Investigation of how redox processes regulate platelet function in health and disease

Background: Reactive oxygen species (ROS) are key signalling components in virtually all cells, acting either as second messengers or oxidants to target proteins. It has been reported that NADPH oxidase 1 (Nox-1) and the thiol isomerase protein disulphide isomerase (PDI) may interact physically and functionally to fine-tune ROS production in vascular cells. Both Nox-1 and PDI are able to individually regulate platelets, which are circulating cells of importance for thrombosis and haemostasis. However, little is known on how Nox-1 and PDI may collaborate to fine-tune platelet activity. Likewise, there is a current need to uncover novel inhibitors of these proteins and understand how ROS in platelets are implicated in metabolic diseases. Aims: The overarching aim of this thesis is to deepen our understanding of how redox processes regulate platelets in health and disease. Redox processes include the consequences of ROS production, as well as interest in specific redox proteins, namely PDI and Nox-1. Results: First, the flavonoid myricetin was studied and shown to be a novel inhibitor of PDI. Myricetin was able to reduce platelet aggregation, fibrinogen binding, granule exposure and in vitro thrombus formation, with no effect on tail bleeding in vivo. It is proposed that this flavonoid binds non-covalently near the active site of PDI and ERp5, which is also a thiol isomerase. Since PDI has been shown to associate with Nox-1 in vascular cells, the functional association of PDI and Nox-1 was studied. Through the use of selective inhibitors and Nox-1 deficient murine platelets, it was evident that PDI and Nox-1 collaborated to GPVI-mediated signalling in a process that involved phosphorylation of p38 mitogen-activated kinase and p47phox, which is a cytosol organizer of Nox-1. Moreover, in a healthy population study (n=137) PDI was positively correlated with waist/hip ratio and insulinaemia, whereas Nox-1 was correlated with body mass index (BMI) and systolic blood pressure. In line with the relevance of redox proteins PDI and Nox-1 to the regulation of platelet function, the redox regulation of platelets was also shown in a murine obesity model, in which it is shown for the first time that maternal high-fat diet intake is able to programme platelets from male offspring in a process associated with higher oxidative stress. Of importance, it is described a novel ‘double-hit’ effect in which maternal and offspring high-fat diet ingestion results in platelet hyperactivity. Conclusions: Altogether, data herein presented advance our understanding of how platelets are fine-tuned by redox processes. Myricetin is proposed as a novel inhibitor of thiol isomerases, while a novel mechanistic association between PDI and Nox-1 is also shown. Furthermore, it is suggested for the first time that platelets can be programmed by maternal insults. Therefore, platelets are highly regulated by redox processes and the study of these processes will contribute to improve the development of more effective antiplatelet therapy

Evolução da notificação de violência sexual no Brasil de 2009 a 2013

O objetivo foi analisar a evolução das notificações de violência sexual no Brasil entre 2009 e 2013, dando especial enfoque ao estupro. Realizou-se um estudo observacional de série temporal, analisando dados entre 2009 e 2013, provenientes do banco de dados secundários coletados do Sistema de Informação de Agravos de Notificação (SINAN). A variável independente referiu-se ao número de notificações de violência sexual entre 2009 e 2013, sendo esta comparada a diversas variáveis sociodemográficas, de violência e de saúde. Os dados foram analisados por modelo de regressão linear generalizada de Prais-Winsten, determinando as variações como estável, crescente ou decrescente pela análise do coeficiente de determinação (R2 de Pearson) e nível de significância (p < 0,05). As notificações de violência sexual tiveram maior variação positiva em: 10-19 anos (364%), indígenas (520%), da Região Sul (414%), sexo feminino (331%) e baixa escolaridade (343%). Mais de 70% das notificações foram estupros, enquanto um terço foi casos de repetição, com decréscimo de notificações acompanhadas de espancamento. As características do agressor mantiveram-se estáveis. O local de ocorrência modificou-se, com menos casos ocorridos em via pública. Por fim, a taxa de notificação estupro aumentou em 590%, com tendência semelhante aos estupros dentro de casa e aumento da resolutividade dos atendimentos. O presente estudo evidencia o perfil sociodemográfico das vítimas de violência sexual notificadas no Brasil e o aumento da notificação de estupros atrelado aos casos em domicílio. Ainda, houve aumento da resolutividade das notificações

