDT and dieldrin were compared over the range of 1 to 50 ppm of the diet. Dieldrin was a more potent inducer at all dietary levels. At low doses both compounds produced greater induction when measured by hexobarbital sleep time than by the in vitro enzyme procedures. At high doses, the in vitro 0-ethyl 0-p-nitrophenyl phenyl-phosphonothioate (EPN) detoxication was a more responsive measure. Dieldrin, DDT, and lindane were fed to guinea pigs at 25 ppm of the diet for 15 days, but only dieldrin stimulated a significant level of induction. DDT antagonism of dieldrin storage seen in rats by Street (Sci. 146:1580, 1964) did not occur in guinea pigs, but rather dieldrin antagonized DDT storage. Some general dietary factors affecting induction in rats were observed. A semipurified diet lowered the baseline microsomal enzyme activity but supported induction as effectively as a conventional diet. Vitamin A at very high dietary levels induced enzyme activity; this induction was apparently additive to that produced by 1 ppm dieldrin. Other fat-soluble Vitamins produced inconsistent responses. Ascorbic acid deficiency in guinea pigs impaired induction by dieldrin. Impairment was seen by the second day on the deficient diet. However, dieldrin was able to produce a small amount of induction at all stages of deficiency. In frank scurvy, induction by DDT and lindane was completely blocked, but there was a moderate level of induction by dieldrin. Maintenance of maximum induction was related to dietary rather than liver levels of ascorbic acid; 50 ppm ascorbic acid in the diet was grossly inadequate while 200 ppm supported about 80% of the induction produced by feeding 2000 ppm. It was concluded that (1) microsomal enzyme induction is important in resistance to organochlorine intoxication, (2) factors found in the normal diet can induce microsomal enzyme activity, (3) high dietary levels of ascorbic acid are necessary to support maximum induction, and (4) dieldrin is an inducer of such high potency that it can stimulate a limited amount of induction in spite of ascorbic acid deficiency.

Este estudo foi estruturado em cinco capítulos. No capítulo I temos um breve referencial teórico sobre a importância da carne de frango, os mecanismos da oxidação lipídica e a utilização de antioxidantes naturais. O capítulo II traz os ensaios da avaliação da atividade antioxidante in vitro do mel e das especiarias orégano (Origamum vulgare L.) e sálvia (Salvia officinalis L.) durante a vida de prateleira. Os resultados de fenólicos totais do orégano indicaram um aumento de 1154,09 a 1611,28 mgEAG/100g (O a 12 meses), na sálvia os valores variaram entre 1309,8 a 2032,4 mgEAG/100g no decorrer do tempo (O a 12 meses) e no meios valores medidos foram 1007,1; 1830,4 e 2129,9 mg/100g/EAG para tempos 0,6 e 12 meses, respectivamente. Os resultados da porcentagem da inibição da oxidação lipídica (% IOL), pelo sistema (β-caroteno/ácido linoléico mostrou que a sálvia inibiu a oxidação em 74,6, 81,3 e 81,3%, nos tempos (O, 6 e 12 meses) e o orégano apresentou valores de inibição de (43,2, 63,3 e 50,7%). Quando se avaliou o índice de atividade antioxidante (IAA) utilizando o aparelho Rancimat, a sálvia apresentou um índice de atividade antioxidante (3,35) superior aos demais, que apresentaram 1,69, 1,25 e 1,08 para o BHT, orégano e mel, respectivamente. Os resultados do ensaio da capacidade de absorbância do radical oxigênio (ORAC) revelou que o orégano apresentou valores de 544,6, 430,7 e 1019,6 ET μmol/g, nos tempos 0, 6 e 12 meses, respectivamente. A sálvia apresentou valores de ORAC de: 610,45(0 mês), 467,44(6 meses) e 822,21(12 meses) e no mel foram de: 47,3; 22,4 e 26,1 ET μmol/g, nos tempos 0, 6 e 12 meses. Comparando estes resultados com os descritos na literatura podemos concluir que as especiarias e o mel possuem alto potencial antioxidante. No capítulo III mostramos a influência dos compostos bioativos da sálvia e de orégano na proteção da oxidação lipídica em substrato microssomal de carne de frango. As concentrações médias de TBARS (μMol de MDA/mg de proteína) observadas nas amostras de peito foram: controle (7,45); BHT (1,91) e orégano + sálvia (3,45) e na carne de sobrecoxa, observou-se os seguintes resultados: controle (9,83); BHT (4,27); orégano + sálvia (3, 15). Os tratamentos (BHT e orégano+sálvia) atingiram o ápice de inibição no tempo de 3 horas (82,42 % e 82,25 %), respectivamente. Porém quando analisamos a inibição da oxidação lipídica na fração microssomal da carne de sobrecoxa, o tratamento BHT apresentou o seu ápice de inibição (66,50 %) no tempo de 1 hora de indução e o tratamento orégano + sálvia alcançou a maior porcentagem de inibição no tempo de 3 horas (82,25 %). Os resultados da inibição da oxidação lipídica durante o período de indução mostram que em relação ao controle, os tratamentos realizados apresentaram influência positiva na proteção da oxidação lipídica em substrato microssomal de carne de peito de frango. No capítulo IV avaliamos o efeito de especiarias e mel na proteção da oxidação lipídica em sistema modelo homogenato de carne de frango refrigerada. Os resultados de atividade de água nos homogenatos de peito e sobrecoxa (crus ou cozidos) o tratamento (orégano+sálvia+10%Mel) reduziu a quantidade de água livre durante o tempo de refrigeração. Em relação aos valores de pH nos homogenatos de peito notou-se uma elevação nos valores de pH durante o período de refrigeração, em todos os tratamentos avaliados. Na carne de sobrecoxa os valores de pH foram maiores aos observados na carne de peito. Nos homogenatos de peito cru observou-se uma perda acentuada de umidade em todos os tratamentos, e particularmente em todos os tempos de refrigeração. Nas amostras de peito cozido não foram observadas diferenças significativas na umidade entre os tratamentos controle e orégano+sálvia+5%Mel. Nos homogenatos de sobrecoxa crua os valores de umidade variaram entre 60,82 e 66,96 g/100 g. Os teores de mioglobina nos homogenatos de peito cru variaram entre 1,95 % a 2,01 % no tempo 0. Ao final de 96 horas de refrigeração, a porcentagem de Mb variou entre 1,85 e 1,96, com redução das concentrações nas amostras tratados com especiarias e mel. Comportamento diferente foi apresentado nos homogenatos de peito cozido, onde após 96 horas de refrigeração observou-se aumento das concentrações de mioglobina em todos os tratamentos avaliados, com exceção do homogenato contendo orégano+sálvia+10% de mel. Em relação aos valores de metamioglobina (% MMb) para os homogenatos de peito crus e cozidos observou-se pequenas variações ao longo do tempo de refrigeração. Nas amostras de peito cru, as concentrações de oximioglobina (%O2Mb) apresentaram-se similares após 96 horas de refrigeração nos tratamentos controle, BHT