Elevated plasma concentrations of lipoprotein(a) [Lp(a)] have been identified as an independent risk factor for vascular diseases including coronary heart disease and stroke. In the current study, we have examined the binding and degradation of recombinant forms of apolipoprotein(a) [r-apo(a)], the unique kringle-containing moiety of Lp(a), using a cultured cell model. We found that the incubation of human hepatoma (HepG2) cells with an iodinated 17 kringle-containing (17K) recombinant form of apo(a) resulted in a two-component binding system characterized by a high affinity (Kd = 12 nM), low capacity binding site, and a low affinity (Kd = 249 nM), high capacity binding site. We subsequently determined that the high affinity binding site on HepG2 cells corresponds to the LDL receptor. In the HepG2 cell model, association of apo(a) with the LDL receptor was shown to be dependent on the formation of Lp(a) particles from endogenous LDL. Using an apo(a) mutant incapable of binding to the high affinity site through its inability to form Lp(a) particles (17KΔLBS7,8), we further demonstrated that the LDL receptor does not participate in Lp(a) catabolism. The low affinity binding component observed on HepG2 cells, familial hypercholesterolemia (FH) fibroblasts and human embryonic kidney (HEK) 293 cells may correspond to a member(s) of the plasminogen receptor family, as binding to this site(s) was decreased by the addition of the lysine analogue epsilon-aminocaproic acid. The lysine-dependent nature of the low affinity binding site was further confirmed in HepG2 binding studies utilizing r-apo(a) species with impaired lysine binding ability. We observed a reduction maximum binding capacity for 17K r-apo(a) variants lacking the strong lysine binding site (LBS) in kringle IV type 10 (17KΔAsp) and the very weak LBS in kringle V (17KΔV). Degradation of Lp(a)/apo(a) was found to be mediated exclusively by the low affinity component on both HepG2 cells and FH fibroblasts. Fluorescence confocal microscopy, using the 17K r-apo(a) variant fused to green fluorescent protein, further confirmed that degradation by the low affinity component on HepG2 cells does not proceed by the activity of cellular lysosomes. Taken together, these data suggest a potentially significant route for Lp(a)/apo(a) clearance in vivo. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-26 02:15:50.754

This study revolves around a family in which 4 male members have metabolic profiles similar to that of atypical 3–methylcrotonyl–CoA carboxylase (MCC) deficiency, an inborn error of leucine catabolism. This profile consists of high urinary 3–hydroxyisovaleric acid (3–HIVA) and trace amounts of 3–methylcrotonylglycine. One of the individuals also had clinical symptoms of chronic fatigue and muscle weakness, symptoms also related to MCC–deficiency. Further investigation showed that these individuals were negative for MCC–deficiency. The inheritance pattern of the abnormal metabolic profile seemed to indicate a link to the X–chromosome. In this study the single nucleotide polymorphism (SNP) and copy number variation (CNV) profiles of the X–chromosomes of participating members of the family were investigated for a possible link to the abnormal metabolic profile, using SNP6 DNA microarrays. The data generated by the SNP6 arrays was of good quality. The small sample size available for this study necessitated an unorthodox method for analysing the SNP6 data. No clear link between the SNP6 data and the abnormal metabolic profile was found. Selected SNP calls made by the SNP6 arrays were verified by sequencing. The origin of the elevated 3–HIVA detected in the urine of the male family members was also investigated. This was done by culturing fibroblasts from case individuals in culture medium supplemented with deuterium labelled leucine. The culture medium was analysed using GC–MS after an organic acid extraction. The resulting data seems to indicate at least two sources of 3–HIVA formation by the cells, one originating from leucine and another from a source other than leucine. The mevalonate shunt is one possible source of 3–HIVA, which does not originate from leucine catabolism. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

