Faculdade de Medicina
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- 3DCellPol: joint detection and pairing of cell structures to compute cell polarityPublication . Narotamo, Hemaxi; Franco, Cláudio A.; Silveira, MargaridaCell polarity is essential for tissue structure and cell migration, and its dysregulation is linked to diseases such as cancer and vascular disorders. Understanding the associations between molecular mechanisms, such as genetic defects, and abnormal cell polarization can provide clinicians with valuable biomarkers for early disease diagnosis and lead to more targeted therapeutic interventions. Here, we present a deep-learning framework for cell polarity computation based on the association between pairs of objects. Our approach, named 3DCellPol, is trained to detect and group the centroids of two distinct objects. To demonstrate the potential of 3DCellPol, we use it to compute cell polarity by pairing two cell organelles: nuclei and Golgi. The vectors between nuclei and Golgi define the front-rear polarity axis in endothelial cells. 3DCellPol was evaluated on 3D microscopy images of mouse retinas. It detected 71% of the nucleus–Golgi vectors and outperformed previous methods while requiring much less supervision. Moreover, incorporating synthetic data generated by a generative adversarial network further improved detection to 78%. We additionally demonstrated our model's adaptability to 2D images by applying it to a public dataset of cervical cytology images, where polarity is defined based on the cytoplasm-nucleus vectors. In this dataset, our model detected over 90% of vectors. 3DCellPol's ability to robustly compute cell polarity is crucial for understanding mechanisms of diseases where abnormal polarity plays a key role, and it may contribute to improved diagnostics and enable targeted therapies. Hence, it is a valuable open-source tool for both biomedical research and clinical practice.
- 3DVascNet: an automated software for segmentation and quantification of mouse vascular networks in 3DPublication . Narotamo, Hemaxi; Silveira, Margarida; Franco, Cláudio A.BACKGROUND: Analysis of vascular networks is an essential step to unravel the mechanisms regulating the physiological and pathological organization of blood vessels. So far, most of the analyses are performed using 2-dimensional projections of 3-dimensional (3D) networks, a strategy that has several obvious shortcomings. For instance, it does not capture the true geometry of the vasculature and generates artifacts on vessel connectivity. These limitations are accepted in the field because manual analysis of 3D vascular networks is a laborious and complex process that is often prohibitive for large volumes. METHODS: To overcome these issues, we developed 3DVascNet, a deep learning–based software for automated segmentation and quantification of 3D retinal vascular networks. 3DVascNet performs segmentation based on a deep learning model, and it quantifies vascular morphometric parameters such as vessel density, branch length, vessel radius, and branching point density. We tested the performance of 3DVascNet using a large data set of 3D microscopy images of mouse retinal blood vessels. RESULTS: We demonstrated that 3DVascNet efficiently segments vascular networks in 3D and that vascular morphometric parameters capture phenotypes detected by using manual segmentation and quantification in 2 dimension. In addition, we showed that, despite being trained on retinal images, 3DVascNet has high generalization capability and successfully segments images originating from other data sets and organs. CONCLUSIONS: Overall, we present 3DVascNet, a freely available software that includes a user-friendly graphical interface for researchers with no programming experience, which will greatly facilitate the ability to study vascular networks in 3D in health and disease. Moreover, the source code of 3DVascNet is publicly available, thus it can be easily extended for the analysis of other 3D vascular networks by other users.
- A 96-wells fluidic system for high-throughput screenings under laminar high wall shear stress conditionsPublication . Fonseca, Catarina Gonçalves; Silvério, Vânia; Barata, David; Giese, Wolfgang; Gerhardt, Holger; Cardoso, Susana; Franco, Cláudio AreiasThe ability of endothelial cells to respond to blood flow is fundamental for the correct formation and maintenance of a functional and hierarchically organized vascular network. Defective flow responses, in particular related to high flow conditions, have been associated with atherosclerosis, stroke, arteriovenous malformations, and neurodegenerative diseases. Yet, the molecular mechanisms involved in high flow response are still poorly understood. Here, we described the development and validation of a 96-wells fluidic system, with interchangeable cell culture and fluidics, to perform high-throughput screenings under laminar high-flow conditions. We demonstrated that endothelial cells in our newly developed 96-wells fluidic system respond to fluid flow-induced shear stress by aligning along the flow direction and increasing the levels of KLF2 and KLF4. We further demonstrate that our 96-wells fluidic system allows for efficient gene knock-down compatible with automated liquid handling for high-throughput screening platforms. Overall, we propose that this modular 96-well fluidic system is an excellent platform to perform genome-wide and/or drug screenings to identify the molecular mechanisms involved in the responses of endothelial cells to high wall shear stress. [Figure not available: see fulltext.].
