abstract: Background: Evidence about the purported hypoglycemic and hypolipidemic effects of nopales (prickly pear cactus pads) is limited. Objective: To evaluate the efficacy of nopales for improving cardiometabolic risk factors and oxidative stress, compared to control, in adults with hypercholesterolemia. Design: In a randomized crossover trial, participants were assigned to a 2-wk intervention with 2 cups/day of nopales or cucumbers (control), with a 2 to 3-wk washout period. The study included 16 adults (5 male; 46±14 y; BMI = 31.4±5.7 kg/m2) with moderate hypercholesterolemia (low density lipoprotein cholesterol [LDL-c] = 137±21 mg/dL), but otherwise healthy. Main outcomes measured included: dietary intake (energy, macronutrients and micronutrients), cardiometabolic risk markers (total cholesterol, LDL-c, high density lipoprotein cholesterol [HDL-c], triglycerides, cholesterol distribution in LDL and HDL subfractions, glucose, insulin, homeostasis model assessment, and C-reactive protein), and oxidative stress markers (vitamin C, total antioxidant capacity, oxidized LDL, and LDL susceptibility to oxidation). Effects of treatment, time, or interactions were assessed using repeated measures ANOVA. Results: There was no significant treatment-by-time effect for any dietary composition data, lipid profile, cardiometabolic outcomes, or oxidative stress markers. A significant time effect was observed for energy, which was decreased in both treatments (cucumber, -8.3%; nopales, -10.1%; pTime=0.026) mostly due to lower mono and polyunsaturated fatty acids intake (pTime=0.023 and pTime=0.003, respectively). Both treatments significantly increased triglyceride concentrations (cucumber, 14.8%; nopales, 15.2%; pTime=0.020). Despite the lack of significant treatment-by-time effects, great individual response variability was observed for all outcomes. After the cucumber and nopales phases, a decrease in LDL-c was observed in 44% and 63% of the participants respectively. On average LDL-c was decreased by 2.0 mg/dL (-1.4%) after the cucumber phase and 3.9 mg/dL (-2.9%) after the nopales phase (pTime=0.176). Pro-atherogenic changes in HDL subfractions were observed in both interventions over time, by decreasing the proportion of HDL-c in large HDL (cucumber, -5.1%; nopales, -5.9%; pTime=0.021) and increasing the proportion in small HDL (cucumber, 4.1%; nopales, 7.9%; pTime=0.002). Conclusions: These data do not support the purported benefits of nopales at doses of 2 cups/day for 2-wk on markers of lipoprotein profile, cardiometabolic risk, and oxidative stress in hypercholesterolemic adults. / Dissertation/Thesis / Ph.D. Nutrition 2013

nÃo hà / A ketamina à usada principalmente como anestÃsico, produzindo um tipo de anestesia, denominada anestesia dissociativa que se caracteriza por um estado catalÃptico. Entretanto, o uso da ketamina nÃo tem sido restrito à prÃtica clÃnica e pesquisa, uma vez que, atualmente, tem sido utilizada como droga de abuso, visto produzir efeitos tanto alucinÃgenos quanto estimulantes. O objetivo deste estudo foi avaliar alteraÃÃes comportamentais tais como ansiedade e depressÃo em camundongos submetidos a diferentes tratamentos com ketamina (agudo, subcrÃnico e abstinÃncia de 24h), bem como investigar o envolvimento do estresse oxidativo em cÃrtex prÃ-frontal de camundongos apÃs tratamento agudo com ketamina. Trata-se de um estudo experimental, onde para avaliar as alteraÃÃes comportamentais (ansiedade e depressÃo) os camundongos foram tratados com ketamina agudamente ou subcronicamente (7 dias consecutivos), sendo realizados os modelos experimentais de Campo Aberto (TCA), Labirinto de Cruz Elevado (LCE), Nado ForÃado (TNF) e SuspensÃo da Cauda (TSC). Os animais tambÃm foram expostos a abstinÃncia de 24h apÃs o tratamento subcrÃnico e submetidos aos modelos experimentais supracitados. Para investigar o envolvimento do estresse oxidativo, os animais foram tratados agudamente com ketamina e utilizado o cÃrtex prÃ-frontal para avaliar atividade da catalase, nÃveis de glutationa, malonialdeÃdo