This study revolves around a family in which 4 male members have metabolic profiles similar to that of atypical 3–methylcrotonyl–CoA carboxylase (MCC) deficiency, an inborn error of leucine catabolism. This profile consists of high urinary 3–hydroxyisovaleric acid (3–HIVA) and trace amounts of 3–methylcrotonylglycine. One of the individuals also had clinical symptoms of chronic fatigue and muscle weakness, symptoms also related to MCC–deficiency. Further investigation showed that these individuals were negative for MCC–deficiency. The inheritance pattern of the abnormal metabolic profile seemed to indicate a link to the X–chromosome. In this study the single nucleotide polymorphism (SNP) and copy number variation (CNV) profiles of the X–chromosomes of participating members of the family were investigated for a possible link to the abnormal metabolic profile, using SNP6 DNA microarrays. The data generated by the SNP6 arrays was of good quality. The small sample size available for this study necessitated an unorthodox method for analysing the SNP6 data. No clear link between the SNP6 data and the abnormal metabolic profile was found. Selected SNP calls made by the SNP6 arrays were verified by sequencing. The origin of the elevated 3–HIVA detected in the urine of the male family members was also investigated. This was done by culturing fibroblasts from case individuals in culture medium supplemented with deuterium labelled leucine. The culture medium was analysed using GC–MS after an organic acid extraction. The resulting data seems to indicate at least two sources of 3–HIVA formation by the cells, one originating from leucine and another from a source other than leucine. The mevalonate shunt is one possible source of 3–HIVA, which does not originate from leucine catabolism. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Hyaluronic acid (HA) is a high molecular weight polysaccharide that is mainly produced by animals and certain bacteria. This polymer is biocompatible and possesses desirable rheological properties that are accentuated by high molecular weight. Diverse therapeutic applications have developed which harness these features. Pharmaceutical grade HA is mostly extracted from animal tissue. The HA derived from this source is suitable for most pharmaceutical preparations but there is growing pressure to avoid animal tissue products. This has provided the incentive to expand microbial-based HA manufacturing. However, the inherent low molecular weight of the polymer derived via this route has hampered widespread acceptance of microbial HA. This thesis examined the ramifications of improving the cellular economy of the HA-producing, gram-positive bacterium, Streptococcus zooepidemicus. Improved cellular economy is believed to be a prerequisite for achieving superior HA yields and molecular weights in this microorganism. This work examined the metabolic variation that accompanied the shift to more efficient modes of carbon utilization. In particular the effect of different sugar sources, uncoupling growth and polymer formation, and changes to the cellular oxidoreduction capacity were studied in more detail. This study utilized different sugar sources to enhance the recovery of energy. Fermenting glucose, fructose and maltose produced contrasting patterns of growth and HA formation. Culturing the organism in maltose caused a shift towards energy-efficient heterofermentative metabolism. Maltose-cultured cells displayed a biphasic pattern of metabolism. The first stage corresponded to a growth phase in which biomass synthesis profited from the increased energy yield. The second stage corresponded to an arginine-deficient stationary phase where the majority of the HA was formed. The fermentation rate was slower during stationary phase but continued to support HA biosynthesis. This bisphasic metabolism proved to be beneficial. A protracted stationary phase led to higher molecular weight HA. Fructose was unable to sustain a comparable polymer yield or molecular weight