e orégano+sálvia. Já nas amostras cozidas, os resultados foram similares entre as amostras controle e BHT. Nas amostras de sobrecoxa cruas, após 96 horas de refrigeração, observou-se redução das concentrações de metamioglobina (% MMb) e elevação dos teores de· mioglobina (% Mb) para todos os tratamentos avaliados. Nas amostras submetidas ao preparo térmico, ao contrário dos homogenatos crus, os teores de metarnioglobina (% MMb) elevaram-se após 96 horas de refrigeração nos tratamentos BHT e orégano+sálvia, permanecendo estáveis nos demais. Nos resultados da inibição da oxidação lipídica da carne de peito cozida durante o tempo de refrigeração, observamos que em relação ao controle, os tratamentos realizados apresentaram influência positiva. O tratamento com BHT inibiução oxidação em 51,4 % no tempo 0 e após 48 horas 74,3 %. Atingiu o ápice de inibição no tempo de 96 horas com 77,7 %. Dentre os tratamentos avaliados, o contendo orégano+sálvia+l0%Mel foi o mais efetivo contra a oxidação em relação aos demais. Nas amostras de sobrecoxa cozida, o tratamento BHT, não inibiu a oxidação em relação ao controle no tempo 0. Entretanto, nos outros tempos ocorreu um efeito protetor de 78,1 após 48 horas e 76,3 % após 96 horas de refrigeração. Nos tratamentos com especiarias, novamente o tratamento com orégano+sálvia+10%Mel foi o mais eficaz, com efeito protetor de 98%. No capítulo V avaliou-se o efeito da combinação de especiarias e mel na estabilidade oxidativa em carne de frango assada e refrigerada. Ao final das 96 horas, as amostras com adição de especiarias e mel apresentaram valores de TBARs mais baixos em relação ao controle e ao BHT, sugerindo que a sálvia, o orégano e o mel tenham exercido efeito antioxidante durante o experimento. Quando avaliamos os resultados da análise de dienos conjugados e hexanal, todas as amostras analisadas apresentaram um acréscimo nos valores dos dienos e de hexanal para todos os tempos de refrigeração. O tratamento que apresentou os menores valores de hexanal após 96 horas de refrigeração foi o (orégano+sálvia+5% de mel), seguido de (orégano+sálvia+10% de mel). Durante o processamento e ao longo do tempo de estocagem foram encontrados apenas traços dos óxidos (25-0H, 7-Ceto, 7α-OH e 7β-OH), com exceção apenas para o tratamento BHT no tempo de 48 horas (sobrecoxa assada) onde foi quantificada a presença de 7α-OH. Quando avaliamos os resultados dos ácidos graxos poli saturados da carne de peito, observamos alterações significativas nos tratamentos BHT, orégano+sálvia e orégano+sálvia+10%mel, provavelmente ocasionada pela refrigeração. Nas amostras de sobrecoxa, foram observados efeitos de interação entre tratamentos e tempos de refrigeração para todas as classes de ácidos graxos (saturados, insaturados, monoinsaturados e polinsaturados). As amostras oferecidas na análise sensorial receberam notas acima da nota de corte, no entanto, a maior parcela dos provadores atribuiu notas altas às amostras submetidas aos tratamentos com especiarias e mel. Os resultados obtidos neste estudo reafirmam a hipótese de que os compostos bioativos da sálvia, orégano e do mel foram capazes de inibir a oxidação lipídica, em todas as amostras. / This study has been structured into five chapters. Chapter I provides a brief historical theoretical reference point regarding the importance of chicken meat, the mechanisms of lipid oxidation and the use of natural antioxidants. Chapter II presents trials evaluating the in vitro antioxidant activity, of honey and the spices oregano (Origanum vulgare L.) and sage (Salvia officinalis L.) over their shelf life. The results for oregano indicated an increase in total phenols from 1154.09 to 1611.28 mg of gallic acid equivalent (GAE)/100 g (0 to 12 months). For sage, the values changed from 1309.8 to 2032.4 mg GAE/100 g over the period (0 to 12 months) and for honey, the values measured were 1007.1, 1830.4 and 2129.9 mg GAE/100 g at the times of 0,6 and 12 months, respectively. The results relating to the percentage inhibition of lipid oxidation(% ILO) by the β-carotene/linoleic acid system showed that sage inhibited oxidation by 74.6, 81.3 and 81.3% at the times of 0,6 and 12 months, while oregano presented inhibition values of 43.2, 63.3 and 50.7%. Evaluation of the antioxidant activity index (AAI) using the Rancimat apparatus showed that the AAI for sage (3.35) was greater than the indices for the other agents, which were 1.69, 1.25 and 1.08 for BHT, oregano and honey, respectively. The results from testing the oxygen radical absorbance capacity (ORAC) showed that oregano presented values of 544.6, 430.7 and 1019.6 TE μmol/g at the times of 0,6 and 12 months, respectively. Sage presented ORAC values of 610.45, 467.44 and 822.21 at the times of 0,6 and 12 months and honey presented 47.3, 22.4 and 26.1 ET μmol/g, at the times of 0,6 and 12 months. Comparing these results with those described in the literature, it can be concluded that these herbs and honey have high potential as antioxidants. Chapter III shows the influence of the bioactive compounds in sage and oregano for protection against lipid oxidation, in a microsomal substrate of chicken meat. The mean concentrations of TBARS (μmol of MDA/mg of protein) observed in the breast meat samples were, for the control, 7.45; for BHT, 1.91; and for oregano+sage, 3.45. In the thigh meat samples, the following results were observed: control (9.83); BHT (4.27); oregano+sage (3.15). The treatments (BHT and oregano+sage) reached their peak inhibition after three hours (82.42% and 82.25%, respectively). However, analysis of the inhibition of lipid oxidation in the microsomal fraction of the thigh meat showed that the BHT treatment reached its peak inhibition (66.50%) after one hour of induction and the oregano+sage treatment reached its peak inhibition after three hours (82.25%). The results relating to inhibition of lipid oxidation during the induction period showed that, in relation to the control, the treatments implemented had a positive influence regarding protection against lipid oxidation in a microsomal substrate of chicken breast meat. Chapter IV evaluates the effect of spices and honey with regard to protecting against lipid oxidation in a system consisting of a homogenate model of chilled chicken meat. The results from water action on the breast and thigh meat homogenates (either, raw or cooked) showed that the treatment (oregano+sage+10%honey) reduced the quantity of free water over the period of refrigeration. With regard to the pH values in the breast meat homogenates, it was seen that they rose over the period of refrigeration, in all the treatments evaluated. In the thigh meat, the pH values were higher than those observed in the breast meat. In the homogenates