Nonmammalian vertebrates possess some unusual features in their hormonal systems/ when compared to mammals. As a consequence, they can make an important contribution in investigations concerning the fundamental mechanisms operating in endocrinology. Such studies concerning androgens include inter alia their effects on developmental aspects in the brain of birds and related singing behaviour; the role of neural enzymes in reproductive processes in fish; and the relation between androgens and the stages of spermatogenesis in amphibia, The present thesis examines the biochemistry of androgens in the Australian lizard Tiliqua rugosa. The major compounds studied were testosterone and epitestosterone, which are known to be present in high concentrations in the plasma of the male animal. Previous investigations are expanded, particularly in the areas of steroid identification and testicular biosynthesis. In addition, preliminary studies on the metabolism in the brain (and other tissues) and plasma protein binding are reported. The presence of epitestosterone as a major free androgen in the plasma of the male lizard was confirmed. Other steroids were found in the sulphate fraction. Testosterone sulphate was the most rigorously identified compound, while some evidence was also found for the presence of conjugated 5-androstene-3β,17-diols, etiocholanolone and dehydroepiandrosterone (DHA). Epitestosterone does not appear to be extensively conjugated in this animal. Steroids were not found to be conjugated as glucuronides. The identification studies employed a novel method of electrochemical detection of steroids. This technique was investigated and extended in the current thesis. Biosynthetic studies were carried out on androgen interconversions in the testis, in vitro. The major enzyme activities detected were 17α-arid 17β-oxidoreductases (17α-OR and l7β-OR) and 3β-hydroxysteroid dehydrogenase (3β-HSD)/isonerase. No evidence was found for the presence of a steroid-17-epimerase that would directly interconvert testosterone and epitestosterone. The 17-oxidoreductases were found to be dependent on the cofactor NBDFH. Testosterone appears to be formed mainly via the 4-ene pathway, whereas epitestosterone is formed from both the 4- and 5-ene routes. The compound 5-androstene-3β, 17α-diol was found to be an intermediate in the synthesis of epitestosterone from DHA. Temperature was found to significantly affect 17α-OR activity (maximum at 32°C). In contrast,17β-OR activity was independent of this factor in the testis. Androgen metabolism in the testis was found to be regulated by cofactors, temperature and season. The major enzyme activities found in the male brain were 17α- and 17β-OR. 3βHSD/isomerase was not found; however a low activity of 5α-reductase was identified. Aromatase activity was not positively identified, but preliminary results suggest that it may be present at low levels. The 17-oxidoreductases were widespread throughout the brain. The 17α-OR was significantly lower in the forebrain than other brain sections. The 170-OR activity did not vary significantly throughout the organ, although there was a trend for its activity to be higher in the midbrain region (containing the hypothalamus in these sections). The concentration of endogenous steroids in brain tissue was estimated by radioimmunoassay. Epitestosterone was found throughout the organ structure, whereas testosterone was found mainly in the midbrain (containing hypothalamic regions in these sections). Correlations between enzyme activities and steroid concentrations in brain regions suggested that the main function of 17α-OR is to produce epitestosterone, whereas the 17β-OR may catalyse a more reversible reaction in vivo. Temperature was found to significantly affect both 17α- and 17β-OR activities in the brain. In contrast to the testis, the maximum activity of the brain enzymes occurred at 37°C. The level of 17α-OR activity in the male lizard (100 nmol/g tissue/h) is the highest reported for this enzyme in vertebrates. Both activities were found to be quantitatively similar in the whole brain homogenates of male and female animals, and did not vary seasonally when examined in the male. The 17-oxidoreductases were also found in most other tissues in T.rugosa, including epididymis, adrenal, kidney and liver (but not blood). This suggests that the high activities of both 17α-OR and 17β-OR are dominant features of the steroid system in this animal. The formation of 11-oxygenated compounds was found in the adrenal, in addition to the formation of polar metabolites in the kidney and liver (possibly polyhydroxylated and conjugated steroids). A preliminary investigation into the plasma binding of androgens was carried out. The insults suggest that there are several binding sites for testosterone; one with high affinity and low capacity; the other with low affinity and high capacity. Binding experiments were carried out at 32°C. At this temperature, specific binding was greater than at 25 or 37°C. From the results of competition studies it was suggested that epitestosterone (with a K(i)= 3 X 10 (-6)M for testosterone binding) regulates the binding of testosterone (K(i)=10(-7)M) and hence the concentrations of the latter steroid as a free compound in plasma. In general, the study has shown that the biochemistry of androgens in the reptile T.rugosa is largely similar to that found in other vertebrates. The major difference is a greatly increased activity of 17α-OR, which causes a higher concentration of 17α-compounds to be present in the tissues of this lizard. The physiological roles for epitestosterone are not yet clear. However it appears from this study that this steroid regulates testosterone concentrations in several tissues by either steroidogenic or binding mechanisms. Several major influences on this regulation include temperature, availability of cofactors and seasonal effects.