Temporal analysis of reported cases of tuberculosis and of tuberculosis-HIV co-infection in Brazil between 2002 and 2012

ABSTRACT Objective: To investigate the reported cases of tuberculosis and of tuberculosis-HIV co-infection in Brazil between 2002 and 2012. Methods: This was an observational study based on secondary time series data collected from the Brazilian Case Registry Database for the 2002-2012 period. The incidence of tuberculosis was stratified by gender, age group, geographical region, and outcome, as was that of tuberculosis-HIV co-infection. Results: Nationally, the incidence of tuberculosis declined by 18%, whereas that of tuberculosis-HIV co-infection increased by 3.8%. There was an overall decrease in the incidence of tuberculosis, despite a significant increase in that of tuberculosis-HIV co-infection in women. The incidence of tuberculosis decreased only in the 0- to 9-year age bracket, remaining stable or increasing in the other age groups. The incidence of tuberculosis-HIV co-infection increased by 209% in the ≥ 60-year age bracket. The incidence of tuberculosis decreased in all geographical regions except the south, whereas that of tuberculosis-HIV co-infection increased by over 150% in the north and northeast. Regarding the outcomes, patients with tuberculosis-HIV co-infection, in comparison with patients infected with tuberculosis only, had a 48% lower chance of cure, a 50% greater risk of treatment nonadherence, and a 94% greater risk of death from tuberculosis. Conclusions: Our study shows that tuberculosis continues to be a relevant public health issue in Brazil, because the goals for the control and cure of the disease have yet to be achieved. In addition, the sharp increase in the incidence of tuberculosis-HIV co-infection in women, in the elderly, and in the northern/northeastern region reveals that the population of HIV-infected individuals is rapidly becoming more female, older, and more impoverished

Protective Effects of a Polyphenol-Rich Extract from Syzygium cumini (L.) Skeels Leaf on Oxidative Stress-Induced Diabetic Rats

Syzygium cumini (L.) Skeels has been reported to exert anti-inflammatory and cardiometabolic activities due to its high content of polyphenols. We characterized the chemical composition and assessed the antidiabetic effects of a novel polyphenol-rich extract (PESc) obtained from S. cumini leaf. Rats were injected with alloxan (150 mg/kg, ip, ALX group) and followed up for 7 days. Some were orally treated with PESc (50 mg/kg/day) for 7 days before and after diabetes induction (ALX-PP) or only for 7 days after alloxan injection (ALX-P). ALX-P and ALX-PP decreased fasting glycemia in 37 and 43%, respectively, as compared to ALX. Triglycerides and total cholesterol serum levels were also significantly reduced in comparison to ALX. PESc presented high polyphenol concentration (71.78 ± 8.57 GAE/100 g), with flavonoid content of 8.21 ± 0.42 QE/100 g. Upon HPLC-MS/MS and MS/MS studies, five main polyphenols—gallic acid, quercetin, myricetin, and its derivatives—were identified. Myricetin was predominant (192.70 ± 16.50 μg/mg PESc), followed by measurable amounts of gallic acid (11.15 ± 0.90 μg/mg PESc) and quercetin (4.72 ± 0.06 μg/mg PESc). Kinetic assessment of total antioxidant capacity revealed PESc high potency, since maximum response was reached within 5 min reaction time in a concentration-dependent manner. Specific antioxidant activity of PESc was assessed against both DPPH• and ABTS•+, showing strong activity (IC50: 3.88 ± 1.09 and 5.98 ± 1.19 μg/mL, resp.). PESc also inhibited lipoxygenase activity (IC50: 27.63 ± 8.47), confirming its antioxidant activity also on biologically relevant radicals. Finally, PESc induced insulin secretion by directly stimulating INS-1E β cells in the absence of any cytotoxic effect. Overall, our results support that PESc is a potent antioxidant phytocomplex with potential pharmacological use as a preventive antidiabetic natural product