Index 03.1
Scientific Platforms 1.1 Advanced Light Microscopy 1.2 Animal Facility 1.3 Biochemical & Biophysical Technologies 1.4 Bioimaging 1.5 Biointerfaces & Nanotechnologies 1.6 Biosciences Screening 1.7 Cell Culture & Genotyping 1.8 Genomics 1.9 Histology & Electron Microscopy 1.10 In vivo CAM assays 1.11 Proteomics 1.12 Translation Cytometry 1.13 X-ray Crystallography
03.2 Open Policy & Training 2.1 Scientific Training 2017
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ANNUAL REPORT 2017 03 SCIENTIFIC PLATFORMS
03.1 Scientific Platforms To set and maintain the highest scientific standards, our strategy relies on cuttingedge Scientific Platforms equipped with state-of-the-art technology and headed by qualified staff. These platforms are acessed on an open policy basis, and investment in new equipment is strategically aligned with the needs of the intramural and extramural scientific community, reinforcing complementarity and networking. Experienced and accomplished specialists coordinate and manage these Scientific Platforms and are available to provide personalized guidance, as well as to help in the processes of experiment design and implementation. Resorting to our platforms means gaining access to expert support and to a range of specialized equipment. While the i3S community is sure to benefit from the Platforms the most, these technological resources are open to the entire scientific community, the industry sector, health services and the entrepreneurial tissue, entities that are strongly encouraged to discover them and make the most of the services they offer. The Scientific Platforms’ facilities and their quality are internationally recognized and the majority of them integrate national and international networks and nodes. Along with the highly qualified and skilled scientists who manage them, this integration in larger networks makes them a valuable asset and act as enhancer hubs for the establishment of synergies and networking activities. Being located in Porto and given its status as the largest research institute operating in the life and health
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sciences in Portugal, i3S and its platforms are easy to reach.
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03.1 Scientific Platforms
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The Scientific Platforms’ facilities and their quality are internationally recognized
03.1.1 Advanced Light Microscopy The Advanced Light Microscopy platform integrates the PPBI-Portuguese Platform of BioImaging and a member of the ELMI-European Light Microscopy Initiative.
The Advanced Light Microscopy (ALM) platform specializes in biological imaging technologies to study cellular systems in all biosciences research areas. The platform provides scientific guiding in project development including experimental planning and data analysis, training, access to equipment, technical support in image acquisition and analysis, and development of new technologies or applications. The ALM is accessible to academy, research institutes and industry on a fee basis open-access policy. The Advanced Light Microscopy platform integrates the PPBI-Portuguese Platform of BioImaging and is a member of the ELMI-European Light Microscopy Initiative.
Resources available The ALM equipment comprises several microscopy systems for analysis of fixed and live samples: • Laser point scanning confocal microscopes (Leica TCS SP5, Leica TCS SP2); • Spinning disk confocal microscope (Andor Revolution XD); • Inverted wide-field microscopes (Zeiss Axiovert 200M, Leica DMI6000 FFW, Leica DMI6000) • TIRF microscope (Nikon Ti TIRF); All systems above have long-term live-cell imaging capacities. • Upright wide-field microscopes (Zeiss Axio Imager Z1, Zeiss Axio Imager Z1 Apotome). The platform also provides workstations running state-of-the-art bioimage analysis software (Huygens suite and Imaris full package) plus open-source software and
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in-house developed algorithms.
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03.1 Scientific Platforms
Applications The high-end advanced microscopy and image data analysis resources available at ALM are suitable to study biomolecular localization, expression and interaction; cell proliferation, cell and tissue morphology; dynamic behaviour of cells and subcellular structures; cell signalling; molecular activity; embryo development and cell response to perturbations (ex. RNAi, CRISPR and drugs). Common samples include bacteria, yeast, plant and animal cells; tissue slices and explants; embryos of model organisms including C. elegans, Drosophila, and Zebrafish; 3D cell cultures, biofilms and biomaterials. The platform has the capacity to analyze fluorescent antibody labelled fixed samples, but is strongly focused on the observation of biological processes in live cells (unstained or expressing fluorescent proteins). Researchers of areas such as cell biology, neurobiology, oncobiology, pharmacology/ toxicology, plant sciences, biomaterials, medicine and chemistry can perform analysis from molecule to organism at nano to macro scale. They can use and benefit from a broad range of basic techniques (brightfield, phase contrast, DIC and fluorescence) and advanced techniques. These comprise multidimensional imaging, optical slicing and 3D reconstruction; spectral imaging, image of Fluorescent Recovery After Photo-bleaching (FRAP) or photo-activation/ conversion to explore molecular and structure dynamics; FĂśrster Resonance Energy Transfer (FRET) for the analysis of molecular interactions and activity; and Total Internal Reflection Fluorescence Microscopy (TIRFM) for single molecule imaging or observation of molecules near the cell surface.
Paula Sampaio sampaio@ibmc.up.pt Paula Sampaio graduated in Biochemistry and has a Ph.D. in Biomedical Sciences from the University of Porto. She is a former visiting researcher in Cayetano Gonzalez's group at EMBL and former postdoctoral fellow at IBMC Molecular Genetics group headed by Prof. Claudio Sunkel where she studied the mechanisms of cell division. Presently, is the coordinator of the Advanced Light Microscopy scientific platform at i3S and national coordinator of the PPBI - Portuguese Platform of BioImaging, a national research infrastructure of strategic interest. At University of Porto, Paula Sampaio is invited Assistant Professor at the Faculty of Sciences and external professor at ICBAS, teaching bioimaging on Master’s courses and PhD programs. She is also the organizer of the course series on Optical Microscopy Imaging for Biosciences and co-organizer of the EMBO course on Biomolecular Interaction Analysis. Paula Sampaio is member of management committees of COST actions Network of European BioImage Analysts (CA15124) and the COMULIS Correlated Multimodal Imaging in Life Sciences (CA17121) and a Portuguese delegate at Euro-Bioimaging ESFRI project.