e nitrito. Os resultados mostraram que no TCA houve aumento da atividade locomotora espontÃnea em todos os tipos de tratamento e em todas as doses utilizadas. No LCE o tratamento agudo, subcrÃnico e abstinÃncia de 24h com ketamina reduziu alguns parÃmetros nos braÃos abertos. No TNF e TSC, o tratamento agudo e subcrÃnico reduziu o tempo de imobilidade em todas as doses utilizadas. Enquanto na abstinÃncia de 24h houve aumento do tempo de imobilidade nas doses de 10 e 20 mg/kg. Quanto ao envolvimento do estresse oxidativo apÃs o tratamento agudo com ketamina em cÃrtex prÃ-frontal de camundongos notou-se um aumento significativo da atividade da catalase, dos nÃveis de malonildialdeÃdo e nitrito. Enquanto nos nÃveis de glutationa reduzida (GSH) observou-se uma diminuiÃÃo significativa. Conclui-se com o trabalho que o tratamento agudo e subcrÃnico causou efeito ansiogÃnico e antidepressivo. Enquanto que a abstinÃncia de 24h apÃs o tratamento subcrÃnico evidenciou um efeito ansiogÃnico e depressor. AlÃm das alteraÃÃes comportamentais, constata-se que o tratamento agudo com ketamina causa aumento do estresse oxidativo, indicando a ocorrÃncia de possÃveis lesÃes neurais em consequÃncia dessas alteraÃÃes. / Ketamine is mainly used as an anesthetic, producing a kind of anesthesia, called dissociative anesthesia characterized by a cataleptic. However, the use of ketamine has been restricted to clinical practice and research, since, currently, has been used as a drug of abuse, since both produce hallucinogenic effects as stimulants. The aim of this study was to evaluate behavioral changes such as depression and anxiety in mice subjected to different treatments with ketamine (acute, subchronic and withdrawal of 24), as well as investigate the involvement of oxidative stress in the prefrontal cortex of mice after acute treatment Ketamine. This is an experimental study to evaluate where behavioral changes (depression and anxiety) mice were treated with ketamine acutely or subcronicamente (7 consecutive days), being conducted experimental models of Open Field (TCA), Labyrinth of High Cross (LCE), Forced Swim (TNF) and Tail Suspension (TSC). The animals also were exposed to 24 hours of abstinence after treatment and subjected to subchronic experimental models above. To investigate the involvement of oxidative stress, the animals were treated acutely with ketamine and used the prefrontal cortex to evaluate the activity of catalase, glutathione, and nitrite malonialdeÃdo. The results showed that the TCA increased spontaneous locomotor activity in all types of treatment and at all doses used. In LCE treating acute and subchronic withdrawal 24h with ketamine reduced some parameters in the open arms. In TNF and TSC, acute and subchronic treatment reduced immobility time at all doses used. While the withdrawal of 24h was increased immobility time in doses of 10 and 20 mg/kg. Regarding the involvement of oxidative stress after acute treatment with ketamine in the prefrontal cortex of mice was noted a significant increase in catalase activity, levels of malondialdehyde and nitrite. While the levels of reduced glutathione (GSH) showed a significant decrease. It concludes with the work that the acute and subchronic treatment caused anxiogenic and antidepressant effects. While abstinence from 24h after treatment showed a subchronic effect anxiogenic and depressant. In addition to behavioral changes, it appears that acute treatment with Ketamine causes increased oxidative stress, indicating the possible occurrence of neural injuries as a result of these changes.