ICVS/3B's - Associate Laboratory Meeting€¦ · 4th ICVS/3B's - Associate Laboratory Meeting Brief...

4th ICVS/3B's - Associate Laboratory Meeting

Index

3

INDEX

Main talk 5

Brief presentations on "out of the box" 6

ICVS/3B’s Grants 2014/Outcomes – Selected presentations 10

Selected oral presentations 18

Poster Session 1 22

Poster Session 2 63


Main Talk

5

SPHERES IN REGENERATIVE MEDICINE

João F. Mano1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal The sphere has been one of the most popular geometry in the design of basic objects and devices in biomedicine. Particles and capsules have been developed from the nano up to the macro scale levels as reservoirs or supports for therapeutic molecules and cells. Besides playing with the size, compartmentalized systems have been proposed in new devices for drug delivery or to induce the local development of new tissues. We have been proposing several methodologies for the processing of such elements using natural-based polymers. Two technologies will be focused in this presentation: (i) layer-by-layer assembling, that was used to produce smart microparticles or hierarchical liquefied capsules containing internal solid microparticules for cells attachment, and (ii) the employment of biomimetic superhydrophobic substrates as support for fabricating hydrogel/polymeric spherical particles under mild conditions and with a high encapsulation efficiency. This presentation overviews a series of examples covering the possibility of using natural-based spherical structures in the context of tissue engineering and regenerative medicine. We invite the researchers from the ICVS/3B’s associate laboratory in joining efforts to apply the general concepts presented in other areas of health sciences and technologies.


Brief presentations on “out of the box”

6

EXPANDING THE COMPLEXITY OF LAYER-BY-LAYER COMPARTMENTAL UNITS WITH

DRUG DELIVERY AND ENZYMATIC DEGRADATION

Rui R. Costa1,2, João F. Mano1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal During the last two years, we were able to extrapolate layer-by-layer (LbL) – a self-assembly-driven surface modification strategy that allows the construction of nanostructured films – to the third dimension. Namely, spherical devices entirely delimited by multilayer polymeric coatings were produced with various dimensions, ranging from a few nanometers to a few millimetres, and organized as hierarchical compartments. Their complexity resembles that of a natural cell, where several organelles with distinct function are found. In the lab, it is possible to encapsulate distinct elements within alginate jellified macrocompartments, such as particles, capsules, drugs and cells. Mimicking the example of cells, one can envisage a platform that allows screening the effectiveness of new drugs and new carriers, as well as controlling the formation of micro-tissues within a miniaturized tunable environment at a laboratorial level. Currently, we propose increasing the complexity of compartmental devices that have been produced so far beyond simple hierarchical organization. Namely, addition of degradation factors, such as enzymes, may provide a more realistic model to study the release of drugs in an environment simulating a specific biological niche. A screening platform of this nature may provide a bioartificial environment bridging in vitro and in vivo experiments. This way, invasive “trial and error” animal studies could be avoided until proper simulations of new therapies are performed.



7

ARTIFICIAL ANTIGEN-PRESENTING CELLS: ARTIFICIAL SOLUTIONS FOR REAL DISEASES

Tírcia C. Santos1,2, Egídio Torrado2,3, Daniel B. Rodrigues1,2, Joaquim M. Oliveira1,2, António G.

Castro2,3, Rui L.Reis1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal Cancer immunotherapy has seen enormous advances in the last decade. One of the most promising approaches is the used of autologous dendritic cells (DCs) loaded with tumor antigens that are injected back into the patient [1]. The rationale behind this approach is to prime and expand anti-tumor T-cell responses that will attack and lyse tumor cells. However, one of the major drawbacks of this approach is the modulation of the DC function by the tumor microenvironment [2], hindering the antigen presenting capacity of the DCs and their cytokine production, critical elements to generate efficient anti-tumor T-cell responses. In this project we propose to develop a novel artificial antigen-presenting cells (aAPCs) system based on natural origin dendronized polymers at the nanoscale that can be loaded with cytokines in their inner core and coated with recombinant MHC molecules at their surface to prime and expand tumor-specific T-cell responses. The advantage of this approach when compared to autologous DC treatments is that the aAPCs are not modulated by the tumor microenvironment. Moreover, the slow and steady release of cytokines from the inner core and the relatively long half-life of the aAPC promote a steady and long lasting activation of cytotoxic T-cell responses. Overall, with this project we expect to develop a novel approach to treat solid tumors but that can also be used for the treatment of other chronic diseases, such as infection. The flexibility of the proposed system allows for the coating of the aAPCs with different molecules that serve not only to expand ongoing T-cell responses but also to modulate the function of the T-cells, preventing the development of their “terminal differentiation” or “exhaustion”. This novel and yet largely unexplored nanomedical approach has a significant translational application in the clinical scenario with important implications in solid tumor treatment. [1] I. Caminschi, E. Maraskovsky, et al, Frontiers in Immunology, 3, 13 (2012). [2] M. Plantinga, H. Hammad, et al, European Journal of Immunology, 40(8), 2112 (2010).



8

PHASE II EXPLORATORY TRIAL OF MESENCHYMAL STEM CELLS IN PRIMARY

PROGRESSIVE MS

João Cerqueira1,2

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho,

Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal

Background Primary progressive MS has no approved treatment and all clinical trials have failed to provide convincing efficacy results. Stem cells have shown promising results in animals models of MS and on preliminary phase I and II studies in MS patients. However, most trials have so far focused on relapsing remitting or secondary progressive forms, just as drug trials have. Thus, while there is a need for PPMS treatments, as far as we know stem cells have never been tested in a randomized trial in this particular group. Research in animal models and patients has suggested that stem cells act mainly as immunomodulators. However, they have also the potential to modulate the activity of other cell types, including astrocytes and microglia, which might be of relevance for PPMS. The present idea addresses aims at exploring the safety and mechanism of action of MSC in PPMS. Idea We propose a phase II trial of autologous MSCs in patients with PPMS. Patients with PPMS (less than 10 years from the beginning of symptoms) and still able to ambulate (EDSS<=6.5), not on any disease modifying therapy or fampridine, would be recruited for the trial. All patients would receive the autologous MSCs, but the trial would be run as a pré and post comparison. After a first year of data collection and prospective follow-up, patients will receive the autologous MSC, administered iV according to standard protocols, and be followed for an additional year (up to 5 years for safety). Patients would be followed at 3 months intervals. Safety measures would be mainly adverse events and serious adverse events. Mechanism of action measures would be: -immunophenotyping of cells in the periphery and the CNS (CSF) -markers of astrocytic activation (S100b, GFAP) in serum and CSF -markers of neurodegeneration (neurofilament light and heavy chains, Abeta) in the CSF Preliminary efficacy measures would be: -progression of timed 25 feet walk from baseline -progression of spinal cord atrophy -progression of SDMT/MSFC score (with SDMT instead of PASAT)



9

BACE-1 AS THE CONVERGING TARGET IN ALZHEIMER´S DISEASE AND DEPRESSIVE

PATHOLOGIES: A NEW NANOPARTICLE DRUG DELIVERY SYSTEM

I. Sotiropoulos1,3, H. Ferreira2,3, M. Martins2,3, H. Leite-Almeida1,3, J. Bessa1,3 R.L. Reis2,3 & N.N. Neves2,3

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Increasingly, depression and Alzheimer´s disease (AD) are recognized as inter-connected disorders with common molecular mediators and neurobiological basis. Many clinical studies indicate the existence of AD-related mechanisms such as amyloid precursor protein (APP) misprocessing and Aβ overproduction in patients suffers from depression. Despite the considerable research progress on both diseases over the past decades, there is still no cure for AD while the depressive treatment in current clinical use is also problematic. While previous AD treatment modalities based on blocking γ-secretase, the last enzyme responsible for Aβ overproduction, proved to be palliative and unable to improve disease clinical symptoms, recently, an increased focus is being given to BACE-1 enzyme, the first enzyme that cleaves leading to Aβ overproduction, as a key regulator protein of AD brain pathology. Indeed, emerging evidence from both clinical and experimental studies, including some of the ICVS ones, suggest that BACE-1 could be the essential mediator of synaptotoxicity found in AD and well as in stress-driven depressive pathology while recent evidence suggests that BACE-1 inhibition may have antidepressant action. While previous clinical trials struggled with inefficient deliver to pathological sites and/or undesirable side effects of increased dosage or peripheral drug effects, this project will take advantage of key nanoparticles (NPs) enabling technologies towards the development of a BACE-1 inhibitor–NP multifunctional biodegradable system that will be able to incorporate and effectively release the BACE-1 inhibitor on the specific hippocampal subpopulation (e.g. dentate gyrus; DG), a key brain area for both AD and depression synaptic pathologies. For that purpose, the NPs surface will be functionalized with specific DG-related molecules while imaging agents will also be included in NPs for diagnosis/disease monitoring. Hippocampal slides will be used for in vitro screening/optimization studies followed by in vivo (animal model) NP validation. These experimental studies will provide new insights towards the development of novel drug deliveries strategies adding to our knowledge about the connecting pathomechanism(s) between depression and AD.


ICVS/3B’s Grants 2014/Outcomes – Selected Presentations

10

EVALUATION OF AUTOPHAGY IN CELL SHEETS OF ADIPOSE STROMAL/STEM CELLS IN

NORMOXIA AND HYPOXIA: CORRELATION WITH STEMNESS

Maria M.G. Azevedo 1,3 and Rogério P. Pirraco,2,3 1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Stromal/stem cells from human adipose tissue (hASC) are increasingly popular in the tissue engineering and regenerative medicine field due to being easily isolated and expanded. Cell sheet engineering has been proposed as an ingenious methodology to deliver such progenitor cells for the treatment of tissue defects or diseases. Using this technology, cells produce their own extracellular matrix, which can be advantageously used as a scaffold to deliver the cells. Concurrently, it has been shown that hASC culture in hypoxic conditions has a positive influence in the maintenance of the cells multidifferentiation potential. Moreover, several authors have recently demonstrated a critical role of autophagy in the homeostatic control of stemness, tissue regeneration and cellular programming abilities of mesenchymal stem cells. The main objective of the present project is to uncover a potential role of autophagy in the modulation of the differentiation potential of hASC cell sheets cultured in hypoxic conditions.