O presente trabalho teve como objetivo avaliar o perfil de metabolização de açúcares por linhagens de S. cerevisiae amplamente utilizadas no setor sucroalcoleiro (CAT-1 e BG-1) e uma linhagem geneticamente modificada (HCJ 003) com o propósito de sobreexpressar invertase intracelular e ainda contém transportadores de sacarose de alta afinidade. Para tal estudo foi realizado 1 ensaio de fermentação simulando as condições industriais de produção de etanol com reciclos de células e mosto misto constituído de melaço e caldo de cana (50% de cada fonte) a 19% de ART. O ensaio de fermentação foi constituído por 4 ciclos sendo os 3 primeiros ciclos destinados a avaliação das cepas CAT -1 e HCJ 003 a qual é diplóide e sobre-expressa somente a invertase intracelular quanto aos diversos parâmetros do processo de fermentação. O 4° ciclo fermentativo foi destinado ao estudo do perfil de metabolização dos açúcares, onde analisou-se os teores de sacarose, glicose e frutose, bem como a formação de glicerol e o teor de trealose na biomassa das linhagens CAT -1, HCJ 003 e BG-1 (ambas com atividades de invertase distintas). No estudo comparativo dos parâmetros fermentativos a cepa HCJ 003 produziu de forma consistente, menos biomassa e mais etanol do que a cepa CAT-1. O rendimento fermentativo em etanol foi maior para a cepa HCJ 003. O fato de transportar a sacarose para o interior da célula faz com que seja gerado apenas 3 moles de ATP (em vez de 4 ATP). Portanto, para repor o ATP que deixa de ser gerado, há um maior direcionamento do metabolismo para as vias fermentativas e conseqüentemente mais etanol é produzido aumentando-se o rendimento da fermentação. No estudo do perfil de metabolização dos açúcares para as cepas CAT -1, HCJ 003 e BG-1 mostrou que a cepa BG -1 hidrolisou mais rapidamente a sacarose devido à alta atividade de invertase e também apresentou quantidades superiores de glicerol em resposta ao aumento da pressão osmótica do meio de fermentação. Já a CAT -1 apresentou maiores níveis de trealose o que pode ser um indicativo da sua maior dominância durante o processo industrial brasileiro. Assim este trabalho resultou no perfil de metabolização de cepas com diferente atividade de invertase e mostrou que a cepa HCJ 003 embora seja uma derivada de uma linhagem laboratorial manteve-se adaptada às condições do processo obtendo-se rendimento superior à CAT-1. / The aim of tis work was to evaluate the metabolic profile of sugars using Saccharomyces cerevisiae strains widely used in industry of sugars and ethanol (CAT-1 e BG-1) and a genetically modified strain in purpose to over-express intracellular invertase and also contains sucrose transporters with high afinity. For this study, 1 test were performed simulating industrial fermentation for ethanol production using cell recycle and mixed must with molasses and sugar cane (50% of each compound) for 19% TRS. The fermentation experiment were composed of 4 recycles and in first 3 cycles were intended for the evaluation of the strains CAT-1 and HCJ 003 wich is diploid and overexpres just the intracellular invertase on the various parameters of the fermentation process. The 4th recycle was designed to study the profile of metabolism of sugars, where were analysed the levels of sucrose, glucose and frutose, as well as the formation of glycerol and trehalose content in biomass of the strains CAT -1, HCJ 003 and BG -1 (all of them with different invertase activity). In the evaluation of the strains on the various parameters of the fermentation process, the strain HCJ 003 produced consistently less biomass and more ethanol than CAT-1. The yield of ethanol fermentation was higher for the strain HCJ 003. The fact that this strain transport sucrose into the cells makes it generated only 3 moles of ATP (instead 4 ATP). So to restore the ATP that is no generated, there is an increase targeting of the metabolism to fermentative pathways and therefore more ethanol is produced increasing the efficiency of fermentation. In the profile of metabolism of sugars to the strains CAT -1 HCJ 003 and BG-1 showed that the strain BG -1 hydrolyzed more rapidly the sucrose due to high invertase activity and also showed higher amounts of glycerol in response to increased osmotic pressure of the fermentation medium. Already CAT -1 showed higher levels of trehalose, which may be indicative of its most dominant during the Brazilian industry. So this work resulted in the profile of metabolism of different strains with invertase activity and showed that strain 003 Hcj although a derivative of a laboratory strain remained adapted to the conditions of the process resulting in higher yield to CAT-1.