of raw breast meat, a marked loss of moisture was observed in all of the treatments and, particularly, at all durations of refrigeration. In the samples of cooked breast meat, no significant differences in moisture were seen between the control and the treatment with oreganó+sage+5%honey. In the homogenates of raw thigh meat, the moisture values ranged from 60.82 to 66.96 g/100 g. The myoglobin content in the homogenates of raw breast meat ranged from 1.95% to 2.01% at time zero. After 96 hours of refrigeration, the percentage myoglobin ranged from 1.85 to 1.96, with lower concentrations in the samples treated with spices and honey. A different pattern of behavior was presented by the homogenates of cooked breast meat, in which after 96 hours of refrigeration, higher concentrations of myoglobin were observed in all the treatments evaluated, with the exception of the homogenate containing oregano+sage+10%honey. With regard to the met myoglobin values (% MMb) for the homogenates of raw and cooked breast meat, small variations were observed over the refrigeration period. In the samples of raw breast meat, the oxymyoglobin concentrations (% O2Mb) were similar after 96 hours of refrigeration in the control and BHT and oregano+sage treatments. On the other hand, in the cooked samples, the results were similar between the control and BHT samples. In the raw thigh meat samples after 96 hours of refrigeration, it was observed that the met myoglobin concentration (% MMb) was lower and the myoglobin content (% Mb) was higher, for all the treatments evaluated. In the samples that were subjected to cooking, contrasting with the raw homogenates, the metmyoglobin content (% MMb) was higher after 96 hours in the BHT and oregano+sage treatments, while it remained stable in the other samples. The results relating to inhibition of lipid oxidation in the cooked breast meat over the refrigeration period showed that, in relation to the control, the treatments had a positive influence. The treatment with BHT inhibited oxidation by 51.4% at time zero and by 74.3% after 48 hours. The peak inhibition was reached after 96 hours, with 77.7%. Among the treatments evaluated, the one containing oregano+sage+10%honey was the most effective treatment against oxidation, in relation to the others. Among the samples of cooked thigh meat, the BHT treatment did not inhibit oxidation in relation to the control at time zero. However, at the other times, there was a protective effect of 78.1% after 48 hours and 76.3% after 96 hours of refrigeration. Among the treatments with spices, the one with oregano+sage+10%honey was again the most effective treatment, with a protective effect of 98%. Chapter V evaluates the effect of combinations of spices and honey on the oxidative stability of roasted and chilled chicken meat. After 96 hours, the samples with added herbs and honey presented TBARS values that were lower than in the control and BHT samples, thus suggesting that sage, oregano and honey had an antioxidant effect during the experiment. Evaluation of the results from analyzing the conjugated dienes and hexanal concentrations showed that all of the samples analyzed presented increased diene and hexanal leveIs at all durations of refrigeration. The treatment that presented the lowest hexanal values after 96 hours of refrigeration was oregano+sage+5%honey, folIowed by oregano+sage+10%honey. During the processing and over the course of storage, only traces of oxides were found (25-OH, 7-keto, 7 α -OH and 7 β-OH), with the sole exception of the BHT treatment on roast thigh meat after 48 hours, in which the presence of 7 α -OH was quantified. Evaluation of the results relating to polyunsaturated fatty acids in the breast meat showed significant changes in the BHT, oregano+sage and oregano+sage+10%honey treatments, probably caused by the refrigeration. In the samples of thigh meat, interaction effects between the treatments and duration of refrigeration were observed for all classes of fatty acids (saturated, unsaturated, monounsaturated and polyunsaturated). The samples offered in the sensory analysis received scores that were above the cutoff score, but most of the testers attributed high scores to the treatments with herbs and honey. The results obtained from this study reaffirm the hypothesis that the bioactive compounds in sage, oregano and honey were capable of inhibiting lipid oxidation in all the samples.

Este estudo foi estruturado em cinco capítulos. No capítulo I temos um breve referencial teórico sobre a importância da carne de frango, os mecanismos da oxidação lipídica e a utilização de antioxidantes naturais. O capítulo II traz os ensaios da avaliação da atividade antioxidante in vitro do mel e das especiarias orégano (Origamum vulgare L.) e sálvia (Salvia officinalis L.) durante a vida de prateleira. Os resultados de fenólicos totais do orégano indicaram um aumento de 1154,09 a 1611,28 mgEAG/100g (O a 12 meses), na sálvia os valores variaram entre 1309,8 a 2032,4 mgEAG/100g no decorrer do tempo (O a 12 meses) e no meios valores medidos foram 1007,1; 1830,4 e 2129,9 mg/100g/EAG para tempos 0,6 e 12 meses, respectivamente. Os resultados da porcentagem da inibição da oxidação lipídica (% IOL), pelo sistema (β-caroteno/ácido linoléico mostrou que a sálvia inibiu a oxidação em 74,6, 81,3 e 81,3%, nos tempos (O, 6 e 12 meses) e o orégano apresentou valores de inibição de (43,2, 63,3 e 50,7%). Quando se avaliou o índice de atividade antioxidante (IAA) utilizando o aparelho Rancimat, a sálvia apresentou um índice de atividade antioxidante (3,35) superior aos demais, que apresentaram 1,69, 1,25 e 1,08 para o BHT, orégano e mel, respectivamente. Os resultados do ensaio da capacidade de absorbância do radical oxigênio (ORAC) revelou que o orégano apresentou valores de 544,6, 430,7 e 1019,6 ET μmol/g, nos tempos 0, 6 e 12 meses, respectivamente. A sálvia apresentou valores de ORAC de: 610,45(0 mês), 467,44(6 meses) e 822,21(12 meses) e no mel foram de: 47,3; 22,4 e 26,1 ET μmol/g, nos tempos 0, 6 e 12 meses. Comparando estes resultados com os descritos na literatura podemos concluir que as especiarias e o mel possuem alto potencial antioxidante. No capítulo III mostramos a influência dos compostos bioativos da sálvia e de orégano na proteção da oxidação lipídica em substrato microssomal de carne de frango. As concentrações médias de TBARS (μMol de MDA/mg de proteína) observadas nas amostras de peito foram: controle (7,45); BHT (1,91) e orégano + sálvia (3,45) e na carne de sobrecoxa, observou-se os seguintes resultados: controle (9,83); BHT (4,27); orégano + sálvia (3, 15). Os tratamentos (BHT e orégano+sálvia) atingiram o ápice de inibição no tempo de 3 horas (82,42 % e 82,25 %), respectivamente. Porém quando