Doctor of Philosophy(PhD) / Atherosclerosis manifests a state of increased oxidative stress characterized by comparable lipid and protein oxidation in the affected arterial wall. While oxidative modification of low density lipoprotein (LDL) has been extensively studied, increasing attention has been focused recently on oxidation of high-density lipoproteins (HDL) and its functional consequences in relation to atherosclerosis. Oxidative modification is thought to generate “dysfunctional” HDL that has lost anti-atherosclerotic activities, including the ability to remove cholesterol from lipid-laden cells. Therefore, there has been much interest in the detection of oxidized HDL. Unfortunately, available methods to detect oxidized HDL are limited at present, in part because oxidative modification of HDL is a complex process and ‘oxidized HDL’ is not a chemically defined entity. What is known however is that conversion of methionine (Met) residues of apolipoprotein (apo) A-I to methionine sulfoxide (MetO) is a process that occurs commonly as HDL undergoes oxidative modification. For example, human apoA-I+16 (containing MetO86 or MetO112) and apoA-I+32 (MetO86 plus MetO112) are generated when apoA-I reacts with lipid hydroperoxides formed as a consequence of the lipoprotein being exposed to 1e−oxidants. The formation of MetO in apoA−I induced by 2e−oxidants (i.e., hydrogen peroxide, hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride system) is associated with an impaired ability of the apolipoprotein to facilitate reactions relevant to reverse cholesterol transport. In addition, a previous study has suggested the plasma content of apoA-I+32 to be increased in certain subjects that have an increased risk to develop cardiovascular disease (CVD). Moreover, the MetO content in circulating, HDL−associated apoA−I is elevated in type 1 diabetes, a disorder commonly associated with increased oxidative stress and a risk factor for atherosclerosis. Therefore, in the present study, an existing HPLC method was applied to HDL samples from the Fletcher−Challenge study, a nested case control study, to test the potential usefulness of MetO-containing apoA-I as a marker of oxidative stress and/or CVD in a general population. Plasma samples whose HDL contained detectable apoA-I+16 and/or apoA-I+32 had significantly elevated levels of F2-isoprostanes, a marker of in vivo lipid oxidation, consistent with MetO-containing apoA-I being a useful marker of in vivo protein oxidation. Despite this however, there was no significant difference between controls and cases in their concentrations of HDL apoA-I+16 and apoA-I+32 or F2-isoprostanes, suggesting that markers of protein and lipid oxidation are not associated with the risk of coronary heart disease (CHD) in this general population. A limitation of the Fletcher−Challenge study was that only 22% of the 534 HDL samples analyzed contained apoA-I+16 and/or apoA-I+32. In addition, the HPLC−based method used is expensive and time−consuming and may lack the sensitivity needed for apolipoproteins to clinical studies. Thus, a mouse monoclonal anti-human apoA-I+32 antibody (MOA−1) was raised using HPLC−purified apoA-I+32 as immunogen. A sensitive ELISA was then developed using a commercial anti-human apoA-I monoclonal antibody as capture and biotinylated MOA−1 as detection antibody, respectively. The assay detected lipid−free HPLC−purified human apoA-I+32 in a concentration-dependent manner and with a significantly lower limit of detection (i.e., 3 ng/mL) than the HPLC method (1 μg/mL). The ELISA also detected lipid-free apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Moreover, the extent of recognition of MetO by MOA−1 increased with increasing numbers of MetO in apoA−I, as assessed by the experiments with H2O2−oxidized forms of apoA−I mutants, in which one, two or three Met residues were replaced with Leu. Their detection was concentration-dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radicals or metal ions, or LDL modified by 2e-oxidants. Furthermore, MOA−1 did not detect other Met−containing proteins oxidized by either hypochlorous acid or hydrogen peroxide. Taken together, the results showed that recognition of oxidized proteins by MOA−1 is limited to MetO contained in apoA−I. Finally, in a pilot study, plasma samples obtained from subjects with coronary artery disease (CAD) proven by angiography, and samples from CAD patients undergoing percutaneous coronary intervention (PCI) were analyzed by the ELISA. The preliminary data obtained showed elevated levels of MetO-containing apoA-I in plasma samples of CAD patients compared to those of corresponding control subjects. Unexpectedly, levels of MetOcontaining apoA-I decreased PCI compared to before PCI. A possible explanation for these results is that HDL−associated apoA−I become displaced by acute phase proteins, such as serum amyloid A, in response to PCI. In summary, the ELISA developed here specifically detects apoA-I containing MetO in HDL and human plasma. As such it may provide a useful tool for investigating the relationship between oxidized HDL and CAD.