THE MOLECULAR MECHANISMS OF IRON AND FERRITIN METABOLISM IN NORMAL AND NEOPLASTIC CELLS

Iron (Fe) is essential for cell growth and replication as many Fe-containing proteins catalyse key reactions involved in energy metabolism (cytochromes, mitochondrial aconitase and Fe-S proteins of the electron transport chain), respiration (hemoglobin and myoglobin) and DNA synthesis (ribonucleotide reductase). If not appropriately shielded, Fe could participate in one-electron transfer reactions that lead to the production of extremely toxic free radicals. The Fe storage protein, ferritin, is essential to protect cells against Fe-mediated oxidative stress by accommodating excess Fe into its protein shell (Xu et al., 2005). However, despite intensive research over the last few decades, many questions relating to intracellular Fe metabolism, e.g. Fe release from ferritin remain unanswered. Therefore, it is important to elucidate the molecular mechanisms of Fe trafficking in cells. At the beginning of my candidature, little was understood regarding the effect of anti-cancer agents, anthracyclines on the Fe-regulated genes, including transferrin receptor-1 (TfR1), N-myc downstream-regulated gene-1 (Ndrg1) and ferritin. Furthermore, the mechanisms of ferritin-Fe release and anthracycline-mediated ferritin-Fe accumulation are unclear. The work presented in Chapters 3 and 4 has addressed these issues. Apart from the studies examining the molecular interactions of anthracyclines with Fe, a mouse model with perturbed Fe metabolism was used and the marked alterations of protein expression in the heart of this knockout mouse model was discussed in Chapter 5. Chapter 3 Anthracyclines are effective anti-cancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron (Fe) and perturb Fe metabolism (Xu et al., 2005). These effects on Fe-trafficking remain poorly understood, but are important to decipher as treatment for anthracycline cardiotoxicity utilises the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the Fe-regulated genes, transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by Fe-depletion, as it was reversed by adding Fe and was prevented by saturating the anthracycline metal-binding site with Fe. However, DOX did not act like a typical chelator, as it did not induce cellular Fe mobilisation. In the presence of DOX and 59Fe-transferrin, Fe-trafficking studies demonstrated ferritin-59Fe accumulation and decreased cytosolic-59Fe incorporation. This could induce cytosolic Fe-deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via HIF-1α-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on Fe metabolism were complex due to its multiple effector mechanisms. Chapter 4 The Fe storage protein, ferritin, can accommodate up to 4500 atoms of Fe in its protein shell (Harrison and Arosio, 1996). However, the underlying mechanism of ferritin-Fe release remains unknown. Previous studies demonstrated that anti-cancer agents, anthracyclines, led to ferritin-59Fe accumulation (Kwok and Richardson, 2003). The increase in ferritin-59Fe was shown to be due to a decrease in the release of Fe from this protein. It could be speculated that DOX may impair the Fe release pathway by preventing the synthesis of essential ferritin partner proteins that induce Fe release. In this study, a native protein purification technique has been utilised to isolate ferritin-associated partners by combining ultra-centrifugation, anion-exchange chromatography, size exclusion chromatography and native gel electrophoresis. In addition to cells in culture (namely, SK-Mel-28 melanoma cells), liver taken from the mouse was used as a physiological in vivo model, as this organ is a major source of ferritin. Four potential partner proteins were identified along with ferritin, e.g. aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). Future studies are required to clarify the relationship of these proteins with cellular Fe metabolism and ferritin-Fe release. Chapter 5 A frequent cause of death in Friedreich’s ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed two dimensional electrophoresis to characterise the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in the protein expression profile were observed in 9-week-old KO mice with severe cardiomyopathy. In contrast, only a few proteins showed altered expression in asymptomatic 4-week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilisation and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse

Effect of Training in Peripheral Blood Lymphocytes and Cytokine Levels in Elite Kayakers