analisamos a inibição da oxidação lipídica na fração microssomal da carne de sobrecoxa, o tratamento BHT apresentou o seu ápice de inibição (66,50 %) no tempo de 1 hora de indução e o tratamento orégano + sálvia alcançou a maior porcentagem de inibição no tempo de 3 horas (82,25 %). Os resultados da inibição da oxidação lipídica durante o período de indução mostram que em relação ao controle, os tratamentos realizados apresentaram influência positiva na proteção da oxidação lipídica em substrato microssomal de carne de peito de frango. No capítulo IV avaliamos o efeito de especiarias e mel na proteção da oxidação lipídica em sistema modelo homogenato de carne de frango refrigerada. Os resultados de atividade de água nos homogenatos de peito e sobrecoxa (crus ou cozidos) o tratamento (orégano+sálvia+10%Mel) reduziu a quantidade de água livre durante o tempo de refrigeração. Em relação aos valores de pH nos homogenatos de peito notou-se uma elevação nos valores de pH durante o período de refrigeração, em todos os tratamentos avaliados. Na carne de sobrecoxa os valores de pH foram maiores aos observados na carne de peito. Nos homogenatos de peito cru observou-se uma perda acentuada de umidade em todos os tratamentos, e particularmente em todos os tempos de refrigeração. Nas amostras de peito cozido não foram observadas diferenças significativas na umidade entre os tratamentos controle e orégano+sálvia+5%Mel. Nos homogenatos de sobrecoxa crua os valores de umidade variaram entre 60,82 e 66,96 g/100 g. Os teores de mioglobina nos homogenatos de peito cru variaram entre 1,95 % a 2,01 % no tempo 0. Ao final de 96 horas de refrigeração, a porcentagem de Mb variou entre 1,85 e 1,96, com redução das concentrações nas amostras tratados com especiarias e mel. Comportamento diferente foi apresentado nos homogenatos de peito cozido, onde após 96 horas de refrigeração observou-se aumento das concentrações de mioglobina em todos os tratamentos avaliados, com exceção do homogenato contendo orégano+sálvia+10% de mel. Em relação aos valores de metamioglobina (% MMb) para os homogenatos de peito crus e cozidos observou-se pequenas variações ao longo do tempo de refrigeração. Nas amostras de peito cru, as concentrações de oximioglobina (%O2Mb) apresentaram-se similares após 96 horas de refrigeração nos tratamentos controle, BHT e orégano+sálvia. Já nas amostras cozidas, os resultados foram similares entre as amostras controle e BHT. Nas amostras de sobrecoxa cruas, após 96 horas de refrigeração, observou-se redução das concentrações de metamioglobina (% MMb) e elevação dos teores de· mioglobina (% Mb) para todos os tratamentos avaliados. Nas amostras submetidas ao preparo térmico, ao contrário dos homogenatos crus, os teores de metarnioglobina (% MMb) elevaram-se após 96 horas de refrigeração nos tratamentos BHT e orégano+sálvia, permanecendo estáveis nos demais. Nos resultados da inibição da oxidação lipídica da carne de peito cozida durante o tempo de refrigeração, observamos que em relação ao controle, os tratamentos realizados apresentaram influência positiva. O tratamento com BHT inibiução oxidação em 51,4 % no tempo 0 e após 48 horas 74,3 %. Atingiu o ápice de inibição no tempo de 96 horas com 77,7 %. Dentre os tratamentos avaliados, o contendo orégano+sálvia+l0%Mel foi o mais efetivo contra a oxidação em relação aos demais. Nas amostras de sobrecoxa cozida, o tratamento BHT, não inibiu a oxidação em relação ao controle no tempo 0. Entretanto, nos outros tempos ocorreu um efeito protetor de 78,1 após 48 horas e 76,3 % após 96 horas de refrigeração. Nos tratamentos com especiarias, novamente o tratamento com orégano+sálvia+10%Mel foi o mais eficaz, com efeito protetor de 98%. No capítulo V avaliou-se o efeito da combinação de especiarias e mel na estabilidade oxidativa em carne de frango assada e refrigerada. Ao final das 96 horas, as amostras com adição de especiarias e mel apresentaram valores de TBARs mais baixos em relação ao controle e ao BHT, sugerindo que a sálvia, o orégano e o mel tenham exercido efeito antioxidante durante o experimento. Quando avaliamos os resultados da análise de dienos conjugados e hexanal, todas as amostras analisadas apresentaram um acréscimo nos valores dos dienos e de hexanal para todos os tempos de refrigeração. O tratamento que apresentou os menores valores de hexanal após 96 horas de refrigeração foi o (orégano+sálvia+5% de mel), seguido de (orégano+sálvia+10% de mel). Durante o processamento e ao longo do tempo de estocagem foram encontrados apenas traços dos óxidos (25-0H, 7-Ceto, 7α-OH e 7β-OH), com exceção apenas para o tratamento BHT no tempo de 48 horas (sobrecoxa assada) onde foi quantificada a presença de 7α-OH. Quando avaliamos os resultados dos ácidos graxos poli saturados da carne de peito, observamos alterações significativas nos tratamentos BHT, orégano+sálvia e orégano+sálvia+10%mel, provavelmente ocasionada pela refrigeração. Nas amostras de sobrecoxa, foram observados efeitos de interação entre tratamentos e tempos de refrigeração para todas as classes de ácidos graxos (saturados, insaturados, monoinsaturados e polinsaturados). As amostras oferecidas na análise sensorial receberam notas acima da nota de corte, no entanto, a maior parcela dos provadores atribuiu notas altas às amostras submetidas aos tratamentos com especiarias e mel. Os resultados obtidos neste estudo reafirmam a hipótese de que os compostos bioativos da sálvia, orégano e do mel foram capazes de inibir a oxidação lipídica, em todas as amostras. / This study has been structured into five chapters. Chapter I provides a brief historical theoretical reference point regarding the importance of chicken meat, the mechanisms of lipid oxidation and the use of natural antioxidants. Chapter II presents trials evaluating the in vitro antioxidant activity, of honey and the spices oregano (Origanum vulgare L.) and sage (Salvia officinalis L.) over their shelf life. The results for oregano indicated an increase in total phenols from 1154.09 to 1611.28 mg of gallic acid equivalent (GAE)/100 g (0 to 12 months). For sage, the values changed from 1309.8 to 2032.4 mg GAE/100 g over the period (0 to 12 months) and for honey, the values measured were 1007.1, 1830.4 and 2129.9 mg GAE/100 g at the times of 0,6 and 12 months, respectively. The results relating to the percentage inhibition of lipid oxidation(% ILO) by the β-carotene/linoleic acid system showed that sage inhibited oxidation by 74.6, 81.3 and 81.3% at the times of 0,6 and 12 months, while oregano presented inhibition values of 43.2, 63.3 and 50.7%. Evaluation of the antioxidant activity index (AAI) using the Rancimat apparatus showed that the AAI for sage (3.35) was greater than the indices for the other agents, which were 