O presente trabalho teve como objetivo avaliar o perfil de metabolização de açúcares por linhagens de S. cerevisiae amplamente utilizadas no setor sucroalcoleiro (CAT-1 e BG-1) e uma linhagem geneticamente modificada (HCJ 003) com o propósito de sobreexpressar invertase intracelular e ainda contém transportadores de sacarose de alta afinidade. Para tal estudo foi realizado 1 ensaio de fermentação simulando as condições industriais de produção de etanol com reciclos de células e mosto misto constituído de melaço e caldo de cana (50% de cada fonte) a 19% de ART. O ensaio de fermentação foi constituído por 4 ciclos sendo os 3 primeiros ciclos destinados a avaliação das cepas CAT -1 e HCJ 003 a qual é diplóide e sobre-expressa somente a invertase intracelular quanto aos diversos parâmetros do processo de fermentação. O 4° ciclo fermentativo foi destinado ao estudo do perfil de metabolização dos açúcares, onde analisou-se os teores de sacarose, glicose e frutose, bem como a formação de glicerol e o teor de trealose na biomassa das linhagens CAT -1, HCJ 003 e BG-1 (ambas com atividades de invertase distintas). No estudo comparativo dos parâmetros fermentativos a cepa HCJ 003 produziu de forma consistente, menos biomassa e mais etanol do que a cepa CAT-1. O rendimento fermentativo em etanol foi maior para a cepa HCJ 003. O fato de transportar a sacarose para o interior da célula faz com que seja gerado apenas 3 moles de ATP (em vez de 4 ATP). Portanto, para repor o ATP que deixa de ser gerado, há um maior direcionamento do metabolismo para as vias fermentativas e conseqüentemente mais etanol é produzido aumentando-se o rendimento da fermentação. No estudo do perfil de metabolização dos açúcares para as cepas CAT -1, HCJ 003 e BG-1 mostrou que a cepa BG -1 hidrolisou mais rapidamente a sacarose devido à alta atividade de invertase e também apresentou quantidades superiores de glicerol em resposta ao aumento da pressão osmótica do meio de fermentação. Já a CAT -1 apresentou maiores níveis de trealose o que pode ser um indicativo da sua maior dominância durante o processo industrial brasileiro. Assim este trabalho resultou no perfil de metabolização de cepas com diferente atividade de invertase e mostrou que a cepa HCJ 003 embora seja uma derivada de uma linhagem laboratorial manteve-se adaptada às condições do processo obtendo-se rendimento superior à CAT-1. / The aim of tis work was to evaluate the metabolic profile of sugars using Saccharomyces cerevisiae strains widely used in industry of sugars and ethanol (CAT-1 e BG-1) and a genetically modified strain in purpose to over-express intracellular invertase and also contains sucrose transporters with high afinity. For this study, 1 test were performed simulating industrial fermentation for ethanol production using cell recycle and mixed must with molasses and sugar cane (50% of each compound) for 19% TRS. The fermentation experiment were composed of 4 recycles and in first 3 cycles were intended for the evaluation of the strains CAT-1 and HCJ 003 wich is diploid and overexpres just the intracellular invertase on the various parameters of the fermentation process. The 4th recycle was designed to study the profile of metabolism of sugars, where were analysed the levels of sucrose, glucose and frutose, as well as the formation of glycerol and trehalose content in biomass of the strains CAT -1, HCJ 003 and BG -1 (all of them with different invertase activity). In the evaluation of the strains on the various parameters of the fermentation process, the strain HCJ 003 produced consistently less biomass and more ethanol than CAT-1. The yield of ethanol fermentation was higher for the strain HCJ 003. The fact that this strain transport sucrose into the cells makes it generated only 3 moles of ATP (instead 4 ATP). So to restore the ATP that is no generated, there is an increase targeting of the metabolism to fermentative pathways and therefore more ethanol is produced increasing the efficiency of fermentation. In the profile of metabolism of sugars to the strains CAT -1 HCJ 003 and BG-1 showed that the strain BG -1 hydrolyzed more rapidly the sucrose due to high invertase activity and also showed higher amounts of glycerol in response to increased osmotic pressure of the fermentation medium. Already CAT -1 showed higher levels of trehalose, which may be indicative of its most dominant during the Brazilian industry. So this work resulted in the profile of metabolism of different strains with invertase activity and showed that strain 003 Hcj although a derivative of a laboratory strain remained adapted to the conditions of the process resulting in higher yield to CAT-1.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal