Estudo da fragmentação do DNA em espermatozoides humanos após separação por MACS

Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2014O espermograma é uma ferramenta fundamental no diagnóstico da infertilidade masculina. Neste exame são avaliados diversos parâmetros dos espermatozoides, como a concentração, motilidade, morfologia, vitalidade e integridade da membrana. Contudo, o espermograma não avalia a integridade do DNA dos espermatozoides, e danos nesta estrutura têm sido relacionados a falhas nos tratamentos de Reprodução Medicamente Assistida (RMA). Com o objetivo de isolar espermatozoides livres de danos no DNA, a técnica Magnetic-Activated Cell Sorting (MACS) foi aplicada para complementar as técnicas clássicas de preparação dos espermatozoides para RMA. No presente trabalho foram processadas 100 amostras de sémen de acordo com diferentes combinações de MACS e técnicas de preparação dos espermatozoides clássicas, incluindo a centrifugação por gradientes de densidade (DGC) e o swim-up (SU). O objetivo consistiu em verificar se, após os protocolos aplicados, ocorria uma diminuição significativa do número de espermatozoides com fragmentação do DNA (sDNAfrag), e determinar qual o protocolo mais eficiente nessa redução. Os resultados obtidos mostraram que todos os grupos apresentaram uma redução significativa da sDNAfrag, com a maior taxa de redução (83,3 ± 15,4%) a verificar-se com o protocolo MACS-DGC-SU. Com este protocolo, verificou-se que pacientes com valores diminuídos para a motilidade progressiva, vitalidade e integridade da membrana dos espermatozoides alcançaram uma maior redução de sDNAfrag. Além disso, pacientes com valores abaixo dos valores de referência para a motilidade progressiva rápida, ou para a motilidade progressiva rápida e morfologia normal, obtiveram também uma maior redução de sDNAfrag do que pacientes com valores abaixo dos valores de referência para a morfologia normal. Tendo em conta estes resultados, a técnica MACS demonstra potencial para otimizar a taxa de redução de sDNAfrag, quando aplicada antes da DGC e do SU, especialmente em amostras de sémen cujos espermatozoides apresentem motilidade progressiva, vitalidade e integridade da membrana diminuídas.Spermiogram analysis is a fundamental tool in the diagnosis of male infertility that evaluates several sperm parameters such as concentration, motility, morphology, vitality and membrane integrity. However, it does not evaluate sperm DNA integrity and sperm DNA injuries have been related to Assisted Reproduction Treatment failures. To isolate sperm without DNA damage, Magnetic-Activated Cell Sorting (MACS) was employed as an adjunct of the classic sperm preparation techniques. In the present study 100 semen samples were processed according to different combinations of MACS with other classic sperm preparation techniques that included density gradient centrifugation (DGC) and swim-up (SU) methods. The aim of the study was to evaluate whether there was a significant decrease in the number of sperm with fragmented DNA (sDNAfrag) after the protocol applied and verify which protocol was the most efficient in reducing sDNAfrag. The results showed that all groups presented a significant decrease in sDNAfrag, with the highest reduction rate (83.3 ± 15.4%) being obtained with the protocol MACS-DGC-SU. With this protocol, patients with worst values in the semen regarding sperm progressive motility, vitality and membrane integrity achieved the best sDNAfrag reduction. Moreover, patients whose semen presented values above the reference for rapid progressive motility, or rapid progressive motility and normal morphology, showed a higher sDNAfrag reduction rate than patients with values above the reference value for normal morphology. Based on the results obtained, MACS showed potential to optimize the sDNAfrag reduction rate when applied before DGC and SU, especially in semen samples with low progressive motility, vitality and membrane integrity

Hemogram as marker of in-hospital mortality in COVID-19.

