Lipid peroxidation is well established to contribute to the etiology of many deteriorative conditions including neurodegeneration, cardiovascular disease, cancer, aging, and recently in ferroptosis—a regulated, necrotic modality of cell death that results from the accumulation of lipid hydroperoxides. Recent high-throughput screening efforts have uncovered ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as two premiere inhibitors of ferroptosis. We propose that these compounds function as radical trapping antioxidants. We employ a systematic methodology of evaluating inherent radical trapping antioxidant (RTA) activity of Lip-1, Fer-1, and various aryl amine and aryl nitroxide analogues to put forward a biologically relevant mechanism of action based on our previous work in the field. Joining these observations with the efficacy of tetrahydronaphthyridinols (THNs), the results support a clear role of autoxidation in the execution of ferroptosis. Next, we expand the THN repertoire by targeting the payload towards the engine of our cells, the mitochondria. Decades of research have implicated mitochondrial dysfunction brought about by the peroxidation of mitochondrial membranes and the leaking of downstream oxidants, in the death of their symbiotic host cells. Isolated successes in the field have been demonstrated academically, though viable drugs remain to be developed, partially due to the lack of effective diagnostic tools. We endeavor to address some of these issues by investigating mitochondrially-targeted THNs (MitoTHNs) as a targeted chain-breaking antioxidant of unparalleled potency. Furthermore, we advance development of the THNs towards therapeutic applications by demonstrating their biodistribution in mice.

Chapter 1 Hydrocarbon autoxidation, a free radical chain reaction, is believed to play a key role in the onset and developments of most degenerative diseases and disorders. The two propagating steps: 1) H-atom abstraction from the hydrocarbon by a hydrocarbon-derived peroxyl radical, and 2) addition of oxygen to the resultant alkyl radical to form a new peroxyl, play a role in determining the rate of hydrocarbon autoxidation, as well as the regio- and stereochemistry of the product hydroperoxides. In the current study, we carried out a set of calculations to provide a detailed framework for understanding the mechanism of the first two steps of autoxidation. Chapter 2 Radical-trapping chain-breaking antioxidants inhibit hydrocarbon autoxidation. Phenols are the prototypical radical-trapping antioxidants and are employed in nature, as well as in industry, to inhibit the autoxidation of hydrocarbons. The mechanism of inhibiting radical chain propagation has recently been suggested to be a PCET on the basis of theoretical calculations. It has been demonstrated that the antioxidant activitiy of phenols is increased in the presence of either protic acids or alcohols, but the basis of this acceleration is not well understood. In the current study, we used computational methods to investigate the effects of acids and alcohols on the PCET pathway for the reaction of phenol with a peroxyl radical. Chapter 3 The antibiotic pyrrolnitrin [3-chloro-4-(2’-nitro-3’-chlorophenyl) pyrrole] (PRN) is biosynthesized from L-tryptophan in four steps, catalyzed by the enzymes PrnA, B, C and D encoded by the prn operon. Two of the four gene products, PrnA and PrnC, are flavin-dependent halogenases, a recently discovered and highly interesting class of enzymatic halogenation catalysts. Their activities have never been unequivocally demonstrated by reconstitution of the activity from a recombinant protein. Herein, we report the results of our efforts to clone the genes encoding PrnA and PrnC, and overexpress, isolate and purify the proteins from E. coli. We were able to successfully reconsistute halogenation activity of both and have obtained the first kinetic data for PrnC, which shows kinetics similar to other flavin-dependent halogenases, along with substrate inhibition. / Thesis (Master, Chemistry) -- Queen's University, 2010-02-03 15:42:39.67