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-09-06T11:32:46Z No. of bitstreams: 1 arquivototal.pdf: 1111473 bytes, checksum: b2da58df331c4ba4ded210b34732682f (MD5) / Made available in DSpace on 2017-09-06T11:32:46Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1111473 bytes, checksum: b2da58df331c4ba4ded210b34732682f (MD5) Previous issue date: 2016-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / In the past decades, the intense genetic selection aiming at the increase of productive efficiency of chickens resulted in changes in the meat quality. The broiler breast meat have been classified in three group of quality, Normal, PSE (Pale, Soft, Exudative) and DFD (Dark, Firm, Dry); however, recently, due to the appearance with pale color and normal firmness, another category of meat quality was suggested as PFN (Pale, Firm, Non-exudative). Considering the absence of studies about the incidence of anomalies of color in broiler breast in the Northeast of Brazil, this work aimed at verifying the incidence of these anomalies in the region and carrying out the chemical and biochemical characterization of these meats. This samples were collected in a commercial slaughterhouse and evaluated the parameters of color (L *, a *, b *), pH, water-holding capacity, cooking loss, shear force, myofibrillar fragmentation index, protein denaturation, mineral and fatty acids content, TBARS values, warmed-over flavor and total carbonyls. The broiler breast meat (n = 838) were classified in three categories: Normal (44 <L * <53, pH> 5.8), PSE (L * ≥53, pH <5.8) and PFN (L * ≥53, pH> 5.8). The incidence of PFN broiler breast meat was 19.8%, almost doubling the incidence of PSE (11.1%); Normal broiler breast meat presented a larger percentage (69.1%). The FPN breasts presented WHC (66.9%) similar to the Normal group and 4.2% larger in the PSE meat. The PFN breasts presented calcium concentrations (373,02 mg/kg), arachidonic acid (84,61 mg/100g) and the MFI (57,4) lesser in relation to PSE meat. We also noticed levels of lipid oxidation (0.23 mg MDA / kg) similar in comparison with Normal and PSE breasts. The PFN and Normal breasts presented a larger concentration of total carbonyls, 8,2 and 7,4 nM/mg of proteins, respectively. The results related confirm the existence of PFN anomaly in broiler breast meat, which present functional properties similar to the Normal group. When added to this, the results related confirm that the PSE syndrome in broiler present a defect in the regulation of calcium causes a fall of the meat pH and consequent compromise of the functional properties. Besides, its less firm texture results from the greater proteolytic activity, which seems to be related not only to the activation of the calpains, due to the excessive calcium ions, but also to the smaller level of protein oxidation. / Nas últimas décadas, a intensa seleção genética visando o aumento da eficiência produtiva de frangos resultou em modificações na qualidade da carne. Os peitos de frango tem sido classificados em três grupos de qualidade, Normal, PSE (Pale, Soft, Exudative) e DFD (Dark, Firm, Dry); porém, recentemente, devido a aparência com cor pálida e firmeza normal, outra categoria de qualidade de carne foi sugerida a PFN (Pale, Firm, Non-exudative). Considerando-se a ausência de estudos sobre a incidência de anomalias de cor em peitos de frango no Nordeste do Brasil, objetivou-se, neste trabalho verificar a incidência dessas anomalias na região e realizar a caracterização química e bioquímicas dessas carnes. Para isso, foram coletadas amostras em um abatedouro comercial e avaliados os parâmetros de cor (L*, a*, b*), pH, capacidade de retenção de água, perda de peso por cozimento, força de cisalhamento, índice de fragmentação miofibrilar, desnaturação proteica, teor de minerais e de ácidos graxos, número de TBARS, aroma requentado e carbonilas totais. Os filés de peito de frango (n=838) foram classificados em três categorias: Normal (44<L*<53; pH>5,8), PSE (L*≥53; pH<5,8) e PFN (L*≥53; pH>5,8). A incidência de peito de frango PFN foi de 19,8%, apresentando-se quase em dobro à incidência de PSE (11,1%); peitos de frango Normal apresentaram-se em maior percentual (69,1%). Os peitos FPN apresentaram CRA (66,9%) similar ao grupo Normal e 4,2% maior à carne PSE. Os peitos PFN apresentaram concentrações de cálcio (373,02 mg/kg), ácido araquidônico (84,61 mg/100g) e o IFM (57,4) menores em relação a carne PSE. Observando-se também níveis de oxidação lipídica (0,23 mg MDA/kg), similar em comparação aos peitos Normal e PSE. Os peitos PFN e Normal apresentaram maior concentração de carbonilas totais, 8,2 e 7, 4 nM/mg de proteínas, respectivamente. Os resultados relatados confirmam a existência da anomalia PFN em peitos de frango, os quais apresentam propriedades funcionais similares ao grupo Normal. Somado a isso, os resultados relatados confirmam que a anomalia PSE em frangos apresenta defeito na regulação de cálcio que acarreta na queda do pH da carne e consequente comprometimento de suas propriedades funcionais; além disso, sua textura menos firme decorre da maior atividade proteolítica, que parece não estar apenas relacionada a ativação das calpaínas, pelo excesso de íons de cálcio, mas ao menor nível de oxidação proteica.