- Acromegaly in humans and cats: pathophysiological, clinical and management resemblances and differencesPublication . Lopes-Pinto, Mariana; Marques, Patrícia Lunet; Lacerda-Nobre, Ema; Miceli, Diego; Leal, Rodolfo Oliveira; Marques, PedroObjective: Acromegaly is a disorder associated with excessive levels of growth hormone (GH) and insulin-like growth factor-1 (IGF-1). In general, GH/IGF-1 excess leads to morphologic craniofacial and acral changes as well as cardiometabolic complications, but the phenotypic changes and clinical presentation of acromegaly differ across species. Here, we review the pathophysiology, clinical presentation and management of acromegaly in humans and cats, and we provide a systematic comparison between this disease across these different species. Design: A comprehensive literature review of pathophysiology, clinical features, diagnosis and management of acromegaly in humans and in cats was performed. Results: Acromegaly is associated with prominent craniofacial changes in both species: frontal bossing, enlarged nose, ears and lips, and protuberant cheekbones are typically encountered in humans, whereas increased width of the head and skull enlargement are commonly found in cats. Malocclusion, prognathism, dental diastema and upper airway obstruction by soft tissue enlargement are reported in both species, as well as continuous growth and widening of extremities resulting in osteoarticular compromise. Increase of articular joint cartilage thickness, vertebral fractures and spine malalignment is more evident in humans, while arthropathy and spondylosis deformans may also occur in cats. Generalized organomegaly is equally observed in both species. Other similarities between humans and cats with acromegaly include heart failure, ventricular hypertrophy, diabetes mellitus, and an overall increased cardiometabolic risk. In GH-secreting pituitary tumours, local compressive effects and behavioral changes are mostly observed in humans, but also present in cats. Cutis verticis gyrata and skin tags are exclusively found in humans, while palmigrade/plantigrade stance may occur in some acromegalic cats. Serum IGF-1 is used for acromegaly diagnosis in both species, but an oral glucose tolerance test with GH measurement is only useful in humans, as glucose load does not inhibit GH secretion in cats. Imaging studies are regularly performed in both species after biochemical diagnosis of acromegaly. Hypophysectomy is the first line treatment for humans and cats, although not always available in veterinary medicine. Conclusion: Acromegaly in humans and cats has substantial similarities, as a result of common pathophysiological mechanisms, however species-specific features may be found.
- Aedes albopictus arrives in Lisbon: an emerging public health threatPublication . Nazareth, Teresa; Seixas, Gonçalo; Lourenço, José; Bettencourt, Paulo J. G.
- Aerocyte specification and lung adaptation to breathing is dependent on alternative splicing changesPublication . Fidalgo, Marta F.; Fonseca, Catarina G.; Caldas, Paulo; Raposo, Alexandre A. S. F.; Balboni, Tania; Henao-Mišíková, Lenka; Grosso, Ana R.; Vasconcelos, Francisca F.; Franco, Cláudio A.Adaptation to breathing is a critical step in lung function and it is crucial for organismal survival. Alveoli are the lung gas exchange units and their development, from late embryonic to early postnatal stages, requires feedbacks between multiple cell types. However, how the crosstalk between the alveolar cell types is modulated to anticipate lung adaptation to breathing is still unclear. Here, we uncovered a synchronous alternative splicing switch in multiple genes in the developing mouse lungs at the transition to birth, and we identified hnRNP A1, Cpeb4, and Elavl2/HuB as putative splicing regulators of this transition. Notably, we found that Vegfa switches from the Vegfa 164 isoform to the longer Vegfa 188 isoform exclusively in lung alveolar epithelial AT1 cells. Functional analysis revealed that VEGFA 188 (and not VEGFA 164) drives the specification of Car4-positive aerocytes, a subtype of alveolar endothelial cells specialized in gas exchanges. Our results reveal that the cell type-specific regulation of Vegfa alternative splicing just before birth modulates the epithelial-endothelial crosstalk in the developing alveoli to promote lung adaptation to breathing.
- An SNF2 helicase-like protein links mitotic transcription termination to sister chromatid resolutionPublication . Carmo, Catarina; Coelho, João; Silva, Rui; Tavares, Alexandra; Boavida, Ana; Gaetani, Paola; Martinho, Rui Gonçalo; Oliveira, Raquel A.Mitotic chromatin is largely assumed incompatible with transcription due to changes in the transcription machinery and chromosome architecture. However, the mechanisms of mitotic transcriptional inactivation and their interplay with chromosome assembly remain largely unknown. By monitoring ongoing transcription in Drosophila early embryos, we reveal that eviction of nascent mRNAs from mitotic chromatin occurs after substantial chromosome compaction and is not promoted by condensin I. Instead, we show that the timely removal of transcripts from mitotic chromatin is driven by the SNF2 helicase-like protein Lodestar (Lds), identified here as a modulator of sister chromatid cohesion defects. In addition to transcriptional termination, we uncovered that Lds cooperates with Topoisomerase 2 to ensure efficient sister chromatid resolution and mitotic fidelity. We conclude that mitotic transcriptional termination is not a passive consequence of cell cycle progression and/or chromosome compaction but occurs via dedicated mechanisms with functional parallelisms to sister chromatid resolution.