Orientador: Admar Costa de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-09T10:09:33Z (GMT). No. of bitstreams: 1 Santos_LiliaZagoFerreirados_D.pdf: 3274551 bytes, checksum: 903add15d9aa93e05454196d09939604 (MD5) Previous issue date: 2007 / Resumo: Ácido linoléico conjugado (CLA) é um conjunto de isômeros geométricos e posicionais do ácido linoléico, encontrado no leite, carnes e seus respectivos derivados. Desde a sua identificação como um agente anticarcinogênico, muitos outros efeitos lhe foram atribuídos; dentre eles, o efeito antioxidante, efeito um tanto quanto intrigante ao considerar que o CLA é um dieno conjugado, ou seja, um ácido graxo em sua fase inicial de autoxidação, mas com uma de suas ligas duplas conjugadas na forma trans. O objetivo desta Tese foi avaliar o efeito da suplementação com CLA, com e sem a presença de antioxidante, sobre o processo de autoxidação dos lípides biológicos, o conteúdo de lípides totais e a morfologia hepática de ratos Wistar saudáveis em crescimento. Foi realizado um ensaio biológico onde sessenta ratos foram divididos em seis grupos (n=10): grupos C (controle), CE (controle + vitamina E), AE (AdvantEdge® CLA), AEE (AdvantEdge® CLA + Vitamina E), CO (CLA One®) e COE (CLA One® + vitamina E). Os grupos controle C e CE receberam ácido linoléico e os grupos suplementados AE, AEE, CO e COE receberam misturas comerciais de CLA distintas cujo conteúdo médio de CLA era de 76,17 % com proporções semelhantes entre os isômeros trans-10, cis-12 e cis-9, trans-11. As condições de suplementação foram padronizadas em ensaio preliminar cujos resultados permitiram concluir que a melhor concentração de CLA a ser administrada, levando em consideração os aspectos operacionais e a resposta biológica, era de 2 % em relação ao consumo de dieta. Sendo assim, os ratos foram suplementados com 2 % de CLA por meio de entubação orogástrica durante 42 dias. Para os animais que receberam vitamina E em associação com o CLA, utilizou-se acetato de a-tocoferol na concentração de 30 mg / dia. Foram determinados índice de peróxido (IP), malondialdeído (MDA), 8-iso-PGF2a isoprostana e atividade da catalase como indicadores da autoxidação lipídica. O conteúdo total de lípides do fígado foi determinado e a morfologia do órgão foi analisada por meio de microscopia eletrônica de transmissão (MET). Os resultados demonstraram que a influência do CLA sobre o processo de oxidação dos lípides biológicos depende do tipo do suplemento e do indicador utilizado e seu respectivo local de determinação (tecido ou fluído corporal). Os valores de MDA sérico (AE: 1,80±0,67 mg MDA / kg; CO: 2,43±0,61 mg MDA / kg) e a atividade sérica da catalase (AE: 4734,23±1078,93 kU / L; CO: 5916,06±2490,71 kU / L) foram significativamente menores (P £ 0,05) em comparação com o controle (MDA: 3,85±0,24 mg MDA / kg; catalase: 10496,52±5121,84 kU / L), já os valores de 8-iso-PGF2a isoprostana foram maiores (P £ 0,05) para o grupo AE (urina: 95,13±20,26 pg / mL; plasma: 18,86±3,41 pg / mL) em relação ao controle (urina: 69,46±16,65 pg / mL; plasma: 13,84±3,55 pg / mL). Quanto ao IP, este foi maior (P £ 0,05) no grupo CO (84,38±10,97mEq / kg) em comparação com o grupo controle (54,75±9,70 mEq / kg). Em ralação à associação do acetato de a-tocoferol à suplementação com CLA, esta influenciou a ação do CLA sobre a oxidação dos lípides biológicos para os indicadores catalase, MDA hepático e 8-iso-PGF2a isoprostana urinária, de forma a diminuí-la. O conteúdo dos lípides hepáticos totais não aumentou nos ratos que receberam CLA (C: 23,93±3,64 %; AE: 21,19±2,05 %; CO: 21,16±0,90 %), embora as imagens obtidas por MET tenham demonstrado que houve aumento dos glóbulos de gordura em número e tamanho, aumento esse que não alterou a morfologia do órgão, visto que tanto o citoplasma quanto as organelas celulares estavam íntegras. Esses achados permitiram concluir que a suplementação com 2 % das misturas comerciais de CLA durante 42 dias influenciou o processo de oxidação dos lípides biológicos, no entanto, não foi possível estabelecer um consenso sobre seu efeito antioxidante/pró-oxidante visto a divergência dos resultados. Quanto às imagens hepáticas obtidas por MET, essas