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The advanced oxidation protein products (AOPPs) are a new class of compounds identified as markers of oxidative damage to proteins. The physiology role of these products is not limited to assess the oxidative stress, and these products may actively participate in the inflammatory process, promoting different cell disorders, including induction of apoptosis and alterations in the processes of cell proliferation and differentiation. Myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) produced by activated neutrophils contribute significantly to AOPP formation, and human serum albumin (HSA) is considered the main protein responsible for the generation of AOPPs However, the molecular composition of AOPPs is unclear. Additional pathways and protein targets for AOPP formation are largely unknown. The aim of this study was to induce the formation of AOPPs in vitro through Fenton reaction and to investigate whether this generation could be counteracted by N-acetylcysteine (NAC) and fructose-1,6-bisphosphate (FBP). In addition, this study aimed to examine whether AOPPs produced by Fenton reaction may induce the activation of the gene transcription of inflammatory molecules, including the nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) in human embryonic kidney cells (HEK 293). Additionally, it was investigated the collagen as a potential source of AOPPs via its exposure to HOCl. The HOCl-modified collagen stimulated the production of oxidants, such as HOCl and superoxide anion radical (O2 -), and pro-inflammatory agents, such as nitric oxide (NO) and AOPPs in human neutrophil. Furthermore, HOCl-modified collagen induced a decrease in cell viability and an increase of apoptosis in these cells. Finally, alpha-tocopherol inhibited the release of oxidants, decreased cell viability and apoptosis, suggesting a therapeutic potential means to prevent deleterious effects caused by AOPPs. Furthermore, these findings indicate that alternative pathways can form AOPPs, and these new products may contribute to the pathogenesis of several clinical conditions related to the accumulation of AOPPs. / Os produtos proteicos de oxidação avançada (AOPPs) são uma nova classe de compostos formados em consequência do estresse oxidativo e considerados biomarcadores úteis na detecção de dano oxidativo proteico. No entanto, o papel desses produtos não se limita apenas a refletir o grau oxidativo de proteínas, mas também podem participar de forma ativa no processo inflamatório, promovendo diferentes perturbações celulares, incluindo a indução de apoptose e alterações nos processos de proliferação e diferenciação celular. O principal mecanismo de formação dos AOPPs é através do ácido hipocloroso (HOCl) produzido pela enzima mieloperoxidase (MPO), sendo a albumina, a principal fonte proteica para a formação desses produtos. No entanto, a via MPO/HOCl parece não ser a única responsável pela formação de AOPPs, e uma série de estudos indicam que há vias alternativas que podem contribuir para formação de AOPPs. Outro fator importante, que também não está totalmente esclarecido, é se a albumina é a única proteína suscetível a formação dos AOPPs, uma vez que a composição molecular desses produtos ainda não está totalmente definida. Além dessas questões, é de interesse avaliar se os AOPPs formados por outra via mantém as características pró-infamatórias e pró-apoptóticas dos AOPPs originados pela via MPO/HOCl. Assim, o presente estudo teve como objetivo investigar se a reação de Fenton, importante geradora de radicais hidroxilas (OH ), é uma potencial via de formação de AOPPs, bem como se o colágeno, principal proteína da matriz extracelular, é uma proteína suscetível a formação desses produtos. Em um primeiro momento, foi demonstrado que a reação de Fenton induziu a formação de AOPPs e esse processo foi inibido por agentes antioxidantes, como N-acetilcisteína (NAC) e frutose-1,6-bisfosfato (FBP). Além disso, os AOPPs gerados através da reação de Fenton induziram um aumento da transcrição gênica de agentes envolvidos no processo inflamatório, incluindo o fator de transcrição κB (NF-κB), a cicloxigenase-2 (COX-2) e a interleucina-6 (IL-6), em células renais embrionárias (HEK 293) através de ensaio de transfecção celular e atividade da Luciferase. Outro importante achado deste estudo foi demonstrar que o colágeno exposto ao HOCl também é uma fonte proteica para a formação de AOPPs. Os AOPPs formados a partir do colágeno estimularam a produção de oxidantes, como o HOCl e radical ânion superóxido (O2 -), e de agentes inflamatórios, como óxido nítrico (NO) e AOPPs, em neutrófilos humanos isolados. Além disso, os AOPPs derivados do colágeno induziram uma diminuição da viabilidade celular e um aumento do processo apoptótico nessas células. Por fim, o alfa-tocoferol inibiu a liberação de oxidantes, preveniu a diminuição da viabilidade celular e o aumento do processo apoptótico, sugerindo que esse composto pode ser uma potencial ferramenta terapêutica para prevenir os efeitos deletérios promovidos pelos AOPPs. Do mesmo modo, esses achados indicam que os AOPPs podem ser formados por vias alternativas, e esses novos produtos podem contribuir na fisiopatogênese de diversas condições clínicas relacionadas com o acúmulo de AOPPs.