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large areas at high resolution; live cell imaging during extend periods of time;
03.1.2 Animal Facility
The aim of the facility is to support scientific projects with animal experimentation ensuring high animal welfare standards by encouraging the application of the 3R’s.
Sofia Lamas sofia.lamas@ibmc.up.pt
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Sofia Lamas graduated in veterinary medicine in 2010, at ICBAS, University of Porto. She is responsible for the IBMC animal facility since 2010, when she completed the FELASA C course. Later on, in 2016 Sofia completed a FELASA D course at the Autonomous University of Barcelona (Master in Science and Welfare of Laboratory Animals). Since 2015 Sofia is doing a PhD in Veterinary Sciences, studying the impact of the microbioma and rederivation techniques on the phenotype of different animal models.
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The i3S animal facility is an AAALAC accredited facility dedicated to the production and maintenance of laboratory animals. The facility provides care and veterinary advice on laboratory animals, mainly mice, rats and rabbits and holds approximately 2500 cages in SPF conditions. Zebrafish and seabass models are also available (associated with specific research groups). The aim of the facility is to support scientific projects with animal experimentation ensuring high animal welfare standards by encouraging the application of the 3R’s. The development of new models (both surgical and genetically modified – CrisprCas9) is supported by the facility team. Biocontainment areas for microbiological risk agents (level 2 and 3) are also available. The facility participates in the training of researchers, users and staff on laboratory animal science and promotes animal welfare practices.
Resources available •
IVIS Lumina series III
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Vevo Ultrasound 2100 – Bioimaging platform
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X-Ray
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Stereomicroscopes (Zeiss Discovery V8 and Olympus SZX 10, Leica S8APO)
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Volatile Anesthesia systems and recovery chamber
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Perfusion pump
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Micromotor K-5 Plus Kavo
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Erbotom coagulator
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Impactor
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Coloview – Small animal endoscopy
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Inverted microscope with microinjection system – only for internal use
03.1 Scientific Platforms
Applications IVIS Lumina series III: this luminescence and fluorescence equipment allows longitudinal studies in which cells, microorganisms or particles can be tagged with fluorescence or luminescence and high sensibility detection is possible with the animal under anesthesia. The same type of acquisition can also be done in vitro before animal testing. The equipment can detect from GFP to near infrared by NIR spectrum. Applications include oncological studies, infectious models, biomaterials, among others. Vevo ultrasound 2100: description included in the Bioimaging platform. X- Ray: an intraoral x-ray is available at the animal facility for 2D images of bone with a resolution of 34 pixel/mm and pixel size of 30 Âľm. This equipment is used mainly in bone studies. Stereomicroscopes: several models are available at the animal facility, including heating base and incident light. This type of equipment can be used for microsurgery and dissection. Volatile anesthesia machines: isoflurane anesthesia machines are available inside procedure rooms. These are used for surgical procedures or procedures that require animal immobilization. Perfusion pump: this perfusion pump with two lines is available for perfusion techniques. Micromotor K-5 Plus Kavo: the micromotor can be used to drill bone areas. Specific drills can be purchased according to the type of lesion researchers need to perform. Erbotom coagulator: This surgical coagulator can be used to perform vessel occlusion through coagulation. It can also be used during surgical procedures to control bleeding. Impactor: the Impactor is a contusion device designed for spinal cord injury in mice and rats. This equipment allows the application of standard forces to produce constant injuries in mice and rats. Applied forces vary between 30 and 200 kDynes. Applications are mainly neurological studies. Coloview Storz endoscope: this system is capable of performing colon imaging in living animals in a simple and quick way. The small probe allows obtaining images from the distal colon in anesthetized animals, collecting biopsy samples and images. It is particularly useful in digestive studies, oncological and immunological studies involving the large intestine. Inverted microscope with microinjection system: this Leica inverted microscope is associated with two micromanipulators and a microinjector, all from Eppendorf. the microinjection of embryos. CrisprCas9 technique is being used for this purpose.
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An eppendorf piezo device is also available. This equipment is used internally for
03.1.3 Biochemical & Biophysical Technologies The team applies off-theshelf methods and designs customized solutions, building on top of the experience and know-how amassed since 2005.
The Biochemical & Biophysical Technologies platform provides expertise and access to state-of-the-art technologies in Protein Production and Molecular Biophysics. The platform’s team implements methodologies and manages resources in the production of proteins for R&D, the structural analysis of biomolecules and their stability and the characterization of molecular interactions. All resources are available to the entire scientific community, health services and industry, at the local, national and international level. The team applies off-the-shelf methods and designs customized solutions, building on top of the experience and know-how accumulated since 2005. The Biochemical & Biophysical Technologies platform integrates the P4EU network – Protein Production and Purification Partnership in Europe and the Association of Resources for Biophysical Research in Europe-Molecular Biophysics in Europe (ARBRE-MOBIEU), in which Frederico Silva coordinates the section on training and human capacity development.
Resources available
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The platform manages 30+ resources including:
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2 high speed centrifuges and 3 ultracentrifuges
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Facilities for the growth and manipulation of microorganisms and insect cells
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Equipment for the homogenization of cells and tissues (sonication, high pressure, beads)
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Ultrafiltration equipment and a freeze dryer
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2 analytic HPLCs
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3 FPLCs and 2 preparative chromatographers
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Fluorimeters and Absorbance spectrometers
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A Circular Dichroism spectrometer (Jasco J815)
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An Isothermal Titration Calorimeter (VP-ITC)
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A surface plasmon resonance biosensor (Biacore X100 Plus)
03.1 Scientific Platforms
Applications Soluble and membrane recombinant proteins or protein complexes are produced mainly in bacteria and insect cells using plasmid vectors or baculovirus. These proteins are then purified by biochemical methods being the lab equipped and having extensive experience in all types of chromatography. Besides preparative chromatography several analytic setups such as RF-HPLC, analytical SEC, IEX and F-SEC are in place to characterize all kinds of biomolecules. The structural characterization of biomolecules can be done in terms of the native size assessment independent from shape (applicable also to crude samples), charge (by IEX) and secondary and tertiary structure of proteins, nucleic acids and other biomolecules by circular dichroism (CD) in solution, lipid phases or suspensions. CD can also be used to monitor conformational changes induced by mutations or external factors (e.g. interaction with other molecules). The stability of proteins can be monitored by differential scanning fluorimeter (DSF, vulgo Thermal shift) or CD. The characterization of interactions can be done with fluorescent probes by DSF or fluorescence anisotropy measurements but the complete kinetic and termodynamic characterization of binding events is done with surface plasmon resonance and isothermal titration calorimetry.
