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Universidade de Lisboa

12 ARTICLES PUBLISHED IN JoVE

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Developmental Biology

In Vitro Differentiation of Mature Myofibers for Live Imaging
Mafalda R. Pimentel 1, Sestina Falcone 2, Bruno Cadot 2, Edgar R. Gomes 1,2
1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 2Myology Research Center, UM76-INSERM U974-CNRS FRE 361, Sorbonne University, UPMC University of Paris 6

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

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Developmental Biology

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm
Marta Figueiredo 1, Hélia Neves 1
1Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina, Universidade de Lisboa

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

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Developmental Biology

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example
Marta Figueiredo 1, Hélia Neves 1
1Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina, Universidade de Lisboa

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

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Medicine

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia
Augusto Ministro 1,2, Paula de Oliveira 1, Raquel J. Nunes 1, André dos Santos Rocha 1, Tiago Ferreira 2, J. Goyri-O'Neill 3, Susana Constantino Rosa Santos 1
1Centro Cardiovascular da Universidade de Lisboa, Lisbon School of Medicine of the Universidade de Lisboa, 2Centro Hospitalar Universitário Lisboa Norte, 3Nova Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests to assess the effectiveness of angiogenic therapies.​

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Medicine

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats
Sónia Ribeiro *1,2, Ana Rita S. Pereira *1, Ana Teresa Pinto 1, Filipe Rocha 1, Augusto Ministro 1,2, Manuela Fiuza 1,2, Fausto Pinto 1,2, Susana Constantino Rosa Santos 1
1Centro Cardiovascular da Universidade de Lisboa, Faculdade de Medicina, Universidade de Lisboa, 2Departamento Coração e Vasos, Centro Hospitalar Universitário Lisboa Norte

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

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Biochemistry

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers
Dalton M. Pessôa Filho *1,2, Danilo A. Massini *2, Leandro O. C. Siqueira *2, Luiz Gustavo A. Santos *2, Camila M. T. Vasconcelos *2, Tiago A. F. Almeida *1,3, Mário A. C. Espada *4,5, Joana F. Reis *3,6, Francisco B. Alves *3, Fred J. DiMenna *7,8
1Department of Physical Education, São Paulo State University (UNESP) at Bauru, 2Institute of Bioscience, Graduate Program in Human Development and Technology, São Paulo State University (UNESP) at Rio Claro, 3Ciper, Faculdade de Motricidade Humana, Universidade de Lisboa, 4Department of Science and Technology, School of Education, Polytechnic Institute of Setúbal, 5Quality of Life Research Center, Polytechnic Institute of Santarem, 6Universidade Europeia at Lisbon, 7Division of Endocrinology, Diabetes and Bone Disease, Icahn School of Medicine at Mount Sinai, 8Department of Biobehavioral Sciences, Teachers College, Columbia University

As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

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Neuroscience

Isolation and Expansion of Neurospheres from Postnatal (P1−3) Mouse Neurogenic Niches
Rita Soares 1,2,3, Filipa F. Ribeiro 1,2, Diogo M. Lourenço 1,2, Rui S. Rodrigues 1,2, João B. Moreira 1,2, Ana M. Sebastião 1,2, Vanessa A. Morais 1,3, Sara Xapelli 1,2
1Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, 2Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, 3Instituto de Biologia Molecular, Faculdade de Medicina, Universidade de Lisboa

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

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Bioengineering

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining
Teresa P. Silva 1,2, Tiago G. Fernandes 1, Diogo E. S. Nogueira 1, Carlos A. V. Rodrigues 1, Evguenia P. Bekman 1,2,3, Yas Hashimura 4, Sunghoon Jung 4, Brian Lee 4, Maria Carmo-Fonseca 2, Joaquim M. S. Cabral 1
1iBB - Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, 2Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, 3The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Universidade de Lisboa, 4PBS Biotech, Inc, Camarillo, CA, USA

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

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Neuroscience

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures
Cláudia A. Valente 1,2, Francisco J. Meda *1,2, Mafalda Carvalho *1,2, Ana M. Sebastião 1,2
1Instituto de Farmacologia e Neurociências, Faculdade de Medicina da Universidade de Lisboa, 2Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

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Chemistry

Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications
Brittany F. Karas 1,2, Cathleen L. Doherty 1, Kristin R. Terez 2, Leonor Côrte-Real 3, Keith R. Cooper 1,2, Brian T. Buckley 1
1Environmental and Occupational Health Sciences Institute, Rutgers University, 2Department of Biochemistry and Microbiology, Rutgers University, 3Centro de Química Estrutural and Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa

The increased rate of pharmaco- and toxicokinetic analyses of metals and metal-based compounds in zebrafish can be advantageous for environmental and clinical translation studies. The limitation of unknown waterborne exposure uptake was overcome by conducting trace metal analysis on digested zebrafish tissue using inductively coupled plasma mass spectrometry.

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Medicine

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis
David Pires 1,2,3, Paulo J. G. Bettencourt 1,2
1Faculty of Medicine, Universidade Católica Portuguesa, 2Center for Interdisciplinary Research in Health, Universidade Católica Portuguesa, 3Host-Pathogen Interactions Unit, Research Institute for Medicines, iMed-ULisboa, Faculty of Pharmacy, Universidade de Lisboa

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

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Engineering

Membraneless Hydrogen Peroxide Fuel Cells as a Promising Clean Energy Source
Fenyang Zhu 1, Guoxiang Chen 1, Aleksei Kuzin 1,2, Dmitry A. Gorin 2, Brij Mohan 3, Gaoshan Huang 1, Yongfeng Mei 1,4,5,6,7, Alexander A. Solovev 1
1Department of Materials Science & State Key Laboratory of Molecular Engineering of Polymers, Fudan University, 2Center for Photonic Science and Engineering, Skolkovo Institute of Science and Technology, 3Centro de Quimica Estrutural, Institute of Molecular Sciences, Instituto Superior Tecnico, Universidade de Lisboa, 4Center for Biomedical Engineering, School of Information Science and Technology, Fudan University, 5International Institute of Intelligent Nanorobots and Nanosystems, Fudan University, 6Shanghai Frontiers Science Research Base of Intelligent Optoelectronics and Perception, Fudan University, 7Yiwu Research Institute of Fudan University

This protocol introduces the design and evaluation of innovative three-dimensional electrodes for hydrogen peroxide fuel cells, utilizing Au-electroplated carbon fiber cloth and Ni-foam electrodes. The research findings highlight hydrogen peroxide's potential as a promising candidate for sustainable energy technologies.

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