The seafood industry is of considerable importance to both the New Zealand and global economies and therefore tissue preservation technologies that increase product quality and/or prolong shelf life have the potential to add significant value. Technologies for maintaining the viability of isolated tissues also have a wide range of other medical and industrial applications. This thesis examines the relationship between metabolic function, oxidation and cell death and the resulting stability of the non-viable tissues during long term storage in chinook salmon (Oncorhynchus tshawytscha) red and white skeletal muscle tissue. This research also looks at the role of the aquatic anaesthetic AQUI-S™, in which the active ingredient is isoeugenol (a lipid soluble antioxidant), and other antioxidant compounds in preserving metabolic function in viable tissues and tissue stability in nonviable tissues. Perfusion of salmon tails at 15℃ over 5 or 10 hours with oxygen saturated saline resulted in significant increases in protein and lipid oxidation (protein carbonyl and TBARS concentrations respectively) in the red muscle, but not the white muscle. The introduction of ascorbic acid and uric acid into the saline did not reduce the oxidation in the red muscle despite significantly increasing their respective concentrations in the tissue. This indicates the difficulties associated with attempting to extend tissue viability by delivering free oxygen to the tissue and also highlights the difference in susceptibility of the two muscle types to oxidation. Tail fillets from salmon harvested in both rested and exhausted physiological states using AQUI-S™, and fillets from exhausted salmon harvested without AQUI-S™, were exposed to air at 15℃ for up to 96 hours. Protein carbonyls increased in a roughly linear fashion over the entire 96 hours in all three groups. Both lipid peroxides (TBARS) and uric acid concentrations began to increase in the exhausted group after 30 hours. In contrast, no significant increases in lipid peroxides or uric acid was seen in the fillets from either group harvested using AQUI-S™. Vitamin E concentrations reduced slowly but did not change significantly despite the oxidation that was evident in the tissue. These processes also occurred in salmon tail fillets during storage at 6℃. The measurement of ATP related compounds provides an effective indicator of both the metabolic state of the tissue post-harvest and the quality. The breakdown of these compounds is also associated with the production of ammonia and hydrogen peroxide. Fresh rested salmon fillets had high concentrations of ATP and creatine phosphate, which were both depleted after 12 hours storage at 15℃. This indicates that cell viability lasted a number of hours following harvesting. These metabolites were depleted in exhausted fillets and metabolic potential appeared to be immediately compromised. The concentration of the taste enhancing compound IMP was significantly reduced in fresh rested tissue, but increased during storage, and was significantly higher than in exhausted tissues following 12 hours of storage at 15℃. This indicates that some properties of rested tissues may improve with limited storage times. The accumulation of uric acid - the metabolic end point for ATP related compounds - was also significantly reduced in rested tissue and increases in K-value were slowed. AQUI-S™ showed an ability to preserve tissue function through its anaesthetic action allowing tissue to be harvested in a rested state, and to reduce late stage lipid oxidation in stored salmon tail fillets. The antioxidant action of isoeugenol in salmon fillets may be mediated through its ability to chelate transition metals released during tissue degradation. This research shows that during reperfusion and during fillet storage there is a significant level of oxidative stress, which needs to be minimized while maintaining basic tissue metabolism to prolong tissue and cellular viability. The development of future technologies to preserve tissue viability may depend on the development of a synthetic oxygen carrying compound with properties similar to red blood cells. This may allow more control over oxygen delivery, potentially reducing the oxidative stress associated with high concentrations of free oxygen in solution. However, preserving cell viability will also require the maintenance of endogenous antioxidant function and there is also the potential to use iron chelating compounds including plant derived flavonoids to preserve non-viable tissues. Future research in these areas is necessary.

O oxigênio molecular singlete (1O2) é formado em sistemas biológicos e reage com diferentes biomoléculas. Proteínas representam um dos principais alvos de oxidação, devido as suas altas concentrações em organismos. Em pH fisiológico 1O2 reage com His, Tyr, Met, Cys e Trp. Neste trabalho investigamos a oxidação causada pelo 1O2 e a formação de dimerização em uma proteína modelo, a lisozima. A identificação dos principais produtos de oxidação e dimerização foi realizada por sequenciamento de peptídeos através de nano cromatografia acoplada a espectrometria de massas (nLC-MS/MS). A geração de 1O2 foi realizada por fotossensibilização utilizando luz e rosa bengala como fotossensibilizador, e pela decomposição térmica de endoperóxidos derivado do naftaleno DHPN16O2 e DHPN18O2, uma fonte limpa de 1O2 no meio reacional. Os resultados demonstraram que a reação do oxigênio singlete com lisozima acarreta oxidação dos resíduos de Met, His e Trp. A caracterização da estrutura primária por nLC-MS/MS dos aminoácidos confirmou a adição de átomos de oxigênio marcado (18O). A lisozima é constituída apenas de um resíduo de histidina (His15) e as oxidações identificadas foram adições de massas de +14 Da (descrita como 2-oxo-histidina), +16 e +32 Da. Os resíduos de metionina (Met12 e Met105) foram identificados como sulfóxidos (MetSO - adição de massas de +16 Da). Para os resíduos de triptofano foram identificados a formação de quinurenina (adição de massas de +4 Da), +16 e +32 Da. As oxidações levaram a formação de dimerização na proteína caracterizada por eletroforese em gel e nLC-MS/MS. O objetivo principal do trabalho foi analisar a ligação cruzada entre o resíduo de histidina 2-oxohistidina na lisozima. Entretanto, foram identificadas ligações cruzadas entre 2- oxo-histidina e resíduos de lisina, além de ligações cruzadas com resíduos de triptofano oxidado. Em consequência dos resultados obtidos com a proteína modelo, avaliamos