as glucose or maltose. There was evidence that the arginine deiminase pathway was responsible for the premature depletion of arginine in maltose-fermenting cultures. The accumulation of biomass exhibited a concentration-dependent response to the amount of glutamine in the medium. A second arginine transporter possessing a low affinity for glutamine could explain this phenomenon. Arginine consumption was slower when the glutamine level was elevated. This may indicate competition for a common transmembrane carrier. An elevated energetic yield and ATP formation rate were features of aerobic maltose metabolism. The relative improvement in biomass and HA yields were substantially greater for cultures fermenting maltose compared to glucose. However, no improvement in molecular weight compared to glucose was observed. A major factor contributing to the success of aerobic maltose fermentation was the particularly high NADH oxidase flux. This enzyme reoxidizes reduction equivalents in a reaction that is physically decoupled from the production of reduced metabolic products. Less lactate and ethanol accumulated in the presence of high NADH oxidase levels but acetate production was stimulated leading to an improved energetic yield. This result prompted an investigation into the effect of elevating the NADH oxidase level. The native NADH oxidase gene was sequenced and cloned into an inducible expression plasmid and introduced into S. zooepidemicus. Overproduction of this enzyme led to the desired improvement in ATP yield. A significant improvement in biomass yield was demonstrated. HA yield and molecular weight were not affected. Lactate and acetate were the main fermentation products. At high induction levels the quantity of lactate and acetate approached limiting levels and pyruvate overflow was more pronounced. This was attributed to insufficient flux capacity of the pyruvate dehydrogenase enzyme complex. The application of metabolic engineering to S. zooepidemicus has provided some insight into the regulation of energy metabolism in this microorganism and its relationship to HA synthesis. This study has observed that the specific rate of HA synthesis is correlated to the sugar uptake rate but is unaffected by the ATP yield. Under present conditions the formation of HA is not limited by the availability of energy. Nonetheless, microbial HA production will benefit from maximizing energetic yield. It was demonstrated that less catabolic carbon was expended to support biomass formation if the energetic yield was high. Therefore more residual carbon was available for HA synthesis.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv., 2007. / Härtill 3 uppsatser.

O objetivo do trabalho foi relacionar a baixa produtividade com perdas corporais lactacionais, caracterizando o perfil das fêmeas propensas ao risco, estudar detalhadamente este grupo de fêmeas e avaliar possíveis alternativas para minimizar os efeitos do catabolismo. O primeiro experimento analisou fêmeas de diferentes ordens de parto (OP) e perda de peso na lactação. Houve efeito da interação entre ordem de parto e perda de peso na taxa de parto das fêmeas (P<0,05) em fêmeas OP1 e OP2. Não houve interação da classe de OP com classe de perda de peso (P>0,05) para IDE e total de leitões nascidos. Fêmeas OP1 apresentaram IDE mais longo e menor tamanho da leitegada no parto subsequente (P<0,05) em comparação às fêmeas OP2 e OP3-5. As perdas corporais na lactação não afetaram o IDE (P>0,05), mas reduziram o tamanho da leitegada do parto seguinte (P<0,05). Os resultados sugerem que as fêmeas mais jovens são mais sensíveis ao catabolismo, afetando o desempenho reprodutivo após o desmame. Além disso, perdas corporais lactacionais reduzem o tamanho da leitegada subsequente. O segundo experimento estudou o efeito do peso ao parto (PP) e consumo energético em relação à mantença (CEM) na lactação de fêmeas OP1 e OP2 no desempenho reprodutivo. O baixo CEM afetou as perdas corporais em ambas as OP (P<0,05). O peso ao parto não afetou o consumo alimentar em fêmeas OP1, mas influenciou a ingestão de OP2. A concentração sérica de NEFA