11

BIORESORBABLE UROLOGICAL STENT SYSTMES BASED ON NATURAL POLYMERS

Ana Rita C. Duarte2,3, Alexandre A. A. Barros2,3, Carlos Oliveira1,3, Ricardo Pires2,3, Estevão Lima1,3,

João F. Mano2,3, Rui L. Reis2,3 1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Urinary stents are commonly applied in urology to prevent blockage in the ureter. The blockage of urine flow is, in most of the cases, temporary and the indwelling time is limited to a short period of time. In this sense, major advantages would be foreseen by the development of bioresorbable stents. The technology proposed relies in the development of bioresorbable urological stents produced from natural origin polymers, and designed to meet the patient needs and to avoiding a second surgery for stent removal. The stents will be formed from an aqueous solution of biopolymer which is gelled using a crosslinking agent and subsequently dried. Although out of the scope of this project we foresee that the mild processing conditions will further allow the incorporation of active compounds and the stents could also be used as drug/protein-delivery platforms. In this project, the development of the stents and in vitro characterization was successfully performed. At the moment, a first assessment of in vivo evaluation of the materials developed was studied using a female pig model. The ureteral stent when implanted was not disrupted when placed through the urethra and the bladder, nonetheless, future work requires the optimization of the mechanical properties of the stent in order to pass the ureteral orifices and settle in the ureter. From the experiments performs we concluded that the biodegradable ureteral stents developed can be implanted following the surgical procedure performed daily in clinics and no major changes to the procedure are required. [1] A. A. Barros, A. R. C. Duarte, R. A. Pires, B. Sampaio-Marques, P. Ludovico, E. Lima, J. F. Mano, R. L. Reis, Journal of Biomedical Materials Research B: Applied Biomaterials, doi:10.1002/jbm.b.33237, 2014



12

3DTUMOURS – DEVELOPMENT OF A 3D CULTURE SYSTEM FOR THE IN VITRO STUDY OF

TUMOURS PATHOPHYSIOLOGY

Albino Martins1,3, Marta Viana-Pereira2,3, Ana S. Nunes2,3, Rui L. Reis1,3, Fátima Baltazar2,3, Nuno M. Neves1,3

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Standard 2D monolayer cultures, which are extensively used in cancer research, poorly represent the in vivo tumour behaviour, since the 3D microenvironment is neglected consistently [1, 2]. Tumour microenvironment is a complex system encompassing several cell types, which collectively perform an active role in carcinogenesis, contributing to tumour initiation, growth, development, invasion and metastasis. Besides its role on tumour progression, the interplay between tumour-proliferating cells and its associated stroma provides a key mechanism through which cancer cells escape the immune system [3, 4]. Hence, studies comprising both neoplastic cells and the surrounding stroma become essential to clearly understand the biology of tumours. Thus, we aim to develop a 3D in vitro tumour model, which recapitulate the cellular complexity and the hierarchical organization of the extracellular matrix (ECM) of tumours in vivo. Different breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT-20, MCF7 and Hs 578t) were tested for their capability to form spheroids. It was assessed the temporal stability and the proliferative state of the formed tumour spheroids, as well as the immunoexpression of hypoxia markers, and of glucose and lactate transporters. The MCF7 and Hs 578t cells were able to form spheroids when in culture in agarose coated well plates, maintaining their physical integrity over 20 days in culture. The MCF7 cells are more proliferative, contrarily to Hs 578t cells, as confirmed by the expression of Ki-67. In general, the immunoexpression of hypoxia markers (i.e. CAIX and LDH), and glucose (i.e. GLUT-1) and lactate transporters (i.e. MCT1, MCT4) is more pronounced in MCF7 cells, suggesting a more aggressive phenotype. Our initial results are critical to enable establishing a more realist in vitro 3D co-culture system involving tumour and endothelial cells, to elucidate the crosstalk and cell-cell interactions developing by those two cell communities in tumours. [1].Burdett, E., et al., Engineering tumors: a tissue engineering perspective in cancer biology. Tissue Eng Part B Rev, 2010. 16(3): p. 351-9. [2].Bissell, M.J. and D. Radisky, Putting tumours in context. Nat Rev Cancer, 2001. 1(1): p. 46-54. [3].Albini, A. and M.B. Sporn, The tumour microenvironment as a target for chemoprevention. Nat Rev Cancer, 2007. 7(2): p. 139-47. [4].Polyak, K., I. Haviv, and I.G. Campbell, Co-evolution of tumor cells and their microenvironment. Trends Genet, 2009. 25(1): p. 30-8.



13

SPIONS-LOADED TROJAN MICROPARTICLES AS DRUG CARRIERS FOR THE TREATMENT

OF TUBERCULOSIS M.T. Rodrigues1,3, R.M.A.Domingues1,3, C. Gonçalves2,3, R.R. Costa1,3, C. Leite2,3, A. Carvalho4, J.F.

Mano1,3, R.L. Reis1,3, J. Pedrosa2,3 and M.E. Gomes1,3 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2-Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal, 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 4- Chemistry Department, University of Minho, Portugal Tuberculosis (TB) is a worldwide infectious disease caused by M. tuberculosis (Mtb), whose currently available treatments require strict and prolonged regimens to be effective. Recently, purine derivatives were reported to have activity against Mtb. Together with Department of Química, we developed several 9-arylpurine derivatives, two of them (P1 and P2) revealing anti-TB activity. Anti-TB drug delivery systems via the pulmonary route would be a vast improvement in: 1) direct drug delivery to the lungs; 2) targeting alveolar Mc infected by Mtb; 3) reducing systemic toxicity; 4) avoiding first pass metabolism; 5) improving patient compliance. Among the various alternatives being explored, dry powders are currently considered the most convenient alternative for this administration route. This project aims to design and develop multifunctional microparticulate carriers for the delivery of the new anti-TB drug into Mtb-infected cells of the lung. Inhalable carriers will facilitate the target delivery of reduced but effective doses of the drug. Also, the carriers will incorporate magnetic nanoparticles (SPIONs) to target the drug release into the site of infection by external application of a magnetic field. We are currently exploring several strategies to produce inhalable microparticle (MPs) carriers based on natural polymers and using porous CaCO3 as sacrificial template. With these approaches we aim to produce MPs with well-defined properties that: i) meet the criteria for inhalable dry powders; ii) be based on biocompatible, biodegradable materials; iii) be able to target drug delivery into alveolar macrophages. As Mtb resides within macrophages, the transport of anti-TB drugs within the carriers also implies an internalization mechanism for a proper deliver of the drug to the infected macrophages. Thus, the proposed carriers will be in vitro and in vivo tested for cell cytotoxicity as well as to determine their potential effectiveness in releasing the drug into healthy and infected alveolar macrophages.



14

SURFACE-ENGINEERED DENDRIMERS AS NOVEL NANOCARRIERS FOR THE DELIVERY OF

TEMOZOLOMIDE CHEMOTHERAPY INTO MALIGNANT BRAIN TUMORS

M. Pojo1,3, M. Carvalho2,3, Nuno Sousa1,3, R.L. Reis2,3, J.M. Oliveira2,3, B.M. Costa1,3 1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Glioblastoma (GBM) is the most common and most malignant tumor of the central nervous system, for which curative therapies are not available partly due to suboptimal access of the different anti-cancer chemotherapeutic drugs to the tumor. Recently, the use of nanoparticles as drug delivery systems (DDS) for cancer treatment has gained particular interest. We previously showed that carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (CMCht/PAMAM NP’s) may be an attractive DDS for these tumors, as GBM cell models can efficiently and rapidly uptake these nanoparticles [1]. Since temozolomide (TMZ) is the standard chemotherapeutic agent used to treat GBM patients, we aim to study the therapeutic value of TMZ-loaded CMCht/PAMAM NP’s, both in vitro and in vivo. We have successfully synthesized and loaded CMCht/PAMAM nanoparticles with TMZ, and characterized its physicochemical properties and in vitro release profile. We also generated preliminary data on the internalization kinetics of both fluorescent-labelled and TMZ-loaded CMCh/PAMAM NP’s by GBM cell lines, and evaluated its possible cytotoxic effect in vitro. Interestingly, it was observed highly efficient (~100%) and rapid (<24 h) internalization of TMZ-loaded CMCh/PAMAM NP’s, accompanied by a decrease in cell viability, globally indicating this DDS may be useful. In the future, we will expand our studies to in vivo mice models of GBM to evaluate TMZ-loaded CMCht/PAMAM NP’s biodistribution and therapeutic effect, particularly at the level of overall survival. Our findings may reveal novel therapeutic strategies for GBM, which can also serve as a proof-of-principle applicable to other human cancers treated with TMZ (e.g., melanoma). [1] M. Pojo, S.R. Cerqueira, T. Mota, A. Xavier-Magalhães, S. Ribeiro-Samy, J.F. Mano, J.M. Oliveira, R.L. Reis, N. Sousa, B.M. Costa, A.J. Salgado. Journal of Nanoparticle Research 15, 1621 (2013)



15

TRACKING CELL-CELL COMMUNICATION IN NEURAL CELLS BY SUGAR LABELING.

J.C. Sousa1,3,#, D. Soares Costa2,3, J. Trigueiro Costa1,3, F. Marques1,3, S. Dá Mesquita1,3, N.

Petkova2,3, R. Reis2,3, N. Sousa1,3, I. Pashkuleva2,3,# 1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal # Project Coordinators This ICVS/3B's project aims at gaining insight on the role of cell surface and extracellular matrix glycans in the modulation of neural cells communication. Glycans are complex carbohydrate molecules composed of variable length chains of simple sugars that are associated with intracellular and cell surface proteins and lipids. In the brain glycans are relevant for neural cells differentiation and function, and were recently implicated in synapse formation and synaptic transmission. We are currently synthetizing peracetylated azido-functionalized analogues of natural sugars (glucose, mannose and fucose) and taking advantage of neural cells glycosylation cellular machinery to present these sugars at the cell surface. We investigated the influence of the chemical modification on the uptake and biosynthetic pathways of the sugars in hippocampal neuronal cells in vitro. We aim at using both in vitro and ex vivo approaches to determine the spatial and temporal distribution of glycans at the cell surface of neural cells by tracking the modified sugars with a labeled phosphine probe. The ultimate goal of this project is to develop this tool to characterize the changes in synaptic plasticity that occur in pathophysiological processes such as the stress response and neuronal regeneration.