- And the quest continues…Publication . Mendes, Lígia
- Arrhythmic risk stratification in patients with left ventricular ring-like scarPublication . Parisi, Vanda; Graziosi, Maddalena; Lopes, Luis R.; Luca, Antonio De; Pasquale, Ferdinando; Tini, Giacomo; Targetti, Mattia; Cueto, Maria R.; Moura, Ana R.; Ditaranto, Raffaello; Torlasco, Camilla; Taglieri, Nevio; Nardi, Elena; Lovato, Luigi; Augusto, João B.; Galiè, Nazzareno; Crotti, Lia; Gasperetti, Alessio; Biffi, Mauro; Autore, Camillo; Merlo, Marco; Olivotto, Iacopo; Sinagra, Gianfranco; Elliott, Perry M.; Biagini, ElenaAims Left ventricular (LV) ring-like scar on cardiac magnetic resonance (CMR) has been linked to malignant arrhythmias in patients with non-ischaemic cardiomyopathy. This study aimed to perform a comprehensive evaluation of this phenotype and to identify risk factors for life-threatening arrhythmic events (LAEs), a composite of sudden cardiac death (SCD), aborted SCD, and sustained ventricular tachycardia. Methods and results One hundred and fifteen patients [median age 39 (interquartile range, IQR, 28–52), 42% females] were identified at 6 referral centres. Inclusion criteria were ring-like LV scar [≥3 contiguous segments with sub-epicardial/midwall late gadolinium enhancement (LGE) in the same slice] and one among: pathogenic/likely pathogenic genetic variant, family history for cardiomyopathy, or arrhythmogenic cardiomyopathy diagnosis. During the study follow-up, survival free from LAEs was 60% (3.8 events/100 patients/year); at a median follow-up of 4.6 years (IQR 1.7–8.4) it was 84%. On multivariable analysis, anterior Q waves [hazard ratio (HR): 1.030, 95% confidence intervals (CI): 1.014–1.046, P < 0.001], QRS width (HR: 4.642, 95% CI: 1.296–16.628, P = 0.018), and LV end-diastolic volume index (LVEDVi; HR: 1.011, 95% CI: 1.001–1.021, per mL/m2 increase, P = 0.040) were independently associated with LAEs; with good discrimination power (Harrell’s C-index = 0.796). Three risk categories were identified: normal electrocardiogram (ECG), abnormal ECG and no LAEs predictive variables, abnormal ECG and ≥1 LAEs predictive variables, with a decreasing survival from 100 to 65% and 49%, respectively (Log-rank test = 0.015). Conclusion In this study, the LV ring-like scar phenotype was associated with a high rate of malignant arrhythmias in presence of anterior Q waves, QRS prolongation, and increased LVEDVi. A normal ECG identified a lower risk sub-group.
- Arthroscopic cam resection reduces femoroacetabular contact pressure: a cadaver studyPublication . Dantas, Pedro; Gonçalves, S. B.; Gonçalves, S. R.; Mascarenhas, V.; Martins, J.; Silva, M. Tavares da; Consciência, J. GuimarãesAims Arthroscopic cam resection in femoroacetabular impingement syndrome leads to clinical improvement, but biomechanical studies on the efect of surgical intervention are scarce. In this study, we compared the femoroacetabular contact pressure (CP) in an intact cam morphology and after arthroscopic cam resection. The hypothesis was that arthroscopic cam resection decreases the femoroacetabular CP. Methods A cadaveric study was performed on nine hips with a cam morphology (? angle > 60°). CP was assessed using a new hip-specifc device and an intracranial pressure (ICP) sensor. These evaluations were performed during hip arthroscopy, in the intact joint and after cam resection, with the joint in diferent positions. These measurements were normalized and reported as a percentage of the native intact joint. Results A statistically signifcant diference in the mean CP measured with the hip-specifc device was observed before and after cam osteoplasty at 0° (41.2% (SD 29.7%); p = 0.014), 30° (54.5% (SD 16.6%); p = 0.011), 60° (39.8% (SD 23.0%); p < 0.001), 80° of fexion (36.3% (SD 22.1%); p < 0.001), and 80° of fexion with 20° of internal rotation (26.0% (SD 22.4%); p < 0.001). The ICP sensor is very fragile and difcult to handle in hip arthroscopy. Consequently, we limited the evaluations using this sensor to fve hips. A statistically signifcant diference in the CP was found before and after cam osteoplasty at 80° of fexion (57.6% (SD 29.1%); p = 0.004). Conclusion This biomechanical study evaluated a new hip-specifc device to intraoperatively measure the CP in arthroscopic surgery. It showed a signifcant decrease in the CP after arthroscopic cam resection with the joint in diferent positions. At 80° of fexion with 20° of internal rotation, a typical position to detect hip impingement, the CP was reduced to 26% after arthroscopic cam resection. The intraoperative measurement of CP provides surgeons with feedback to evaluate the efectiveness of the osteoplasty.