Calcium mobilisation in human sperm and its effects on motility

Dissertação de mestrado em Biologia Celular e Molecular apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.O Ca2+ presente nos espermatozóides pode provenir de duas fontes: uma extracelular, mediada por canais de Ca2+ na membrana plasmática e uma intracelular devido à abertura de canais libertadores de Ca2+ em dois reservatórios de Ca2+ - o acrossoma e o envelope nuclear redundante (RNE). Existem dois tipos de canais membranares de Ca2+ presentes nos espermatozoides: uns responsáveis pelo seu influxo tais como CatSper, VOCC, SOC e CNG e outros pela sua dispersão para o meio envolvente como PMCA e NCX. Os reservatórios intracelulares de Ca2+ também possuem canais para o seu efluxo (IP3R e RyR) e para a dispersão de Ca2+ citoplasmática / inclusão nos reservatórios (SERCAs e SPCAs). Estudos em reservatórios, canais, bombas e permutadores de Ca2+ bem como as suas interacções internas e efeitos em actividades do espermatozoide requerem o uso de moduladores farmacológicos quer para inibir ou induzir transporte de Ca2+ ou simplesmente afectar directamente a mobilidade ou reacção acrossómica do espermatozóide. No presente estudo, seis moduladores foram usados por forma a esclarecer a intercomunicação entre CatSper e os reservatórios intracelulares de Ca2+ (especialmente o RNE) e os seus efeitos na motilidade do espermatozóide humano; progesterona - um indutor geral da concentração de Ca2+ intracelular), Mibefradil dihydrochloride hydrate e NNC 55-0396 dihydrochloride - inibidores de CatSper e, consequentemente, influxo de Ca2+, thimerosal e 4-aminopyridine - mobilizadores de Ca2+ armazenado e indutores de influxo de Ca2+ e trimethylamine hydrochloride - um indutor alcalino do influxo de Ca2+. Com este trabalho, foi possível concluir que o TMA induz eficientemente o influxo de Ca2+ através de CatSper, enquanto que o Mibefradil e o NNC actuam negativamente nestes canais, interferindo com o influxo bem como a resposta induzida pela progesterona; ademais, o thimerosal e a 4-aminopyridine causam um aumento do Ca2+ intracelular que parece ser parcialmente mediado pelos reservatórios de Ca2+, nomeadamente o RNE. Nos estudos de motilidade, os espermatozóides tratados com TMA, Mibefradil e NNC apresentaram uma mudança no seu movimento: velocidade aumentada quando estimulados com TMA e Mibefradil e velocidade VI diminuída com NNC. As diferenças encontradas entre o controlo (com sEBSS) e o tratamento com thimerosal ou 4-aminopyridine não foram significativas, segundo o teste t de Student. Assim sendo, existe uma intercomunicação relevante entre CatSper e reservatórios intracelulares de Ca2+ que previne a descida de Ca2+ intracelular para níveis anormais e também afecta a mobilidade espermática, quando interrompida.Ca2+ in sperm can come from two sources: an extracellular one, mediated by plasma membrane Ca2+ channels and an intracellular one due to the opening of Ca2+-release channels in the two Ca2+ stores - the acrosome and the RNE. There are two types of plasma membrane channels present in sperm: the ones responsible for Ca2+ influx such as CatSper, VOCC, SOC and CNG and those accountable for Ca2+ clearance to the surrounding medium like PMCA and NCX. Intracellular Ca2+ stores also possess channels for Ca2+ efflux (IP3R and RyR) and for cytoplasmic Ca2+ clearance/inclusion into the stores (SERCAs and SPCAs). Studies of Ca2+ stores, channels, pumps and exchangers as well as its internal interactions and effects on sperm activities require the use of pharmacological modulators either to inhibit or induce Ca2+ transportation or simply affect directly sperm motility or acrosome reaction. In the present work, six modulators were used in order to clarify the intercommunication between CatSper channels and intracellular Ca2+ stores (especially the RNE) and its effects on human sperm motility. Progesterone - a general inducer of [Ca2+]i, Mibefradil dihydrochloride hydrate and NNC 55-0396 dihydrochloride - inhibitors of CatSper and thus Ca2+ influx, thimerosal and 4-aminopyridine - mobilisers of stored Ca2+ and inducers of Ca2+ influx and trimethylamine hydrochloride – an alkaline inducer of Ca2+ influx. With this work, it was possible to conclude that TMA efficiently induces Ca2+ influx through CatSper whilst Mibefradil and NNC act negatively on them hence interfering with the influx and also with the progesterone-induced response; moreover, thimerosal and 4-aminopyridine cause a rise of intracellular Ca2+ which appears to be partially mediated by intracellular Ca2+ stores, namely RNE. With the motility studies, TMA, Mibefradil and NNC-treated cells presented a change in swimming pattern: increased velocity when stimulated with TMA and Mibefradil and decreased velocity when with NNC. No statistical significance was attributed to the differences seen between thimerosal and 4-aminopyridine treatment the control (with sEBSS), accordingly to test t of Student. Therefore, there is a relevant intercommunication between CatSper and ICS that prevents the decrease of intracellular Ca2+ to abnormal levels and also affects sperm motility, when interrupted