16

UNIVERSAL VACCINE FOR TUBERCULOSIS BASED ON LIPOSOMAL DELIVERY OF

HYPERVARIABLE ANTIGENS

M. Martins2,3, H. Ferreira2,3, E. Torrado1,3,F. Rodrigues1,3, J. Pedrosa1,3, R.L. Reis2,3, A.G. Castro1,3, N.M. Neves2,3, M. Saraiva1,3, N.S. Osório1,3

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Tuberculosis(TB) kills yearly more than 1 million people and the poor efficacy of the only TB-vaccine in use, BCG, contributes to this devastating impact. Different Mycobacterium tuberculosis strains are associated with differences in immune response, virulence and clinical TB outcomes. However, ongoing trials for TB vaccines still do not take into account the strain diversity and evolution of M. tuberculosis. By analysing genomes of 283 M. tuberculosis clinical isolates from different worldwide locations, we found positive selection sites varying between BCG and M. tuberculosis[1]. These sites are likely to be evolving in response to host immune system-driven pressures and provide good candidates for the development of more effective vaccines[1]. We have now extended the analysis of the M. tuberculosis sites under positive selection to predict its binding affinity to a broad set of class I and II Human Leukocyte Antigen (HLA) molecules. This analysis allowed the selection of the peptides which we aim at delivering as TB vaccine candidates. Nanotechnology-based carriers, namely liposomes (LIP), have emerged in recent years as potentially advantageous vaccine delivery systems due to their biosafety, ability to ensure a sustained release and enabling multi-functionalization versatility. We started by developing two peptide-liposomal formulations (F1 and F2) using a validated M. tuberculosis antigen. The particles exhibit a mean size of 822.1 ± 13.5 nm (F1) and 314.2 ± 8.7 nm (F2) and a ζ- potential close to neutrality for F1 and negative for F2. The peptide encapsulation efficacies were of 65.6% (F1) and 66.9% (F2). The efficacy of these formulations in the delivery of M. tuberculosis antigens will be now tested in vitro using lung cell cultures from TB infected mice. Ultimately, by combining both M. tuberculosis antigen variability and LIP delivery systems we expect to develop a TB vaccine efficacious against a broad range of M. tuberculosis strains. [1] N.S. Osório, F. Rodrigues, S. Gagneux, J. Pedrosa, M. Pinto-Carbó, A.G. Castro, D. Young, I. Comas, M. Saraiva, Molecular biology and evolution 30, 1326 (2013).



17

ARTHYMUS - ARTIFICIAL THYMUS CONCEPTION: A TISSUE ENGINEERING STRATEGY

Cláudia Serre-Miranda1,3, Ana Costa-Pinto2,3, Bruno Cerqueira-Rodrigues1,3, Albino Martins2,3, Marta

Silva2,3, Nuno M. Neves2,3, Margarida Correia-Neves1,3

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal T cell differentiation is dependent on a functional thymus [1]. Several diseases cause premature thymic atrophy or its total absence [2]. Since T cells are essential for the protection against pathogens, it is crucial to promote de novo differentiation of T cells in individuals affected by those diseases. Nowadays, the solution available is the transplantation of a thymus, a protocol that can be just applied to complete athymic infants [3,4]. This procedure is extremely expensive and can lead to the production of autoreactive T cells due to the mismatch between donor and receptor tissues [5]. Given that scenario we proposed exploring the development of strategies enabling the production of T cells in vitro and in vivo by culturing thymic epithelial cells (TECs) with T cell precursors [6]. For that, we are developing protocols in vitro to obtain TECs [7]. We are testing biomaterial structures and supports that are intended to mimic the thymic structure to develop new stable and functional artificial thymi. This strategy is intended to facilitate the development of T cells to be used in different conditions involving absence or reduced thymic function. The first stage of this project involved the establishment of collaboration with Hospital São João to obtain samples of infant thymus collected during pediatric cardio-thoracic surgeries. Next stage, we established the protocols to isolate and develop stable primary cultures of human TECs. Currently, we are evaluating the interaction of TECs with biomaterials, namely polycaprolactone nanofiber meshes (PCL NFMs), by expanding TECs into relevant numbers and by optimizing the cell seeding methods. The next step will involve the study of the interaction between TECs cultured in the scaffolds and human hematopoietic stem cells to evaluate their ability to differentiated into T cells. The last task of this project will validate the hypothesis that TECs can be obtained by the differentiation of various adult stem cells sources in co-culture with adult functional TECs. [1] J.C. Zuniga-Pflucker, Nat Rev Immunol 4, 67-72 (2014) [2] A. Nunes-Alves, C. Nobrega et al, Trends in Immunology 34, 502-10 (2013) [3] R. Hong, Clinical Reviews in Allergy & Immunology 20, 43-60 (2001) [4] M.L. Markert, B. Devlin, I. Chinn, E. McCarthy, Immunologic Research 44, 61-70 (2009) [5] M.L. Markert, Google Patents (2006) [6] M.C, Poznansky, Nat Biotech 18, 729-34 (2000) [7] A.V. Parent, Cell stem cell 13, 219-29 (2013)


Selected Oral Presentations

18

SCARRING IMPAIRMENT BY POLYHYDROXYBUTYRATE-CO-HYDROXYVALERATE

STRUCTURES-LADEN ADIPOSE STEM CELLS

Alessandra Zonari 1-3, A.C Paula1, T. Martins1, J.N Boleoni4, S. Novikoff5, A.P. Marques2,3, V.M. Correlo2,3, R.L. Reis2,3, A.M. Goes1

1- Department of Biochemistry and Immunology, Federal University of Minas Gerais, Brazil 2- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 4-Department of Veterinary Medicine, Federal University of Espírito Santos, Brazil 5-Departament of Nephrology, Federal University of São Paulo, Brazil The limited outcomes of commercially available skin substitutes reinforce the fact that the regeneration of functional skin remains elusive. Recently, our group developed a scaffold based on poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) with properties that favor skin cells performance thus representing a promising strategy to improve wound healing(1). Therefore, this work aimed to further advance this knowledge by assessing the regenerative potential of the PHBV-laden adipose stem cells in a full-thickness rat wound model. Adipose stem cells (ASCs) isolated from GFP transgenic rats (Lewis F455.5/Rrrc) were seeded (1x106 cells) on PHBV structures prepared as previously described3. Constructs were transplanted to rat full-thickness wounds according to the following groups (n=4): empty wound (control), scaffold (PHBV) and scaffold plus cells (PHBV+ASCs). Tissue explants were retrieved on days 7, 14, 21 and 28 post-surgery. Wound closure was followed along the implantation time and correlated with histological (localization of GFP-ASCs, matrix deposition/organization, neovascularization) and molecular analysis (GFP, VEGF, bFGF, TGF-β1 and α-SMA). A significant different percentage of wound closure was only observed between control and PHBV groups at day 14. At the endpoint of the experiment all the wounds were closed and the PHBV structure was completely degraded. GFP-positive ASCs were only detected at the wound area up to 7days. An up-regulation of VEGF and bFGF was also detected at this time-point, consequently leading to a significant higher vessel density in the PHBV+ASC group. Additionally, in the presence of the PHBV structure a down-regulation of TGF-β1 and α-SMA was detected. The PHBV structure allowed ASCs to provide the necessary factors for enhanced vascularization and acted as template for guiding the wound healing process impairing myofibroblasts differentiation and consequently scarring. Therefore, we can conclude that PHBV-laden ASCs represent a promising approach for skin regeneration. [1] A.Zonari et al., Macromol Biosci, 14, 977–990 (2014)



19

EXTRACELLULAR VESICLES: CELL-DERIVED TOOLBOX ENABLING DESIGNING NOVEL

THERAPEUTIC STRATEGIES

M. Martins1,2, A. Martins1,2, R.L. Reis1,2, N.M. Neves1,2

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Extracellular vesicles (EVs) are nanosized vesicles carrying proteins and nucleic acids that are secreted by all cells. EVs have attracted much interest since their involvement on intercellular communication and their identification in biological fluids. These landmarks pointed the way to a new era of biomedical research. In a quickly expanding field, EVs are being increasingly explored in immunology, microbiology, neurosciences, oncology and stem cell biology. EVs mediate a wide variety of functions that range from the regulation of gene expression, regulation of signaling pathways, to distribution of catalytic activity. Due to their relevance, the list of potential medical applications is expanding, envisioning EVs application as next generation non-invasive diagnostic and therapeutic tools. We are particularly interested in understanding the role of human bone marrow mesenchymal stem cells (hBMSCs) EVs as modulators of stem cells behavior, to exploit their application as vectors for advanced cell therapies. Here we show that hBMSCs secrete EVs. These EVs have a slightly negative charge, approximately 30 nm in diameter and are enriched in the well-established EVs protein markers CD9, CD63 and CD81. Additionally, it was observed that the yield of EVs increased over cell culture time. We also observed that homotypic EVs derived from osteogenic stimulated cells are able to potentiate the osteogenic fate of uncommitted hBMSCs. In the absence of other stimuli, EVs stimulated cells exhibited an increased expression of osteogenic early markers (Runx2, Bmp2, Sp7) and extracellular matrix markers (Bglap, Spp1) in comparison with non-stimulated cells. Our results suggest the ability of hBMSCs to secrete EVs that are able to communicate specific cues towards osteogenic commitment. Furthermore, we have established a toolbox of EVs isolation and characterization methods that can be applied to many fields of research and to a wide range of applications.



