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The main objective of this study was to determine the applicability of advanced water treatment processes namely granular activated carbon (GAC) adsorption, ultraviolet (UV) light disinfectant and ozone in the management of deteriorating water quality in the Mid-Vaal River system for drinking purposes. Both the scarcity and the deteriorating quality of water in South Africa can be addressed by investigating advanced water treatment processes such as GAC adsorption, UV light disinfectant and ozone. Previously disregarded water resources have the potential to be purified and advanced treatments can improve water quality where conventional water treatments have failed. In addition, advanced treatment processes can be applied to treat used water. The two sampling sites selected for the study, Rand Water Barrage (RWB) and Midvaal Water Company (MWC), are both located in the Middle Vaal Water Management Area with RWB upstream of MWC. RWB uses GAC adsorption and UV light disinfection and MWC uses ozone as pre- and intermediate treatment process steps for water purification. The quality of the source water at both sampling sites was determined by analysing the physical and chemical characteristics as well as the algal and invertebrate compositions of the source water. The physical and chemical water quality variables measured included pH, conductivity, turbidity, dissolved organic carbon (DOC), total organic carbon (TOC), total photosynthetic pigments (TPP), microcystin and geosmin. The source water of both sites was characterised as hypertrophic on account of high chlorophyll concentrations. The water quality of the two sites was distinctly different and a downstream change was observed. The source water of RWB was characterised by high microcystin, geosmin, DOC, TOC and conductivity measurements and dominated by Bacillariophyceae (diatoms) and Cyanophyceae (blue-green bacteria). Problematic species that were present in the source water of RWB included Aulacoseira sp., other unidentified centric diatoms, Pandorina sp., Anabaena sp., Microcystis sp., Oscillatoria sp., Cryptomonas sp., Ceratium sp. and Trachelomonas sp. The source water of MWC was characterised by high pH, turbidity and TPP measurements and was dominated by Chlorophyceae (green algae) and Bacillariophyceae (diatom) species. Problematic algal species that were present in the source water of MWC included Cyclotella sp., Coelastrum sp., Pediastrum sp. and Scenedesmus sp. The source water of MWC was deemed to be of a better quality due to the lower Cyanophyceae concentrations and lower microcystin levels. The invertebrate composition of both sites was similar with Rotatoria as the dominant invertebrate group. The efficacy of GAC adsorption/UV light disinfection/ozonation on restoring the physical and chemical characteristics of the source water at both sampling sites as well as the algal and invertebrate compositions was determined by ascertaining the nature of the change in or the percentage removal of a water quality variable. The potable water of both sites complied with the standards of water intended for domestic use except for the conductivity at RWB that was slightly elevated. The phytoplankton was removed effectively from the source water of both sites but the removal of invertebrates was unsatisfactory. GAC adsorption and filtration proved to be more effective in the removal of TPP, turbidity, DOC, microcystin and geosmin than ozone. Ozone effected an increase in DOC. UV light disinfection had no or little effect on restoring the water quality variables investigated in this study. / M (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The main objective of this study was to determine the applicability of advanced water treatment processes namely granular activated carbon (GAC) adsorption, ultraviolet (UV) light disinfectant and ozone in the management of deteriorating water quality in the Mid-Vaal River system for drinking purposes. Both the scarcity and the deteriorating quality of water in South Africa can be addressed by investigating advanced water treatment processes such as GAC adsorption, UV light disinfectant and ozone. Previously disregarded water resources have the potential to be purified and advanced treatments can improve water quality where conventional water treatments have failed. In addition, advanced treatment processes can be applied to treat used water. The two sampling sites selected for the study, Rand Water Barrage (RWB) and Midvaal Water Company (MWC), are both