Frederico Silva ffsilva@ibmc.up.pt
the structure and intracellular targeting of proteins, using a multidisciplinary approach covering diverse fields such as Yeast cell biology, biochemistry and protein crystallography. Back in 2005 he was involved in the set up of a Scientific Platform dedicated to the production and purification of recombinant proteins, that later broadened its scope developing work in Molecular Biophysics. He has been actively involved in the development of pan European communities in the fields of protein production and molecular biophysics. Frederico coordinates several postgraduate activities such as an EMBO practical course running every other year focusing in the study of molecular interactions and a yearly practical course on protein expression, purification and characterization of the MCBiology PhD program.
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Frederico Silva got his PhD from the University of Porto in 2006, studying
03.1.4 Bioimaging
Bioimaging operates on the bases of core projects and programmes at the bioimaging/ biomaterials and/ or regeneration interface.
Bioimaging brings together a set of solutions for in vitro and in vivo imaging in order to advance in the development, improvement, integration and use of bioimaging solutions through research, technology development, training and education with focus on the fields of Biomaterials, Nano- and Regenerative Medicine, in biology and medical science. The Bioimaging operates on the bases of core projects and programmes at the bioimaging/ biomaterials and/ or regeneration interface. It is open to all academic researchers and industry on a fee-for-service basis, with specialized technical staff supporting its daily activities. In particular, we provide support to our users on project planning, experimental design, sample preparation, equipment operation and optimization, and image analysis. Besides training for the users in operating the systems for their scientific goal, the Bioimaging also offers advanced workshops and participates in different outreach activities. Bioimaging integrates the PPBI-Portuguese Platform of BioImaging.
Resources available • The collection of resources available allow for the imaging/ characterization from molecules to organisms: • Inverted wide-field microscope (Axiovert 200M, Zeiss) • Confocal Raman microscope (LabRAM HR800 UV, Horiba Jobin-Yvon) • Critical point dryer (CPD 7501, Polaron Range) • Imaging flow cytometer (ImageStreamX, Amnis, Merck Millipore) • Laser point scanning confocal microscope (TCS-SP5 AOBS, Leica) • In vivo micro-ultrasounds imaging (Vevo 2100 (230 V), Fujifilm) • In vivo microCT (Skyscan 1276, Bruker) • Software locally developed: MIQuant; Cell File Analyzer; LCFs - Local Convergence Filters, and support in image analysis (including development of new algorithms and computational tools). • In vivo micro-ultrasounds imaging, Vevo 2100 (230 V) • Spectral Confocal Laser Scanning Microscope Leica TCS-SP5 AOBS
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• Software locally developed: MIQuant; Cell File Analyzer; LCFs - Local Convergence Filters
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Applications Resources available for imaging of biological events at different scales include: At the molecular level, non-destructive chemical imaging (confocal Raman microscopy), which provides information on the structure and chemistry of samples with a resolution down to ~200 nm. Applications include: chemical characterization of biological and synthetic samples; compound distribution within a sample; disease detection and drug design. At the cellular level high-resolution multidimensional imaging for cells, tissues, biomaterials, functional imaging, live cell and high-throughput imaging (imaging flow cytometry, wide-field microscopy and laser scanning confocal microscopy). Applications include: multimodal imaging of biological samples; acquisition of series of overlapping tiled images acquired over a defined area; localization and colocalization analysis; cell cycle and mitosis; morphological changes; spot count, live cell imaging; FRAP; and FRET amongst others. At the organism level, in vivo anatomical, functional and molecular imaging for small animals (micro-ultrasounds and microCT). Applications for the micro-ultrasounds include: cardiovascular phenotyping; image-guided cardiac injection; pregnancy research; tumour volume quantification, in real-time and with a resolution down to 30 µm. While microCT generates 3D images of samples showing morphology and internal microstructure with resolution down to the micrometric level, in a non-destructive and non-invasive fashion for true longitudinal studies. Applications include: cardiovascular phenotyping; imaging of body fat and lungs amongst others.
Maria Gómez Lázaro maria.glazaro@ineb.up.pt María Gómez Lázaro got her PhD from the University of Castilla-La Mancha, Spain, in 2007, studying the involvement of the mitochondrial in different neurodegenerative diseases. She came to Portugal as a postdoc to investigate the mechanisms of secretory granule biogenesis, apical sorting and regulated secretion of digestive enzymes in the acinar cells of the exocrine Pancreas. In 2011 María returned to the University of Castilla-La Mancha to study the synaptic plasticity mechanisms in a mouse model of Parkinson's disease. In 2012 she became a research technician in the Bioimaging Platform where she is the Team Coordinator of the platform and is also involved in the training of researchers and in the development and set up of new techniques.
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characterization of bone morphometrics; tumour and blood vessel imaging;
03.1.5 Biointerfaces & Nanotechnologies A Quality System is implemented at The Biointerfaces and Nanotechnology platform, according to ISO standards and Good Laboratory Practices. This platform integrates the Association of Resources for Biophysical Research in Europe (ARBRE)Molecular Biophysics in Europe (MOBIEU).
The Biointerfaces and Nanotechnology platform aims to study materials, surfaces and interfaces of materials with cells and tissues, going down to the micrometric and nanometric level through the development and the improvement of advanced physical, chemical, mechanical and structural characterization techniques. The platform focuses particularly on the fields of Biomaterials and Nano- and Regenerative Medicine. Expert technical staff provides advanced training and education in specialized areas and support to all users. Academic researchers and industry can use the platform on a fee-for-service basis. A Quality System is implemented at the Biointerfaces and Nanotechnology platform, according to ISO standards and Good Laboratory Practices. This platform integrates the Association of Resources for Biophysical Research in Europe (ARBRE)-Molecular Biophysics in Europe (MOBIEU).