1.69, 1.25 and 1.08 for BHT, oregano and honey, respectively. The results from testing the oxygen radical absorbance capacity (ORAC) showed that oregano presented values of 544.6, 430.7 and 1019.6 TE μmol/g at the times of 0,6 and 12 months, respectively. Sage presented ORAC values of 610.45, 467.44 and 822.21 at the times of 0,6 and 12 months and honey presented 47.3, 22.4 and 26.1 ET μmol/g, at the times of 0,6 and 12 months. Comparing these results with those described in the literature, it can be concluded that these herbs and honey have high potential as antioxidants. Chapter III shows the influence of the bioactive compounds in sage and oregano for protection against lipid oxidation, in a microsomal substrate of chicken meat. The mean concentrations of TBARS (μmol of MDA/mg of protein) observed in the breast meat samples were, for the control, 7.45; for BHT, 1.91; and for oregano+sage, 3.45. In the thigh meat samples, the following results were observed: control (9.83); BHT (4.27); oregano+sage (3.15). The treatments (BHT and oregano+sage) reached their peak inhibition after three hours (82.42% and 82.25%, respectively). However, analysis of the inhibition of lipid oxidation in the microsomal fraction of the thigh meat showed that the BHT treatment reached its peak inhibition (66.50%) after one hour of induction and the oregano+sage treatment reached its peak inhibition after three hours (82.25%). The results relating to inhibition of lipid oxidation during the induction period showed that, in relation to the control, the treatments implemented had a positive influence regarding protection against lipid oxidation in a microsomal substrate of chicken breast meat. Chapter IV evaluates the effect of spices and honey with regard to protecting against lipid oxidation in a system consisting of a homogenate model of chilled chicken meat. The results from water action on the breast and thigh meat homogenates (either, raw or cooked) showed that the treatment (oregano+sage+10%honey) reduced the quantity of free water over the period of refrigeration. With regard to the pH values in the breast meat homogenates, it was seen that they rose over the period of refrigeration, in all the treatments evaluated. In the thigh meat, the pH values were higher than those observed in the breast meat. In the homogenates of raw breast meat, a marked loss of moisture was observed in all of the treatments and, particularly, at all durations of refrigeration. In the samples of cooked breast meat, no significant differences in moisture were seen between the control and the treatment with oreganó+sage+5%honey. In the homogenates of raw thigh meat, the moisture values ranged from 60.82 to 66.96 g/100 g. The myoglobin content in the homogenates of raw breast meat ranged from 1.95% to 2.01% at time zero. After 96 hours of refrigeration, the percentage myoglobin ranged from 1.85 to 1.96, with lower concentrations in the samples treated with spices and honey. A different pattern of behavior was presented by the homogenates of cooked breast meat, in which after 96 hours of refrigeration, higher concentrations of myoglobin were observed in all the treatments evaluated, with the exception of the homogenate containing oregano+sage+10%honey. With regard to the met myoglobin values (% MMb) for the homogenates of raw and cooked breast meat, small variations were observed over the refrigeration period. In the samples of raw breast meat, the oxymyoglobin concentrations (% O2Mb) were similar after 96 hours of refrigeration in the control and BHT and oregano+sage treatments. On the other hand, in the cooked samples, the results were similar between the control and BHT samples. In the raw thigh meat samples after 96 hours of refrigeration, it was observed that the met myoglobin concentration (% MMb) was lower and the myoglobin content (% Mb) was higher, for all the treatments evaluated. In the samples that were subjected to cooking, contrasting with the raw homogenates, the metmyoglobin content (% MMb) was higher after 96 hours in the BHT and oregano+sage treatments, while it remained stable in the other samples. The results relating to inhibition of lipid oxidation in the cooked breast meat over the refrigeration period showed that, in relation to the control, the treatments had a positive influence. The treatment with BHT inhibited oxidation by 51.4% at time zero and by 74.3% after 48 hours. The peak inhibition was reached after 96 hours, with 77.7%. Among the treatments evaluated, the one containing oregano+sage+10%honey was the most effective treatment against oxidation, in relation to the others. Among the samples of cooked thigh meat, the BHT treatment did not inhibit oxidation in relation to the control at time zero. However, at the other times, there was a protective effect of 78.1% after 48 hours and 76.3% after 96 hours of refrigeration. Among the treatments with spices, the one with oregano+sage+10%honey was again the most effective treatment, with a protective effect of 98%. Chapter V evaluates the effect of combinations of spices and honey on the oxidative stability of roasted and chilled chicken meat. After 96 hours, the samples with added herbs and honey presented TBARS values that were lower than in the control and BHT samples, thus suggesting that sage, oregano and honey had an antioxidant effect during the experiment. Evaluation of the results from analyzing the conjugated dienes and hexanal concentrations showed that all of the samples analyzed presented increased diene and hexanal leveIs at all durations of refrigeration. The treatment that presented the lowest hexanal values after 96 hours of refrigeration was oregano+sage+5%honey, folIowed by oregano+sage+10%honey. During the processing and over the course of storage, only traces of oxides were found (25-OH, 7-keto, 7 α -OH and 7 β-OH), with the sole exception of the BHT treatment on roast thigh meat after 48 hours, in which the presence of 7 α -OH was quantified. Evaluation of the results relating to polyunsaturated fatty acids in the breast meat showed significant changes in the BHT, oregano+sage and oregano+sage+10%honey treatments, probably caused by the refrigeration. In the samples of thigh meat, interaction effects between the treatments and duration of refrigeration were observed for all classes of fatty acids (saturated, unsaturated, monounsaturated and polyunsaturated). The samples offered in the sensory analysis received scores that were above the cutoff score, but most of the testers attributed high scores to the treatments with herbs and honey. The results obtained from this study reaffirm the hypothesis that the bioactive compounds in sage, oregano and honey were capable of inhibiting lipid oxidation in all the samples.