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A doenÃa de Parkinson (DP) à uma desordem neurodegenerativa caracterizada pela destruiÃÃo dos neurÃnios nigroestriatais dopaminÃrgicos. O tratamento atual à apenas sintomÃtico e, atà o presente momento, nÃo existem drogas capazes de inibir a degeneraÃÃo neuronal. Assim à grande a procura por agentes neuroprotetores que possam impedir ou retardar a progressÃo desta doenÃa. A Camellia sinensis à uma espÃcie da famÃlia Theaceae, popularmente conhecida como chÃ-verde (CV). VÃrios estudos tÃm demonstrado os efeitos benÃficos desta planta e de seus princÃpios bioativos contra doenÃas neurodegenerativas. O objetivo deste trabalho foi avaliar o efeito neuroprotetor do extrato padronizado do chÃ-verde e de suas catequinas, epicatequina (EC) e epigalocatequina 3-galato (EGCG), no modelo de DP induzida por 6-OHDA em ratos, bem como estudar o efeito do CV em modelos de inflamaÃÃo e nocicepÃÃo. Os animais (ratos Wistar machos, 180-250 g) foram tratados por via oral com CV (25 ou 50 mg/kg), catequinas (EC ou EGCG, na dose de 10 mg/kg), a associaÃÃo de CV (25 mg/kg) + L-DOPA (25 mg/kg) e com L-DOPA sozinha (nas doses de 25 ou 50 mg/kg) durante 14 dias consecutivos; os tratamentos foram iniciados 1h antes da lesÃo estriatal unilateral por 6-OHDA (24 Âg/2ÂL). Nos testes comportamentais, os resultados mostraram que houve um aumento significativo do nÃmero de rotaÃÃes induzidas por apomorfina, diminuiÃÃo da atividade locomotora no teste do campo aberto, aumento do tempo de imobilidade no teste no nado forÃado, aumento do tempo de encontro da plataforma no teste do labirinto aquÃtico e aumento do nÃmero de quedas no teste do rota rod nos animais lesionados com 6-OHDA, em comparaÃÃo com os animais falso-operados. Essas modificaÃÃes foram, em parte ou totalmente, revertidas apÃs os tratamentos com CV, CV + L-DOPA, EC, EGCG e L-DOPA sozinha. A 6-OHDA provocou morte neuronal, evidenciada pela reduÃÃo dos nÃveis de dopamina e metabolitos (DOPAC e HVA) no estriado direito lesionado quando comparado com o estriado esquerdo nÃo-lesionado (contralateral). Essa depleÃÃo foi significativamente atenuada nos animais lesionados e tratados com CV, L-DOPA, EC, EGCG e associaÃÃo CV 25 + L-DOPA 25; estas mesmas drogas diminuÃram os nÃveis de TBARS (indicador de peroxidaÃÃo lipÃdica) e nitrito/nitrato (indicador de estresse oxidativo) no estriado de ratos submetidos a lesÃo estriatal por 6-OHDA. Experimentos in vitro demonstraram que o CV (0,1-100 Âg/Âl) apresentou um forte efeito antioxidante, ao reduzir a produÃÃo de substÃncias oxidantes em neutrÃfilos humanos estimulados por PMA. Na coloraÃÃo de Nissl, observou-se que nos animais lesionados e tratados com CV, EGCG e CV + L-DOPA houve uma preservaÃÃo dos neurÃnios hipocampais. Os tratamentos com CV e CV+L-DOPA aumentaram a imunorreatividade para TH e diminuÃram a imunomarcaÃÃo para COX-2 no estriado, bem como o CV e EGCG atenuaram a marcaÃÃo para iNOS no hipocampo dos animais tratados, em relaÃÃo ao grupo controle. Observou-se que o CV potencializou os efeitos da L-DOPA, evidenciando um possÃvel efeito sinÃrgico entre essas drogas. Em outra etapa do estudo, avaliou-se a atividade anti-inflamatÃria/antinociceptiva do CV. Assim, no modelo de edema de pata, induzido por carragenina ou dextrano, verificou-se uma diminuiÃÃo do volume do edema da pata dos ratos, apÃs tratamento com CV, em relaÃÃo aos do grupo controle (tratado apenas com Ãgua destilada). O CV produziu efeito antinociceptivo, nos testes da formalina e das contorÃÃes abdominais, induzidas por Ãcido acÃtico, como tambÃm nos testes da placa quente e de Hargreaves, possivelmente atravÃs da ativaÃÃo de receptores opioides e da reduÃÃo do processo inflamatÃrio. Nestes testes de nocicepÃÃo e inflamaÃÃo, o CV potencializou os efeitos da morfina, indometacina e dexametasona. Portanto, nossos resultados evidenciaram os efeitos neuroprotetores do CV e de seus princÃpios bioativos, EC e EGCG, possivelmente decorrentes de suas propriedades anti-inflamatÃrias e antioxidantes, tornando-as opÃÃes terapÃuticas futuras para a prevenÃÃo ou tratamento de doenÃas neurodegenerativas, como a doenÃa de Parkinson.