Resources available •
Atomic Force Microscope coupled with Inverted Fluorescence Microscope (PicoPlus5500, Keysight Technologies/ Axiovert Z1, Zeiss)
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Optical Contact Angle Measurement Device (OCA, Data Physics)
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Dynamical Mechanical Analyzer (Tritec 2000, Triton Technology)
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Ellipsometer with image (EP3, Nanofilm)
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Fourier Transformed Infra-Red Spectrometer (Frontier, Perkin Elmer)
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Zetasizer (Nano ZS, Malvern)
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Rheometer (Pro Kinexus, Malvern)
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Zeta Potential Electro Kinetic Analyzer (EKA, Antoon Paar)
Manuela Brás
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mbras@i3s.up.pt Manuela Brás graduated in Metallurgical and Materials Engineering (1995) and obtained her MSc in Biomedical Engineering (2001) both at the Faculdade de Engenharia da Universidade do Porto. She headed the Quality department at A. Brito Engrenagens, Lda (mechanical industry) and from 1996 onwards was a lab technician at INEB. Presently, she is the Chief-technician of INEB. Since 2005, she has been the Team Coordinator (Head) of Biointerfaces and Nanotechnology platform and the development of new applications using Atomic Force Microscopy coupled to Inverted Fluorescence Microscopy. Manuela Brás is also a PhD student at Faculdade de Engenharia da Universidade do Porto, studying the mechanobiology of human cancer tissues using Atomic Force microscopy.
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Applications Atomic Force Microscope (AFM) coupled with Inverted Fluorescence Microscope (IFM): Life and Health sciences (topographic imaging of molecules, cells, tissues; force spectroscopy of binding forces in ligand-receptor and cell-cell adhesion; mechanical properties quantification in biological samples; roughness and morphometric parameters quantification; polymer industry and biosensors and electronic industry (imaging and roughness measurements of surface). Contact Angle Measurement Device (OCA) Wettability and surface tension. Can be used in several fields: Life and Health sciences; polymer industry; agrochemical industry (the effectiveness of a sprayed liquid will be determined by the degree to which it coats and adheres to the surface); shoes and clothes industry (wettability of leather and tissues surface). Dynamic Mechanical Analyzer (DMA): Materials science (bulk solid, film, fiber, …) with different deformation modes: tension, compression, shear and 3D point bending, resulting from changes in five experimental variables: temperature, time, frequency, force, and strain. Ellipsometer with image: Biology (thickness of film proteins and natural polymers); biomaterials (quantification of thickness of thin oxide films formed on metallic biomaterials); refractive substrates (films in a nanometric scale, namely self-assembled monolayers - SAMs); semiconductors physics to microelectronics; quality control (films metrology); flat panel display industry; optical lenses; photovoltaics thin films. Fourier Transformed Infra-Red Spectrometer (FTIR): Biology (adsorption of proteins, …); materials (adsorption of organic layers in ceramics, metals, ...); geology; identification of contaminants; food industry (identification of fat content); forensic sciences (identification of chemical information in residues of fibers, coatings, paints, ….); pharmaceutical field (formulation, validation and quality control). Zetasizer Nano ZS: Study of nanoparticles (biomaterials, liposomes, …); formulation for health therapies: size and charge; protein conformation: size determination; environmental strategies to address pollution: size; formulation stability; proteins and polymer characterization: size, charge and isoelectric point; quantum dots analysis: the size determination. Rheometer: It allows the application of four different modes of assays: viscometry, oscillation, creep and recovery and stress and relaxation in different areas: Life sciences and Health (mechanics of biological tissues); materials science (hydrogels,…); coatings and food industry (paints and chocolate, respectively); pharmaceutical and cosmetic industry (liquids, ointments, creams, pastes, ….). Zeta Potential Electro Kinetic Analyzer (EKA): EKA is used to perform the membranes, granules, plates, …)
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quantification of Zeta potential at different pH (applied to solid materials: powders,
03.1.6 Biosciences Screening The Biosciences Screening platform provides state-ofthe-art technology and expertise to solve challenging questions with high throughput and high content technologies.
The Biosciences Screening platform provides state-of-the-art technology and expertise to solve challenging questions with high throughput and high content technologies. Highly qualified scientists with experience on project evaluation, assay and development, liquid handling, automated microscopy, multimode microplate readers, image and data analysis, work with project teams to successfully run mediumto-high throughput screens. Examples are genetic and chemical screens for the purpose of target and/or drug discovery. The platform facilitates access to genetic and compound screening libraries and is actively establishing collaborations in order to receive and expand its own libraries. Training is provided to all users and regular courses are organized for the general scientific community. The Biosciences Screening platform integrates the PPBI - Portuguese Platform of BioImaging, participates in the NEUBIAS – Network of European BioImage Analysts and in the GENiE - Collaborative European Network of C.elegans early-stage researchers and young investigators.
Resources available Assay Preparation: •
MultidropTM Combi Reagent Dispenser (Thermo-Scientific)
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JANUS with 4 Tip Arm and MDT Arm (PerkinElmer)
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Liquidator 96 (Metler-Toledo)
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ALPS 50 V-Manual Heat Sealer (Thermo-Scientific)
For Image/Data Acquisition: André Maia
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andre.maia@i3s.up.pt André Maia graduated in Biology (2004) and has a PhD in Biomedical Sciences (2010) from the University of Porto. He is a former postdoctoral fellow in René Medema´s group at UMC Utrecht/NKI and Claudio Sunkel´s lab at IBMC, where he studied how cells accurately segregate their chromosomes during mitosis, resorting to human cell lines and animal models such as Drosophila melanogaster and Caenorhabditis elegans. Since 2014, coordinates and manages a Scientific Platform dedicated to High-throughput and High-Content screening. He also co-supervises PhD students and develops bioimage analysis protocols. André organizes and participates in postgraduate courses such as the annual course on High Throughput and Image Analysis for Biosciences. He is a member of the management committee of the COST action GENiE (BM1408) and participates in the COST action NEUBIAS (CA15124).