foi influenciada pelo CEM em OP1 e OP2. Alto CEM de OP1 implicou em aumento de ureia. Em OP1, o tamanho da leitegada não foi afetado pelo PP ou CEM, mas foi reduzida em OP2 com baixo PP ou CEM. O terceiro trabalho investigou o efeito de atrasar o primeiro serviço de OP1 após o desmame com tratamento com altrenogest (ALT) ou cobertura do segundo estro após o desmame (SKIP), comparado à inseminação no primeiro estro (CON). A restrição alimentar de 60% na última semana de lactação induziu uma perda média de peso corporal de 17kg. Quanto maior foi o intervalo desmame-serviço, maior foi a recuperação do peso à inseminação. O grupo ALT foi mais síncrono na entrada ao estro após a retirada do produto. A taxa de prenhez foi maior no SKIP e CON. ALT teve maior peso de corpora lutea e níveis de progesterona até 120h pós-ovulação. Não foi observada diferenças na taxa de ovulação, embriões viáveis, sobrevivência embrionária, tamanho dos embriões ou volume de fluido placentário. Dependendo do sistema de produção, estas estratégias podem trazer benefícios econômicos, quando aplicadas com critério em fêmeas com maior risco à baixa produtividade, uma vez que custo deve ser considerado. / The aim of this work was to relate low productivity to lactational weight loss, identifying the profile of females with more risk, study in details this cohort of sows and evaluate possible alternatives to minimize the effects of catabolism. The first trial evaluated females of different parity order (PO) and weight loss during lactation. There was interaction effect between parity order and weight loss on farrow rate (P<0.05) in PO1 and PO2 females. There was no interaction between PO and weight loss class (P>0.05) on WEI and subsequent total born. PO1 females presented longer WEI and lower litter size on subsequent farrowing compared to PO2 and PO3-5 females. Weight loss did not affect WEI (P>0.05), but it was related to a decrease of litter size in the subsequent farrowing (P<0.05). Results suggest young females are more sensitive to catabolism, affecting reproductive performance post weaning. The second experiment studied the effect of body weight at farrowing (BWF) and energy intake related to maintenance (MEIM) during lactation on subsequent reproductive performance of PO1 and PO2 sows. Low MEIM affected body weight loss in both PO (P<0.05). BWF did not affect energy intake in PO1 sows but influenced the consumption in PO2 sows. Serum NEFA concentration was influenced by MEIM in PO1 and PO2 sows. High MEIM PO1 sows showed higher urea concentration. In PO1, litter size was not affected by BWF or MEIM but was reduced in PO2 with Low BWF or MEIM. Third trial investigated the effect of delayed breeding in weaned PO1 sows with altrenogest treatment (ALT) or breeding at second estrus after weaning (SKIP), compared to breeding at first estrus after weaning (CON). Feed restriction at 60% during last week of lactation induced 17kg of body weight loss. The longer weaning to service interval resulted in greater recover of body weight at breeding. ALT group was more synchronized in estrus after altrenogest withdrawal. Pregnancy rate was greater in SKIP and CON. ALT had higher corpora lutea weight and progesterone levels at 120h post-ovulation. No differences in ovulation rate, live embryos, embryo survival, embryo size, or placental fluid volume were detected. Depending on the production system, these strategies may offer economic benefits, when carefully applied in a cohort of females with more risk to low productivity, since costs must be considered.