Prolonged strenuous exercise has been associated with impaired immune function. The objective of this study was to investigate the differences in the peripheral blood cell counts and cytokynes in kayakers at different moments of training. The sample consisted of eight elite male kayakers, 22± 4 years old, 77.2 kg±6.7 body mass and 177.5±5.6 cm stature. The initial VO2max was 61.2±5.5 mL.kg.min-1. The control group consisted of six healthy males, 18±1 years old, 81.3± 13.8 kg body mass and 171.9±4.5cm stature. Blood samples were collected, at rest, from the kayakers at 4 time points of the training season: t0 early November (beginning of the training season), t1 late February (after an increased volume in training load), t2 early April (after an increase in training intensity) and t4 in June (after a major competition). Blood samples from the controls were taken at 3 equivalent time-points. Lymphocyte subpopulations were determined by flow cytometry. Cytokine concentrations were determined by ELISA. Statistical analysis was done using Friedman\u27s ANOVA- Repeated measures and the Mann-Whitney test for comparing the kaykers and control groups. The total nº of lymphocytes decreased and the natural killer (NK) CD3+CD56+CD8+ cells increased at t2 when compared to baseline (t0). No changes between time points for the lymphocyte subpopulations were found in the control group. When comparing both groups lower levels of NK cells were found at baseline and througth out the season in the kayakers. When looking at plasma cytokine concentration an increase in intensity training and the competitive period were able to elevate the concentrations of pro and anti-inflammatory cytokines (IL1-Ra, IL-6, Il-1beta and IL-18), even after 24hours of rest. However IFN-Υ, TNF-α and IL-10 were not significantly affected in the kayakers. When comparing both groups at t0, kayakers showed lower levels of IL-1β, IL-18, IL-1ra and IFN-γ. Changes in training load intensity were able to affect the number of lymphocytes and their subpopulations in the circulation periphery. Significant reductions in circulating NK CD3-CD56+ and CD3-CD56+CD8+, appears to be associated with increased risk of URS. In spite of the increase of cytokines after the competition period, the kayakers maintained lower levels of pro and anti-inflammatory cytokines when compared with the control group. Despite extreme levels of exercise being associated with reduced immune function and increased susceptibility to infections, a reduction in inflammation due to training may represent a beneficial effect of the long term exercise practice

Mesenchymal stem/stromal cells : disrupting cell therapy storage and distribution with hypothermic preservation of adipose-derived mesenchymal stromal cells

Background & Aim: Cell and gene therapies (CGT) have reached new therapeutic targets but have noticeably high prices. Solutions to reduce production costs might be found in CGT storage and transportation since they typically involve cryopreservation, which is a heavily burdened process. Encapsulation at hypothermic temperatures (e.g.,2–8°C) could be a feasible alternative. In this study, we aim to determine the ability of alginate encapsulation to maintain cell viability, identity, and function in the context of MSC-based therapy manufacturing. Methods, Results & Conclusion: Adipose tissue-derived mesenchymal stromal cells (MSC(AT)) expanded using fetal bovine serum (FBS)- (MSC-FBS) or human platelet lysate (HPL)-supplemented mediums (MSC-HPL) were encapsulated in alginate beads (BeadReady™ kits kindly provided by Atelerix) for 30 min, 5 days, and 12 days. After bead release, cell recovery and viability were determined to assess encapsulation performance. MSC identity and functional immunophenotype, MSC tri-lineage differentiation potential, metabolic activity, and hematopoietic support capacity were determined and compared between timepoints. MSC(AT) were able to survive encapsulated for a standard transportation period of 5 days, with recovery values of 56 ± 5% for MSC-FBS and 77 ± 6% for MSC-HPL (which is a negligible drop compared to earlier timepoints). Importantly, MSC function did not suffer from encapsulation, with recovered cells showing robust differentiation potential, expression of immunomodulatory molecules, and hematopoietic support capacity. MSC(AT) encapsulation was proven possible for a remarkable 12 day period. There is currently no solution to completely replace cryopreservation in CGT logistics and supply chain, although encapsulation has shown potential to act as a serious competitor.info:eu-repo/semantics/publishedVersio

Frequency and functional activity of Th17, Tc17 and other T-cell subsets in Systemic Lupus Erythematosus