located in the Middle Vaal Water Management Area with RWB upstream of MWC. RWB uses GAC adsorption and UV light disinfection and MWC uses ozone as pre- and intermediate treatment process steps for water purification. The quality of the source water at both sampling sites was determined by analysing the physical and chemical characteristics as well as the algal and invertebrate compositions of the source water. The physical and chemical water quality variables measured included pH, conductivity, turbidity, dissolved organic carbon (DOC), total organic carbon (TOC), total photosynthetic pigments (TPP), microcystin and geosmin. The source water of both sites was characterised as hypertrophic on account of high chlorophyll concentrations. The water quality of the two sites was distinctly different and a downstream change was observed. The source water of RWB was characterised by high microcystin, geosmin, DOC, TOC and conductivity measurements and dominated by Bacillariophyceae (diatoms) and Cyanophyceae (blue-green bacteria). Problematic species that were present in the source water of RWB included Aulacoseira sp., other unidentified centric diatoms, Pandorina sp., Anabaena sp., Microcystis sp., Oscillatoria sp., Cryptomonas sp., Ceratium sp. and Trachelomonas sp. The source water of MWC was characterised by high pH, turbidity and TPP measurements and was dominated by Chlorophyceae (green algae) and Bacillariophyceae (diatom) species. Problematic algal species that were present in the source water of MWC included Cyclotella sp., Coelastrum sp., Pediastrum sp. and Scenedesmus sp. The source water of MWC was deemed to be of a better quality due to the lower Cyanophyceae concentrations and lower microcystin levels. The invertebrate composition of both sites was similar with Rotatoria as the dominant invertebrate group. The efficacy of GAC adsorption/UV light disinfection/ozonation on restoring the physical and chemical characteristics of the source water at both sampling sites as well as the algal and invertebrate compositions was determined by ascertaining the nature of the change in or the percentage removal of a water quality variable. The potable water of both sites complied with the standards of water intended for domestic use except for the conductivity at RWB that was slightly elevated. The phytoplankton was removed effectively from the source water of both sites but the removal of invertebrates was unsatisfactory. GAC adsorption and filtration proved to be more effective in the removal of TPP, turbidity, DOC, microcystin and geosmin than ozone. Ozone effected an increase in DOC. UV light disinfection had no or little effect on restoring the water quality variables investigated in this study. / M (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Apesar de extensa investigação das modificações oxidativas irreversíveis sofridas pelas proteínas in vitro e in vivo, os produtos formados pela oxidação de resíduos de triptofano ainda permanecem apenas parcialmente conhecidos. Recentemente, nosso grupo caracterizou uma ligação cruzada de ditriptofano produzida pela recombinação de radicais hSOD1-triptofanila gerados pelo ataque do radical carbonato produzido durante a atividade peroxidásica da enzima superóxido dismutase humana (hSOD1). Neste trabalho, examinamos se a ligação ditriptofano pode ser formada em outras proteínas, além da hSOD1 e por outros oxidantes, além do radical carbonato. A lisozima da clara do ovo e a beta cristalino bovina foram utilizadas como alvos de oxidação. A lisozima foi utilizada por ser uma enzima pequena (129 aminoácidos) e de estrutura bem conhecida, contendo seis resíduos de Trp. Os resultados mostraram que o radical carbonato, gerado enzimatica ou fotoliticamente, promove a oxidação, dimerização e inativação da lisozima. Os principais produtos de oxidação caracterizados por análise de nano-ESI-Q-TOF-MS/MS foram hidroxi-triptofano e N-formilquinurenina juntamente com um dímero de lisozima (lisozima-Trp28-Trp28-lisozima) e um hetero dímero lisozima-hSOD1 (lisozima-Trp28-Trp32-hSOD1), ambos ligados por uma ligação ditriptofano. Também demonstramos que a irradiação da lisozima com luz UVC leva à formação do dímero lisozima-Trp28-Trp28-lisozima. Em consequência, resolvemos tratar a beta cristalino bovina com radical carbonato gerado fotoliticamente ou com luz UVC, e a proteína também sofreu oxidação, dimerização e agregação. Os principais produtos de oxidação caracterizados por nano-ESI-Q-TOF-MS/MS foram hidroxi-triptofano, N-formilquinurenina, DOPA e um dímero de beta cristalino (βB2-Trp151-Trp151-βB2). A irradiação com luz UVC também levou à formação de um dímero intra-cadeia, caracterizado como βA2-Trp78-Trp81. Quando a beta cristalino foi irradiada com um simulador de luz solar (UVA e UVB) também foi possível observar um dímero, caracterizado como βA2-Trp150-Trp150-βA2. A presença de produtos de oxidação de resíduos de Trp, dentre eles a ligação cruzada ditritpofano, também foi avaliada in vivo, utilizando o cristalino de pacientes que foram submetidos a cirurgia para remoção de catarata. Beta, alfa e gama cristalino foram as principais proteínas identificadas nas frações solúvel e insolúvel do cristalino. A principal modificação pós-traducionais identificada foi deamidação. Um alto conteúdo de resíduos de metionina e triptofano oxidados foram identificados nas proteínas presentes na fração insolúvel. Os principais produtos de oxidação de Trp identificados por nano-ESI-Q-TOF-MS/MS foram quinurenina e N-formilquinurenina. A presença de dímeros covalentes no cristalino com catarata foi confirmada por análises de massas. A completa caracterização desses dímeros (βB1-Trp127-Trp127-βB1 e βB1-Trp193-Trp193-βB1) confirmou que as cadeias polipeptídicas foram ligadas por uma ligação ditriptofano. Em síntese, nossos dados demonstraram que o radical carbonato e a luz UV podem produzir dímeros de ditriptofano em diferentes proteínas. Também, a presença da ligação cruzada de ditriptofano in vivo (catarata humana) foi pela primeira vez detectada. / Despite extensive investigation of irreversible oxidative modifications suffered by proteins in vitro and in vivo, the products formed by oxidation of tryptophan residues remain partially characterized. Our group recently described a ditryptophan cross-link produced by recombination of hSOD1-tryptophanyl radicals generated by attack of the carbonate radical produced during the peroxidase activity of the human superoxide dismutase (hSOD1) enzyme. Here, we examine whether the ditryptophan cross-link can be produced in others proteins besides the hSOD1 and by other oxidants, in addition to the carbonate radical. The egg white lysozyme and bovine beta crystalline were used as targets. Lysozyme was used because it is a small enzyme (129 amino acids) with a well-known structure, containing six Trp residues. The results showed that the carbonate radical, generated enzymatically or photolytically, promotes lysozyme oxidation, inactivation and dimerization. The major oxidation products characterized by nano-ESI-Q-TOF-MS/MS analysis were hydroxy-tryptophan and N-formylkynurenine together with a dimer of lysozyme (lysozyme-Trp28-Trp28-lysozyme) and a hetero dimer hSOD1-lysozyme (lysozyme-Trp28-Trp32-hSOD1), both bound by a ditryptophan cross-link. Also, it was demonstrated that lysozyme irradiation with UVC light leads to the formation of the dimer lysozyme-Trp28-Trp28-lysozyme. In view of these results, we decided to treat beta crystalline bovine with photolytically generated carbonate radical and UVC. Beta crystalline also suffered oxidation, dimerization and aggregation. The major oxidation products characterized were hydroxy-tryptophan, N-formylkynurenine, DOPA and a beta crystalline dimer (βB2-Trp151-Trp151-βB2) by nano-ESI-Q-TOF-MS/MS. Irradiation with UVC light also led to the formation of an intra-chain dimer, which was characterized as βA2-Trp78-Trp81. When beta crystalline was irradiated with a solar simulator (UVA and UVB), it was also possible to observe a dimer which was characterized as βA2-Trp150-Trp150-βA2. The presence of oxidized tryptophan products, including the ditryptophan cross-link, was also evaluated in vivo in the lenses of patients submitted to cataract removal. Beta, alpha and gamma crystalline were the main proteins identified in soluble and insoluble fractions of the lenses. The main post translational modification identified was deamidation. A high content of oxidized methionine and tryptophan residues were identified in proteins present in the insoluble fraction. The main tryptophan oxidation products identified by nano-ESI-Q-TOF-MS/MS were kynurenine and N-formylkynurenine. The presence of covalent dimers in the lenses with cataract was demonstrated by mass analysis. Full MS/MS characterization of the dimers βB1-Trp127-Trp127-βB1 and βB1-Trp193-Trp193-βB1 confirmed that they were linked by a ditryptophan bond. In summary, our data demonstrate that the carbonate radical and UV light can produce ditryptophan dimers in different proteins. Also, the presence of the ditryptophan cross-link was first detected in vivo (human cataract).