20

PROTEOMIC ANALYSIS OF THE ACTION OF THE MYCOBACTERIUM ULCERANS TOXIN

MYCOLACTONE: TARGETING HOST CELLS CYTOSKELETON AND COLLAGEN(1)

José B. Gama1,2, Steffen Ohlmeier3, Teresa G. Martins1,2, Alexandra G. Fraga1,2,

Belém Sampaio-Marques1,2, Maria A. Carvalho4, Fernanda Proença4, Manuel T. Silva5, Jorge Pedrosa1,2*, Paula Ludovico1,2*

1 - Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2 - ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3 - Proteomics Core Facility, Biocenter Oulu, Department of Biochemistry, University of Oulu, Oulu, Finland 4 - Chemistry Research Center, School of Sciences, University of Minho, Braga, Portugal 5 - Institute for Molecular and Cell Biology, Porto, Portugal Buruli ulcer (BU) is a slowly progressive, but very destructive and crippling disease, caused by the environmental pathogen Mycobacterium ulcerans, whose pathogenicity is mainly mediated by the lipid toxin mycolactone. There is a recommended antibiotic treatment. However 25% of the patients suffer from long-term sequelae caused by scarring and contracture of the wound, leading to functional limitations, psychosocial distress and stigma. In order to better characterize the cytopathogenesis of this disease, we applied two-dimensional electrophoresis (2-DE) to identify cellular processes affected upon exposure to mycolactone, on L929 mouse fibroblast. One of the main clusters of proteins changed were collagen biosynthesis enzymes (Plod1, Plod3, P4ha1), which were found down-regulated in mycolactone-exposed L929 cells. These endoplasmic reticulum-resident collagen modifying enzymes are essential for the formation and stabilization of collagen fibers. Consistent with a mycolactone impact upon the collagen fibers stability, in vivo analyses in a BU mouse model revealed mycolactone-dependent dermal collagen fibers degeneration upon infection with M. ulcerans. Our results demonstrate that the M. ulcerans toxin impact upon the extracellular matrix biosynthesis. This feature may cause fibroblasts and myofibroblasts repopulating the lesion during the healing process, when mycolactone levels decrease, to overcome the collagen-deficiency through abnormal- or over-production of collagen leading to the extreme contractures characteristic of BU. In this way, collagen stands out as an attractive therapeutic target and the value of collagen-based interventions in BU should be addressed in the future. * These authors are joint senior authors on this work (1) Gama JB, Ohlmeier S, Martins TG, Fraga AG, Sampaio-Marques B, Carvalho MA, Proença F, Silva MT, Pedrosa J, Ludovico P, PLoS Negl Trop Dis 8: e3066 (2014)



21

DOPAMINE D2 RECEPTOR IN THE NUCLEUS ACCUMBENS REGULATES MOTIVATION

C. Soares-Cunha 1,2, B. Coimbra 1,2, A. David-Pereira1,2,L. Pinto1,2, S. Sousa1,2, A.J. Rodrigues1,2

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Dopaminergic neurons in the mesolimbic circuit are relevant for motivational drive, but less is known about the role of the different types of dopamine receptors in this process. Herein, we observed a marked activation of dopamine receptor 2 (D2) expressing neurons in the nucleus accumbens (NAc) in a progressive ratio (PR) schedule of reinforcement paradigm and subsequently demonstrated that optogenetic phasic activation of D2 expressing neurons in this brain region was sufficient to significantly boost motivation; to further complement these studies, we showed that specific D2-neuronal activation in the NAc normalized motivational deficits in an animal model of accumbal D2 dysfunction. Altogether these results demonstrate that D2-dependent dopaminergic activation in the NAc is sufficient to modulate motivated behaviours.


Poster Session 1

22

Poster number

First Author Title

P1_.01 Nuno Alves UNDERCOVERING THE INVOLVEMENT OF ADULT NEURO- GLIOPLASTICITY

TO THE COURSE OF DEPRESSION – A BEHAVIORAL AND STRUCTURAL

ANALYSIS

P1_.02 Diana Amorim GALANIN IN THE DORSOMEDIAL NUCLEUS OF THE HYPOTHALAMUS

ACTIVATES TWO INDEPENDENT DESCENDING PRONOCICEPTIVE

PATHWAYS

P1_.03 Sara Amorim MOLECULAR WEIGHT OF SURFACE IMMOBILIZED HYALURONIC ACID

INFLUENCES CD44-MEDIATED BINDING OF AGS GASTRIC CANCER CELLS

P1_.04 Mafalda Martins de Araújo IMPACT OF CHRONIC STRESS ON BRAIN SPLICEOSOME – IMPLICATIONS

FOR NEURODEGENERATION

P1_.05 Ana Araújo CORK ANTI-OXIDANT COMPOUNDS AS A NOVEL THERAPEUTIC STRATEGY

FOR MACHADO-JOSEPH DISEASE

P1_.06 Ana Rita Araújo INFLUENCE OF THE SULFATION DEGREE ON FIBRONECTIN BINDING AND

STEM CELL ADHESION

P1_.07 Ivo Aroso NATURAL DEEP EUTECTIC SOLVENTS – EXPLORING A NEW CLASS OF

GREEN SOLVENTS FOR ENGINEERING PROCESSES

P1_.08 Pedro Babo DEVELOPMENT OF PLATELETS LYSATE-BASED MATERIALS TARGETING

PERIODONTAL TISSUES REGENERATION

P1_.09 Carlos Bessa LACK OF H3K4 DEMETHYLASE RBR-2/KDM5C LEADS TO GABA-RELATED

BEHAVIORAL DEFECTS IN C. ELEGANS

P1_.10 Sónia Borges ROLE OF GLUCOCORTICOIDS IN THE PROGRAMMING OF THE ASCENDING

CHOLINERGIC SYSTEM

P1_.11 Raphael Canadas ACELLULAR BILAYERED SCAFFOLDS FOR OSTEOCHONDRAL TISSUE

REGENERATION

P1_.12 Ramon Carballal OXIME CLICK CHEMISTRY TO MIMIC MULTIVALENT INTERACTIONS OF

GLYCOSAMINOGLYCANS

P1_.13 Flávia Castro IMPACT OF MYELOID RESTRICTED SIRT2 ABSENCE DURING

MYCOBACTERIUM TUBERCULOSIS INFECTION

P1_.14 Flávia Castro IL-10 MEDIATES SUSCEPTIBILITY TO LISTERIA MONOCYTOGENES

INFECTION BY MODULATING THE ACCUMULATION AND DIFFERENTIATION

OF CD11B+ LY6C+ MONOCYTES

P1_.15 Fatih Cengiz 3D CELL DENSITY OF HUMAN KNEE MENISCUS

P1_.16 Mariana Cerqueira PRE-CLINICAL POTENTIAL OF CELL SHEETS OF ADIPOSE STEM CELLS FOR

SKIN REGENERATION

P1_.17 Ana Coelho TOWARDS AN IMPROVED SET OF EXPRESSION VECTORS FOR

PARACOCCIDIOIDES SPP.


Poster Session 1

23

P1_.18 Clara Correia PARACRINE SIGNALLING BETWEEN ADIPOSE TISSUE STEM AND

MICROVASCULAR ENDOTHELIAL CELLS WITHIN MULTILAYERED

CAPSULES TRIGGER OSTEOBLASTOGENESIS

P1_.19 Joana Correia DEVELOPMENT OF ANATOMICAL SILK/METHACRYLATED GELLAN GUM

SCAFFOLDS FOR CONTROLLING SEGMENTAL VASCULARIZATION IN

MENISCUS TISSUE REGENERATION

P1_.20 Ana Margarida Costa FABRICATION OF HIERARCHICAL POLYMERIC CARRIERS FOR DRUG

DELIVERY AND CELL ENCAPSULATION USING SUPERHYDROPHOBIC

SURFACES

P1_.21 Marina Costa STROMAL VASCULAR FRACTION FROM ADIPOSE TISSUE AND CELL SHEET

ENGINEERING TO BUILD VASCULARIZATION UNITS FOR TISSUE

ENGINEERING AND REGENERATIVE MEDICINE

P1_.22 Catarina Custódio CELL SELECTIVE CHITOSAN MICROPARTICLES AS INJECTABLE CELL

CARRIERS FOR TISSUE REGENERATION

P1_.23 Rui Domingues EXPLORING MULTIPLE APPLICATIONS OF CELLULOSE NANOCRYSTALS IN BIONANOCOMPOSITE SCAFFOLDS DEVELOPMENT

P1_.24 Ângela Fernandes PROTEOLYTIC SYSTEMS AND AMP-ACTIVATED PROTEIN KINASE AS IMPORTANT TARGETS FOR ACUTE MYELOID LEUKEMIA THERAPEUTIC APPROACHES

P1_.25 Helena Ferreira DEVELOPMENT OF NANOPARTICLE CARRIERS FOR THERAPEUTIC, IMAGING AND THERANOSTIC APPLICATIONS

P1_.26 Alexandra Fraga SPONTANEOUS HEALING OF MYCOBACTERIUM ULCERANS INDUCED LESIONS IN THE GUINEA PIG MODEL

P1_.27 Albina Franco ANTIBACTERIAL PROPERTIES OF BIOACTIVE STARCH-SCAFFOLDS FUNCTIONALIZED WITH SILANOL GROUPS

P1_.28 Joana Gaifem MOLECULAR MACHINERY INVOLVED IN PLASMA MEMBRANE REPAIR DURING MYCOBACTERIUM TUBERCULOSIS INFECTION

P1_.29 Ana Rita Gomes THE IMPACT OF IMMUNE-RELATED GENE POLYMORPHISMS IN HUMAN MYCOBACTERIUM ULCERANS INFECTION

P1_.30 Ana Gonçalves ISOLATION AND CHARACTERIZATION OF DISTINCT SUB-POPULATIONS OF ADIPOSE STEM CELLS PREDISPOSED TO TENOGENIC DIFFERENTIATION

P1_.31 Sara Granja TARGETING CD147/BASIGIN SENSITIZES A549 LUNG CARCINOMA CELLS TO PHENFORMIN

P1_.32 Marta Guedes THE ROLE OF TET ENZYMES AND DNA HYDROXYMETHYLATION IN NEURONAL DIFFERENTIATION

P1_.33 Marco Rafael Guimarães PERIPHERAL NERVE INJURY – IMPACT ON BEHAVIOR IN PAINFUL AND SILENT CONDITIONS

P1_.34 Alvaro Leite SYNTHESIS AND CHARACTERIZATION OF BIOACTIVE BIODEGRADABLE CHITOSAN COMPOSITE SPHERES WITH SHAPE MEMORY CAPABILITY

P1_.35 Diogo Lobo-Silva MOLECULAR MECHANISMS REGULATING IL-10 PRODUCTION IN INNATE IMMUNE CELLS ACTIVATED VIA TLRS


Poster Session 1

24

P1_.36 Henrique Machado MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM TUBERCULOSIS PHYLOGENETIC LINEAGES IN A GROUP OF LOW RISK TUBERCULOSIS PATIENTS FROM THE NORTH OF PORTUGAL