O Trypanosoma cruzi, agente etiológico da doença de Chagas, possui um ciclo de vida complexo, deve lidar com diversas condições do ambiente e depende dos hospedeiros para suprir suas necessidades nutricionais. Uma delas é a necessidade de captar a molécula de heme (Fe-protoporfirina IX) que será utilizada como fator de crescimento. Os mecanismos envolvendo o metabolismo de heme são cruciais para a sobrevivência do T. cruzi pois o parasito não possui várias enzimas de biossíntese dessa porfirina e o heme livre pode apresentar citotoxicidade para célula. Na tentativa de perseguir o destino final do heme no parasito, nós estudamos essa via inexplorada no T. cruzi. Nessa tese, nós demonstramos que epimastigotas cultivados com heme, produziram os compostos, &#945;-meso hidroxiheme, verdoheme e biliverdina (identificados por HPLC acoplado á espectrofotômetria). Além disso, nós observamos através de análise dos extratos de epimastigotas no espectrômetro de massas (LQT Orbitrap), espécies iônicas de m/z 583,4 e m/z 619,3. A fragmentação subsequente desses íons originaram espécies filhas típicas das moléculas de biliverdina e verdoheme, respectivamente. Nós observamos também, espécies iônicas de m/z 1397,4 e m/z 1135,4. A fragmentação dessas espécies produziram íons, sendo um deles com a mesma massa molecular de heme (m/z 616,3). Essa espécie iônica por sua vez, gerou fragmentos iônicos idênticos a uma molécula de heme, confirmando que esses intermediários são produtos da modificação da porfirina. Baseado nesses resultados, nós propomos um modelo onde o catabolismo de heme em T. cruzi, envolveria a conjugação da bis(glutationil)spermina, um derivado da tripanotiona presente em tripanossomatídeos, à porfirina (m/z 1137,4), seguido da remoção de dois resíduos de ácidos glutâmicos (m/z 1135,4). Embora o significado bioquímico e fisiológico da adição desse resíduo tiol na molécula de heme ainda é pouco compreendido, alguns trabalhos demonstram a abilidade desses compostos em ligar na porfirina, sem contar também, que esse heme conjugado poderia resultar em uma forma efetiva de prevenção de danos à membrana e a célula ocasionados pelo acúmulo de heme livre. Em conjunto, esses resultados fornecem novas abordagens do metabolismo de heme em T. cruzi, revelando possíveis alvos de intervenção quimioterápica futuros. Nossa proposta está direcionada para uma via ativa de catabolismo de heme que inclui a adição de grupos tiol (derivado da tripanotiona) à heme e a clivagem do anel porfirínico originando a molécula de biliverdina. / Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and they must cope with diverse environmental conditions and depends on hosts for its nutritional needs. One of the nutritional characteristic is that they need a heme compound (Fe-protoporphyrin IX) as a growth factor. The mechanisms involved in these processes are crucial for their survival mainly because of trypanosomatids lack of the complete heme biosynthetic pathway and the cytotoxic activity of free heme. Following the fate of this porphyrin in the parasite we studied this missing pathway in T. cruzi. Here, we show that epimastigotes cultivated with heme yielded the compounds, &#945;-meso hydroxyheme, verdoheme and biliverdin (as determined by HPLC with diode array detector). Furthermore, we observed ion species of m/z 583.4 and m/z 619.3 from epimastigotes extracts detected by direct infusion on LQT Orbitrap platform. A tipical biliverdin and verdoheme doughter-ion species were generated by m/z 583.4 and m/z 619.3 fragmentations, respectively. We also observed an ion species at m/z 1397.4 and m/z 1135. The subsequent fragmentation of this species produced a daughter-ions whose one with the same molecular mass as heme (m/z 616.4). This species, in turn, generated daughter species identical to an authentic heme, confirming that these intermediates were modified heme products. Based on these findings, we propose that heme catabolism in T. cruzi involves a additions of Bis(glutathionyl)spermine, a low molecular mass thiols occurring in trypanosomatids, to heme (m/z 1397.4), followed by removal of the glutamic residues (m/z 1135). Although the biochemical and physiological significance of the addition of thiol residues to heme molecule is underexplored, some works, already demonstrated their ability to bind heme and also this modified heme may resulting in the effective prevention of membrane damage and cytotoxicity by the heme accumulation. Taken together, these results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets. We propose an active heme catabolism pathway that includes a trypanotione derivate additions and cleavage of the heme porphyring ring to biliverdin.