as oxidações e formação de dímeros em proteínas extraídas do cristalino do olho bovino. Diferentes tipos de modificações foram observados, além da formação de dímeros entre resíduos de histidina (2-oxoHis-His) caracterizados por nLC-MS/MS e bioinformática. Os dados obtidos neste trabalho, fornecem evidências da ocorrência simultânea de formação de ligações cruzadas entre diferentes proteicas após exposição a 1O2. O trabalho resultou na identificação e sequenciamento através de nLC-MS/MS de peptídeos oxidados por 1O2 a partir de uma proteína modelo. Esses resultados reiteram o importante papel do 1O2 em reações com proteínas além do seu envolvimento no desenvolvimento de condições patológicas. A dimerização formada na ligação cruzada em 2-oxo-His-His representa um possível novo biomarcador para o 1O2 em sistemas biológicos / Singlet molecular oxygen (1O2) can be generated in biological systems, reacting with different biomolecules. Proteins are major target for oxidants due to higher concentration in organisms. At physiological pH, 1O2 may react with the following aminoacids: His, Tyr, Met, Cys and Trp. Here, we investigated oxidation and dimerization reactions of proteins exposed to 1O2 using lysozyme as a model. Modifications of lysozyme by 1O2 were investigated using mass spectrometry approaches. Identification of the main oxidation and dimerization products were performed by peptide sequencing by nano-chromatography coupled to mass spectrometry (nLC-MS/MS). Singlet oxygen was generated using visible light and rose Bengal as photosensitizer, and from the decomposition of thermolabile endoperoxides DHPN16O2 e DHPN18O2, clean sources of 1O2. Experimental findings showed oxidation of Met, His, and Trp residuesin lysozyme. Structural characterization by nLC-MS/MS of the oxidative modifications in lysozyme tryptic peptides showed the addition of [18O]-labeled atoms in different amino acid residues. Lysozyme has in its structure a single histidine residue (His15). We identified shifts of +14 Da (described as oxohistidine), +16 and +32 Da in this residue. Methionine residues (Met12 and Met105) were oxidized to sulfoxides (MetSO mass shift of +16 Da). Modifications in tryptophan residues were identified as kynurenine (shift mass of +4 Da), +16 and + 32 Da. Oxidized lysozyme subjected to SDS-Page showed dimmers formation. The main aim was to analyze cross-linking formation between 2-oxo-histidine residues in lysozyme. However, cross-links between 2- oxo-histidine and lysine residues, and cross-links between oxidized tryptophan residues have been identified. Following results obtained with the protein model, we evaluated oxidation and the dimers formation in proteins extracted from the lens of the bovine eye. Analysis performed in nLC-MS/MS and bioinformatics identified different types of modifications, including formation of dimers with histidine residues (2-oxo-His-His). The data provided evidence for simultaneous occurrence of protein cross-linking on exposure 1O2. These results demonstrated the important role of 1O2 in protein reactions beyond its involvement in developing of pathological conditions. In conclusion, dimerization of proteins through 2-oxo-His residues may be a possible new biomarker for 1O2 in biological systems.

O oxigênio molecular singlete (1O2) é formado em sistemas biológicos e reage com diferentes biomoléculas. Proteínas representam um dos principais alvos de oxidação, devido as suas altas concentrações em organismos. Em pH fisiológico 1O2 reage com His, Tyr, Met, Cys e Trp. Neste trabalho investigamos a oxidação causada pelo 1O2 e a formação de dimerização em uma proteína modelo, a lisozima. A identificação dos principais produtos de oxidação e dimerização foi realizada por sequenciamento de peptídeos através de nano cromatografia acoplada a espectrometria de massas (nLC-MS/MS). A geração de 1O2 foi realizada por fotossensibilização utilizando luz e rosa bengala como fotossensibilizador, e pela decomposição térmica de endoperóxidos derivado do naftaleno DHPN16O2 e DHPN18O2, uma fonte limpa de 1O2 no meio reacional. Os resultados demonstraram que a reação do oxigênio singlete com lisozima acarreta oxidação dos resíduos de Met, His e Trp. A caracterização da estrutura primária por nLC-MS/MS dos aminoácidos confirmou a adição de átomos de oxigênio marcado (18O). A lisozima é constituída apenas de um resíduo de histidina (His15) e as oxidações identificadas foram adições de massas de +14 Da (descrita como 2-oxo-histidina), +16 e +32 Da. Os resíduos de metionina (Met12 e Met105) foram identificados como sulfóxidos (MetSO - adição de massas de +16 Da). Para os resíduos de triptofano foram identificados a formação de quinurenina (adição de massas de +4 Da), +16 e +32 Da. As oxidações levaram a formação de dimerização na proteína caracterizada por eletroforese em gel e nLC-MS/MS. O objetivo principal do trabalho foi analisar a ligação cruzada entre o resíduo de histidina 2-oxohistidina na lisozima. Entretanto, foram identificadas ligações cruzadas entre 2- oxo-histidina e resíduos de lisina, além de ligações cruzadas com resíduos de triptofano oxidado. Em consequência dos resultados obtidos com a proteína modelo, avaliamos as oxidações e formação de dímeros em proteínas extraídas do cristalino do olho bovino. Diferentes tipos de modificações foram observados, além da formação de dímeros entre resíduos de histidina (2-oxoHis-His) caracterizados por nLC-MS/MS e bioinformática. Os dados obtidos neste trabalho, fornecem evidências da ocorrência simultânea de formação de ligações cruzadas entre diferentes proteicas após exposição a 1O2. O trabalho resultou na identificação e sequenciamento através de nLC-MS/MS de peptídeos oxidados por 1O2 a partir de uma proteína modelo. Esses resultados reiteram o importante papel do 1O2 em reações com proteínas além do seu envolvimento no desenvolvimento de condições patológicas. A dimerização formada na ligação cruzada em 2-oxo-His-His representa um possível novo biomarcador para o 1O2 em sistemas biológicos / Singlet molecular oxygen (1O2) can be generated in biological systems, reacting with different biomolecules. Proteins are major target for oxidants due to higher concentration in organisms. At physiological pH, 1O2 may react with the following aminoacids: His, Tyr, Met, Cys and Trp. Here, we investigated oxidation and dimerization reactions of proteins exposed to 1O2 using lysozyme as a model. Modifications of lysozyme by 1O2 were investigated using mass spectrometry approaches. Identification