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / The Systemic Inflammatory Response Syndrome (SIRS) is characterized by an excessive release of inflammatory mediators as a systemic inflammatory response to a serious clinical injuries. The use of glutamine in nutraceutical doses has been studied as a strategy in tissue protection and preservative of tissue metabolic function in stressful situations, helping to improve the immune response of patients. The effects of enteral glutamine supplementation in nutraceutical doses on the inflammatory markers, of glycolytic metabolism, of immune system and of oxidative stress were studied in adult and elderly patients with SIRS in a prospective, clinical, randomized, controlled, double-blind crossover study. Thirty six moderately severe patients admitted to the Intensive Care Unit were selected according to pre-defined criteria, diagnosis of SIRS and the APACHE II score (>10<20), distributed into two groups and submitted to the supplementation with 1 litre of enteral nutrition with addition of 30g of L-glutamine or calcium caseinate or 1 litre of enteral nutrition with addition of 30g of calcium caseinate or L-glutamine for two days, pause for one day only with diet, followed by four days of supplementation. Blood samples were collected before (T0) and after (T1) each supplementation. For evaluation blood parameters (hematocrit, leukocytes, lymphocytes, monocytes, prealbumin, blood urea nitrogen, creatinine, glucose, lactate, C-peptide and insulin), IL-1, IL-6, IL-10 and TNF&#945; were also assayed. Glutathione, TBARS, and glutamine and glutamate amino acids were measured. Six patients died during the study. Thirty patients finished the study, 16 men (53%) and 14 (47%) women, median age 74.4 years (30-92 years) in moderately severe state of health (APACHE II 13.1 - range 10-19). All patients developed SIRS and were given enteral nutrition supplemented with L-glutamine or calcium caseinate, 1464kcal/day (range 792-1914kcal/day). The use of L-glutamine in nutraceutical dose of 30g/day showed no changes in blood parameters. All laboratory parameters remained within normal values except the blood urea [Calcium Caseinate T1=47.0mg/dL (range 34.0-69.0 mg/dL) versus Glutamine T1=50.0mg/dL (36.75-75.0mg/dL); p=0.030]. Creatinine concentrations were not statistically different. There was no statistically significant difference in assessment of inflammatory parameters (IL-1, IL-6, IL-10 E TNF&#945;). Leukocytes count decreased significantly in both groups [Calcium Caseinate T0=13.650 1/mm3 (10.148-18.250 1/mm3) versus T1=11.500 1/mm3 (8.050-29.100 1/mm3); p=0,019] and [Glutamine T0=12.850 1/mm3 (11.155-15.550 1/mm3) versus T1=11.000 1/mm3 (9.200-16.325 1/mm3); p=0.046]. There was increase statistically significant difference in lymphocytes count between groups [Calcium Caseinate T1=1085 1/mm3 (range 805-1363 1/mm3) versus Glutamine T1=1916 1/mm3 (1301-2517 l/mm3); p<0.0001] and Calcium Caseinate group decreases [T0=1288 1/mm3 (range 834-2209 1/mm3) versus T1=1085 1/mm3 (range 805-1363 1/mm3); p=0.0324] and Glutamine group increases [T0=954 1/mm3 (range 785-1442 1/mm3) versus T1=1916 1/mm3 (range 1301-2517 l/mm3); p<0.0001]. Blood concentration of TBARS decreased significantly in both groups [Calcium Caseinate T0=20.56&#61549;mol MDA/ml (range 13.64-20.56&#61549;mol MDA/ml); p=0.001] and [Glutamine T0=17.67 &#61549;mol MDA/ml (range 8.11-34.98 &#61549;mol MDA/ml) versus T1=16.52 &#61549;mol MDA/ml (range 5.41-21.86 &#61549;mol MDA/ml); p=0.020]. The blood concentrations of Gluthatione showed a statistically significant reduction in caseinate group (T0=486.0&#61549;mol/ml (range 486.0Â165.8&#61549;mol/ml versus T1=451.0Â167.4&#61549;mol/ml; p=0.047) and no statistically significant difference in the glutamine group, nor between groups. However, there were no differences between groups. Glutamine and glutamate were not statistically different. Enteral nutrition supplemented with glutamine in nutraceutical doses of 30g/day increase lymphocyte count, helps to reduce lipid peroxidation and maintains the antioxidant glutathione capacity, interfering beneficially modulating the inflammatory response and stress, but present no effect upon cytokines concentrations or glycolytic parameters. / A SÃndrome da Resposta InflamatÃria SistÃmica (SRIS) caracteriza-se por uma liberaÃÃo excessiva de mediadores inflamatÃrios a uma sÃrie de