Amifostine is the only approved radioprotective agent by the Food and Drug Administration for reducing the damaging effects of radiation on healthy tissues. In this study, the effects of ionizing radiation on rat liver microsomal membrane and brain tissue and the protecting effects of amifostine on these systems were investigated at molecular level. Sprague-Dawley rats, which were administered amifostine or not, were whole-body irradiated and liver microsomal membranes and different regions of the brain of these rats were analyzed using FTIR spectroscopy, FTIR microspectroscopy and synchrotron FTIR microspectroscopy. The first part of this study revealed that ionizing radiation caused a decrease in the total lipid content and CH2 groups of lipids, an increase in the carbonyl esters, olefinic=CH and CH3 groups of lipids in the white matter and grey matter regions of the brain, which could be interpreted as a result of lipid peroxidation. In addition, radiation altered the protein structure of the brain. Amifostine caused significant protective effect against all the radiation induced damages in the brain. In the second part of the study, FTIR results showed that radiation induced a decrease in the lipid/protein ratio and a degradation of lipids into smaller fragments that contain less CH2 and more carbonyl esters, olefinic=CH and CH3 groups in microsomal membranes. In addition, radiation caused an alteration in the secondary structure of proteins, an increase in lipid order and a decrease in the membrane dynamics. Amifostine prevented all the radiation induced compositional, structural and functional damages in the liver microsomal membranes.

High blood glucose levels induce metabolic disorders that initiate a sequence of events including renal, arterial, cardiac and retinal disorders. Diabetes mellitus increases oxidative stress in tissues of animals including humans. The resulting oxidative stress might play role in the development of diabetic complications. In the present study, 36 male Wistar rats (250-300g) were divided into 5 groups as Control (n=6), Diabetic (n=7), Diabetic + Vit C (n=7), Diabetic + &amp / #945 / -Lipoic acid (n=6) and Diabetic + Combination of Vit C and &amp / #945 / -Lipoic acid (n=10). From the livers of all groups cytoplasmic and microsomal membrane fractions were prepared from liver and antioxidant enzymes namely, superoxide dismutase, glutathione peroxidase, catalase and glutathione S-transferase activities were measured. Microsomal lipid peroxidation, total lipid, total protein, reduced glutathione levels of each group was determined and compared. Microsomal fractions were also analyzed by FT-IR spectroscopy. The total protein levels of diabetic rats were found to be decreased significantly (p&lt / 0.05) compared to controls and the &amp / #945 / -lipoic acid and vitamin C supplemented groups tend to compensate the decreased levels of total proteins. Decreased catalase activity in diabetic group compared to control was restored by &amp / #945 / -lipoic acid, vitamin C treatment and/or combination of both. Increased glutathione peroxidase activity was decreased to control levels by the treatement of both &amp / #945 / -lipoic acid and vitamin C. Superoxide dismutase activities of diabetic rats were increased (p&lt / 0.05) compared to control group. Whereas glutathione S-transferase activities though showed some fluctuations, the results were not statistically significant. Total glutathione levels decreased in all groups significantly (p&lt / 0.0.5) compared to control group but any of the agent failed to compensate the reduced levels of glutathione. As an index of lipid peroxidation, TBA-reactivity (MDA) levels increased significantly in all diabetic groups and only combination group&rsquo / s TBARS levels decreased significantly compared to diabetic group. FT-IR study of rat liver microsomal membranes was carried out in order to understand the effects of diabetes on membrane order, dynamics and lipid peroxidation status. For this purpose CH2 symmetric wavenumber, CH2 antisymmetric bandwidth, =CH olefinic band area were compared. In temperature dependent FT-IR studies microsomal membrane phase behavior, order and dynamics were analyzed. Diabetic samples showed apparent decrease in both frequency and bandwidth. =CH olefinic band integrated area was increased for diabetic samples compared to controls. Alpha-lipoic acid and vitamin C supplemented groups showed similar effects. They tend to restore decreased levels of band frequency and bandwidth. Additive effect between &amp / #945 / -lipoic acid and vitamin C was seen in some cases that only the combination group achieved to restore control values while &amp / #945 / -lipoic acid and vitamin C were failed to restore alone. In conclusion, STZ-induced diabetes mainly caused an increase in antioxidant enzyme activities. Also, increase in lipid peroxidation caused a decrease in the fluidity and order of the membrane resulting in more rigid membrane structures. The loss of cooperation between the antioxidant network may play a role in the secondary complications of diabetes.

The Salmonella typhimurium/mammalian microsome test has provided a simple and sensitive short‐term assay for the detection of environmental mutagens. Metabolic activation of precarcinogens is usually achieved by incubating the compound to be tested, the bacterial strain and mammalian liver homogenates with NADPH. The results presented here utilize Salmonella typhimurium strain TA100, the precarcinogen 2‐aminofluorene and microsomal enzyme preparations prepared from liver homogenate of carp (Cyprinus carpio) taken from aquatic environments of northeastern Tennessee. Those environments range from virtually unpolluted to extremely polluted. The results show that the precarcinogen 2‐aminofluorene is activated either partially or totally in the presence of liver homogenates of carp taken from polluted aquatic environments (e.g., microsomal enzyme preparations made from rat liver with 2‐aminofluorene produced 808 revertants; whereas the liver preparations made from carp, taken from the Pigeon River, with 2‐aminofluorene produced 2,786 revertants). Revertant colony results correlated well with the degree of pollution within those waters. An increase (data were statistically different at the 0.05 level of significance) of TA100 revertant colonies was observed as aquatic contamination worsened. All data pairs of collecting sites in their order of increasing contamination, as well as those between collecting sites, were statistically different at the 0.05 level of significance.