Effect of bio-functional MAO layers on the electrochemical behaviour of highly porous Ti

Ti foams are attractive for orthopaedic applications due to reduced Young's modulus and ability of bone ingrowth. However, poor corrosion behaviour and lack of bioactivity are yet to be overcome. In the present work, highly porous Ti samples were processed by powder metallurgy with space holder technique and bio-functionalized by micro-arc oxidation, resulting in nano/micro structured TiO2 surfaces containing bioactive elements. The electrochemical behaviour of these bio-functionalized highly porous Ti surfaces was evaluated through potentiodynamic polarization and EIS in physiological solution at body temperature. Results showed that bio-functionalization improved the corrosion behaviour of highly porous Ti. However, increased macro-porosity led to an increased corrosion rate.This work is supported by FCT with the reference project UID/EEA/04436/2019, M-ERA NET/0001/2015 project, and Programa de Accoes Universitarias Integradas Luso-Francesas' (PAUILF TC-12_14). CQE is financed by FCT under Pluriannual contract 2020-2023. The authors also gratefully acknowledge the "Investissements d'avenir" programs (nos. ANR-11-IDEX-0003-02 and ANR-10-EQPX-37 MATMECA Grant) for financial support on Micro-CT analysis

Resistance profile of osimertinib in pre-treated patients with EGFR T790M-mutated non-small cell lung cancer

Background: Osimertinib efficacy in pre-treated patients with epidermal growth factor receptor (EGFR) T790M-mutated non-small cell lung cancer (NSCLC) has been demonstrated in clinical trials, but real-world data, particularly regarding resistance profile, remains limited. This study aims to analyze the resistance mechanisms acquired after treatment with Osimertinib. Methods: Clinical outcomes and molecular results from re-biopsies at the time of osimertinib progression of EGFR T790M-mutated NSCLC patient were analyzed. Results: Twenty-one patients with stage IV adenocarcinoma were included [median 69 years; 57.1% female; 85.7% never-smokers; 23.8% ECOG performance status (PS) >= 2]. Median PFS and OS were 13.4 (95% CI: 8.0-18.9) and 26.4 (95% IC: 8.9-43.8) months, respectively. At the time of analysis, 10 patients had tumor progression (47.6%). T790M loss occurred in 50%, being associated with earlier progression (median PFS 8.1 vs. 21.4 months, p = 0.011). Diverse molecular alterations were identified, including C797S mutation (n = 1), PIK3CA mutation (n = 2), MET amplification (n = 1), CTNNB1 mutation (n = 1), and DCTN1-ALK fusion (n = 1). Histological transformation into small cell carcinoma occurred in one patient. Conclusions: This real-world life study highlights the relevance of re-biopsy at the time of disease progression, contributing to understand resistance mechanisms and to guide treatment strategies