P1_.37 Carlos Magalhães GENETIC DIVERSITY IN MYCOBACTERIUM TUBERCULOSIS AND ITS IMPACT ON HOST/PATHOGEN CO-EVOLUTION

P1_.38 Fernanda Marques THE CHOROID PLEXUS IS MODULATED BY VARIOUS STIMULI: IMPLICATIONS TO DISEASES OF THE CENTRAL NERVOUS SYSTEM


Poster Session 1

25

UNDERCOVERING THE INVOLVEMENT OF ADULT NEURO- GLIOPLASTICITY TO THE

COURSE OF DEPRESSION – A BEHAVIORAL AND STRUCTURAL ANALYSIS

N. D. Alves1,2, A. R. Machado-Santos1,2, P. Patrício1,2, A. Mateus-Pinheiro1,2, M. Morais1,2, J. Correia1,2, J. Bessa1,2, N. Sousa1,2, L. Pinto1,2

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Depression is a complex neuropsychiatric disorder that presents a huge impact in the modern society. This long-lasting disease encloses several and distinct stages, in which, following the manifestation of symptoms, antidepressant (AD) treatment may lead to complete recovery and achievement of remission. However, in a large number of cases, symptoms may return, giving rise to relapses and recurrence episodes. Although the pathophysiology of this disease is still poorly understood, several studies indicate that alterations on neuroplastic mechanisms including remodelling of neuronal branching and hippocampal cytogenesis (neurogenesis and gliogenesis) may be involved in both development and treatment of depression. In this context, using an animal model of depression, we focused on understanding the importance of these events along the course of the disease, namely during manifestation, treatment, remission and recurrence. These phases were modelled by exposure to an unpredictable chronic mild stress (uCMS) protocol, followed by AD treatment – imipramine and fluoxetine. Afterwards, animals followed a period of recovery, without stress exposure. Subsequently, a second stress protocol was applied to precipitate the return of depressive-like phenotype. In terms of behaviour, we evaluated mood, anxiety and cognition, dimensions typically affected in depression. Moreover, cell fate was also analysed, using an in vitro approach. Regarding adult neuroplastic mechanisms, distinct markers of proliferation and differentiation of neural stem cells were analysed as well as the morphology of granular neurons from the dentate gyrus. Results have revealed a peculiar impact of neuroplastic mechanisms at different timepoints, particularly in what concerns to the involvement on the AD action. While adult neurogenesis may not be important for the immediate effects of antidepressants, it is essential for a sustained recovery of depression. Additionally, these studies revealed that the action of typical ADs as fluoxetine and imipramine depend differently of the glio and neurogenic processes.


Poster Session 1

26

GALANIN IN THE DORSOMEDIAL NUCLEUS OF THE HYPOTHALAMUS ACTIVATES TWO

INDEPENDENT DESCENDING PRONOCICEPTIVE PATHWAYS

Diana Amorim1,2,3, Hanna Viisanen3, Hong Wei3, Armando Almeida1,2, Antti Pertovaara3, Filipa Pinto-Ribeiro1,2

1Life and Health Sciences Research Institute (ICVS), School of Health Sciences (ECS), Campus of Gualtar, University of Minho, 4750-057 Braga, Portugal 2ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland The activation of the dorsomedial nucleus of the hypothalamus (DMH) by galanin (GAL) induces behavioural hyperalgesia. Since DMH neurones do not project directly to the spinal cord, we hypothesized that the medullary dorsal reticular nucleus (DRt), a pronociceptive region that strongly projects to the spinal dorsal horn (SDH) and/or the serotoninergic raphe-spinal pathway acting on the spinal 5-HT3 receptor (5HT3R) could relay descending nociceptive facilitation induced by GAL in the DMH. Heat-evoked paw-withdrawal latency (PWL) and activity of SDH neurones were assessed in monoarthritic (ARTH) and control (SHAM) animals after pharmacological manipulations of the DMH, DRt and spinal cord. The results showed that GAL in the DMH and glutamate in the DRt lead to behavioural hyperalgesia in both SHAM and ARTH animals, which is accompanied by increased stimulus-evoked activity of SDH nociceptive neurones. Facilitation of pain behaviour induced by GAL in the DMH was reversed by lidocaine in the DRt and by ondansetron, a 5HT3R antagonist, in the spinal cord. However, the hyperalgesia induced by glutamate in the DRt was not blocked by spinal ondansetron. In addition, in ARTH but not SHAM animals PWL was increased after lidocaine in the DRt and ondansetron in the spinal cord. Our data demonstrate that GAL in the DMH activates two independent descending facilitatory pathways: (i) one relays in the DRt and (ii) the other one involves 5-HT neurones acting on spinal 5HT3Rs. In experimental ARTH, the tonic pain-facilitatory action is increased in both of these descending pathways.


Poster Session 1

27

MOLECULAR WEIGHT OF SURFACE IMMOBILIZED HYALURONIC ACID

INFLUENCES CD44-MEDIATED BINDING OF AGS GASTRIC CANCER CELLS

S. Amorim1,2, D. Soares da Costa1,2, R. L. Reis1,2, I. Pashkuleva1,2 and R. A. Pires1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal CD44, a transmembrane glycoprotein, is a major cell surface receptor for hyaluronic acid (HA) [1]. HA is a negatively charged polysaccharide composed of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine and is a naturally occurring glycosaminoglycan. It has been studied as a targeting motif in drug delivery systems for cancer therapy because various tumour cells are known to overexpress CD44 [2]. The influence of HA molecular weight (Mw) on the HA-CD44 interaction are reported for HA nanoparticles [3] and for solubilized HA [4]. However, this Mw influence has not been reported for surface immobilized HA, e.g. by Layer-by-Layer (LbL). Herein, we prepared LbL assemblies, on gold-coated surfaces, using poly-L-lysine (PLL, Mw=30-70kDa) and HA of different Mws, i.e. 6.4kDa, 752kDa and 1.5MDa. LbL films of (HA-PLL)5 presenting HA in the surface were analysed using Quartz Crystal Microbalance with dissipation (QCM-D) for their capacity to adsorb CD44. We observed a higher capacity of the 1.5MDa HA to adsorb more CD44. We also cultured AGS cells (a CD44 up-regulated cell line) on the same LbL films. The adhesion of the cells and the influence of the HA Mw on the CD44 expression (immunostaining) were evaluated. Surprisingly, and in contrast with the QCM adsorption study, the results showed that cell adhesion and CD44 expression was higher for the surface presenting HA with lower Mw, i.e. 6.4kDa. Our data suggests that CD44 expression on the AGS cell surface is not directly linked with the chemical affinity between CD44 and HA. Acknowledgments: FP7 project POLARIS (REGPOT-2012-2013-1-316331) is acknowledged. References: 1. Wolny et al. J. Biol. Chem. 285, 30170 (2010). 2. Choi et al. J. Mater. Chem. 19, 4102 (2009). 3. Mizrahy et al. J. Control. Release 156, 231–8 (2011). 4. Fuchs et al. Cell Death Dis. 4, e819 (2013).


Poster Session 1

28

IMPACT OF CHRONIC STRESS ON BRAIN SPLICEOSOME – IMPLICATIONS FOR

NEURODEGENERATION

Mafalda Martins de Araújo1,2, Vítor Pinto1,2, Cristina Mota1,2, Paula Silva1,2, Tiago Gil Oliveira1,2, Nuno Sousa1,2

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal

The spliceosome, a macromolecular machinery composed of five small nuclear ribonucleoprotein complexes (snRNPs) and hundreds of accessory proteins, is responsible for pre-mRNA splicing, the removal of non-coding intervening regions (introns) present in primary transcripts, in order to generate mature mRNA messages that can be translated into proteins 1. More than 90% of human genes are alternatively spliced and accumulating evidence suggests dysfunctional alternative splicing as a potential contributor to neurodegenerative disorders (e.g. SMA, ALS, AD) 2. In this project we want to understand whether chronic stress, a major risk factor for the development of neurodegenerative conditions, has an impact on spliceosome function, i.e. whether a dysfunction of this gene expression machinery is implicated on the molecular mechanisms taking place during neurodegeneration. We show here that mice under 6 weeks of chronic stress have alterations in the expression of key spliceosome components, namely the U1, U2, U4 and U6 snRNAs. These alterations were mainly restricted to brain regions involved in regulating emotional behavior, a behavioral dimension known to be affected by stress. In the amygdala, both snRNAs from the U4/U6-di-snRNP were altered in the stressed group indicating a possible functional consequence for spliceosome activity. In the ventral hippocampus we detected reduced levels of U1 snRNA, a snRNA found to be enriched in the detergent-insoluble fraction of human AD brains3. In the cerebellum, a brain region where the effect of stress is poorly understood, we detected downregulation of U2 and U6 snRNAs, two snRNAs with a fundamental role in the formation of the catalytically active spliceosome. Interestingly, dysfunction of one of the mouse multicopy U2 snRNA genes was previously shown to cause neurodegeneration in the cerebellum4. [1] Wahl M.C., Will C.L. and Luhrmann R. The Spliceosome: design principles of a dynamic RNP machine. Cell. 136(4):701-18 (2009). [2] Mills, J. D. & Janitz, M. Alternative splicing of mRNA in the molecular pathology of neurodegenerative diseases. Neurobiology of aging 33, 1012.e11–24 (2012). [3] Bai B., et al. U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’sdisease. PNAS.110(41):16562-7 (2013). [4] Jia Y., Mu. J.C & Ackerman S.L. Mutation of a U2 snRNA gene causes global disruption of alternative splicing and neurodegeneration. Cell. 148(1-2):296-308.