To compare frequency and functional activity of peripheral blood (PB) Th(c)17, Th(c)1 and Treg cells and the amount of type 2 cytokines mRNA we recruited SLE patients in active (n = 15) and inactive disease (n = 19) and healthy age- and gender-matched controls (n = 15). The study of Th(c)17, Th(c)1 and Treg cells was done by flow cytometry and cytokine mRNA by real-time PCR. Compared to NC, SLE patients present an increased proportion of Th(c)17 cells, but with lower amounts of IL-17 per cell and also a decreased frequency of Treg, but with increased production of TGF-b and FoxP3 mRNA. In active compared to inactive SLE, there is a marked decreased in frequency of Th(c)1 cells, an increased production of type 2 cytokines mRNA and a distinct functional profile of Th(c)17 cells. Our findings suggest a functional disequilibrium of T-cell subsets in SLE which may contribute to the inflammatory process and disease pathogenesis

Perfil socioeconômico, demográfico e obstétrico de gestantes com Síndrome Hipertensiva de uma maternidade pública

Objetivo: Descrever o perfil socioeconômico, demográfico e obstétrico de gestantescom Síndrome Hipertensiva Gestacional.Métodos: Estudo descritivo e correlacional, realizado em unidade Maternidade EscolaAssis Chateaubriand, com 120 gestantes, mediante questionário analisado por estatísticadescritiva e analítica.Resultados: Prevaleceu gestantes com hipertensão crônica (60,83%). Quanto ao perfilsocioeconômico e demográfico, prevaleceram gestantes com idade média de 30,9 ± 6,9anos, católicas, pardas, com vínculo empregatício, união estável, ensino médiocompleto e renda até R$ 954,00 reais. Quanto ao perfil obstétrico, eram gestantes comÍndice de Massa Corporal até 66, pressão arterial levemente elevada, média de cincoconsultas pré-natais, duas gestações, um parto e nenhum aborto. As mulheres comhipertensão crônica eram mais velhas (p=0,0024), tinham menor idade gestacional(p=0,0219) e maior número de abortos (p=0,0140).Conclusões: as gestantes apresentam sobrepeso/obesidade, idade média de 30,9 anos evulnerabilidade social. As gestantes com hipertensão arterial crônica, são mais velhas ecom maior número de abortos.Palavras-chave: Enfermagem. Epidemiologia. Saúde da mulher. Hipertensão induzidapela gravidez

Telhado verde: desempenho do sistema construtivo na redução do escoamento superficial

Na atualidade, metade da população mundial reside em centros urbanos, e os impactos negativos decorrentes de eventos hidrológicos têm sido recorrentes, visto que, com o aumento da impermeabilização, há redução nas taxas de infiltração, levando à diminuição da recarga dos aquíferos e à diminuição do escoamento de base. Consequentemente, o escoamento superficial é intensificado, aumentando a frequência e a magnitude dos picos de cheia, o que pode resultar na ocorrência de inundações, especialmente nos centros urbanos. Por outro lado, verifica-se já há algum tempo o emprego de telhados verdes em várias partes do mundo para contribuição arquitetônica estética e melhoria do conforto ambiental. Além desses enfoques, esta solução vem sendo tratada, também, como uma estrutura de controle do escoamento pluvial. Considerando características reais e os dados obtidos em campo, foi realizada a simulação da dinâmica da água em dois telhados verdes, com o emprego do código computacional Hydrus-1D, para diferentes intensidades de precipitação, visando verificar o desempenho desse sistema construtivo na redução do escoamento superficial. Os hidrogramas de saída mostraram-se qualitativamente adequados e quantitativamente coerentes e pode-se concluir que os telhados verdes constituem importantes dispositivos no amortecimento do escoamento superficial oriundo dos telhados, para as condições climáticas da área investigada