The clinical impact of COVID-19 disease calls for the identification of routine variables to identify patients at increased risk of death. Current understanding of moderate-to-severe COVID-19 pathophysiology points toward an underlying cytokine release driving a hyperinflammatory and procoagulant state. In this scenario, white blood cells and platelets play a direct role as effectors of such inflammation and thrombotic response. We investigate whether hemogram-derived ratios such as neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio and the systemic immune-inflammation index may help to identify patients at risk of fatal outcomes. Activated platelets and neutrophils may be playing a decisive role during the thromboinflammatory phase of COVID-19 so, in addition, we introduce and validate a novel marker, the neutrophil-to-platelet ratio (NPR). Two thousand and eighty-eight hospitalized patients with COVID-19 admitted at any of the hospitals of HM Hospitales group in Spain, from March 1 to June 10, 2020, were categorized according to the primary outcome of in-hospital death. Baseline values, as well as the rate of increase of the four ratios analyzed were significantly higher at hospital admission in patients who died than in those who were discharged (p<0.0001). In multivariable logistic regression models, NLR (OR 1.05; 95% CI 1.02 to 1.08, p=0.00035) and NPR (OR 1.23; 95% CI 1.12 to 1.36, p<0.0001) were significantly and independently associated with in-hospital mortality. According to our results, hemogram-derived ratios obtained at hospital admission, as well as the rate of change during hospitalization, may easily detect, primarily using NLR and the novel NPR, patients with COVID-19 at high risk of in-hospital mortality.pre-print300 K

Alignment and Proficiency of Virgin Olive Oil Sensory Panels: The OLEUM Approach

A set of 334 commercial virgin olive oil (VOO) samples were evaluated by six sensory panels during the H2020 OLEUM project. Sensory data were elaborated with two main objectives: (i) to classify and characterize samples in order to use them for possible correlations with physical\u2013chemical data and (ii) to monitor and improve the performance of panels. After revision of the IOC guidelines in 2018, this work represents the first published attempt to verify some of the recommended quality control tools to increase harmonization among panels. Specifically, a new \u201cdecision tree\u201d scheme was developed, and some IOC quality control procedures were applied. The adoption of these tools allowed for reliable classification of 289 of 334 VOOs; for the remaining 45, misalignments between panels of first (on the category, 21 cases) or second type (on the main perceived defect, 24 cases) occurred. In these cases, a \u201cformative reassessment\u201d was necessary. At the end, 329 of 334 VOOs (98.5%) were classified, thus confirming the eectiveness of this approach to achieve a better proficiency. The panels showed good performance, but the need to adopt new reference materials that are stable and reproducible to improve the panel\u2019s skills and agreement also emerged

Naive and Stem Cell Memory T Cell Subset Recovery Reveals Opposing Reconstitution Patterns in CD4 and CD8 T Cells in Chronic Graft vs. Host Disease

The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of hematological malignancies remains hampered by life-threatening chronic graft vs. host disease (cGVHD). Although multifactorial in nature, cGVHD has been associated with imbalances between effector and regulatory T cells (Treg). To further elucidate this issue, we performed a prospective analysis of patients undergoing unrelated donor allo-HSCT after a reduced intensity conditioning (RIC) regimen containing anti-thymocyte globulin (ATG) and the same GVHD prophylaxis, at a single institution. We studied T cell subset homeostasis over a 24-month follow-up after HSCT in a comparative analysis of patients with and without cGVHD. We also quantified naive and memory T cell subsets, proliferation and expression of the apoptosis-related proteins Bcl-2 and CD95. Finally, we assessed thymic function by T cell receptor excision circle (TREC) quantification and T cell receptor (TCR) diversity by TCRVβ spectratyping. While the total number of conventional CD4 (Tcon) and CD8 T cells was similar between patient groups, Treg were decreased in cGVHD patients. Interestingly, we also observed divergent patterns of Naive and Stem Cell Memory (SCM) subset recovery in Treg and Tcon compared to CD8. Patients with cGVHD showed impaired recovery of Naive and SCM Tcon and Treg, but significantly increased frequencies and absolute numbers of Naive and SCM were observed in the CD8 pool. Markedly increased EMRA CD8 T cells were also noted in cGVHD. Taken together, these results suggest that Naive, SCM and EMRA CD8 play a role in the emergence of cGHVD. Reduced Naive and recent thymic emigrant Tcon and Treg in cGVHD was likely due to impaired thymic output, as it was accompanied by decreased CD4 TREC and TCR diversity. On the other hand, CD8 TCR diversity was similar between patient groups. Furthermore, no correlation was observed between CD8 TREC content and Naive CD8 numbers, suggesting limited thymic production of Naive CD8 T cells in patients after transplant, especially in those developing cGVHD. The mechanisms behind the opposing patterns of CD4 and CD8 subset cell recovery in cGVHD remain elusive, but may be linked to thymic damage associated with the conditioning regimen and/or acute GVHD