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IN Cell Analyzer 2000 (GE Healthcare)
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Synergy 2 (BioTek)
For Image/Data Analysis and Visualization: The scientific platform has powerful computer workstations to perform digital image analysis. Examples of common applications are: image processing, segmentation and quantification.
03.1 Scientific Platforms
Applications Information generated using high throughput and high content technologies is highly valuable to gain a deeper understanding of complex diseases, including cancer, neurology, immunology, and infectious diseases. The Biosciences Screening scientific platform has 3 main areas of activity:
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High Throughput and/or High Content Screening
2. Bioimage data analysis 3. Stand-alone usage of equipment 1. HTS/HCS 1.1 Compound screening The platform has access to compound libraries allowing to screen thousands of small molecules on cells or even all organisms. Typical read-outs are luminescence, fluorescence, absorvance or microscopy. 1.2. Genetic screening The platform is equipped to deal with siRNA, dsRNA, CRISPR/CAS9 screens in high-throughput.
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Bioimage data analysis
2.1 Digital image analysis "Image analysis is a process of discovering, identifying, and understanding patterns that are relevant to the performance of an image-based task. One of the principle goals of image analysis by computer is endow a machine the capability to approximate, in some sense, a similar capability in human beings" in Gonzales, R.C. and Woods, R.E. (1992). 2.2. Data Analysis Proprietary and open-source data analysis software is available (GraphPad, Spotfire, CellProfiler Analyst, shinyHTM) 3. Equipment 3.1 Assay preparation The platform is equipped with liquid handling equipment allowing for medium to high throughput sample preparation 3.2. Data acquisition The platform reading instruments include multimode microplate readers
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and high content screening microscope
03.1.7 Cell Cultures & Genotyping At the CCGen Cell Culture Lab we make available tailormade conditions for a variety of cell lines, primary cells and virus cultures, including lentivirusbased transfection service.
The Cell Culture and Genotyping Service (CCGen) intends to facilitate the implementation of state-of-the-art advances in cell culture, genotyping and gene expression technology, by providing researchers with selected services, expert consultation and training in cell culture, genotyping and gene expression techniques. At the CCGen Cell Culture Lab we make available tailor-made conditions for a variety of cell lines, primary cells and virus cultures, including lentivirus-based transfection service. The training we provide is mandatory. We also collaborate with interested groups in the implementation and optimization of mouse genotyping protocols including DNA extraction, DNA fragment amplification, detection, analysis and interpretation of results. Through the Animal Facility researchers may access our online ordering of mouse genotyping service request (about 100 mouse genotyping protocols in routine). In addition, we offer urgent genotyping results for all investigators requiring it to do a select primary culture or in need of a specific time point. A qPCR equipment, with thermal validation and quality control, is available for Gene Expression. We also support the analysis of RNA integrity by automated electrophoresis and collaboration in all interpretation data.
Resources available Gene Expression •
iCycler iQ5 Real-Time PCR
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CFX Real Time System
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Experion, Automated Electrophoresis System
Genotyping •
TProfessional Basic Gradient 96 thermocycler
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Termomixer compacto
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Dry Bath FB15103
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Centrifuges 5415R / MULTIFUGE X1R 230V RESEARCH
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Gel Tray, 15x20 cm and 10x15 cm
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GelDoc EZ System
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Maxwell 16 DNA/RNA/Protein Automated Extractor
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Cell Culture
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Six CO2, heated incubators, distributed by 3 independent rooms
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Three laminar flow hoods
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Inverted microscope (Zeiss, Axiovert 25)
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Heated water bath
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Refrigerated centrifuge
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ZOE Fluorescent Cell Imager
03.1 Scientific Platforms
Applications Gene Expression The qPCR was developed to rapidly and accurately determine the relative amount of RNA or DNA in a sample. Real-time PCR or quantitative PCR (qPCR) is routinely used in laboratories to help identify pathogens, genotype infectious agents and for cancer diagnostics. The Experion performs multiple electrophoresis steps in one and is used for analysis (quantity and quality) of RNA, DNA and protein. This instrument serves as a complement to the qPCR, allowing for fast and economic assessment of RNA quality. Genotyping The platform implements and optimizes animal (mouse, zebrafish…) genotyping protocols, including DNA extraction, DNA fragment amplification, detection, analysis and interpretation of results (about 100 mouse genotyping protocols in routine). Cell Culture The platform’s team offers expert consultation and training in cell culture with establishment of tailor-made conditions for culture of a variety of cell lines and primary cells, maintenance of hygienic conditions of the cell culture lab and mandatory training of all new users.
Paula Magalhães Paula Magalhães graduated in Aquatic Sciences from the Institute of Biomedical Sciences Abel Salazar and completed a Master’s degree in Quality Control from the Faculty of Pharmacy of the University of Porto. Since 2001 and until 2006 she worked as a Molecular Geneticist in CGPP - Centro de Genetica Preditiva e Preventiva. Currently, Paula Magalhães coordinates the Cell Culture and Genotyping platform at i3S. Her research interests focus on genomic methodologies, particularly genotyping, sequencing and gene expression and she has more than 15 publications in international peer reviewed journals.
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paulam@ibmc.up.pt
03.1.8 Genomics
The Genomics Platform integrates the GenomePT consortium and an Ion Torrent certified service provider.
The Genomics platform was established in 2006 at Ipatimup. Since then, the facility has been steadily growing, both in human resources and equipment. Today, it is a full service facility dedicated to providing researchers state-of-the-art technological solutions in the field of genomics and high throughput analysis. The platform offers technical expertise and support to experimental design, protocol development, and data analysis guidance, as well as training. Due to the very dynamic and rapidly evolving field of genomics, the platform’s team actively collaborate with different companies in developing NGS-related products both for sample preparation and for bioinformatics data analysis. The Genomics Platform integrates the GenomePT consortium and an Ion Torrent certified service provider.