A Cross-Sectional Survey-Based Study of Medical Oncologists

Funding Information: We would like to thank Andrea Bothwell who wrote the manuscript outline and first draft on behalf of Springer Healthcare Communications. We also thank Prof. Carina Silva (ESTEsL – Escola Superior de Tecnologias de Saúde de Lisboa) who performed the preliminary statistical analysis of this study. This medical writing assistance and statistical analysis was funded by CUF Oncologia. Funding Information: Diogo Alpuim Costa has received honoraria from the Portuguese Navy, CUF Oncologia, and NTT DATA, and has served as a speaker, advisory board member, or has received research or education funding from CUF Oncologia, AstraZeneca, Hoffmann-La Roche, Merck KGaA, Novartis, Pfizer, Uriage, Daiichi Sankyo, Gilead, Merck Sharp & Dohme, Nanobiotix, Puma Biotechnology Inc., Sanofi, and Seagen Inc. Margarida Brito has participated as advisory board member for Roche, Novartis, Merck Sharp & Dohme, and Pfizer. Mário Fontes-Sousa has served as a speaker or advisory board member for Bristol Myers Squibb, Daiichi Sankyo, Gilead, Lilly, Merck Sharp & Dohme, Novartis, Pfizer, Roche, and Servier. Diogo Martins-Branco received honoraria and advisory board fees from Janssen, Pfizer, Merck Sharp & Dohme, Angelini, AstraZeneca, and Novartis, meeting and travel grants from LEO Farmacêuticos, Merck Sharp & Dohme, Ipsen, Janssen, and Roche, and institutional grants from F. Hoffmann-La Roche Ltd. José Guilherme Gonçalves Nobre, João Paulo Fernandes, Marta Vaz Batista, Ana Simas, Carolina Sales, Helena Gouveia, Leonor Abreu Ribeiro, Andreia Coelho, Mariana Inácio, André Cruz, Mónica Mariano, Joana Savva-Bordalo, Ricardo Fernandes, André Oliveira, Andreia Chaves, Mafalda Sampaio-Alves, and Noémia Afonso have nothing to declare. Publisher Copyright: © 2022, The Author(s).Introduction: Cancer care providers have faced many challenges in delivering safe care for patients during the COVID-19 pandemic. This cross-sectional survey-based study investigated the impact of the pandemic on clinical practices of Portuguese medical oncologists caring for patients with breast cancer. Methods: An anonymous online survey comprising 42 questions gathered information regarding COVID-19 testing, treatment in (neo)adjuvant and metastatic settings, and other aspects of breast cancer management. Practices before and during the pandemic were compared, and potential differences in outcomes according to respondents’ regions, case volumes, and practice type were explored. Results: Of 129 respondents, 108 worked in the public health system, giving a representative national picture of the impact of the COVID-19 pandemic on breast cancer management. Seventy-one percent of respondents reported a reduction in visits for new cases of breast cancer, and there was a shift towards increased use of telemedicine. Clinical decision-making was largely unaffected in the most aggressive indications (i.e., triple-negative, HER2-positive, visceral crisis). The use of neoadjuvant therapy increased when access to surgery was difficult, whereas dose-dense regimens decreased, and cyclin-dependent kinase 4/6 inhibitor treatment decreased for less aggressive disease and increased for more aggressive disease. The use of oral formulations and metronomic chemotherapy regimens increased, and clinical trial participation decreased. Some differences by respondents’ region and case volume were noted. Conclusion: Medical oncologists in Portugal implemented many changes during the COVID-19 pandemic, most of which were logical and reasonable responses to the current healthcare emergency; however, the true impact on patient outcomes remains unknown.publishersversionepub_ahead_of_prin

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Management of Costa Vicentina as tourism destination for foreign surf practitioner and beginners

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and EconomicsBeing a country with such remarkable conditions for tourism activity and a strong contribution of this sector to the national GDP, Portugal, has also been growing fast in the attraction of Surf practitioners and beginners from all over the world, due to its amazing conditions for all surf levels and to its large extent of shore. Costa Vicentina is an example of how surf can be an attraction to visitants. In this project I will analyze this region, study the segment “foreign surf beginners and practitioners”, and we will see how Costa Vicentina can be improved in terms of tourism destination management, regarding this specific target

Effects of single vs mixed EDCs in mouse testes

11 p.-5 fig.-2 tab.Endocrine-disrupting chemicals (EDCs) generate reproductive dysfunctions affecting the biosynthesis of steroid hormones and genes of the steroidogenic pathway. EDCs effects are mainly reported as a result of exposure to single compounds. However, humans are environmentally exposed to a mixture of EDCs. Herein, we assess chronic exposure to single alkylphenols and phthalates versus a mixture in mouse testes histology and steroidogenesis. Pregnant mice were exposed through drinking water to: 0.3 mg/Kg-body weight (BW)/day of each phthalate (DEHP, DBP, BBP), 0.05 mg/Kg-BW/day of each alkylphenol (NP, OP), or their mixture, covering from 0.5 post-coital day to weaning, continuing in the male offspring each exposure until adulthood (60 days old). Body and relative testis weight were increased in mixture-exposed mice along with histological alterations. Intratesticular testosterone (T) changed only in mice exposed to DBP, whereas estradiol (E2) levels were altered in all groups (except BBP). mRNA levels of genes encoding hormones of the steroid pathway (Cyp11a1, Hsd3b1, Cyp17a1 and Cyp19a1), cholesterol transporters (Star), and transcriptional factors (Sp1) showed that mice exposed to single or mixed compounds had alterations in at least two transcripts. However, none of the different types of exposure induced changes in all transcripts. In addition, changes at the mRNA or protein levels with single compounds were not always the same as those with a mixture. In conclusion, the effects of a chronic exposure to a mixture of EDCs on the expression of genes and proteins of the steroidogenic pathway and hormonal status were different from those exposed to single EDC.This work was supported by grants from FONDECYT (1150352) and CONICYT (21120505),Chile, and MINECO (BFU2013-42164-R), Spain.Peer reviewe

Cuidar a educação em São Tomé e Príncipe: primeiros estudos de uma rede de investigação

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