Copper-64 Chloride Exhibits Therapeutic Potential in Three-Dimensional Cellular Models of Prostate Cancer

Prostate cancer (PCa) is the second most common cancer type in men, and in advanced metastatic stages is considerable incurable. This justifies the need for efficient early diagnostic methods and novel therapies, particularly radiopharmaceuticals with the potential for simultaneous diagnosis and therapy (theranostics). We have previously demonstrated, using monolayer-cultured cells, that copper-64 chloride, a promising theranostic agent for PCa, has the potential to induce significant damage in cancer cells while having minimal side effects in healthy tissues. Here, we further explored this compound for its theranostic applications using more advanced PCa cellular models, specifically multicellular spheroids. Namely, we evaluated the cellular uptake of 64CuCl2 in three human PCa spheroids (derived from 22RV1, DU145, and LNCaP cells), and characterized the growth profile and viability of those spheroids as well as the clonogenic capacity of spheroid-derived cells after exposure to 64CuCl2. Furthermore, the populations of cancer stem cells (CSCs), known to be important for cancer resistance and recurrence, present in the spheroid models were also evaluated using two different markers (CD44 and CD117). 64CuCl2 was found to have significant detrimental effects in spheroids and spheroid-derived cells, being able to reduce their growth and impair the viability and reproductive ability of spheroids from both castration-resistant (22RV1 and DU145) and hormone-naïve PCa (LNCaP). Interestingly, resistance to 64CuCl2 treatment seemed to be related with the presence of a CSC population, since the most resistant spheroids, derived from the DU145 cell line, had the highest initial percentage of CSCs among the three cell lines under study. Altogether, these results clearly highlight the theranostic potential of 64CuCl2

Evaluation of the theranostic potential of [64Cu]CuCl2 in glioblastoma spheroids

Abstract Background Glioblastoma is an extremely aggressive malignant tumor with a very poor prognosis. Due to the increased proliferation rate of glioblastoma, there is the development of hypoxic regions, characterized by an increased concentration of copper (Cu). Considering this, 64Cu has attracted attention as a possible theranostic radionuclide for glioblastoma. In particular, [64Cu]CuCl2 accumulates in glioblastoma, being considered a suitable agent for positron emission tomography. Here, we explore further the theranostic potential of [64Cu]CuCl2, by studying its therapeutic effects in advanced three-dimensional glioblastoma cellular models. First, we established spheroids from three glioblastoma (T98G, U373, and U87) and a non-tumoral astrocytic cell line. Then, we evaluated the therapeutic responses of spheroids to [64Cu]CuCl2 exposure by analyzing spheroids' growth, viability, and cells' proliferative capacity. Afterward, we studied possible mechanisms responsible for the therapeutic outcomes, including the uptake of 64Cu, the expression levels of a copper transporter (CTR1), the presence of a cancer stem cell population, and the production of reactive oxygen species (ROS). Results Results revealed that [64Cu]CuCl2 is able to significantly reduce spheroids' growth and viability, while also affecting cells' proliferation capacity. The uptake of 64Cu, the presence of cancer stem-like cells and the production of ROS were in accordance with the therapeutic response. However, expression levels of CTR1 were not in agreement with uptake levels, revealing that other mechanisms could be involved in the uptake of 64Cu. Conclusions Overall, our results further support [64Cu]CuCl2 potential as a theranostic agent for glioblastoma, unveiling potential mechanisms that could be involved in the therapeutic response