situaÃÃes clÃnicas graves. A utilizaÃÃo da glutamina em doses nutracÃuticas tem sido estudada como uma estratÃgia de proteÃÃo tecidual e metabÃlica em situaÃÃes de estresse, melhorando a resposta imune de pacientes. Os efeitos da nutriÃÃo enteral suplementada com 30g/dia de glutamina sobre os marcadores inflamatÃrios, do metabolismo glicolÃtico, da funÃÃo imune e do estresse oxidativo foram estudados em pacientes adultos e idosos com SRIS. Foi realizado estudo clÃnico prospectivo, randomizado, controlado, duplo-cego, cruzado. Trinta e seis pacientes internados em Unidade de Terapia Intensiva foram selecionados pelos critÃrios do estudo, diagnÃstico da SRIS e score APACHE II (>10<20), distribuÃdos em dois grupos e submetidos à suplementaÃÃo com 1 litro de dieta enteral suplementada com 30g de L-glutamina ou caseinato de cÃlcio ou 1 litro de dieta enteral suplementada com 30g de caseinato de cÃlcio ou L-glutamina por dois dias, intervalo de um dia somente com dieta, perfazendo quatro dias de dieta com suplementaÃÃo. Amostras de sangue foram coletadas antes (T0) e apÃs (T1) cada suplementaÃÃo. Foram realizadas anÃlises do hematÃcrito, leucÃcitos, linfÃcitos, monÃcitos, prÃ-albumina, urÃia, creatinina, glicose, lactato, peptÃdeo-C e insulina, das IL-1, IL-6, IL-10, TNF&#945;, glutationa, TBARS e dos aminoÃcidos glutamina e glutamato. Seis pacientes foram a Ãbito durante o estudo e trinta pacientes concluÃram o estudo, sendo 16(53%) homens e 14(47%) mulheres, mediana de idade 74,4 anos (30-92 anos), moderadamente graves, mediana de APACHE II 13,1 (10-19) e mediana de ingestÃo calÃrica de 1464kcal/dia (792-1914kcal/dia). O uso L-glutamina em dose nutracÃutica de 30g/dia nÃo mostrou alteraÃÃes nos parÃmetros hematolÃgicos. Houve aumento da urÃia [Caseinato T1=47,000mg/dL (34,000-69,000mg/dL) versus Glutamina T1=50,000mg/dL (36,750-75,000mg/dL); p=0,030] na comparaÃÃo intergrupos, mas nÃo houve diferenÃa estatisticamente significante de creatinina em nenhum dos grupos. NÃo houve alteraÃÃo estatisticamente significante nos parÃmetros inflamatÃrios (IL-1, IL-6, IL-10 e TNF&#945;). A contagem de leucÃcitos diminuiu significantemente em ambos os grupos [Caseinato T0=13.650 1/mm3 (10.148-18.250 1/mm3) versus T1=11.500 1/mm3 (8.050-29.100 1/mm3); p=0,019] e [Glutamina T0=12.850 1/mm3 (11.155-15.550 1/mm3) versus T1=11.000 1/mm3 (9.200-16.325 1/mm3); p=0,046]. Houve aumento estatisticamente significante na contagem de linfÃcitos na comparaÃÃo intergrupos [Caseinato T1=1.085 1/mm3 (805-1.363 1/mm3) versus Glutamina T1=1.916 1/mm3 (1.301-2.517 l/mm3); p<0,0001], uma diminuiÃÃo estatisticamente significante no grupo Caseinato [T0=1.288 1/mm3 (834-2.209 1/mm3) versus T1=1.085 1/mm3 (805-1.363 1/mm3); p=0,0324] e aumento no grupo Glutamina [T0=954 1/mm3 (785-1.442 1/mm3) versus T1=1.916 1/mm3 (1.301-2.517 l/mm3); p<0,0001]. Observou-se reduÃÃo estatisticamente significante na dosagem do TBARS na comparaÃÃo intragrupos [Caseinato T0=20,56&#61549;mol MDA/ml (13,64-20,56&#61549;mol MDA/ml) versus T1=15,08 &#61549;mol MDA/ml (13,64-20,56 &#61549;mol MDA/ml); p=0,001] e [Glutamina T0=17,67 &#61549;mol MDA/ml (8,11-34,98 &#61549;mol MDA/ml) versus T1=16,52 &#61549;mol MDA/ml (5,41-21,86 &#61549;mol MDA/ml); p=0,020], mas nÃo houve diferenÃas intergrupos. A concentraÃÃo sanguÃnea de glutationa apresentou uma reduÃÃo estatisticamente significante no grupo Caseinato (T0=486,00&#61549;mol/mlÂ165,80&#61549;mol/ml) versus T1=451,00Â167,40&#61549;mol/ml; p=0,047) e nÃo houve diferenÃa no grupo Glutamina, tampouco entre os grupos. Glutamina e glutamato nÃo demonstraram diferenÃas estatisticamente significantes. Conclui-se que a nutriÃÃo enteral suplementada com glutamina em dose nutracÃutica de 30g/dia em pacientes moderadamente graves promove um aumento dos linfÃcitos, contribui para reduzir a peroxidaÃÃo lipÃdica e mantÃm a capacidade antioxidante da glutationa, interferindo de forma benÃfica na modulaÃÃo da resposta inflamatÃria e do estresse, mas nÃo apresenta nenhum efeito sobre a concentraÃÃo de citocinas ou parÃmetros glicolÃticos.

Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterized by motor symptoms such as tremor, rigidity, akinesia and postural instability. Approximately 90% of the cases are due to unknown causes however a familial inheritance has been shown for about 10% of cases. Loss-of-function mutations in DJ-1 cause early-onset PD. Originally identified as an oncogene, DJ-1 has since had many functions attributed to it but its major role in the cell seems to be oxidative stress handling. We have previously demonstrated that DJ-1 deficiency results in hypersensitivity of cells to oxidative stress. Additionally, mitochondria from DJ-1 null mice are fragmented and produce more ROS. To better understand the relationship between DJ-1, cell survival and mitochondria, we investigated the possible interaction between DJ-1 and the mitochondrial protein voltage dependent anion channel 1 (VDAC1). Here we show mitochondrial translocation of DJ-1 following oxidative stress in murine embryonic fibroblasts (MEFs) and primary cortical neurons, a process dependent on Pink1 and Parkin. Additionally, we confirm that DJ-1 and VDAC1 interact and that stress-induced mitochondrial translocation of DJ-1 depends on VDAC1. Deficiency of VDAC1 in primary cortical neurons results in decreased survival, increased ROS production following extended stress, fragmented mitochondria and decreased mitochondrial ATP production. We also demonstrate that there is substantially less matrix-localized DJ-1 in VDAC1 deficient cells. Finally, we demonstrate that decreased mitochondria ATP production can signal for DJ-1 translocation to mitochondria. Taken together, we suggest that mitochondrial translocation of DJ-1 is a two-step process. First, a signal, perhaps decreased mitochondrial ATP production, induces DJ-1 translocation to mitochondria. Second, DJ-1 localizes to the matrix in a VDAC1-dependent manner. Our work suggests that stress-induced mitochondrial localization of DJ-1, specifically to the matrix, is regulated by VDAC1 to promote survival possibly by promoting ATP production.

Peroxiredoxins are ubiquitous, thiol-specific proteins that have multiple functions in stress protection, including oxidative stress. Tsa1 is the major yeast peroxiredoxin and we show that it functions as a specific antioxidant to protect against oxidative stress caused by nascent protein misfolding and aggregation. Yeast mutants lacking TSA1 are sensitive to misfolding caused by exposure to the proline analogue azetidine-2-carboxylic acid (AZC). AZC promotes protein aggregation and its toxicity to a tsa1 mutant is caused by reactive oxygen species (ROS). Generation of [rho0] cells lacking mitochondrial DNA rescues the tsa1 mutant AZC sensitivity indicating that mitochondria are the source of ROS. Inhibition of nascent protein synthesis with cycloheximide prevents AZC-induced protein aggregation and abrogates ROS generation confirming that aggregate formation causes ROS production. Protein aggregation is accompanied by mitochondrial fragmentation and we show that Tsa1 localizes to the sites of protein aggregation, which are formed adjacent to mitochondria. Further investigation reveals that AZC-induced protein aggregation leads to an inhibition of mitochondrial respiration and the depolarisation of the mitochondrial membrane. Remarkably, this was entirely dependent on the presence of Tsa1. We show that the effects of protein aggregation on mitochondrial function are mediated by the Ras/PKA pathway and that Tsa1 appears to influence the activity of this pathway through its effects on the yeast phosphodiesterase, Pde2. Together, these data indicate a new role for peroxiredoxins in the response to ROS, generated as a result of protein misfolding and aggregate formation. Finally, we analysed the characteristics of proteins found within protein aggregates, isolated from different conditions during the course of the study. Our results highlight the differences between proteins that aggregate under normal, mid-exponential growth conditions (physiological aggregates) and those which aggregate during cellular stress. We were able to establish the characteristics of an archetypical physiological aggregate, through an assessment of a range of properties, identifying factors that significantly differed from genomic expectations. Furthermore, our observations indicate that, in general, cellular stress reduces the threshold of metrics associated with protein aggregation propensity. We also found that different stresses result in the aggregation of proteins that are, largely, physicochemically indistinct from one another, regardless of the mode of toxicity. Finally we show that a significant number of proteins, identified in our protein aggregates, were also present in protein aggregates isolated from aged C. elegans. This suggests that the factors and components of protein aggregates are conserved.

