Conselho Nacional de Desenvolvimento Científico e Tecnológico / In the first study examined ammonia, urea, creatinine, protein, nitrite, nitrate, and phosphorus (P) excretion at different water hardness, humic acid or pH levels in silver catfish (Rhamdia quelen) juveniles. The fish were exposed to different levels of water hardness (4, 24, 50, or 100 mg CaCO3 L-1), humic acid (0, 2.5 or 5.0 mg L-1) or pH (5.0, 6.0, 7.0, 8.0, or 9.0) for 10 days. The overall measured nitrogen excretions were 88.1% (244 423 μmol kg-1 h-1) for ammonia, 10.9% (30 52 μmol kg-1 h-1) for creatinine, 0.02% (0.05 0.08 μmol kg-1 h-1) for protein, 0.001% (0.002 0.004 μmol kg-1 h-1) for urea, 0.5% (0.64 3.6 μmol kg-1 h-1) for nitrite, and 0.5% (0.0 6.9 μmol kg-1 h-1) for nitrate, and these proportions were not affected by water hardness or humic acid levels. The overall P excretion in R. quelen was 0.14 2.97 μmol kg-1 h-1. Ammonia excretion in R. quelen usually was significantly higher in the first 12 h after feeding, and no clear effect of water hardness, humic acid levels and pH on this daily pattern of ammonia excretion could be observed. Water hardness only affected the ammonia and P excretion of R. quelen juveniles in the initial and fifth days after transfer, respectively. The exposure of this species to humic acid decreased ammonia excretion after 10 days of exposure but did not affect P excretion. An increase in pH decreased ammonia and increased creatinine excretion but did not change P excretion in R. quelen. Therefore, when there is any change on humic acid levels or pH in the culture of this species nitrogenous compounds must be monitored because their excretion rates are variable. On the other hand, P excretion rates determined in the present study are applicable to a wide range of fish culture conditions. The aim of the second study was to determine the preferred pH in silver catfish Rhamdia quelen acclimated to different water hardness and the effect of shelters and infection by Ichthyophthirius multifiliis. Fish were acclimated for two weeks at different water hardness levels (4, 24, 50, or 100 mg CaCO3 L-1) and then transferred to a polyethylene tube with a pH gradient ranging from 3.5 to 11.7. The position of the fish in the pH gradient was observed at 1, 2, 4, 6, 8, 10, and 12 h after transference. Acclimation to different water hardness did not change pH preference of uninfected silver catfish (pH 7.30-7.83), and the presence of a shelter at the preferred pH or outside this preferred pH did not change the chosen pH range, either. Consequently silver catfish favored the acid-base regulation over shelter seeking tendency. Juveniles infected with I. multifiliis acclimated to water hardness of 24 mg CaCO3 L-1 preferred alkaline pH (9.08-9.79). This choice is not explained by the higher Na+ levels at alkaline pH compared to neutral pH because infected and uninfected fish choose the same waterborne Na+ levels in a Na+ gradient with the same pH. / No primeiro estudo analisou-se a excreção de amônia, ureia, creatinina, proteína, nitrito, nitrato e fósforo (P) em diferentes níveis de dureza da água, ácido húmico ou pH em juvenis de jundiás (Rhamdia quelen). Os peixes foram submetidos a diferentes níveis de dureza da água (4, 24, 50 ou 100 mg CaCO3 L-1), ácido húmico (0, 2,5 ou 5,0 mg L-1) ou pH (5,0, 6,0, 7,0, 8,0 ou 9,0) durante 10 dias. A excreção nitrogenada global medida foi de 88,1% (244 423 μmol kg-1 h-1) para amônia, 10,9% (30 52 μmol kg-1 h-1) para creatinina, 0,02% (0,05 0,08 μmol kg-1 h-1) para proteína, 0,001% (0,002 0,004 μmol kg-1 h-1) para ureia, 0,5% (0,64 3,6 μmol kg-1 h-1) para nitrito e 0,5% (0,0 6,9 μmol kg-1 h-1) para nitrato, e estas proporções não foram afetadas pelos níveis de dureza da água ou ácido húmico. A excreção global de P em R. quelen foi 0,14 2,97 μmol kg-1 h-1. A excreção de amônia em R. quelen, em geral, foi significativamente maior nas primeiras 12 horas após a alimentação e nenhum efeito claro dos níveis de dureza da água, ácido húmico e do pH pode ser observado sobre este padrão diário de excreção de amônia. A dureza da água afetou apenas a excreção de amônia e de P de juvenis de R. quelen no primeiro e quinto dias após a transferência, respectivamente. A exposição desta espécie ao ácido húmico diminuiu a excreção de amônia após 10 dias de exposição, mas não afetou a excreção de P. Um aumento no pH diminuiu a excreção de amônia e aumentou a excreção de creatinina, mas não alterou a excreção de P em R. quelen. Portanto, quando houver qualquer alteração nos níveis de ácido húmico ou pH na cultura desta espécie os compostos nitrogenados devem ser monitorados, pois suas taxas de excreção são variáveis. Por outro lado, as taxas de excreção de P determinados no presente estudo são aplicáveis a uma ampla gama de condições na cultura de peixes. O objetivo do segundo estudo foi determinar o pH preferido em jundiá Rhamdia quelen aclimatados a diferentes durezas da água e o efeito de abrigos e infecção por Ichthyophthirius multifiliis. Os peixes foram aclimatados durante duas semanas em diferentes níveis de