a hospital-based cohort study

Publisher Copyright: © Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.OBJECTIVES: Healthcare workers (HCWs) were the first to be prioritised for COVID-19 vaccination. This study aims to estimate the COVID-19 vaccine effectiveness (VE) against SARS-CoV-2 symptomatic infection among HCWs in Portuguese hospitals. DESIGN: Prospective cohort study. SETTING AND PARTICIPANTS: We analysed data from HCWs (all professional categories) from three central hospitals: one in the Lisbon and Tagus Valley region and two in the central region of mainland Portugal, between December 2020 and March 2022. VE against symptomatic SARS-CoV-2 infection was estimated as one minus the confounder adjusted HRs by Cox models considering age group, sex, self-reported chronic disease and occupational exposure to patients diagnosed with COVID-19 as adjustment variables. RESULTS: During the 15 months of follow-up, the 3034 HCWs contributed a total of 3054 person-years at risk, and 581 SARS-CoV-2 events occurred. Most participants were already vaccinated with a booster dose (n=2653, 87%), some are vaccinated with only the primary scheme (n=369, 12.6%) and a few remained unvaccinated (n=12, 0.4%) at the end of the study period. VE against symptomatic infection was 63.6% (95% CI 22.6% to 82.9%) for HCWs vaccinated with two doses and 55.9% (95% CI -1.3% to 80.8%) for HCWs vaccinated with one booster dose. Point estimate VE was higher for individuals with two doses taken between 14 days and 98 days (VE=71.9%; 95% CI 32.3% to 88.3%). CONCLUSION: This cohort study found a high COVID-19 VE against symptomatic SARS-CoV-2 infection in Portuguese HCWs after vaccination with one booster dose, even after Omicron variant occurrence. The small sample size, the high vaccine coverage, the very low number of unvaccinated individuals and the few events observed during the study period contributed to the low precision of the estimates.publishersversionpublishe

Graphic interpretation and ventilatory monitoring: the knowledge of physiotherapist

Objetivos: avaliar o conhecimento do fisioterapeuta em relação à interpretação gráfica e à monitorização ventilatória. Métodos: estudo quantitativo realizado entre março e abril de 2018, em três hospitais de Fortaleza/CE, com 41 fisioterapeutas intensivistas sobre seu conhecimento no que diz respeito à interpretação gráfica e monitorização ventilatória, bem como sua abordagem terapêutica frente aos achados clínicos. Os dados foram colhidos por meio de um questionário com 10 questões objetivas e analisados por meio do software Statistical Package for Social Sciences (SPSS) 17.0. Resultados: a maioria dos fisioterapeutas tinham entre 6 e 10 anos de atuação na UTI (43,9%). Quando questionados sobre os conhecimentos básicos de pressão e parâmetros de normalidade do Índice de Oxigenação, 80,5% e 70,6% fisioterapeutas ofereceram respoatas corretasrespectivamente. Acerca do conhecimento de monitorização ventilatória, o conceito de drive pressure foi o que apresentou melhor índice de assertiva com 73,2% acertos. Já sobre a análise gráfica, a interpretação da apneia (87,8%) e a abordagem na auto-PEEP (58,5%) foram as questões com maior número de acertos. Conclusão: houve assertividades relevantes no tocante ao conhecimento dos conceitos básicos, da monitorização dos parâmetros ventilatórios e da interpretação gráfica por parte dos profissionais entrevistados, porém sugere-se que haja mais estudos sobre a temática por meio de uma estratégia de educação continuada a fim de gerar um maior suporte teórico-prático para os fisioterapeutas intensivistas.Objectives: To evaluate the knowledge of the physiotherapist in relation to the graphic interpretation and the ventilation monitoring. Methods: A quantitative study was performed between March and April 2018 in three hospitals in Fortaleza / CE, with 41 intensivist physiotherapists about their knowledge regarding graphic interpretation and ventilation monitoring, as well as their therapeutic approach to clinical findings. The data were collected through a questionnaire with 10 objective questions and analyzed through the software Statistical Package for Social Sciences (SPSS) 17.0. Results: The majority of physiotherapists had between 6 and 10 years of ICU performance 43.9%. When questioned about the basic knowledge of pressure and parameters of normality of the Oxygenation Index, 80.5% and 70.6% physiotherapists answered correctly. Regarding the knowledge of ventilation monitoring, the concept of drive pressure was the one that presented the best assertive index with 73.2% correct answers. On the graphical analysis, the interpretation of apnea 87.8% and the auto-PEEP approach with 58.5% were the questions with the highest number of correct answers. Conclusion: There were relevant assertions regarding the knowledge of the basic concepts, the monitoring of ventilation parameters and the graphic interpretation by the professionals interviewed, but it is suggested that there be more studies on the subject through a strategy of continuing education in order to generate more theoretical-practical support for intensivist physiotherapists

Proinflammatory and endothelial activation profiles in hospitalized COVID-19 patients: impact of arterial hypertension and previous treatment with renin-angiotensin-aldosterone inhibitors

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