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Resources available
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Capillary electrophoresis sequencers (Applied Biosystems 3130xl and Applied Biosystems 3500 genetic analyzers)
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Real-time PCR systems (Applied Biosystems 7500 Fast Real-Time PCR Systems)
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Digital PCR system (QuantStudio 3D digital PCR system)
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RNA and DNA sample QC and quantification (Agilent 2200 TapeStation system, Bioanalyzer, Qubit 3.0 Fluorometer)
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Automated system for next generation sequencing library and template preparation (Ion Chef)
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Next generation sequencers (Ion PGM system, Ion S5 system, Ion S5xl system)
03.1 Scientific Platforms
Applications - Sanger sequencing is the gold-standard DNA sequencing method, which continues to generate highly accurate, reliable sequencing data. Applications include detection of variants (SNPs) and mutations, detection of methylation events, verification of clone constructs, confirmation of NGS results. - Fragment analysis applications are those in which fluorescent fragments of DNA are separated using capillary electrophoresis and sized by comparison to a size standard. Applications include microsatellite analysis (for example, human, animal and plant typing), SNP genotyping. - Digital PCR is an alternate method to conventional real-time quantitative PCR for absolute quantification and rare allele detection. It does not rely on a calibration curve for sample target quantification and no reference standards or endogenous controls are needed. Applications include copy number variation, differential gene expression, low-level pathogen detection. - Quality control of biological samples applications include verification of RNA and DNA integrity; sizing and quantity control for next generation sequencing libraries. - Next generation sequencing (NGS) performs sequencing of millions of small fragments of DNA in parallel, providing high depth to deliver accurate data. Applications include exome sequencing, targeted DNA and RNA sequencing, small genome sequencing, metagenome sequencing, whole transcriptome sequencing.
Ana Mafalda Rocha sequencing@ipatimup.pt
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Ana Mafalda Rocha graduated in Biology at the University of Aveiro in 2005 and received a Master's degree in Forensic Genetics in 2012 from the University of Porto. Since 2006 she coordinates the Genomics platform. Ana Mafalda Rocha was also involved in the certification process, equipment management, among other tasks, as a former member of the Risk Management System and of the Technical Body. Presently, as Team Coordinator of the i3S Genomics platform, her main responsibility is managing the platform.
03.1.9 Histology & Electron Microscopy The Histology and Electron Microscopy platform is focused on Electronic Microscopy and Optical Microscopy.
The Histology and Electron Microscopy platform is focused on Electronic Microscopy and Optical Microscopy. The Histopathology service, integrated in the platform, offers a full range of macroscopic and histopathology services, able to support research groups using animal models or human tissues in histopathology studies. The unit provides the equipment, technical support and guidelines to researchers looking for high level electron and optical microscopy to tackle studies either of cells, tissues or material sciences, not only for i3S and the University of Porto, but also for outside institutions, industry and companies. The platform can help to define optimal experimental conditions for the research, while also taking part in the projects. Besides organizing training courses for researchers, Master’s and PhD programs, the unit also offers internships for higher education students, and engages in workshops and exhibitions for the general public in collaboration with the i3S Communication Unit. The Histology and Electron Microscopy platform integrates the PPBI - Portuguese Platform of BioImaging and participates in the COST-Action COMULIS - Correlated Multimodal Imaging in Life Sciences.
Resources available Histology Equipment •
Cryostat Leica CM 3050S
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Light microscope Olympus DP 25 Camera Software Cell B
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Paraffin microtome Microm HM335E
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Modular embedding system Microm STP 120-1
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Tissue Chopper McILwain EC350
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Paraffin tissue processor Microm STP 120
Electron Microscopy Equipment •
Transmission Electron Microscope Jeol JEM 1400 with STEM detector and EDS
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system
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Transmission Electron Microscope Zeiss model EM 902 A
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Ultramicrotome Leica Reichert SuperNova
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Ultramicrotome LKB Bromma Ultratome Nova
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Ultramicrotome PT RMC RMC PowerTome PC=XL
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Light microscope Zeiss Primo-Star
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Knifemaker Leica Glass
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Applications The main application of the histology is to provide Grossing & Processing & Embedding & Sectioning, Special stains (monochromatic / polychromatic), histology image analysis, cryosectioning, cytobloks / histogel, optimization of immunohistochemistry and/or immunocytochemistry, tissue sections for DNA extraction. Transmission electron microscopy (TEM) has been widely applied to characterize morphology, crystalline structure, and elemental information of several elements. The main application of a transmission electron microscope is to provide conventional ultrastructure, immunoelectromicroscopy, negative and cytochemistry stains, Energy Dispersive X-ray Spectrometry (EDS), Grossing & Processing & Embedding & Sectioning, Consultation in tissue analysis.
Rui Fernandes rfernand@ibmc.up.pt
Rui Fernandes has participated in numerous studies, namely in conventional Ultrastructure, immunoelectromicroscopy, elemental analysis EDS, STEM, among others. He has more than 35 publications in international peer reviewed journals.
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Rui Fernandes graduated in Biology in 1996 from the Faculty of Sciences, University of Porto. In 1995 he worked with Professors Isabel Santos and Roberto Salema at Centro de Citologia Experimental. In 1996, he started working at Centro de Estudos de Paramiloidose, Hospital Santo António. Afterwards he began a research project in the area of Familial Amyloidotic Polyneuropathy, with Prof. Maria João Saraiva at IBMC. In 2002, he became the head of IBMC’s Advanced Tissue Analysis Facility. Currently, he coordinates the Histology and Electron Microscopy platform at i3S.
03.1.10 In vivo CAM assays
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The In vivo CAM assays platform’s work is validated by our publication track record, and by the national and international network of collaborators and clients, both from academia and industry.