dureza da água (4, 24, 50 ou 100 mg de CaCO3 L-1) e então transferidos para um tubo de polietileno com um gradiente de pH de 3,5 a 11,7. A posição do peixe no gradiente de pH foi observada 1, 2, 4, 6, 8, 10 e 12 h após a transferência. A aclimatação a diferentes durezas da água não afetou o pH preferido de jundiás não infectados (pH 7,30-7,83) e a presença de um abrigo no pH preferido ou fora deste pH também não alterou a faixa de pH preferida. Portanto, jundiás favorecem a regulação ácido-base em detrimento a uma tendência de procurar abrigo. Em juvenis infectados com I. multifiliis aclimatados à dureza da água de 24 mg de CaCO3 L-1 o pH preferido é alcalino (9,08-9,79). Esta escolha não é explicada pelos maiores níveis de Na+ em pH alcalino que em pH neutro porque peixes infectados e não infectados escolheram os mesmos níveis de Na+ na água em um gradiente de Na+ com o mesmo pH. Palavras-chave: piscicultura, gradiente de pH, ácido húmico, dureza da água

Nous démontrons qu'il est possible de former des bicouches fluides non phospholipides en milieu aqueux avec un mélange d'acide palmitique (PA), cholestérol (Chol) et sulfate de cholestérol (Schol) avec une proportion molaire de 30/28/42. Ces liposomes non phospholipidiques peuvent maintenir un gradient de pH (pHinterne 8 / pHexterne 6) sur une période 100 fois plus longue que les liposomes faits de 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphocholine (POPC) et de cholestérol (60/40 mol/mol). De plus, ces LUV non phospholipidiques protègent l'acide ascorbique d'un milieu oxydant (1 mM de fer (III)). Une fois piégé dans les liposomes, l'acide ascorbique présente une vitesse de dégradation similaire à celle obtenue en l'absence de fer(III). Ces performances illustrent la perméabilité exceptionnellement limitée de ces liposomes, ce qui implique qu'ils peuvent présenter des avantages comme nanocontenants pour certaines applications. D'autre part, des vésicules unilamellaires géantes (GUV pour Giant Unilamellar Vesicles) ont été formées à partir d'un mélange d'acide palmitique et de cholestérol (30/70 mol/mol). Ces GUV sont stables sur l'échelle de temps de semaines, elles ne s'agrègent pas et elles sont sensibles au pH. Afin d'établir la formation des GUV, l'imagerie par microscopie confocale à balayage laser a été utilisée. Deux sondes fluorescentes ont été utilisées: le rouge du Nile, une sonde hydrophobe qui s'insère dans le cœur hydrophobe des bicouches lipidiques, et la calcéine, une sonde hydrophile qui a été emprisonné dans le réservoir interne des GUV. Cette approche a permis l'observation des parois des GUV ainsi que de leur contenu. Ces résultats montrent la possibilité de former de nouveaux microcontenants à partir d'un mélange d'un amphiphile monoalkylé et de stérol. / First, we demonstrate that it is possible to form non-phospholipid fluid bilayers in aqueous milieu with a mixture of palmitic acid (PA), cholesterol (Chol), and cholesterol sulfate (Schol) in a molar proportion of 30/28/42. These non-phospholipid liposomes can sustain a pH gradient (pHinternal 8 / pHexternal 6) 100 times longer than LUVs made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (60/40 mol/mol). These non-phospholipid LUVs are shown to protect ascorbic acid from an oxidizing environment (1 mM Iron (III)). Once entrapped in these liposomes, ascorbic acid displays a degradation rate similar to that obtained in the absence of Iron (III). This ability illustrates the exceptionally limited permeability of these liposomes, indicating that they can present advantages as nanocontainers for some applications. Second, Giant Unilamellar Vesicles (GUVs) were formed from a mixture of palmitic acid and cholesterol (30/70 mol/mol). These GUVs were stable over weeks, did not aggregate, and were pH-sensitive. In order to establish their formation, confocal laser scanning microscopy imaging was carried out. Two fluorescent probes were used: Nile Red, a hydrophobic probe that inserted in the hydrophobic core of lipid bilayers, and calcein, a hydrophilic probe that was trapped in the GUV internal pool. This approach allowed observation of both the walls of the GUVs as well as their entrapped content. These results show the possibility to form novel microcontainers from a mixture of a monoalkylated amphiphile and sterols.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009. / Bibliography: p. 200-212. / Introduction -- Shotgun proteomic analysis of rat liver membrane proteins -- A combination of immobilised pH gradients improve membrane proteomics -- Affects of tumor-induced inflammation on membrane proteins abundance in the mouse liver -- Affects of tumor-induced inflammation on biochemical pathways in the mouse liver -- General discussion -- References. / The aim of this thesis was to develop a proteomics methodology that improves the identification of membrane proteomes from mammalian liver. Shotgun proteomics is a method that allows the analysis of proteins from cells, tissues and organs and provides comprehensive characterisation of proteomes of interest. The method developed in