Global climate change imposes physiological constraints on marine ecosystems that can alter the distribution of intertidal organisms. In one such instance, the native cold-adapted mussel Mytilus trossulus is being replaced along its southern range by the invasive warm-adapted Mytilus galloprovincialis. These blue mussels occur throughout rocky intertidal zones where they are subjected to greatly varying environmental conditions known to induce oxidative stress. We hypothesize that while under acute stress, related Mytilus congeners undergo a shift in redox potential from NADH-fueled respiratory pathways to pathways producing NADPH as a way to decrease the production of reactive oxygen species (ROS) and provide reducing equivalents to detoxify ROS. Additionally, we hypothesize that sirtuins (SIRT; a family of NAD-dependent deacetylases) might be involved in the regulation of this metabolic transition. To test the latter, a discovery approach will be used to analyze the proteomic response of M. galloprovincialis and M. trossulus to the pro-oxidant menadione, and sirtuin-inhibitors nicotinamide and suramin. Menadione can induce oxidative stress by increasing endogenous peroxide and superoxide radicals, while suramin and nicotinamde both inhibit sirtuin activity. Organisms were exposed to these compounds in filtered seawater for 8 h, followed by a 24.5 h recovery period under constant aeration. A multivariate analysis utilizing 2D-gel electrophoresis and protein identification via mass spectrometry showed that 18% and 17% of all identified protein spots detected demonstrated changes in abundance in M. galloprovincialis and M. trossulus, respectively. Using matrix-assisted laser desorption ionization (MALDI) tandem time-of-light mass spectrometry, we were able to identify 32-41% of proteins, depending on the species. The two Mytilus congeners showed the greatest differences in changes of protein abundance for oxidative stress proteins (including NADP-dependent isocitrate dehydrogenase). Both congeners showed similar effects in response to simultaneous sirtuin inhibition and MIOS for proteins involved in protein degradation (proteasome), cytoskeletal modifications (actin and tubulin), proteins regulating actin filament growth (F-actin capping protein), amino acid metabolism and stress signaling (G-proteins, small G-proteins and MAPK). Results indicate that protein acetylation plays an important role in the oxidative stress response of M. galloprovincialis. More specifically this suggests that sirtuins play an important role in regulating the general stress response in M. galloprovincialis and thus contribute to the greater stress resistance of this species. Furthermore, changes in the abundance of several molecular chaperones suggest a greater effect of sirtuins in regulating the cellular response to heat stress, which could in part explain why this species is more heat-tolerant than the native M. trossulus.

L’argument selon lequel les antibiotiques bactéricides stimulent la formation d'espèces réactives de l’oxygène (ROS pour reactive oxygen species) chez certaines bactéries en augmentant leur activité respiratoire est sujet à discussions. Enterococcus faecalis a des propriétés intéressantes pour tester cette hypothèse. La respiration ne s’observe qu’en présence d'hème ou de fumarate. En l'absence d'hème, E. faecalis produit du superoxyde extracellulaire par l’autoxydation d’une demethylmenaquinone (DMK), un composant de sa chaîne respiratoire associé à sa membrane. En raison de cette propriété, E. faecalis est soupçonné de jouer un rôle dans la cancérogenèse colorectale. Nous montrons dans cette étude que les β–lactamines amplifient significativement la production de ROS. Cette augmentation, dépendante de DMK, n'est observée qu'en l'absence de respiration. Nos résultats pourraient également fournir une explication quant au risque accru de cancer colorectal observé chez des patients traités par des β–lactamines, comme le montrent de récentes études cliniques. / The proposal that bactericidal antibiotics stimulate the formation of reactive oxygen species (ROS) by increasing respiration is still a matter of debate. Enterococcus faecalis has interesting properties to test this hypothesis. Respiration occurs only in the presence of heme or fumarate. In the absence of heme, E. faecalis produces extracellular superoxide through autoxidation of demethylmenaquinone (DMK), a component of its respiratory chain. Due to this ability, E. faecalis is suspected to play a role in colorectal carcinogenesis. We show in this study that β–lactam antibiotics increase significantly the production of ROS. This boost of ROS formation is DMK–dependent and only observed in the absence of respiration. Our results could provide a mechanistic explanation for the observed increased risk of colorectal cancer by β–lactam antibiotics in several recent nested case–control studies.