Poster Session 1

29

CORK ANTI-OXIDANT COMPOUNDS AS A NOVEL THERAPEUTIC STRATEGY FOR

MACHADO-JOSEPH DISEASE

Ana R. Araújo,1,2 Ana Jalles,2,3 Patrícia Maciel,2,3 Rui L. Reis,1,3 Andreia Teixeira-Castro,2,3 Ricardo A. Pires1,3

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Machado-Joseph disease (MJD) is a neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) tract within the C-terminal of the ataxin-3 (ATXN3) protein. Mutant ATXN3 acquires the ability to self-associate and enter an aggregation process, which is associated with several pathophysiological consequences for neurons. An imbalance in the oxidative state of the neuronal cells has been linked to the onset and progression of MJD [1]. In order to control disease progression, polyphenols have been tested as compounds that combine anti-oxidant (AO) properties with the capacity to inhibit protein aggregation. Cork polyphenolic extracts usually present AO activity [2], however, there is no report on their capacity to inhibit ATXN3 aggregation, nor to minimize the MJD symptoms. In this context, we collected the cork water extract (WE) and fractionated it (by HPLC) in order to test their activity towards MJD. We tested these compounds in the C. elegans MJD animal model due to the fact that this nematode presents several of the MJD characteristic symptoms in humans, namely, motor neuron dysfunction and the accumulation of ATXN3 aggregates in specific neurons. The toxic evaluation of the cork compounds (food clearance assay, 7 days) leads us to conclude that they are not toxic towards MJD and wild-type (WT) animals, at concentrations up to 1mg/mL (WE) or 500μg/mL (HPLC fractions). Next, we evaluated the impact of WE on the motility of MJD animals, and we observed a 63% improvement on the mutant ATXN3-mediated neurological dysfunction. In addition, WE-treated WT animals did not present any improvement in locomotion, suggesting that the WE action may be specific to mutant ATXN3-mediated pathogenesis. Treatment with WE was also able to suppress mutant ATXN3 aggregation in vivo. These results suggest that WE show neuroprotective properties probably through the enhancement of cell proteostasis. Acknowledgements: We acknowledge FP7-REGPOT-CT2012-316331-POLARIS for funding. [1] Butterfield, D.A. and J. Kanski, Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins. Mechanisms of Ageing and Development, 2001. 122(9): p. 945-962. [2] Santos, S.A.O., et al., Chemical composition and antioxidant activity of phenolic extracts of cork from Quercus suber L. Industrial Crops and Products, 2010. 31(3): p. 521-526.


Poster Session 1

30

INFLUENCE OF THE SULFATION DEGREE ON FIBRONECTIN BINDING AND STEM CELL

ADHESION

A.R. Araújo1,2, D. Soares da Costa1,2, S. Amorim1,2, R.L. Reis1,2, R.A. Pires1,2 and I. Pashkuleva1,2 13B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2ICVS/3B's PT Government Associate Laboratory, Guimarães/Braga, Portugal Glycosaminoglycans (GAGs) represent a fundamental part of the extracellular matrix (ECM). They are anionic linear polysaccharides whose negative charge is due to the presence of sulfate groups. These have a crucial role in GAG interaction with proteins and therefore in key biochemical processes/signaling related to cell functionality and survival. Cell surface GAGs also act as receptors for soluble bioactive molecules such as growth factors and cytokines. Nowadays, it is well established that GAGs and proteins act in synergism, both on the cell surface and in the ECM, being equally critical for the development, growth, function or survival of an organism [1]. We have previously developed a platform based on self-assembled monolayers to allow precise control on the surface exposed sulfonic groups (-SO3H) and proved them as reliable tools in proteins and cell studies [2, 3]. In this work, we used this platform to further investigate the influence of sulfonic-functional groups on the adhesion and morphology of adipose-derived stem cells (ASCs) and the role of fibronectin in these events. We used quartz crystal microbalance with dissipation (QCM-D) and live-imaging to follow in situ the cell adhesion in the presence or absence of fibronectin and immunofluorescence to check the effect on cell morphology. The presence of fibronectin causes an opposite effect on the studied surfaces: delays cell adhesion on –SO3H functionalised surfaces and accelerates it on –OH ones. Different cell morphology was also observed: cells spread after 7h on –SO3H surfaces and on –OH surfaces coated with fibronectin. These results suggested different mechanisms of adhesion on both studied surfaces. Live-imaging and QCM-D confirmed this hypothesis. Based on these results, we propose a model according to which fibronectin interacts with the underlying –SO3H groups causing both passivation of the surface and change in the conformation of the protein. Acknowledgments: FP7 project POLARIS (REGPOT-2012-2013-1-316331) is acknowledged. 1.Pashkuleva, I. and R.L. Reis, J Mater Chem 20, 8803 (2010) 2.Amorim, S., et al., Langmuir 29, 7983 (2013) 3.Soares da Costa, D., et al., J Mater Chem 22, 7172 (2012)


Poster Session 1

31

NATURAL DEEP EUTECTIC SOLVENTS – EXPLORING A NEW CLASS OF GREEN SOLVENTS

FOR ENGINEERING PROCESSES

Ivo M. Aroso1,2, Rui L. Reis1,2, Ana RitaC. Duarte1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Deep eutectic solvents (DES) systems are in essence constituted by two or more solid or liquid components that, when combined in a particular composition, form a mixture that has a melting point significantly lower than that of the individual components.[1, 2] When the DES components are of natural origin they are defined as NADES (Natural Deep Eutectic Solvent).[3] Hayan and coworkers [4] report, for example, the properties of fruit sugar-based NADES. The most common DES are based on choline chloride (ChCl), carboxylic acids and other hydrogen-bond donors, e.g. urea, citric acid, succinic acid and glycerol. Much attention is nowadays devoted to its development as demonstrated by the increasing number of publications in recent years.[5, 6] The diversity of NADES that can be produced is enormous and the ability to tailor their properties for particular applications is one of their biggest advantages. However, due to insufficient knowledge at the fundamental level, these are still being explored following trial and error schemes. It is crucial to understand the physical and chemical phenomena that underlay the ability of specific compounds to form NADES and characterize the fundamental properties of such systems. In this work, NADES based on choline, organic acids, and sugars were prepared and characterized in terms of thermophysical properties such as density, thermal behavior, polarity and rheological properties as function of water content. Their application for extraction of natural components and polymer processing [7] was evaluated and found to successfully replace the use of organic solvents, in many cases, with process advantages. The results obtained, far contribute to the increase of the scientific knowledge in this field and contribute towards novel application developments. Thus, this new class of solvents will definitely play an important role towards a more sustainable industrial development. [1] Stott PW, Williams AC, Barry BW. J Control Release 50 297-308 (1998) [2] Russell SF, Norris BN. Abstr Pap Am Chem S 243 (2012). [3] Choi YH, van Spronsen J, Dai YT, Verberne M, Hollmann F, Arends IWCE, et al., Plant Physiol 156 1701-5 (2011) [4] Hayyan A, Mjalli FS, AlNashef IM, Al-Wahaibi T, Al-Wahaibi YM, Hashim MA., Thermochim Acta 541 70-5 (2012) [5] Carriazo D, Serrano MC, Gutierrez MC, Ferrer ML, del Monte F., Chem Soc Rev 41 4996-5014 (2012) [6] Paiva A, Craveiro R, Aroso I, Martins M, Barreiros S, Reis RL, Duarte ARC, ACS Sustainable Chemistry & Engineering DOI: 10.1021/sc500096j (2014) [7] Martins M, Aroso I, Craveiro R, Reis RL, Paiva A, Duarte ARC, AIChE Journal, DOI: 10.1002/aic.14607 (2014)


Poster Session 1

32

DEVELOPMENT OF PLATELETS LYSATE-BASED MATERIALS TARGETING PERIODONTAL

TISSUES REGENERATION

Pedro Babo1,2, Rui L. Reis1,2, Manuela E. Gomes1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal The research on new biomaterials incorporating biochemical cues and well-defined architectures has opened horizons for the regeneration of complex tissues, such as the periodontium. The use of Platelet Lysates (PLs) have shown a great potential for tissue regeneration, as the growth factors (GFs) derived from platelets are involved in essential stages of wound healing and regenerative processes such as chemotaxis, cell proliferation and differentiation. Moreover, platelets release numerous cell adhesion molecules (fibrin, fibronectin and vitronectin) that are important for the formation of ECM and for the adhesion and migration of cells. Achieve periodontal regeneration is very challenging as multiple tissues are involved, namely cement, periodontal ligament (PDL) and alveolar bone that have distinct requirements. In this work we proposed the development of a dual system aiming the treatment of a periodontal defect, incorporating 1) a PL-based membrane for the PDL layer restoration, and 2) calcium phosphate cement (CPC) for the bone regeneration, incorporating hyaluronan (HA) microspheres loaded with PL The PL were shown to be very versatile and liable to be processed into materials with different characteristics. PL membranes were produced either by self-crossliking, or by incorporation in photocrosslinkable hydrogels. Moreover, the incorporation of PL loaded HA microspheres in a CPC originated an injectable cement capable of setting in situ for alveolar bone regeneration. All, the developed materials, were able to release PL soluble factors in a controlled manner and positively influenced periodontal origin cells. The bi-layered system was tested in a periodontal defect model in rat and, while the injectable cement didn’t promote significant alveolar bone augmentation, the PL-based membrane showed to be osteoconductive and able to promote significant functional ligament regeneration. Furthermore, the mechanical properties of the materials obtained based on PL and the biological relevance of the released factors for wound repair, suggest their great potential to be applied in further tissue engineering strategies.


Poster Session 1

33

LACK OF H3K4 DEMETHYLASE RBR-2/KDM5C LEADS TO GABA-RELATED BEHAVIORAL

DEFECTS IN C. ELEGANS

Bessa C.1,3, Rodrigues A.J.1,3, Marques F.1,3, Amorim M.1,3, Lopes F.1,3, Maciel P.1,3 1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Intellectual disability (ID) is one of the most frequent and disabling neurological impairments with an estimated prevalence of 1.5-2% in Western countries. Technological advances such as the use of array comparative genomic hybridization (aCGH) and massive parallel sequencing have allowed the identification of novel genetic causes of ID. This large-scale analysis originates an incredible number of novel genetic associations that most often require functional validation. We are using the simple round worm Caenorhabditis elegans (C. elegans) as a platform to functionally validate ID genetic associations and to better understand the importance of target genes and proteins in the nervous system and neuronal function. So far, we have studied 35 mutant strains that correspond to 29 orthologues of human genes previously linked to ID. Absence of one candidate, rbr-2, the orthologue of human KDM5C/JARID1C, leads to anatomical reproductive defects, developmental delay (larval arrest), increased embryonic lethality and decreased life span. Interestingly, rbr-2 mutant worms also present several behavioural deficits which suggest an impairment of neuronal functions. Of notice, worms exhibit increased motor uncoordination and abnormal sensory chemotactic responses. By crossing the rbr-2 knock-out strain with reporter strains expressing GFP in specific neuronal sub-types, we found an increased occurrence of GABAergic network defects – namely abnormal neuronal process positioning and migration. In addition, these animals were more sensitive to Pentylenetetrazol (PTZ – a GABA antagonist) than wild-type worms. This correlates well with expression analysis of genes related to GABA metabolism or transport, some of which were found be altered in mutant worms. Overall, our data suggests that rbr-2, which encodes for a histone demethylase, plays a relevant role in nervous system development and specifically in GABA-dependent neuronal functions.