Made available in DSpace on 2014-06-11T19:33:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-16Bitstream added on 2014-06-13T19:23:22Z : No. of bitstreams: 1 santos_ag_dr_botfm_prot.pdf: 1292610 bytes, checksum: 023b1ade3c67039310cf3b6cd129631a (MD5) / A colestase crônica por ligadura e ressecção do ducto biliar em ratos jovens é freqüentemente utilizada como modelo experimental de atresia biliar. Na colestase ocorre má absorção de vitamina E com resultante estresse oxidativo. Objetivos: Sendo a vitamina E-TPGS hidromiscível, e portanto absorvível mesmo na colestase, testamos os seus efeitos sobre as conseqüências nutricionais, sobre as alterações do metabolismo lipídico e sobre a lesão hepática da colestase obstrutiva crônica no modelo experimental acima. Métodos: Quarenta ratos machos da raça Wister com 21 dias de vida (P21) foram divididos em 4 grupos de 10 animais e submetidos a um dos seguintes tratamentos: 1) LA-ligadura e ressecção do ducto biliar comum e administração diária de água, por gavagem, num volume de 0,02ml por grama de peso do animal; 2) LELigadura e ressecção do ducto biliar comum e administração diária, por gavagem, de 25UI/kg num volume de 0,02ml de vitamina E-TPGS por grama de peso do animal de uma solução a 20% de vitamina E-TPGS; 3) SA-operação simulada e administração diária de água, por gavagem, num volume de 0,02ml por grama de peso do animal; 4) SE-operação simulada e administração diária, por gavagem, de 25Ul/kg num volume de 0,02ml de vitamina E-TPGS por grama de peso do animal de uma solução a 20% de vitamina E-TPGS. Durante o experimento foi determinado o ganho de peso, a quantidade de ração ingerida, o aproveitamento nutricional (P21 a P49) e balanço nitrogenado (P42 aoP49). No P48, foram submetidos ao teste do tempo de sono após pentobarbital. No P49, foram sacrificados e colhido sangue e órgãos para seguintes determinações: peso fresco, conteúdo de água e gordura da carcaça, fígado e baço, concentrações séricas de colesterol-T, triacilglicerol, LOL-colesterol, VLDL-colesterol, HDL-colesterol, albumina, globulinas totais, vitamina A e E, atividade sérica das aminotransferases (ALT e AST). / Chronic cholestasis by bile duct ligature and resection in young rats is a commonly used experimental model of biliary atresia. Vitamin E absorption is poor in cholestasis causing oxidative stress. Objectives: As Vitamin E-TPGS dissolves in water, and is therefore absorbable even in cholestasis, we tested its effects on nutritional outcome, Iipid metabolism alterations, and hepatic lesion from chronic obstructive cholestasis in the above mode!. Methods: Forty 21-day-old male Wistar rats (P21) were divided into four groups of 10 and submitted to the following treatments: 1) LA - ligature and common bile duct resection with daily administration of water by gavage (0.02ml/g animal weight); 2) LE- ligature and common bile duct resection with daily administration of 251U/Kg Vitamin E-TPGS in water by gavage (0.02mLlg wt of 20% Vitamin E-TPGS solution); 3) SA - sham operation and daily administration of water by gavage (0.02ml/g wt); and 4) SE - sham operation and daily administration of 251U/Kg Vitamin E-TPGS in water by gavage (0.02ml/g wt of 20% Vitamin E-TPGS solution). During the experiment we measured weight gain, ingested food, diet utilization (P21 to P49), and nitrogen balance (P42 to P49). On P48, pentobarbital sleeping time was measured. On P49, eutanasia was carried out and blood and organs were collected to determine: body,liver, and spleen fresh weight, and water and fat content, serum levels of total cholesterol, triacylg Iycerols, LDL-cholesterol, VLDL -cholesterol, HOL-cholesterol, albumin, total globulins, Vitamin A & E, and ALT & AST activity. Also liver histological sections were analyzed for fibrosis intensity, duct proliferation, necrosis, steatosis, hydropic degeneration, inflammation, and mitosis frequency. The effects of cholestasis, Vitamin E-TPGS, and their interactions were tested by ANOVA with two factors, and multiple paired comparisons... (Complete abstract click electronic access below)