of the main oxidation and dimerization products were performed by peptide sequencing by nano-chromatography coupled to mass spectrometry (nLC-MS/MS). Singlet oxygen was generated using visible light and rose Bengal as photosensitizer, and from the decomposition of thermolabile endoperoxides DHPN16O2 e DHPN18O2, clean sources of 1O2. Experimental findings showed oxidation of Met, His, and Trp residuesin lysozyme. Structural characterization by nLC-MS/MS of the oxidative modifications in lysozyme tryptic peptides showed the addition of [18O]-labeled atoms in different amino acid residues. Lysozyme has in its structure a single histidine residue (His15). We identified shifts of +14 Da (described as oxohistidine), +16 and +32 Da in this residue. Methionine residues (Met12 and Met105) were oxidized to sulfoxides (MetSO mass shift of +16 Da). Modifications in tryptophan residues were identified as kynurenine (shift mass of +4 Da), +16 and + 32 Da. Oxidized lysozyme subjected to SDS-Page showed dimmers formation. The main aim was to analyze cross-linking formation between 2-oxo-histidine residues in lysozyme. However, cross-links between 2- oxo-histidine and lysine residues, and cross-links between oxidized tryptophan residues have been identified. Following results obtained with the protein model, we evaluated oxidation and the dimers formation in proteins extracted from the lens of the bovine eye. Analysis performed in nLC-MS/MS and bioinformatics identified different types of modifications, including formation of dimers with histidine residues (2-oxo-His-His). The data provided evidence for simultaneous occurrence of protein cross-linking on exposure 1O2. These results demonstrated the important role of 1O2 in protein reactions beyond its involvement in developing of pathological conditions. In conclusion, dimerization of proteins through 2-oxo-His residues may be a possible new biomarker for 1O2 in biological systems.

The seafood industry is of considerable importance to both the New Zealand and global economies and therefore tissue preservation technologies that increase product quality and/or prolong shelf life have the potential to add significant value. Technologies for maintaining the viability of isolated tissues also have a wide range of other medical and industrial applications. This thesis examines the relationship between metabolic function, oxidation and cell death and the resulting stability of the non-viable tissues during long term storage in chinook salmon (Oncorhynchus tshawytscha) red and white skeletal muscle tissue. This research also looks at the role of the aquatic anaesthetic AQUI-S™, in which the active ingredient is isoeugenol (a lipid soluble antioxidant), and other antioxidant compounds in preserving metabolic function in viable tissues and tissue stability in nonviable tissues. Perfusion of salmon tails at 15℃ over 5 or 10 hours with oxygen saturated saline resulted in significant increases in protein and lipid oxidation (protein carbonyl and TBARS concentrations respectively) in the red muscle, but not the white muscle. The introduction of ascorbic acid and uric acid into the saline did not reduce the oxidation in the red muscle despite significantly increasing their respective concentrations in the tissue. This indicates the difficulties associated with attempting to extend tissue viability by delivering free oxygen to the tissue and also highlights the difference in susceptibility of the two muscle types to oxidation. Tail fillets from salmon harvested in both rested and exhausted physiological states using AQUI-S™, and fillets from exhausted salmon harvested without AQUI-S™, were exposed to air at 15℃ for up to 96 hours. Protein carbonyls increased in a roughly linear fashion over the entire 96 hours in all three groups. Both lipid peroxides (TBARS) and uric acid concentrations began to increase in the exhausted group after 30 hours. In contrast, no significant increases in lipid peroxides or uric acid was seen in the fillets from either group harvested using AQUI-S™. Vitamin E concentrations reduced slowly but did not change significantly despite the oxidation that was evident in the tissue. These processes also occurred in salmon tail fillets during storage at 6℃. The measurement of ATP related compounds provides an effective indicator of both the metabolic state of the tissue post-harvest and the quality. The breakdown of these compounds is also associated with the production of ammonia and hydrogen peroxide. Fresh rested salmon fillets had high concentrations of ATP and creatine phosphate, which were both depleted after 12 hours storage at 15℃. This indicates that cell viability lasted a number of hours following harvesting. These metabolites were depleted in exhausted fillets and metabolic potential appeared to be immediately compromised. The concentration of the taste enhancing compound IMP was significantly reduced in fresh rested tissue, but increased during storage, and was significantly higher than in exhausted tissues following 12 hours of storage at 15℃. This indicates that some properties of rested tissues may improve with limited storage times. The accumulation of uric acid - the metabolic end point for ATP related compounds - was also significantly reduced in rested tissue and increases in K-value were slowed. AQUI-S™ showed an ability to preserve tissue function through its anaesthetic action allowing tissue to be harvested in a rested state, and to reduce late stage lipid oxidation in stored salmon tail fillets. The antioxidant action of isoeugenol in salmon fillets may be mediated through its ability to chelate transition metals released during tissue degradation. This research shows that during reperfusion and during fillet storage there is a significant level of oxidative stress, which needs to be minimized while maintaining basic tissue metabolism to prolong tissue and cellular viability. The development of future technologies to preserve tissue viability may depend on the development of a synthetic oxygen carrying compound with properties similar to red blood cells. This may allow more control over oxygen delivery, potentially reducing the oxidative stress associated with high concentrations of free oxygen in solution. However, preserving cell viability will also require the maintenance of endogenous antioxidant function and there is also the potential to use iron chelating compounds including plant derived flavonoids to preserve non-viable tissues. Future research in these areas is necessary.

