University of Porto

7 ARTICLES PUBLISHED IN JoVE

Developmental Biology

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Ana C. Lima^*1,2,3,4, Min Jung^*1, Jannette Rusch¹, Abul Usmani¹, Alexandra M. Lopes^3,4, Donald F. Conrad¹

¹Department of Genetics, Washington University School of Medicine, ²Graduate Program in Areas of Basic and Applied Biology (GABBA), Abel Salazar Institute of Biomedical Sciences, University of Porto, ³Instituto de Investigação e Inovação em Saúde, University of Porto, ⁴IPATIMUP - Instituto de Patologia e Imunologia Molecular da Universidade do Porto, ⁵Department of Pathology & Immunology, Washington University School of Medicine

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Bioengineering

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Ana C. Silva^1,2,3,4, Maria J. Oliveira^1,2,5, Todd C McDevitt^4,6, Mário A. Barbosa^1,2,3, Diana S. Nascimento^1,2, Perpétua Pinto-do-Ó^1,2,3

¹i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, ²INEB - Instituto de Engenharia Biomédica, Universidade do Porto, ³Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, ⁴Gladstone Institute of Cardiovascular Disease, ⁵Faculty of Medicine, University of Porto, ⁶University of California San Francisco

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Cancer Research

Yeast As a Chassis for Developing Functional Assays to Study Human P53

Paola Monti¹, Bartolomeo Bosco², Sara Gomes³, Lucilia Saraiva^*3, Gilberto Fronza^*1, Alberto Inga^*2

¹Mutagenesis and Cancer Prevention Unit, Ospedale Policlinico San Martino, ²Department CIBIO, University of Trento, ³LAQV/REQUIMTE, Laboratory of Microbiology, Faculty of Pharmacy, University of Porto

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

Medicine

A Minimally Invasive Model of Aortic Stenosis in Swine

Rui Cerqueira^*1,2, Liliana Moreira-Costa^*1, Evangelia Beslika^1,3, André Leite-Moreira¹, Joana Silva^3,4, Paula A. da Costa Martins³, Adelino Leite-Moreira^1,2, André Lourenço¹, Pedro Mendes-Ferreira¹

¹UnIC@RISE, Department of Surgery and Physiology, Faculty of Medicine, University of Porto, ²Department of Cardiothoracic Surgery, Hospital Universitário de São João, ³CARIM School for Cardiovascular Diseases, Maastricht University, ⁴Mirabilis Therapeutics

This protocol describes a minimally invasive surgical procedure for ascending aortic banding in swine.

Behavior

Eye Movements in Visual Duration Perception: Disentangling Stimulus from Time in Predecisional Processes

Dinis Catronas¹, Nathércia Lima Torres¹, Susana Silva¹

¹Center for Psychology at the University of Porto, Faculty of Psychology and Educational Sciences, University of Porto

We present a protocol that employs eye tracking to monitor eye movements during an interval comparison (duration perception) task based on visual events. The aim is to provide a preliminary guide to separate oculomotor responses to duration perception tasks (comparison or discrimination of time intervals) from responses to the stimulus itself.

Biochemistry

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

Patrícia Correia¹, Iva Fernandes¹

¹REQUIMTE/LAQV, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

Immunology and Infection

Isolation, Fixation and Characterization of Juvenile Gilthead Seabream Head Kidney Leukocytes by Flow Cytometry

Isa Marmelo^1,2,3, Zélia Silva^4,5, Daniel Bolotas², Ricardo N. Alves⁶, Paula A. Videira^4,5,7, António Marques^2,3, Mário Sousa Diniz^1,5, Ana Luísa Maulvault^1,2,5

¹UCIBIO - Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, NOVA University of Lisbon, ²IPMA DivAV - Division of Aquaculture, Upgrading and Bioprospection, Portuguese Institute for the Sea and Atmosphere, ³CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, ⁴UCIBIO - Applied Molecular Biosciences Unit, Department of Life Sciences, NOVA School of Science and Technology, NOVA University of Lisbon, ⁵Associate Laboratory i4HB - Institute for Health and Bioeconomy, NOVA School of Science and Technology, NOVA University of Lisbon, ⁶Bioscience Core Lab, King Abdullah University of Science and Technology, ⁷CDG & Allies - Professionals and Patient Associations International Network (CDG & Allies - PPAIN), Department of Life Sciences, NOVA School of Science and Technology, NOVA University of Lisbon

This manuscript describes the isolation and fixation of leukocytes extracted from gilthead seabream's head-kidney and the assessment of their viability by flow cytometry. This work contributes to the standardization of protocols and leverages the processing of a higher number of samples without compromising sample quality, promoting advancements in fish immunology knowledge.