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The In vivo CAM assays platform provides scientific expertise and services using the chick embryo model, more specifically, assays based on the chorioallantoic membrane – the CAM. Created in 2012, the platform offers researchers additional or alternative in vivo tools (complying with the 3Rs policy) that are reliable, as well as cost and time efficient. The platform ensures protocol design, experimental execution, analysis of the results and data interpretation and is fully equipped to address new challenges and widen the model applications in a wide range of research topics. The In vivo CAM assays platform’s work is validated by our publication track record, and by the national and international network of collaborators and clients, both from academia and industry.
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Applications Due to the CAM structure and its easy access, CAM assays constitute attractive preclinical in vivo tools for drug screening and/or vascular growth studies. Associated with the chick natural immunoincompetence, CAM assays can also be used to study complex cancer features and the effect of potential therapeutic molecules. Assays are available for all scientific community to analyze functional features such as angiogenesis, tumorigenesis, cell invasion, metastasization, vascular permeability. The available collection of functional assays can be applied to a diversity of test conditions including cells previously grown in culture, biomaterials, drugs, tested directly on the CAM or on CAM xenograft tumours, conditioned mediums, supernatants enriched in exosomes or other vesicles or components, extracts from plants or microorganisms.
Marta Teixeira Pinto mtpinto@ipatimup.pt
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Marta Teixeira Pinto is responsible for the In vivo CAM assays platform at i3S. She graduated in Biochemistry (FCUP, 1999), completed her MSc degree in Neurosciences (FMUL, 2001) and obtained her PhD in Human Biology (FMUP, 2008). Marta Teixeira Pinto joined Ipatimup as a researcher (CiĂŞncia2008) of the Cancer Genetics Group and was mainly dedicated to study cancer angiogenesis, invasion and metastasis. She authors 24 papers with more than 230 citations and h-Index=10. After setting up the laboratory and implementing several important assays, in 2012, Marta Teixeira Pinto established the In vivo CAM assays platform.
03.1.11 Proteomics
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The Proteomics platform integrates the RNEM Portuguese Mass Spectrometry Network. RNEM is included in the FCT's Research Infrastructures Roadmap.
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i3S Proteomics platform provides access to mass spectrometry analysis of protein samples from extracts, solutions and gel bands. The Proteomics platform is prepared to work in a variety of experimental workflows in order to provide answers to scientific questions and to address researchers’ needs. The team offers scientific and technical expertise with protocol design, experimental strategy, results/data interpretation and consultancy including assistance in grant proposals and project setup. We also provide training in workshops and pre-/ postgraduate courses. The Proteomics platform integrates the RNEM - Portuguese Mass Spectrometry Network. RNEM is included in the FCT's Research Infrastructures Roadmap.
Resources available •
Hybrid Quadrupole-Orbitrap mass spectrometer (Q-Exactive, Thermo);
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NanoUHPLC (Ultimate 3000, Thermo) and nanoESI source;
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SpeedVac sample concentrator (Thermo);
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GS800 densitometer (Bio-rad).
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Applications The Proteomics platform is able to perform nanoLC / high-resolution accuratemass spectrometry approaches for protein characterization of biological and clinical samples. Examples include molecular mass determination of biomolecules including metabolites, peptides or proteins; high-throughput protein identification; labeled or label free quantitation of protein expression levels; targeted protein quantitative analysis and characterization of protein post-translational modifications including phosphorylation, acetylation, glycosylation, among others.
Hugo Osรณrio hosorio@i3s.up.pt
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Hugo Osรณrio is the coordinator of the Proteomics platform at i3S, researcher in the Glycobiology in Cancer group and Affiliated Professor at the Faculty of Medicine of the University of Porto. He graduated in Biochemistry at University of Porto in 1999 and finished his PhD in Biomedical Sciences in 2004 at ICBAS-UP. He is specialized in proteomics and mass spectrometry applied to health and life sciences. His major research activity is focused on the search for novel protein biomarkers with diagnostic application in cancer with an emphasis in protein post-translational modifications. Hugo Osรณrio has a total of 50 publications in international peer-reviewed journals and two book chapters. His research work has over 778 citations and an h-index of 14.
03.1.12 Translational Cytometry
The platform provides training and consulting for researchers that intend to use the flow cytometry in their projects and helps analyzing and interpreting data with the FlowJo software.
The mission of the Translational Cytometry Scientific Platform in i3S is to offer efficient and reliable flow cytometry services with the highest standards of quality control and productivity. Translational Cytometry provides equipment and support for acquisition and analysis of flow cytometry data and cell sorting (separation) from single cell suspension using fluorescence. Also, the platform organizes hands-on training for researchers who intend to use the flow cytometry in their projects. Furthermore, we offer consulting in analyzing and interpreting data with the FlowJo software.
Resources available Cell • • • • • •
analyzers: BD FACSCalibur Lasers: Blue (488nm); Red (635nm) BD Accuri C6 Lasers: Blue (488nm); Red (640nm) BD FACSCANTO II Lasers: Violet (405nm); Blue (488nm); Red (633nm)
Cell sorters: • BD FACSARIA II • Lasers: Blue (488nm); yellow/green (561nm); Red (633nm)
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Data analysis software: • FlowJo v9 and v10
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Applications The Flow Cytometers provide analysis of multiple parameters in individual particles within heterogenous population by passing thousands of cells per second, one by one, through a laser beam and collecting the resulting scatter or refracted light and fluorescence. Therefore, a number of applications, including multicolor analysis of cell phenotype, gene expression, membrane potential, Ca2+ and Mg2+ influx and cell cycle can be performed. Moreover, the Cell Sorter allows the separation of a population of cells into subpopulations based on fluorescent labeling. Individual cells can then be diverted from the fluid stream and collected into viable and homogeneous fractions at exceptionally high speeds and a purity near 100%.
Catarina Meireles catarina.meireles@ibmc.up.pt
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Catarina Meireles got her Bachelor’s degree in Microbiology from Escola Superior de Biotecnologia, Universidade Católica do Porto (2008), Master’s from Universidade do Minho (2010) and she did her Ph.D. thesis at IBMC of Universidade do Porto (2014-2018). Since 2016, Catarina Meireles holds the position of coordinator of the Translational Cytometry platform at i3S.