This diploma thesis was focused on the possibilities of extending the shelf-life of fish products by dipping containing seven commercial additives. ANTIBAK, MIC STAB, Bakont, SEA-F75, Misocarine LR, SAFE A Plus and AMX liquid. The effectiveness of these substances on the extending of shelf-life was evaluated on the basis of tests of TVC (total viable count), level of fat and protein oxidation, determination of nutritional parameters of muscle, and finaly sensory analysis. Experimental species were 2 important commodities for the Czech aquaculture rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio). The project was divided into 6 sub-stages. During testing was the initial number of aditives (7) limited by stepwise selection for 2 preparations. Namely Bakont and AMX liquid, which were tested further on trout (filet with skin and scales) and carp (filets with skin without scales) chilled bulk and chilled packaged under vakuum. For trout was studied antimicrobial effect of dipping on eviscerated fish with the head with- or without gills. Treated trout fillets in bulk and vakuum-packed showed significantly less abundance of muscle mikroflora. In the case of carp fillets chilled bulk we can not say that the bath had influence on the CMP in meat. However, the combination of dipping and vakuum packaging was singnificantly different between the control and product Bakont. AMX liquid was not applied in this case in sufficient dose or in sufficient time to carp muscle. A positive finding is that the application of the aditives to the product "eviscerated trout with head" it does not matter, whether the gills are left in fish or not. However, in all cases the analysis of the presence of pathogens Escherichia coli, Salmonella spp. or Listeria monocytogenes were negative. From the results of sensory analysis can be concluded, that the substances contained in aditives are not reflected in the sensory properties of tested fish.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / This study aimed to evaluate the effect of the loquat seed extract on oxidative stability of catfish fillets, pate and meatballs, during storage. We evaluated the antioxidant and antimicrobial in vitro activity of loquat seed extracts obtained by different extraction using different solvents and ultrasound treatment. This was followed by evaluating the applicability of loquat seed extracts on oxidative stability of frozen catfish fillets (extracts obtained with 70% acetone and rotaevaporated; AE); on catfish based-pates refrigerated stored (hydroethanolic extract; EE) and on catfish based-meatballs, pre-cooked and frozen stored. Acetone extracts showed the highest total phenolics and total tannins content. Ultrasound treatment improved total phenolic content in 70% acetone extraction and FRAP activity in 35% and 70% acetone extracts. No antimicrobial activity was observed in extracts. The 70% acetone extract was indicated as the one with more significant antioxidant properties. In fish fillets treated with AE, ascorbic acid containing formulation had higher conjugated dienes (CD) than the control and AE-treated pates at 6 months of storage, and presented higher thiobarbituric acid reactive substances (TBARS) than the 400 ppm treated fish fillet at 9 months of storage. However, CD and TBARS had similar values among treatments at 12 months. The protein carbonyl (PC) content increased until 12 months compared to 0 months, however, had no treatment effect. There was a decrease of a* values in all periods and an increase of b* values at 9 and 12 months. The evaluated concentrations of AE were not able to slow lipid and protein oxidation in fish fillets frozen stored, however, did not alter chemical composition or acceptability of fish fillets at 0 months of storage. In catfish based-pates treated with EE, the CD and peroxide values (PV) increased during storage, however, were similar in all treatments after 35 days. TBARS content was not affected by EE. There was a linear increase in PC content in pates over storage. At evaluated concentrations, EE was not able to inhibit or reduce the lipid and protein oxidation in fish pates refrigerated stored. In catfish based-meatballs prepared with AE-treated fish fillets, PV and TBARS decreased over storage time due to high values at initial times assigned to cooking and mincing. Ascorbic acid and 800 ppm containing formulations had higher PV than control formulation, decrease of a* values and increase of b* values over frozen storage. PC content increased over storage time, paralleled to the hardness, with no treatment effect. In the tested concentrations, AE was not able to inhibit the lipid and protein oxidation or to prevent the color change of pre-cooked fish meatballs, and ascorbic acid and 800 ppm formulations showed pro-oxidant effects. / Este trabalho teve como objetivo avaliar o efeito de extratos de semente de nêspera sobre a estabilidade oxidativa em filés, patê e almôndega à base de jundiá, ao longo do armazenamento. Foi avaliada a capacidade antioxidante e atividade antimicrobiana in vitro de extratos de semente de nêspera obtidos através de diferentes extrações, utilizando diferentes solventes e ultrassom. Seguiu-se com a avaliação da aplicabilidade dos extratos de semente de nêspera na estabilidade oxidativa de filés de jundiá congelados (extrato obtido com acetona 70% e rotaevaporado; EA); em patês refrigerados à base de pescado refrigerado (extrato hidretanólico; EE) e de almôndegas à base de pescado, pré-cozidas e armazenadas congeladas (EA). Os extratos acetônicos apresentaram o