Poster Session 1

34

ROLE OF GLUCOCORTICOIDS IN THE PROGRAMMING OF THE ASCENDING CHOLINERGIC

SYSTEM Borges S.1,2, Coimbra B.1,2, Soares-Cunha C.1,2, Ventura-Silva A.P.1,2, Pinto L.1,2, Carvalho M.M.1,2,

Pego J.M.1,2, Sousa N.1,2 and Rodrigues A.J.1,2

1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal 2- ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Adverse events occurring during the prenatal period, such as exposure to high levels of glucocorticoids (GCs) have long-term effects on brain development, increasing the risk for developing neuropsychiatric disorders in adulthood, such as depression, anxiety or addiction. We have previously shown that prenatal exposure to synthetic GC dexamethasone in utero (iuGC), strongly modulates the mesolimbic dopaminergic circuit by altering the expression pattern of the dopamine receptor 2 (Drd2) gene and dopamine levels, resulting in depressive- and anxious-like behavior, social interaction deficits and drug-seeking behavior Since the cholinergic circuitry is highly responsive to GCs, we decided to evaluate the condition of this system in iuGC animals. In fact, iuGC treatment resulted in an increased expression of choline acetyltransferase (ChAT) and decreased expression of acetylcholinesterase (AChE), enzymes involved in the cholinergic pathway, in a brain-region specific manner. At a behavioral level, we found that iuGC exposure is able to exacerbate fear behavior in a contextual fear conditioning paradigm and induce a hyperanxiety state coupled with increased emission of 22 kHz negative USVs. These results were consistent with the observed hypercholinergic status in the ascending mesopontine cholinergic system, a key modulator of negative USV emission. Furthermore, and in accordance with the amplified response to an adverse stimulus of iuGC animals, we found a differential cholinergic activation pattern of the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nucleus (PPT) after an adverse stimulus. Altogether, our results suggest that GCs may program cholinergic nuclei in a region-specific manner and eventually lead to altered behavior. [1] Borges, S., Coimbra, B., Soares-Cunha, C., Pêgo, J.M., Sousa, N., Rodrigues, A.J., 2013. Dopaminergic modulation of affective and social deficits induced by prenatal glucocorticoid exposure. Neuropsychopharmacol 38, 2068-2079. [2] Rodrigues, A.J., Leão, P., Pêgo, J.M., Cardona, D., Carvalho, M.M., Oliveira, M., Costa, B.M, Carvalho, A.F., Morgado, P., Araújo, D., Palha, J.A., Almeida, O.F.X., Sousa, N., 2012. Mechanisms of initiation and reversal of drug-seeking behavior induced by prenatal exposure to glucocorticoids. Mol Psychiatry 17, 1295-1305. [3] Brudzynski SM., Barnabi F., 1996. Contribution of the ascending cholinergic pathways in the production of ultrasonic vocalization in the rat. Behav Brain Res 80,145–5210.


Poster Session 1

35

ACELLULAR BILAYERED SCAFFOLDS FOR OSTEOCHONDRAL TISSUE REGENERATION

R.F. Canadas1,2, A.P. Marques1,2, R.L. Reis1,2 and J.M. Oliveira1,2

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Last decade 9.6% of the men and 18% of the woman with more than 60 years presented symptomatic Osteoarthritis [1]. Despite the progress already achieved in osteochondral (OC) repair, some limitations still need to be overcome. A promising strategy involves bilayer scaffolds presenting different composition and microarchitecture that intend to mimic the tissue to be regenerated. The use of an acellular structure capable of recruiting the cells during the in vivo OC repair could be a faster and more efficient approach to translate into the clinics. Under this context, the present work aims to develop bilayered gellan gum (GG)/GG-hydroxyapatite (HAp) scaffolds for OC regeneration. The bilayered scaffolds were obtained by means of freeze-drying. The cartilage-like layer was obtained from a 2 wt% LAGG solution while the bone-like layer consisted of 2 wt% LAGG with HAp (20 and 30 wt%). Bioactivity tests were performed by means of soaking the scaffolds in a simulated bofy fluid solution (pH 7.4). The scaffolds microarchitecture and surface morphology were investigated by means of micro-computed tomography and scanning electron microscopy. Potential inflammatory reaction to the developed structures was assessed after subcutaneous implantation in mice for the period of 2 weeks. The freeze-dried bilayered scaffolds present two integrated layers, with a gradient of HAp in the bone-like layer that, unlike cartilage-like layer, showed to be bioactive. Moreover, bilayered scaffolds with about 90% porosity, 500 µm of mean pore size and 85% interconnectivity were obtained. The developed scaffolds absorbed about 120% of its weight in water and lost 10% of its mass after 30 days of immersion in phosphate buffered saline solution. In vivo subcutaneous implantation studies revealed that the bilayered scaffolds do not elicit any acute inflammatory response in mice. Good host tissue integration and cells infiltration further confirmed the non-inflammatory potential of the scaffolds. Ultimately, the developed bilayered scaffolds will provide new therapeutic possibilities for the repair of OC defects. [1] AD Wol, B Pfleger. Bulletin of the World Health Organization 81 (9): 646-656, 2003


Poster Session 1

36

OXIME CLICK CHEMISTRY TO MIMIC MULTIVALENT INTERACTIONS OF

GLYCOSAMINOGLYCANS

Ramon Novoa-Carballal1,2, Carla Silva,1, Rui. L. Reis1,2 and Iva Pashkuleva1,2

1- 3B´s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal; 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal

Glycosaminoglycans (GAGs) are negatively charged polysaccharides that span different biological functions ranging from pure structural role in the extracellular matrix to their involvement in cell signaling pathways.[1] Because of their biofunctions, GAGs have been proposed as therapeutics for a variety of diseases such as thrombosis, amyloid diseases, arthritis and cancer among others.1 However, the complex nano organization in which they are naturally involved, have impeded the elucidation of the exact mechanisms of GAGs interactions and therefore led to underestimation of their real therapeutic value. Thus, efforts have been made to mimic GAGs and their multivalent interactions by preparing glycopolymers.2 These mimics are usually synthesized by modification of synthetic polymers with mono or oligosaccharides.3 Herein we present an alternative approach in which polymer-GAG hybrids are synthesized by Oxime Click Chemistry.4 We have chosen hyperbranched polyglycerols and PEG-b-hydroxyethylmethacrylate as synthetic cores to obtain tree-like and brush-like structures. 5 These cores were aminooxy functionalized and then conjugated with GAGs at the sugar reducing end. We have assayed their electrostatic interaction with model positively charged proteins by dynamic light scattering. The new family of bio-hybrids has enormous potential in the pharmaceutical field (by modulating their natural biological roles with tree-like architectures), tissue engineering (mimicking the natural nanoscale organization found in natural tissues) and drug delivery.

[1] Pashkuleva, I.; Reis, R. L. J Mat Chem, 20, 8803. (2010) [2] Papp, I.; Sieben, C.; Sisson, A. L.; Kostka, J.; Böttcher, C.; Ludwig, K.; Herrmann, A.; Haag, R. ChemBioChem, 12, 887 (2011). [3] Sunasee, R.; Narain, R. Macromol. Biosci., 13, 9 (2013) [4] Novoa-Carballal, R.; Müller, A. H. E. Chem. Commun., 48, 3781 (2012). [5] Khandare, J.; Calderon, M.; Dagia, N. M.; Haag, R. Chem. Soc. Rev., 41, 2824, (2012). This work was supported by POLARIS (REGPOT-2012-2013-1-316331)


Poster Session 1

37

IMPACT OF MYELOID RESTRICTED SIRT2 ABSENCE DURING MYCOBACTERIUM

TUBERCULOSIS INFECTION

Flávia Castro1,2,*; Ana Filipa Cardoso1,2,*; Lúcia Moreira-Teixeira1,2; Jorge Pedrosa1,2; Egídio Torrado1,2; António Gil Castro1,2; Teresa Pais3,#; Margarida Saraiva1,2,#

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 3- Instituto de Medicina Molecular, 1649-028 Lisbon, Portugal;Instituto de Fisiologia, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisbon, Portugal *,# These authors contributed equally to the study Regulation of the immune response, mediated by numerous mechanisms, is critical for infection control and host survival. Sirtuin 2 (SIRT2) is a deacetylase associated with cytoskeletal regulation and NF-kB regulation, with impact in the expression of a broad number of immune and inflammatory genes. The role of SIRT2 in infection is not fully understood, but recent evidence showed that it mediates histone deacetylation, being essential for resistance to infection by Listeria monocytogenes. In the present study, we investigated the role of myeloid-restricted SIRT2 expression during infection with Mycobacterium tuberculosis (Mtb). Our results show that the absence of SIRT2 in the myeloid lineage did not impact the control of Mtb in the lung, cytokine expression or lung pathology. However, an increased susceptibility was observed in the liver of Mtb-infected SIRT2-/- animals. This was correlated with a decrease in the accumulation and activation of monocytes and a decrease in T cell response in the liver. These data suggest a transient effect of SIRT2 during infection with Mtb. Funding: This work is co-funded by Programa Operacional Regional do Norte (ON.2 – O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER). MS is an Associate FCT-Investigator. LMT was supported by FCT grant SFRH/BPD/77399/2011. Grant number PTDC/SAU-ORG/114083/2009 to TP.