03.1.13 X-Ray Crystallography
The platform has an openaccess policy and its resources are available to both the scientific community and the industry (pay-per-use or contract services).
The X-ray Crystallography platform provides access to state-of-the-art facilities and instrumentation for crystallization and crystallographic data collection of small molecules and macromolecules. The platform has an open-access policy and its resources are available to both the scientific community and the industry (pay-peruse or contract services). While specialized training is required for access, it can be provided on demand.
Resources available Dual walk-in temperature-controlled crystallization chambers (4 °C and 20 °C) Douglas Instruments Oryx4 LCP crystallization robot: •
sub-microliter scale (minimum 100 nL) setups in 96-well format
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sitting-drop, hanging-drop, and microbatch configurations
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random and regular microseeding
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LCP dispensing with lipidic cubic phase attachment
Rigaku/Oxford Diffraction Gemini PX Ultra single-crystal X-ray diffractometer •
Dual X-ray source: Enhance Mo K-alpha with channel cut graphite monochromator & Enhance Ultra Cu K-alpha with multi-layer optics
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Four-circle high angular resolution (0.37 Å for Mo, 0.78 Å for Cu) kappa goniostat
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Onyx CCD 165 mm area detector
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Oxford Cryosystems Cryostream 700 sample cryostat
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Applications The platform’s equipment and expertise can be used in the crystallization of small molecules, biomolecules and biomolecular complexes, including membrane proteins. It allows the collection and processing of crystallographic data from small molecule crystals and biomolecular crystals, and the optimization of cryoprotection conditions.
Pedro J. B. Pereira ppereira@ibmc.up.pt
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Pedro Pereira graduated in Biochemistry from the University of Porto in 1993 and was a PhD student of Programa Gulbenkian de Doutoramento em Biologia e Medicina from 1993 to 1999. From 1994 to 1999 he was a PhD fellow at the Max-Planck-Institut fuer Biochemie (Martinsried, Germany) under the supervision of Prof. Dr. Dr. hc mult. Robert Huber. Pedro Pereira attained a PhD in Biomedical Sciences (Biochemistry) from the University of Porto (1999). He received FEBS Long-Term (1999-2000) and FCT (20002001) post-doctoral fellowships while at the Institut de Biologia Molecular de Barcelona. From 2002 to 2009 he worked as an Assistant Researcher at IBMC and since 2009 he is a Principal Investigator at the institute. Pedro Pereira leads the Macromolecular Structure group and is the coordinator of the X-ray crystallography platform at i3S.
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03.2 Open Policy & Training
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03.2 Open Policy & Training
Our open access policy renders the scientific platforms’ resources available to both internal and external scientific communities, as well as to the industry. Notwithstanding, their use requires training, so the coordinator of each platform defines the necessary training scheme for each user and provides, on demand, specialized training. In particular, Animal Facility’s legal and internal rules require a license to work with laboratory animals provided by the competent authority in Portugal (DGAV), training in laboratory animal science (FELASA or FELASA equivalent) and the completion of the internal training program. Upon training, users can book and use the equipments at their own convenience, although some restrictions may apply. The i3S scientific personnel puts forth advanced and highly skilled training targeted at academic and industry researchers, PhD students, technicians and other members of the scientific community. Courses and workshops, mainly focused on biomedical and life sciences, cover a wide range of disciplines, such as Laboratory Animal Science, Advanced Light Microscopy, Statistical Analysis of Biological Data, Biomolecular Interactions, Biosciences Screening, and Cancer Research, among others. Some training activities have been developed in collaboration with EMBL and EMBO. The added value of these platforms lies in their integrative and collaborative approach by providing consultancy during project writing and evaluation, assay development and validation, as well as pilot experiment prior to project submission. Moreover, when it comes to project implementation, platforms train the project team to use the laboratory facilities and equipment, perform data acquisition, image and data analysis. In addition, non-listed services can be requested, in which case the platform will not only assist in project evaluation and development, assay development and validation, but also oversee experimental work and data analysis along with reporting. The cost is determined according to the platform in question and the type of usage or service required. For more details please contact the Team Coordinator of the scientific
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platform you are interested in or visit our website at www.i3s.up.pt.
03.2.1 Technical Training 2017 01
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Training Session: Blood sampling and intravenous injection in laboratory mice 24 February 2017
16 January 2017 Workshop on Responsible conduct of research
8, 15 & 22 February 2017 Workshop: A different way of managing references
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9-10 February 2017 GENiE High content screening Workshop
Training Session: Administration techniques in laboratory rats
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22 May 2017
Workshop on neuroengineering: revealing, repairing & enhancing brains 27-28 April 2017
Introduction to applied statistics with R - Module 1
23rd Laboratory Animal Science Category C
2-3 June 2017
8-19 May 2017
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Course: Introduction to digital image analysis with ImageJ / Fiji 19-21 September
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2017
Session: EndNote & Web of Science & InCities 12 September 2017
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Hands-on workshop on image analysis for imaging flow cytometry 28-30 September 2017
20th Introductory course in laboratory animal science Oct-Dec 2017
03.2 Open Policy & Training
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Optical Microscopy Imaging for Biosciences
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27-31 March
20 March 2017
Training Session: Administration techniques in laboratory mice 21 April 2017
3rd Edition Introduction to data analysis & image processing with MATLAB
Training Session: Blood sampling and intravenous injection in laboratory rats
19-21 April 2017
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3rd Course on high content screening and image analysis for biosciences
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6-9 June 2017
Hands-on course: Introduction to biological data analysis with R 26-28 June 2017
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9,10, 11 October 2017
July-Sept 2017
24th Laboratory animal science Category C 11-22 September 2017
Workshop on Introduction to epidemiological research and the methods of systematic reviews 23 November 2017
1st Edition: Workshop in atomic force microscopy coupled to inverted fluorescence microscopy 16-17 November
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Course in experimental design and analysis of data for research with animals
19 Introductory course in laboratory animal science th