ABSTRACT Effects of Milk Processing on the Milk Fat Globule Membrane Constituents Xiomara E. Elías-Argote The milk fat globule membrane (MFGM) is avidly studied by many groups of scientists around the world due to its unprecedented nutritional and functional properties; however, limited research has been performed on the effects of milk processing on the chemical changes of the MFGM. Thus, this study highlights the changes that lipids and proteins undergo from the time milk leaves cow’s udders. Cooling (4 °C) was included along with subsequent pasteurization by different traditional thermal processes and cold pasteurization by pulse light ultra violet treatment. Cooling milk to 4 °C had a measureable effect on the MFGM composition, resulting in protein alterations, particularly to butyrophilin and adipophilin. Thermal treatments disturbed the native structures of molecules and increased the adsorption of milk and whey proteins on the globules, especially a-lactalbumin and b-lactoglobulin. As the heat intensity increased, the aggregation of fat globules became more pronounced due to protein interactions. Intrinsic MFGM proteins also varied in relative abundance during the processing steps. The concentrations of polar lipids did not change during processing, with the exception of phosphatidylserine, which decreased during the cooling and thermal treatments. Cold pasteurization (UV treatment) had a minimal effect on fat globules and MFGM proteins. Since the MFGM promises to deliver nutritional effects and more when included in food products, currently HTST pasteurization was shown to be the best method to process milk and obtain MFGM isolates for further supplementation.

Apesar de extensa investigação das modificações oxidativas irreversíveis sofridas pelas proteínas in vitro e in vivo, os produtos formados pela oxidação de resíduos de triptofano ainda permanecem apenas parcialmente conhecidos. Recentemente, nosso grupo caracterizou uma ligação cruzada de ditriptofano produzida pela recombinação de radicais hSOD1-triptofanila gerados pelo ataque do radical carbonato produzido durante a atividade peroxidásica da enzima superóxido dismutase humana (hSOD1). Neste trabalho, examinamos se a ligação ditriptofano pode ser formada em outras proteínas, além da hSOD1 e por outros oxidantes, além do radical carbonato. A lisozima da clara do ovo e a beta cristalino bovina foram utilizadas como alvos de oxidação. A lisozima foi utilizada por ser uma enzima pequena (129 aminoácidos) e de estrutura bem conhecida, contendo seis resíduos de Trp. Os resultados mostraram que o radical carbonato, gerado enzimatica ou fotoliticamente, promove a oxidação, dimerização e inativação da lisozima. Os principais produtos de oxidação caracterizados por análise de nano-ESI-Q-TOF-MS/MS foram hidroxi-triptofano e N-formilquinurenina juntamente com um dímero de lisozima (lisozima-Trp28-Trp28-lisozima) e um hetero dímero lisozima-hSOD1 (lisozima-Trp28-Trp32-hSOD1), ambos ligados por uma ligação ditriptofano. Também demonstramos que a irradiação da lisozima com luz UVC leva à formação do dímero lisozima-Trp28-Trp28-lisozima. Em consequência, resolvemos tratar a beta cristalino bovina com radical carbonato gerado fotoliticamente ou com luz UVC, e a proteína também sofreu oxidação, dimerização e agregação. Os principais produtos de oxidação caracterizados por nano-ESI-Q-TOF-MS/MS foram hidroxi-triptofano, N-formilquinurenina, DOPA e um dímero de beta cristalino (βB2-Trp151-Trp151-βB2). A irradiação com luz UVC também levou à formação de um dímero intra-cadeia, caracterizado como βA2-Trp78-Trp81. Quando a beta cristalino foi irradiada com um simulador de luz solar (UVA e UVB) também foi possível observar um dímero, caracterizado como βA2-Trp150-Trp150-βA2. A presença de produtos de oxidação de resíduos de Trp, dentre eles a ligação cruzada ditritpofano, também foi avaliada in vivo, utilizando o cristalino de pacientes que foram submetidos a cirurgia para remoção de catarata. Beta, alfa e gama cristalino foram as principais proteínas identificadas nas frações solúvel e insolúvel do cristalino. A principal modificação pós-traducionais identificada foi deamidação. Um alto conteúdo de resíduos de metionina e triptofano oxidados foram identificados nas proteínas presentes na fração insolúvel. Os principais produtos de oxidação de Trp identificados por nano-ESI-Q-TOF-MS/MS foram quinurenina e N-formilquinurenina. A presença de dímeros covalentes no cristalino com catarata foi confirmada por análises de massas. A completa caracterização desses dímeros (βB1-Trp127-Trp127-βB1 e βB1-Trp193-Trp193-βB1) confirmou que as cadeias polipeptídicas foram ligadas por uma ligação ditriptofano. Em síntese, nossos dados demonstraram que o radical carbonato e a luz UV podem produzir dímeros de ditriptofano em diferentes proteínas. Também, a presença da ligação cruzada de ditriptofano in