Orientador: Cláudio Antônio Rabello Coelho / Banca: Cilmery S. Kurokwa / Banca: Rosangela Maria Barone / Banca: Ana Paula R. Battochio / Banca: Maria Ângela / Resumo: A colestase crônica por ligadura e ressecção do ducto biliar em ratos jovens é freqüentemente utilizada como modelo experimental de atresia biliar. Na colestase ocorre má absorção de vitamina E com resultante estresse oxidativo. Objetivos: Sendo a vitamina E-TPGS hidromiscível, e portanto absorvível mesmo na colestase, testamos os seus efeitos sobre as conseqüências nutricionais, sobre as alterações do metabolismo lipídico e sobre a lesão hepática da colestase obstrutiva crônica no modelo experimental acima. Métodos: Quarenta ratos machos da raça Wister com 21 dias de vida (P21) foram divididos em 4 grupos de 10 animais e submetidos a um dos seguintes tratamentos: 1) LA-ligadura e ressecção do ducto biliar comum e administração diária de água, por gavagem, num volume de 0,02ml por grama de peso do animal; 2) LELigadura e ressecção do ducto biliar comum e administração diária, por gavagem, de 25UI/kg num volume de 0,02ml de vitamina E-TPGS por grama de peso do animal de uma solução a 20% de vitamina E-TPGS; 3) SA-operação simulada e administração diária de água, por gavagem, num volume de 0,02ml por grama de peso do animal; 4) SE-operação simulada e administração diária, por gavagem, de 25Ul/kg num volume de 0,02ml de vitamina E-TPGS por grama de peso do animal de uma solução a 20% de vitamina E-TPGS. Durante o experimento foi determinado o ganho de peso, a quantidade de ração ingerida, o aproveitamento nutricional (P21 a P49) e balanço nitrogenado (P42 aoP49). No P48, foram submetidos ao teste do tempo de sono após pentobarbital. No P49, foram sacrificados e colhido sangue e órgãos para seguintes determinações: peso fresco, conteúdo de água e gordura da carcaça, fígado e baço, concentrações séricas de colesterol-T, triacilglicerol, LOL-colesterol, VLDL-colesterol, HDL-colesterol, albumina, globulinas totais, vitamina A e E, atividade sérica das aminotransferases (ALT e AST). / Abstract: Chronic cholestasis by bile duct ligature and resection in young rats is a commonly used experimental model of biliary atresia. Vitamin E absorption is poor in cholestasis causing oxidative stress. Objectives: As Vitamin E-TPGS dissolves in water, and is therefore absorbable even in cholestasis, we tested its effects on nutritional outcome, Iipid metabolism alterations, and hepatic lesion from chronic obstructive cholestasis in the above mode!. Methods: Forty 21-day-old male Wistar rats (P21) were divided into four groups of 10 and submitted to the following treatments: 1) LA - ligature and common bile duct resection with daily administration of water by gavage (0.02ml/g animal weight); 2) LE- ligature and common bile duct resection with daily administration of 251U/Kg Vitamin E-TPGS in water by gavage (0.02mLlg wt of 20% Vitamin E-TPGS solution); 3) SA - sham operation and daily administration of water by gavage (0.02ml/g wt); and 4) SE - sham operation and daily administration of 251U/Kg Vitamin E-TPGS in water by gavage (0.02ml/g wt of 20% Vitamin E-TPGS solution). During the experiment we measured weight gain, ingested food, diet utilization (P21 to P49), and nitrogen balance (P42 to P49). On P48, pentobarbital sleeping time was measured. On P49, eutanasia was carried out and blood and organs were collected to determine: body,liver, and spleen fresh weight, and water and fat content, serum levels of total cholesterol, triacylg Iycerols, LDL-cholesterol, VLDL -cholesterol, HOL-cholesterol, albumin, total globulins, Vitamin A & E, and ALT & AST activity. Also liver histological sections were analyzed for fibrosis intensity, duct proliferation, necrosis, steatosis, hydropic degeneration, inflammation, and mitosis frequency. The effects of cholestasis, Vitamin E-TPGS, and their interactions were tested by ANOVA with two factors, and multiple paired comparisons... (Complete abstract click electronic access below) / Doutor