maior conteúdo de compostos fenólicos totais e também de taninos totais. O tratamento com ultrassom melhorou a extração de compostos fenólicos totais na extração com acetona 70% e a atividade FRAP nos extratos de acetona 35% e 70%. Nenhuma atividade antimicrobiana foi observada nos extratos. O extrato de acetona 70% foi apontado como o extrato com propriedades antioxidantes mais expressivas. Nos filés tratados com o EA, a formulação contendo ácido ascórbico apresentou maiores valores de dienos conjugados (DC) que o controle e que os filés tratados com EA aos 6 meses de armazenamento, além de apresentar valores maiores de substâncias reativas ao ácido tiobarbitúrico (TBARS) que a formulação tratada com o extrato 400 ppm aos 9 meses de armazenamento. Contudo, os valores de DC e TBARS foram semelhantes entre os tratamentos aos 12 meses de armazenamento. O teor de proteínas carboniladas (PC) aumentou até os 12 meses, contudo, não teve influência dos tratamentos. Houve diminuição dos valores de a* em todos os períodos avaliados e aumento de b* nos tempos 9 e 12 meses. Nas concentrações avaliadas o EA não foi capaz de retardar a oxidação lipídica e proteica em filés armazenados congelados, contudo, não alterou a composição centesimal ou aceitabilidade dos filés avaliadas no tempo 0 meses. Nos patês tratados com o EE, os teores de DC e peróxidos (PV) aumentaram ao longo do armazenamento, contudo, foram similares entre todos os tratamentos aos 35 dias. O conteúdo TBARS não foi afetado pelo EE e houve aumento linear no conteúdo de PC nos patês ao longo do armazenamento. Nas concentrações avaliadas, o EE não foi capaz de inibir ou reduzir as oxidações lipídicas e proteicas em patês à base de pescado armazenados refrigerados. Nas almôndegas desenvolvidas com filés previamente tratados com o EA, PV e TBARS diminuíram ao longo do tempo de armazenamento devido aos altos valores nos tempos iniciais atribuídos à cocção e manipulação. As formulações contendo ácido ascórbico e 800 ppm apresentaram maiores valores de PV que a formulação controle, decréscimo da tendência ao vermelho e aumento da tendência ao amarelo ao longo do armazenamento congelado. O conteúdo de proteínas carboniladas aumentou ao longo do tempo de armazenamento, similar ao ocorrido com a dureza das almôndegas cozidas, sem efeito dos tratamentos. Nas concentrações avaliadas, o EA não foi capaz de inibir a oxidação de lípidos e proteínas ou de prevenir a alteração de cor das almôndegas à base de pescado, sendo que as formulações contendo ácido ascórbico e 800 ppm apresentaram efeitos pró-oxidantes.

Widespread evidence indicates that exposure of cell cultures to α particles results in significant biological changes in both the irradiated and non-irradiated bystander cells in the population. The induction of non-targeted biological responses in cell cultures exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation and to radiotherapy. Here, we investigated the mechanisms underlying the induction of stressful effects in confluent normal human fibroblast cultures exposed to low fluences of 1000 MeV/u iron ions (linear energy transfer (LET) ~151 keV/µm), 600 MeV/u silicon ions (LET ~50 keV/µm) or 290 MeV/u carbon ions (LET ~13 keV/µm). We compared the results with those obtained in cell cultures exposed, in parallel, to low fluences of 0.92 MeV/u α particles (LET ~109 keV/µm).Induction of DNA damage, changes in gene expression, protein carbonylation and lipid peroxidation during 24 h after exposure of confluent cultures to mean doses as low as 0.2 cGy of iron or silicon ions strongly supported the propagation of stressful effects from irradiated to bystander cells. At a mean dose of 0.2 cGy, only ~1 and 3 % of the cells would be targeted through the nucleus by an iron or silicon ion, respectively. Within 24 h post-irradiation, immunoblot analyses revealed significant increases in the levels of phospho-TP53 (serine 15), p21Waf1 (also known as CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation. The magnitude of the responses suggested participation of non-targeted cells in the response. Furthermore, when the irradiated cell populations were subcultured in fresh medium shortly after irradiation, greater than expected increases in the levels of these markers were also observed during 24 h. Together, the results imply a rapidly propagated and persistent bystander effect. In situ analyses in confluent cultures showed 53BP1 foci formation, a marker of DNA damage, in more cells than expected based on the fraction of cells traversed through the nucleus by an iron or silicon ion. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure to a mean absorbed dose of 0.2 cGy of 3.7 MeV α particles, but not after 0.2 cGy of 290 MeV/u carbon ions.Analyses in dishes that incorporate a CR-39 solid state nuclear track detector bottom identified the cells irradiated with iron or silicon ions and further supported the participation of bystander cells in the stress response. Mechanistic studies indicated that gap junction intercellular communication, DNA repair, and oxidative metabolism participate in the propagation of the induced effects.We also considered the possible contribution of secondary particles produced along the primary particle tracks to the biological responses. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cells cultures exposed to HZE particles comprise <1 % of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10-20 µm Thus, the latter are unlikely to significantly contribute to the stressful effects in cells not targeted by primary HZE particles.