vivo (catarata humana) foi pela primeira vez detectada. / Despite extensive investigation of irreversible oxidative modifications suffered by proteins in vitro and in vivo, the products formed by oxidation of tryptophan residues remain partially characterized. Our group recently described a ditryptophan cross-link produced by recombination of hSOD1-tryptophanyl radicals generated by attack of the carbonate radical produced during the peroxidase activity of the human superoxide dismutase (hSOD1) enzyme. Here, we examine whether the ditryptophan cross-link can be produced in others proteins besides the hSOD1 and by other oxidants, in addition to the carbonate radical. The egg white lysozyme and bovine beta crystalline were used as targets. Lysozyme was used because it is a small enzyme (129 amino acids) with a well-known structure, containing six Trp residues. The results showed that the carbonate radical, generated enzymatically or photolytically, promotes lysozyme oxidation, inactivation and dimerization. The major oxidation products characterized by nano-ESI-Q-TOF-MS/MS analysis were hydroxy-tryptophan and N-formylkynurenine together with a dimer of lysozyme (lysozyme-Trp28-Trp28-lysozyme) and a hetero dimer hSOD1-lysozyme (lysozyme-Trp28-Trp32-hSOD1), both bound by a ditryptophan cross-link. Also, it was demonstrated that lysozyme irradiation with UVC light leads to the formation of the dimer lysozyme-Trp28-Trp28-lysozyme. In view of these results, we decided to treat beta crystalline bovine with photolytically generated carbonate radical and UVC. Beta crystalline also suffered oxidation, dimerization and aggregation. The major oxidation products characterized were hydroxy-tryptophan, N-formylkynurenine, DOPA and a beta crystalline dimer (βB2-Trp151-Trp151-βB2) by nano-ESI-Q-TOF-MS/MS. Irradiation with UVC light also led to the formation of an intra-chain dimer, which was characterized as βA2-Trp78-Trp81. When beta crystalline was irradiated with a solar simulator (UVA and UVB), it was also possible to observe a dimer which was characterized as βA2-Trp150-Trp150-βA2. The presence of oxidized tryptophan products, including the ditryptophan cross-link, was also evaluated in vivo in the lenses of patients submitted to cataract removal. Beta, alpha and gamma crystalline were the main proteins identified in soluble and insoluble fractions of the lenses. The main post translational modification identified was deamidation. A high content of oxidized methionine and tryptophan residues were identified in proteins present in the insoluble fraction. The main tryptophan oxidation products identified by nano-ESI-Q-TOF-MS/MS were kynurenine and N-formylkynurenine. The presence of covalent dimers in the lenses with cataract was demonstrated by mass analysis. Full MS/MS characterization of the dimers βB1-Trp127-Trp127-βB1 and βB1-Trp193-Trp193-βB1 confirmed that they were linked by a ditryptophan bond. In summary, our data demonstrate that the carbonate radical and UV light can produce ditryptophan dimers in different proteins. Also, the presence of the ditryptophan cross-link was first detected in vivo (human cataract).

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21428号 / 農博第2306号 / 学位論文||H30||N5156(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 近藤 直, 准教授 小川 雄一, 教授 飯田 訓久 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Metabolism is a subset of chemistry that allows cells to defy thermodynamic equilibrium, a fundamental process that must have been in place from the very beginning of biology. Before evolution produced efficient catalysts in the form of complex protein machinery, short metal binding peptides might have preceded modern metalloproteins. Such prebiotic, metal-binding motifs have been hypothesized to have existed through analyses of extant protein sequences. However, it is unclear how metal-binding motifs might have evolved in the harsh prebiotic environment. Here, we show how certain environments, in particular seawater-like environments rich in divalent cations and especially Mg2+, support the survival of short peptides upon extreme temperatures as high as 150 °C. Moreover, while Mg2+ does not offer the same protection from UV light, peptides are protected from both heat and irradiation when bound to a metal ion. The results suggest that specific environments rich in metal ions may be better suited for the emergence of complex systems in the path toward life. Additionally, the conditional degradation of peptides depending on their ability of binding metals might have enabled a selection mechanism that would favor the survival of metal-binding motifs which resemble the motifs found in modern proteins. These short sequences could have acted as early, simple catalysts able to facilitate a restricted set of chemical reactions, which would shape the emergence and biology of the Last Universal Common Ancestor.