La sclérodermie (sclérose systémique, ScS) est une maladie auto-immune du tissu conjonctif caractérisée  par  l’épaississement  de  la  peau,  l’apparition  spontanée de lésions cicatricielles, des maladies   des   vaisseaux   sanguins,   divers   degrés   d’inflammation,   en   association   avec   un   système   immunitaire hyperactif. La pathogénèse exacte de cette maladie est inconnue et aucun traitement approprié   n’est   disponible.   La fibrose est un élément distinctif de la maladie de ScS et est considérée   résulter   d’une   incapacité   à   mettre   fin   de   façon   appropriée   à   la   réponse   normale   de   réparation   des   plaies.   L’analyse   histologique   du   stade   initial   de   la   ScS   révèle   une   infiltration   périvasculaire de cellules mononucléaires dans le derme, associée à une synthèse accrue de collagène dans les fibroblastes environnants. Ainsi, la compréhension des moyens de contrôler le stade inflammatoire de la ScS pourrait être bénéfique pour contrôler la progression de la maladie peu après son apparition. La mPGES-1 est une enzyme inductible qui agit en aval de la cyclo- oxygénase (COX) pour catalyser spécifiquement la conversion de la prostaglandine (PG) H2 en PGE2. La mPGES-1  joue  un  rôle  clé  dans  l’inflammation,  la  douleur  et  l’arthrite;;  toutefois,  le   rôle de la mPGES-1 dans les mécanismes de fibrose, spécifiquement en rapport avec la ScS humaine, est inconnu. Mon laboratoire a précédemment montré que les souris à mPGES-1 nulle sont résistantes à la fibrose   cutanée   induite   par   la   bléomycine,   à   l’inflammation,   à   l’épaississement  cutané,  à  la  production  de  collagène  et  à  la  formation  de  myofibroblastes.  Sur  la   base  de  ces  résultats,  j’ai  formulé  l’hypothèse  que  l’inhibition pharmacologique de la mPGES-1 régulera à la baisse la production de médiateurs pro-inflammatoires et pro-fibreux au cours de la maladie   de   ScS.   Afin   d’explorer   le   rôle   de   la   mPGES-1   dans   l’inflammation   et   la   fibrose   associées  à  la  maladie  de  ScS,  j’ai  d’abord  examiné  l’expression  de  la  mPGES-1 dans la peau normale comparativement à des biopsies de peau extraites de patients atteints de ScS. Mes résultats ont montré que la mPGES-1 est nettement élevée dans la peau de patients atteints de ScS en comparaison avec la peau humaine normale. De plus, les niveaux de PGE2 dérivés de la mPGES-1 étaient également significativement plus élevés dans les fibroblastes cutanés isolés de patients  atteints  de  ScS  comparativement  aux  fibroblastes  isolés  de  témoins  sains.  J’ai  également   étudié  l’effet  de  l’inhibition pharmacologique de la mPGES-1  sur  l’expression  de  marqueurs  pro- fibreux.   Mes   études   ont   montré   que   l’expression   de   médiateurs   pro-fibreux clés (α-SMA, endothéline-1, collagène de type 1 et facteur de croissance du tissu conjonctif (FCTC)) est élevée dans les fibroblastes cutanés ScS en comparaison avec les fibroblastes cutanés normaux. Un traitement avec un inhibiteur de la mPGES-1 a eu pour effet de réduire significativement l’expression  de  l’α-SMA,  de  l’endothéline-1, du collagène de type 1 mais pas du FCTC dans les fibroblastes  ScS,  sans  effet  significatif  sur  les  fibroblastes  normaux.  J’ai  en  outre  examiné  l’effet   de  l’inhibition  de  la  mPGES-1 sur des cytokines pro-inflammatoires clés impliquées dans la pathologie de la ScS, incluant IL-6, IL-8 et MCP-1.  L’inhibition  pharmacologique  de  la  mPGES- 1 a eu pour effet de réduire significativement les niveaux de production de cytokines pro- inflammatoires IL6, IL8 et MCP-1 dans les fibroblastes avec lésion ScS comparativement à des fibroblastes non traités. De plus, les patients atteints de ScS ont présenté des niveaux plus élevés de p-AKT, de p-FAK et de p-SMAD3 en comparaison avec les fibroblastes cutanés normaux. L’inhibiteur  de  la  mPGES-1 a pu réguler à la baisse cette expression accrue de p-AKT et de p- FAK, mais pas de p-SMAD3,  dans  les  fibroblastes  ScS.  Ces  résultats  ont  suggéré  que  l’inhibition   de la mPGES-1 pourrait être une méthode viable pour réduire le développement de sclérose cutanée et constituent une cible thérapeutique potentielle pour contrôler les mécanismes fibreux et inflammatoires associés à la pathophysiologie de la maladie de ScS. L’un   des   autres   processus   critiques   reliés   à   l’évolution de la réponse fibreuse associée à la maladie de ScS est la différenciation des fibroblastes en des cellules activées spécialisées iii iv appelées myofibroblastes, responsables de déclencher une signalisation adhésive excessive et le dépôt excessif de matrice extracellulaire,   conduisant   à   la   destruction   de   l’architecture   de   l’organe.   Ainsi,   l’identification   des   facteurs   endogènes   qui   initient/   favorisent   la   différenciation   fibroblaste-myofibroblaste peut mener à des stratégies thérapeutiques prometteuses pour contrôler  l’excès  de  signalisation  adhésive  et  de  fibrose  associé  à  la  maladie  de  ScS.  Des  études   antérieures  dans  le  domaine  de  la  biologie  du  cancer  ont  suggéré  que  l’éphrine  B2,  une  protéine   transmembranaire appartenant à la famille des éphrines, est impliquée dans la signalisation adhésive   et   le   remodelage   extracellulaire.   Cependant,   son   rôle   dans   la   fibrose   n’a   jamais   été   exploré.   Dans   la   deuxième   partie   de   mon   étude,   j’ai   donc   étudié   le   rôle   de   l’éphrine   B2   dans   la   fibrose.   Mes   études   montrent   que   l’expression   de   l’éphrine   B2   est   significativement   augmentée   dans la peau humaine ScS comparativement à la peau normale. Plus important encore, le traitement in vitro de   fibroblastes   de   la   peau   humaine   normale   avec   de   l’éphrine   B2   recombinante est capable de transformer des fibroblastes en cellules myofibroblastiques manifestant toutes les caractéristiques myofibroblastiques typiques, incluant la formation accrue de  fibres  de  tension,  des  adhérences  focales,  l’activation  accrue  de  la  FAK,  un  accroissement  de   l’expression  et  de  la  migration  de  fibroblastes  et  de  leur  adhérence  à  la  fibronectine  à  la  fois  chez   les   fibroblastes   cutanés   normaux   et   ScS.   En   outre,   j’ai   traité   des   souris   avec   de   l’éphrine   B2   recombinante et montré que ces souris ont développé une fibrose cutanée significative associée à une épaisseur dermique et à une synthèse de collagène augmentées, une teneur en hydroxyproline (teneur en collagène) accrue et un nombre accru de myofibroblastes exprimant de   l’α-SMA, une activation augmentée de la FAK et de marqueurs pro-fibreux incluant le collagène de type 1 et le FCTC. Dans  l’ensemble,  mes  études  ont  identifié  deux  médiateurs  endogènes  cruciaux  impliqués  dans  la   propagation  de  l’inflammation  et  de  la  fibrose  associées  à  la  maladie  de  ScS.  L’inhibition  de  la   mPGES-1   pourrait   représenter   une   bonne   stratégie   alternative   pour   contrer   l’inflammation   et   la   fibrose au moins durant les stades précoces de la maladie de ScS. De plus, une signalisation excessive   de   l’éphrine B2 favorise la signalisation adhésive et fibreuse en déclenchant la différenciation   de   fibroblastes   en   myofibroblastes   par   l’activation   de   la   voie   de   signalisation   de   la  FAK.  Ainsi,  l’inhibition  d’éphrine  B2  bloquera  la  formation  de  fibroblastes-myofibroblastes et régulera à la baisse la fibrose associée à la maladie de ScS. En somme, la mPGES-1  et  l’éphrine   B2 semblent toutes deux des cibles attrayantes pour le traitement de la ScS et des troubles fibreux qui y sont reliés. / Scleroderma (Systemic sclerosis, SSc) is an autoimmune disease of the connective tissue featuring skin thickening, spontaneous scarring, and blood vessel disease, varying degrees of inflammation, associated with an overactive immune system. The exact pathogenesis of this disease is unknown and there is no appropriate treatment available. Fibrosis is a hallmark of SSc disease and is considered to arise due to an inability to appropriately terminate the normal wound repair response. Histological analysis of the initial stage of SSc reveals perivascular infiltrates of mononuclear cells in the dermis, which is associated with increased collagen synthesis in the surrounding fibroblasts. Thus understanding how to control the inflammatory stage of SSc may be of benefit in controlling the progression of early onset disease. mPGES-1 is an inducible enzyme that acts downstream of cyclooxygenase (COX) to specifically catalyze the conversion of prostaglandin (PG) H2 to PGE2. mPGES-1 plays a key role in inflammation, pain and arthritis; however, the role of mPGES-1 in fibrotic mechanisms especially with respect to human SSc is unknown. My laboratory has previously shown that mPGES-1-null mice are resistant to bleomycin-induced skin fibrosis, inflammation, cutaneous thickening, collagen production and myofibroblast formation. Based on these results I hypothesized that pharmacological inhibition of mPGES-1 will downregulate the production of pro-inflammatory and pro-fibrotic mediators during SSc disease. To explore the role of mPGES-1 in inflammation and fibrosis associated with SSc disease, I first investigated the expression of mPGES-1 in normal skin compared to skin biopsies extracted from SSc patients. My results showed that mPGES-1 is markedly elevated in SSc skin compared to normal human skin. In addition, the levels of mPGES-1- derived PGE2 were also significantly higher in skin fibroblasts isolated from SSc patients compared to fibroblasts isolated from healthy controls. I further investigated the effect of pharmacological inhibition of mPGES-1 on the expression of pro-fibrotic markers. My studies showed the expression of key pro-fibrotic mediators (α-SMA, endothelin-1, collagen type 1 and connective tissue growth factor) are elevated in SSc skin fibroblasts compared to normal skin fibroblasts. Treatment with mPGES-1 inhibitor resulted in significant reduction in the expression of α-SMA, endothelin-1, collagen type 1 but not CTGF in SSc and normal fibroblasts. Further, I investigated the effect of mPGES-1 inhibition on key pro-inflammatory cytokines implicated in SSc pathology including IL-6, IL-8 and MCP-1. Pharmacological inhibition of mPGES-1 resulted in significant reduction in the production levels of pro-inflammatory cytokines, IL6, IL8 and MCP-1 in SSc-lesioned fibroblasts compared to untreated fibroblasts. In addition, SSc patients exhibited higher levels of p-AKT, p-FAK and p-SMAD3 compared to normal skin fibroblasts. mPGES-1 inhibitor was able to down regulate this increased expression of p-AKT, p-FAK but not p-SMAD3 in SSc fibroblasts. These results suggested that inhibition of mPGES-1 may be a viable method to alleviate the development of cutaneous sclerosis and is a potential therapeutic target to control fibrotic and inflammatory mechanisms associated with the pathophysiology of SSc disease. One of the other critical processes associated with the evolution of fibrotic response associated with SSc disease is the differentiation of fibroblasts into specialized activated cells called myofibroblasts responsible for triggering excessive adhesive signaling and deposition of excessive extracellular matrix (ECM) leading to the destruction of organ architecture. Thus identifying endogenous factors which initiate/promote fibroblast-myofibroblast differentiation can lead to promising therapeutic strategies to control excessive adhesive signaling and fibrosis associated with SSc disease. Previous studies in cancer biology have suggested that ephrin B2, a transmembrane protein belonging to the family of ephrins, is involved in adhesive signaling and extracellular remodeling. However its role in fibrosis has never been explored. Therefore, in second part of my study, I investigated the role of ephrin B2 in fibrosis. My studies show ephrin v vi B2 expression is significantly enhanced in human SSc skin versus normal skin. Most importantly, in vitro treatment of normal human skin fibroblasts with recombinant ephrin B2 is able to transform fibroblasts into myofibroblastic cells exhibiting all typical myofibroblastic- characteristics including increased stress fibre formation, focal adhesions, increased activation of FAK, increased expression of and enhanced fibroblast migration and adhesion to fibronectin in both normal and SSc skin fibroblasts. Further, I treated mice with recombinant ephrin B2 and showed that these mice developed significant skin fibrosis associated with enhanced dermal thickness and collagen synthesis, increased hydroxyproline content (collagen content) and increased number of α-SMA-expressing myofibroblasts, enhanced activation of FAK and pro- fibrotic markers including type-I collagen and CTGF. Overall, my studies have identified two crucial endogenous mediators involved in propagating inflammation and fibrosis associated with SSc disease. mPGES-1 inhibition may present a good alternative strategy to counteract inflammation and fibrosis at least during early stages of SSc disease. Further, excessive ephrin B2 signaling promotes adhesive and fibrotic signaling by triggering fibroblast to myofibroblast differentiation via activation of the FAK signaling pathway. Thus, inhibition of ephrin B2 will block fibroblast-myofibroblast formation and downregulate fibrosis associated with SSc disease. Overall, both mPGES-1 and ephrin B2 seems to be attractive targets for treatment of SSc and related fibrotic disorders.