Parkinson disease (PD) is an incurable disease, second only to Alzheimer’s disease as the most common neurodegenerative disease in adults. Unfortunately, the course of disease is significantly longer for individuals diagnosed at an early age (20-40 years of age). Although early-onset, recessively inherited cases represent a small subset of individuals with PD (~5- 10%), their clinical presentation is unique, with symptoms being almost exclusively motor-related. The expressivity of early-onset PD is partially explained by post-mortem neuropathological findings, which demonstrate a specific loss of dopamine synthesizing cells in brainstem nuclei that also produce neuromelanin (i.e. Substantia nigra and Locus coeruleus). With the majority of early-onset PD cases being caused by homozygous and biallelic heterozygous mutations in the PRKN gene, its gene product, parkin, has been extensively studied. It is generally accepted that loss of its E3 ligase function leads to neurodegeneration by either one of the following two mechanisms: i) toxic substrate accumulation from the loss of target protein ubiquitination (and related degradation), or ii) accumulation of dysfunctional mitochondria due to impaired mitophagy initiation. However, whether these mechanisms lead to selective neuronal loss within the human brain remains unknown. This thesis represents a body of work that supports a novel role for parkin as a thiol-based anti-oxidant and redox homeostasis regulator, which helps explain the cell-specificity observed in recessive, PRKN-linked PD. These findings include: i) evidence that human brain parkin uniquely and natively undergoes age-associated aggregation beginning at 40 years of age (Chapter 2); ii) identification of multiple, reversible and irreversible oxidative modifications of parkin cysteines, both in cells and tissues, including dopamine-adduct conjugation on primate sequence-specific cysteine 95 (Chapter 2); iii) the demonstration that irreversible oxidation of parkin cysteines causes aggregate formation ii in cells and mice exposed to exogenous and/or endogenous sources of oxidative and dopamine stress (Chapter 2 and 3); iv) evidence that parkin functions as a thiol-dependent anti-oxidant similar to glutathione (Chapter 2), which lowers oxidation state in cells and tissues under native and stress conditions (Chapter 2 and 3); v) the demonstration that parkin cysteines, notably C95, directly bind glutathione and regulate glutathione redox homeostasis in cells and tissues in a dynamic fashion (Chapter 3); and vi) the development of novel, human-specific, anti-parkin monoclonal antibodies that preferentially detect oxidized and aggregated forms of parkin found associated with neuromelanin and lysosomal storage vesicles within neurons of human Substantia nigra (Chapter 2 and 4). Future studies focusing on further validation of in situ oxidative modifications of parkin cysteines and their effect on protein structure, notably the poorly studied linker region that contains C95, will provide insight into how these oxidative modifications affect the function of parkin in vivo, including in adult human brain. Also, identifying the bona fide intracellular redox partners of parkin will be crucial to understanding how this protein regulates cellular redox state. Of clinical importance, the findings presented here indicate a potential, human-specific link between parkin and neuromelanin formation, which deserves to be further explored, such as with parkin mouse models engineered to produce neuromelanin. Finally, designing clinical trials using anti-oxidants specifically in individuals affected by PRKN-associated PD represents a logical, translational treatment approach to explore.

Background: Although alcohol intake has been positively associated with breast cancer risk in epidemiologic studies, the mechanisms mediating this association are speculative. Objective: The Postmenopausal Women's Alcohol Study was designed to explore the effects of moderate alcohol consumption on potential risk factors for breast cancer. In the present analysis, we evaluated the relationship of alcohol consumption with antioxidant nutrients and a biomarker of oxidative stress. Design: Participants (n = 53) consumed a controlled diet plus each of three treatments (15 or 30 g alcohol/day or a no-alcohol placebo beverage), during three 8-week periods in random order. We measured the antioxidants, vitamin E (alpha (α)- and gamma (γ-tocopherols), selenium, and vitamin C in fasting blood samples which were collected at the end of diet periods, treated and frozen for assay at the end of the study. We also measured 15-F2t-IsoP isoprostane, produced by lipid peroxidation, which serves as an indicator of oxidative stress and may serve as a biomarker for conditions favorable to carcinogenesis. Results: After adjusting for BMI (all models) and total serum cholesterol (tocopherol and isoprostane models) we observed a significant 4.6% decrease (P=0.02) in α-tocopherol and a marginally significant 4.9% increase (P = 0.07) in isoprostane levels when women consumed 30 g alcohol/day (P = 0.06 and 0.05 for overall effect of alcohol on α-tocopherol and isoprostanes, respectively). The other antioxidants were not significantly modified by the alcohol treatment. Conclusions: These results suggest that moderate alcohol consumption increases some biomarkers of oxidative stress in postmenopausal women.