O Trypanosoma cruzi, agente etiológico da doença de Chagas, possui um ciclo de vida complexo, deve lidar com diversas condições do ambiente e depende dos hospedeiros para suprir suas necessidades nutricionais. Uma delas é a necessidade de captar a molécula de heme (Fe-protoporfirina IX) que será utilizada como fator de crescimento. Os mecanismos envolvendo o metabolismo de heme são cruciais para a sobrevivência do T. cruzi pois o parasito não possui várias enzimas de biossíntese dessa porfirina e o heme livre pode apresentar citotoxicidade para célula. Na tentativa de perseguir o destino final do heme no parasito, nós estudamos essa via inexplorada no T. cruzi. Nessa tese, nós demonstramos que epimastigotas cultivados com heme, produziram os compostos, &#945;-meso hidroxiheme, verdoheme e biliverdina (identificados por HPLC acoplado á espectrofotômetria). Além disso, nós observamos através de análise dos extratos de epimastigotas no espectrômetro de massas (LQT Orbitrap), espécies iônicas de m/z 583,4 e m/z 619,3. A fragmentação subsequente desses íons originaram espécies filhas típicas das moléculas de biliverdina e verdoheme, respectivamente. Nós observamos também, espécies iônicas de m/z 1397,4 e m/z 1135,4. A fragmentação dessas espécies produziram íons, sendo um deles com a mesma massa molecular de heme (m/z 616,3). Essa espécie iônica por sua vez, gerou fragmentos iônicos idênticos a uma molécula de heme, confirmando que esses intermediários são produtos da modificação da porfirina. Baseado nesses resultados, nós propomos um modelo onde o catabolismo de heme em T. cruzi, envolveria a conjugação da bis(glutationil)spermina, um derivado da tripanotiona presente em tripanossomatídeos, à porfirina (m/z 1137,4), seguido da remoção de dois resíduos de ácidos glutâmicos (m/z 1135,4). Embora o significado bioquímico e fisiológico da adição desse resíduo tiol na molécula de heme ainda é pouco compreendido, alguns trabalhos demonstram a abilidade desses compostos em ligar na porfirina, sem contar também, que esse heme conjugado poderia resultar em uma forma efetiva de prevenção de danos à membrana e a célula ocasionados pelo acúmulo de heme livre. Em conjunto, esses resultados fornecem novas abordagens do metabolismo de heme em T. cruzi, revelando possíveis alvos de intervenção quimioterápica futuros. Nossa proposta está direcionada para uma via ativa de catabolismo de heme que inclui a adição de grupos tiol (derivado da tripanotiona) à heme e a clivagem do anel porfirínico originando a molécula de biliverdina. / Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and they must cope with diverse environmental conditions and depends on hosts for its nutritional needs. One of the nutritional characteristic is that they need a heme compound (Fe-protoporphyrin IX) as a growth factor. The mechanisms involved in these processes are crucial for their survival mainly because of trypanosomatids lack of the complete heme biosynthetic pathway and the cytotoxic activity of free heme. Following the fate of this porphyrin in the parasite we studied this missing pathway in T. cruzi. Here, we show that epimastigotes cultivated with heme yielded the compounds, &#945;-meso hydroxyheme, verdoheme and biliverdin (as determined by HPLC with diode array detector). Furthermore, we observed ion species of m/z 583.4 and m/z 619.3 from epimastigotes extracts detected by direct infusion on LQT Orbitrap platform. A tipical biliverdin and verdoheme doughter-ion species were generated by m/z 583.4 and m/z 619.3 fragmentations, respectively. We also observed an ion species at m/z 1397.4 and m/z 1135. The subsequent fragmentation of this species produced a daughter-ions whose one with the same molecular mass as heme (m/z 616.4). This species, in turn, generated daughter species identical to an authentic heme, confirming that these intermediates were modified heme products. Based on these findings, we propose that heme catabolism in T. cruzi involves a additions of Bis(glutathionyl)spermine, a low molecular mass thiols occurring in trypanosomatids, to heme (m/z 1397.4), followed by removal of the glutamic residues (m/z 1135). Although the biochemical and physiological significance of the addition of thiol residues to heme molecule is underexplored, some works, already demonstrated their ability to bind heme and also this modified heme may resulting in the effective prevention of membrane damage and cytotoxicity by the heme accumulation. Taken together, these results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets. We propose an active heme catabolism pathway that includes a trypanotione derivate additions and cleavage of the heme porphyring ring to biliverdin.