caracterizaram uma informação de natureza qualitativa, mas não menos importante, pois permitiu concluir que esse protocolo de suplementação com CLA não promeveu danos morfológicos ao órgão visto a integridade dos hepatócitos / Abstract: Conjugated linoleic acid (CLA) is a set of geometrical and positional isomers of the linoleic acid, found in milk, meats and their products. Since its identification as an anticarcinogenic agent, other effects have been attributed to it since, among them an antioxidant action -- a rather intriguing claim in view of the fact that CLA is a conjugated diene, i.e., a fatty acid in an early stage of autoxidation, but with one of its conjugated double bonds in the trans form. The objective of this Thesis was to assess the effect of CLA supplementation, both in the presence and in the absence of an antioxidant, on: (a) the process of biological lipid autoxidation, (b) the total lipid content, and (c) the hepatic morphology of healthy growing Wistar rats. A biological assay on sixty rats divided into six groups (n=10) was realized: C (control), CE (control + vitamin E), AE (AdvantEdge® CLA), AEE (AdvantEdge® CLA + Vitamin E), CO (CLA One®), and COE (CLA One®+ vitamin E). Control groups C and CE received linoleic acid, and supplemented groups AE, AEE, CO and COE received commercial CLA mixtures with a mean CLA content 76.17% and similar proportions of trans-10, cis-12 and cis-9, trans-11 isomers. A preliminary assay was conducted in order to standardize supplementation conditions; its results indicated that the best CLA concentration for administration, taking into account operational aspects and biological response, was 2% of diet intake. Rats were supplemented with that concentration of CLA by orogastric intubation for 42 days. For animals receiving vitamin E in combination with CLA, alpha-tocopherol acetate in the concentration of 30 mg/day was used. Peroxide index (IP), malondialdehyde (MDA), 8-iso-PGF2 alpha isoprostane, and catalase activity were determined as lipid autoxidation indicators. Total liver lipid content was determined, and organ morphology was examined by transmission electronic microscopy (TEM). Results demonstrated that the influence of CLA on the process of biological lipid oxidation depends on supplement type, on the chosen indicator, and on whether it is determined in a tissue or in a body fluid. Serum values of MDA (AE: 1.80±0.67 mg MDA / kg; CO: 2.43±0.61 mg MDA / kg) and catalase serum activity (AE: 4734.23±107893 kU / L; CO: 5916.06±2490.71 kU / L) were significantly lower (P <0,05) than in the controls (MDA: 3.85±0.24 mg MDA / kg; catalase: 10496.52±5121.84 kU / L), whereas 8-iso-PGF2 alpha isoprostane were higher (P <0.05) for AE (urina: 95.13±20.26 pg / mL; plasma: 18.86±3.41 pg / mL) than in the controls (urina: 69.46±16.65 pg / mL; plasma: 13.84±3.55 pg / mL). IP values were higher (P<0,05) for CO (84,.38±1097mEq / kg) than in the control (54.75±9.70 mEq / kg). Regarding the combination of alpha-tocopherol acetate and CLA supplementation, the presence of this antioxidant influenced the action of CLA on biological lipid oxidation as indicated by catalase, hepatic MDA and urinary 8-iso-PGF2 alpha isoprostane. Total hepatic lipid content did not increase in rats receiving CLA (C: 23.93±3,.64 %; AE: 21.19±2.05 %; CO: 21.16±0.90 %), although TEM images showed an increase in size and amount of fat globules; this, however, did not change organ morphology, as indicated by the fact that cell cytoplasm and organelles were undamaged. These findings allowed to conclude that supplementation with 2% of commercial CLA mixtures for 42 days influenced the process of biological lipid oxidation. However, no conclusion could be reached as to its anti-oxidant or prooxidant effect, because of inconsistencies in the results. Hepatic TEM images, even being a qualitative information, were nevertheless important, as they allowed to conclude that this CLA supplementation protocol did not cause any morphological damage to the organ, as indicated by the integrity of hepatocytes / Doutorado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Doutor em Alimentos e Nutrição