this thesis uses separation of peptides from trypsin digested membrane proteins by immobilised pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two dimensional shotgun proteomics. In this thesis, peptide IPG-IEF was shown to be a highly reproducible, high resolution analytical separation that provided the identification of over 4,000 individual protein identifications from rat liver membrane samples. Furthermore, this shotgun proteomics strategy provided the identification of approximately 1,100 integral membrane proteins from the rat liver. The advantages of using peptide IPG-IEF as a shotgun proteomics separation dimension in conjunction with label-free quantification was applied to a biological question: namely, does the presence of a spatially unrelated benign tumor affect the abundance of mouse liver proteins. IPG-IEF shotgun proteomics provided comprehensive coverage of the mouse liver membrane proteome with 1,569 quantified proteins. In addition, the presence of an Englebreth-Holm-Swarm sarcoma induced changes in abundance of proteins in the mouse liver, including many integral membrane proteins. Changes in the abundance of liver proteins was observed in key liver metabolic processes such as fatty acid metabolism, fatty acid transport, xenobiotic metabolism and clearance. These results provide compelling evidence that the developed shotgun proteomics methodology allows for the comprehensive analysis of mammalian liver membrane proteins and detailed some of the underlying changes in liver metabolism induced by the presence of a tumor. This model may reflect changes that could occur in the livers of cancer patients and has implications for drug treatments. / Mode of access: World Wide Web. / 609 p. ill. (some col.)

Nous démontrons qu'il est possible de former des bicouches fluides non phospholipides en milieu aqueux avec un mélange d'acide palmitique (PA), cholestérol (Chol) et sulfate de cholestérol (Schol) avec une proportion molaire de 30/28/42. Ces liposomes non phospholipidiques peuvent maintenir un gradient de pH (pHinterne 8 / pHexterne 6) sur une période 100 fois plus longue que les liposomes faits de 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphocholine (POPC) et de cholestérol (60/40 mol/mol). De plus, ces LUV non phospholipidiques protègent l'acide ascorbique d'un milieu oxydant (1 mM de fer (III)). Une fois piégé dans les liposomes, l'acide ascorbique présente une vitesse de dégradation similaire à celle obtenue en l'absence de fer(III). Ces performances illustrent la perméabilité exceptionnellement limitée de ces liposomes, ce qui implique qu'ils peuvent présenter des avantages comme nanocontenants pour certaines applications. D'autre part, des vésicules unilamellaires géantes (GUV pour Giant Unilamellar Vesicles) ont été formées à partir d'un mélange d'acide palmitique et de cholestérol (30/70 mol/mol). Ces GUV sont stables sur l'échelle de temps de semaines, elles ne s'agrègent pas et elles sont sensibles au pH. Afin d'établir la formation des GUV, l'imagerie par microscopie confocale à balayage laser a été utilisée. Deux sondes fluorescentes ont été utilisées: le rouge du Nile, une sonde hydrophobe qui s'insère dans le cœur hydrophobe des bicouches lipidiques, et la calcéine, une sonde hydrophile qui a été emprisonné dans le réservoir interne des GUV. Cette approche a permis l'observation des parois des GUV ainsi que de leur contenu. Ces résultats montrent la possibilité de former de nouveaux microcontenants à partir d'un mélange d'un amphiphile monoalkylé et de stérol. / First, we demonstrate that it is possible to form non-phospholipid fluid bilayers in aqueous milieu with a mixture of palmitic acid (PA), cholesterol (Chol), and cholesterol sulfate (Schol) in a molar proportion of 30/28/42. These non-phospholipid liposomes can sustain a pH gradient (pHinternal 8 / pHexternal 6) 100 times longer than LUVs made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (60/40 mol/mol). These non-phospholipid LUVs are shown to protect ascorbic acid from an oxidizing environment (1 mM Iron (III)). Once entrapped in these liposomes, ascorbic acid displays a degradation rate similar to that obtained in the absence of Iron (III). This ability illustrates the exceptionally limited permeability of these liposomes, indicating that they can present advantages as nanocontainers for some applications. Second, Giant Unilamellar Vesicles (GUVs) were formed from a mixture of palmitic acid and cholesterol (30/70 mol/mol). These GUVs were stable over weeks, did not aggregate, and were pH-sensitive. In order to establish their formation, confocal laser scanning microscopy imaging was carried out. Two fluorescent probes were used: Nile Red, a hydrophobic probe that inserted in the hydrophobic core of lipid bilayers, and calcein, a hydrophilic probe that was trapped in the GUV internal pool. This approach allowed observation of both the walls of the GUVs as well as their entrapped content. These results show the possibility to form novel microcontainers from a mixture of a monoalkylated amphiphile and sterols.