Poster Session 1

38

IL-10 MEDIATES SUSCEPTIBILITY TO LISTERIA MONOCYTOGENES INFECTION BY

MODULATING THE ACCUMULATION AND DIFFERENTIATION OF CD11B+ LY6C+ MONOCYTES

Flávia Castro1,2, Vânia Cardoso1,2, Ricardo Silvestre1,2, Jorge Pedrosa1,2, Margarida Saraiva1,2,

António Gil Castro1,2 and Egídio Torrado1,2 1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal IL-10 is an important immunomodulatory cytokine with a broad impact in the immune response. During infection, IL-10 has been shown to have protective roles, by modulating the intensity of the protective immune response or pathological roles, by preventing the expression of these responses. However, the mechanisms underlying these effects are still incompletely known. To investigate the mechanisms whereby IL-10 mediates pathological responses during infection, we used a genetically modified mouse strain (pMT-10) to allow the temporal control of IL-10 overexpression during infection. We chose Listeria monocytogenes as an infection model, because IL-10 has been shown to prevent the efficient control of infection by yet unidentified mechanisms. We show that, in the presence of high levels of IL-10 mice are extremely susceptible to L. monocytogenes infection and display clear signs of pathology in the infected organs. Mechanistically, we found that high levels of IL-10 during the early stages of L. monocytogenes infection prevent the accumulation of CD11b+ Ly6C+ monocytes and their differentiation into CD11c+ MHC-II+ dendritic cells. Since we did not find any difference in the emigration of CD11b+ Ly6C+ monocytes from the bone-marrow or alterations in the percentage of these cells in the blood, our data suggest that IL-10 inhibits the proliferation or differentiation of CD11b+ Ly6C+ cells in the infection site. We are currently investigating the cellular and molecular mechanisms by which IL-10 modulates the activity of CD11b+ Ly6C+ monocytes.


Poster Session 1

39

3D CELL DENSITY OF HUMAN KNEE MENISCUS

Ibrahim Fatih Cengiz1,2, Hélder Pereira1,2,4,5, José Miguel Pêgo2,3,, João Espregueira-Mendes1,2,4,

Nuno Sousa2,3, Joaquim Miguel Oliveira1,2, Rui Luís Reis1,2 1- 3B's Research Group - Biomaterials, Biodegradables and Biomimetics, Univ. Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, S. Cláudio de Barco, 4806-909 Taipas, Guimarães, Portugal; 2- ICVS/3B’s - PT Government Associated Laboratory, Portugal; 3- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; 4- Clínica Espregueira-Mendes F.C. Porto Stadium – FIFA Medical Centre of Excellence, Portugal; 5- Orthopedic Department Centro Hospitalar Póvoa de Varzim – Vila do Conde, Portugal. The knee menisci have important roles in the knee joint. The complete healing of meniscus remains as a challenge in the clinics. Cellularity is one of the most important biological parameters that must be taken into account in regenerative strategies. However, the knowledge on the 3D cellularity of meniscus lacks in the literature. The aim of this study is to quantify the 3D cellular density of the human meniscus in a segmental and regional manner with respect to the laterality. Human lateral menisci were histologically processed, and stained with Giemsa for the histomorphometric analysis. The cells were counted in an in-depth fashion based on their roundish or flattened morphologies. The cell density in the vascular region (27,199 cells/mm3) was significantly higher as compared to avascular region (12,820 cells/mm3). The fibrochondrocytes density (14,705 cells/mm3) was significantly greater than the fibroblast-like cells density (5,539 cells/mm3). A strong positive correlation was found between fibrochondrocytes and fibroblast-like cells densities. The best-fit equation for the prediction of the fibrochondrocytes density from the fibroblast-like cell density was found to be: "Fibrochondrocytes density = 1.22 × Fibroblastlike cell density + 7750". The obtained data shows that the meniscus tissue is heterogeneous in a segmental and regional manner in terms of 3D cellular density. The present study revealed the segmental and regional 3D cellular density of human knee meniscus with respect to the laterality. This crucial but so far missing information will empower the cellular strategies aiming meniscus tissue regeneration.


Poster Session 1

40

PRE-CLINICAL POTENTIAL OF CELL SHEETS OF ADIPOSE STEM CELLS FOR SKIN

REGENERATION

M. T. Cerqueira1,3, J. Moreira-Pinto2,3, A. Miranda2,3, J. Correia-Pinto2,3, R. L. Reis1,3, A. P. Marques1,3

1-3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Stem Cells have emerged as powerful tools for skin regeneration, not only as building blocks but also as central mediators of a well-orchestrated signaling interplay [1, 2], frequently compromised with the current clinically available skin substitutes. Cell sheet (CS) engineering, taking advantage of particular cell-cell/cell-extracellular matrix interactions, and subsequent cellular milieu, has been successfully used to create 3D tissue engineered constructs to promote full-thickness skin wound regeneration. In our previous work [3], we proposed human adipose tissue stem cells (hASCs)-based constructs obtained through CS Engineering, to target full-thickness skin wound regeneration. Evidences that the proposed strategy yield promising outcomes relevant for skin regeneration were provided after transplantation into mice full-thickness excisional skin wounds. hASCs embedded in their natural extracellular matrix (ECM) lead to a prolonged cell engraftment, improved neovascularization, but more importantly showed a significant impact over neoepidermis morphogenesis by interacting with host keratinocytes in a paracrine manner. Due to these encouraging results but also to the dissimilarities between humans and rodents’ healing nature [4], this work aims at confirming the results into a porcine wound model [5], validating this rationale in a pre-clinical set. Considering an autologous approach, we have so far optimized the isolation of ASCs from porcine subcutaneous adipose tissue as well as the creation of the respective cell sheets in order to proceed with a pilot test. Following the evidences described in the mice full-thickness excisional wound model, among other aspects such as neovascularization, neodermal arrangement and re-epithelialization, the formation of rete-ridges structures and hair follicles and subsequent long-term functionality will be a major focus of study. 1 Wu, Y., et al., Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells, 2007. 25(10): p. 2648-59. 2 Altman, A.M., et al., IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. Stem Cells, 2009. 27(1): p. 250-8. 3 Cerqueira, M.T., et al., Human adipose stem cells cell sheet constructs impact epidermal morphogenesis in full-thickness excisional wounds. Biomacromolecules, 2013. 14(11): p. 3997-4008. 4 Wong, V.W., et al., Surgical approaches to create murine models of human wound healing. J Biomed Biotechnol, 2011. 2011: p. 969618. 5 Sullivan, T.P., et al., The pig as a model for human wound healing. Wound Repair Regen, 2001. 9(2): p. 66-76.


Poster Session 1

41

TOWARDS AN IMPROVED SET OF EXPRESSION VECTORS FOR PARACOCCIDIOIDES SPP.

Coelho A. 1,2, Rezende J. 1,2, Ferreira R. 1,2, Menino JF. 1,2, Rodrigues F. 1,2

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Paracoccidioidomycosis (PCM) is a neglected tropical disease caused by Paracoccidioides spp. estimated to affect around 10 million individuals in countries from Latin America. The currently available genetic tool to uncover the molecular and cellular biology of this fungus is based on an Agrobacterium tumefaciens-mediated transformation (ATMT) using T-DNA binary vectors. This system allows for genomic integration of a single copy of genetic constructs either for gene silencing or gene overexpression. One of the major limitations of this system is that it relies on the usage of exogenous promoters. Therefore, and taking into account that Paracoccidioides is thermodimorphic and that the infectious process comprises a temperature-dependent morphological shift, it reveals of major importance to use endogenous phase-specific promoters for genetic manipulation. In this sense, a set of endogenous promoters was selected from gene expression data in Paracoccidioides spp., which comprised those of genes showing constitutive highly expression in both yeast and mycelium forms, as well as those associated with genes being highly expressed in either yeast or mycelium forms. These included promoters of heat shock protein 70 (HSP70) and ribossomal protein L35, yeast specific genes YL1 and GTPase RHO1 and mycelium specific gene aminopeptidase Y (APE3). T-DNA binary vectors were constructed harboring GFP under the control of each of the selected promoters. GFP expression in both yeast and mycelium form was evaluated by epifluorescence microscopy observation of cells, flow cytometry analysis of the fluorescent populations and quantitative real-time PCR of GFP transcripts. Our results demonstrate that the usage of endogenous promoters can represent an improvement to the currently ATMT-based protocol, which can ultimately lead to modifications of Paracoccidioides spp. phenotypes by e.g. gene overexpression and gene silencing, as well as explore gene tracking of fluorescently-labelled proteins and cells in vitro and in vivo.


Poster Session 1

42

PARACRINE SIGNALLING BETWEEN ADIPOSE TISSUE STEM AND MICROVASCULAR

ENDOTHELIAL CELLS WITHIN MULTILAYERED CAPSULES TRIGGER OSTEOBLASTOGENESIS

Clara R. Correia1,2, Rogério P. Pirraco1,2, Mariana T. Cerqueira1,2, Alexandra P. Marques1,2, Rui L. Reis1,2, João F. Mano1,2

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Bone regeneration remains a challenging milestone in orthopedic research. Inspired by the synergic angio-osteogenesis resulting from the co-existence of vascular and stem cells in the native environment of cancellous bone [1], we propose a stem-cell based strategy that provides an immunoprivileged atmosphere to induce osteoblastogenesis. We developed a hierarchical system to create a bottom-up tissue engineering (TE) approach combining (i) microparticles as cell adhesion sites, and (ii) cells of two different phenotypes isolated from adipose tissue, encapsulated in (iii) multilayered liquified capsules. Our hypothesis is that in the capsules confined and controlled environment, the paracrine interaction of co-cultured endothelial and human adipose-derived stem cells (hASCs) would trigger osteogenic differentiation and the generation of osteoblasts. For that, liquified multilayered poly(L-lysine)-alginate-chitosan capsules containing collagen I functionalized PLLA microparticles, produced as previously described [2], were used to encapsulate hASCs alone or co-cultured with human adipose microvascular endothelial cells (1:1). The two formulations of capsules were cultured in EndoGroTM-MV-VEGF complete kit media only containing β-glycerophosphate (basal medium) or containing all the osteogenic differentiation factors, namely dexamethasone, ascorbic acid, and β-glycerophosphate (osteogenic medium). The osteogenic phenotype of the cells in capsules was assessed by ALP/DNA quantification, alizarin red staining, calcium deposits quantification and expression of osteogenic markers by PCR and immunofluorescence stainings up to 21 days post-encapsulation. Results showed that the proposed co-culturing strategy supported osteoblastogenesis even in basal medium. Moreover, the developed system can be transplanted by minimal invasive procedures. Capsules can be directly injected into the lesion site due to the flexible multilayered membrane. We believe that our results provide new insight into the importance of co-culture systems in engineering bioencapsulation systems for bone TE. [1] C.J. Kirkpatrick, S. Fuchs, R.E. Unger, Advanced Drug Delivery Reviews 63, 291 (2011) [2] C.R. Correia, R.L. Reis, J.F. Mano, Biomacromolecules 14, 743 (2013)


Poster Session 1

43

DEVELOPMENT OF ANATOMICAL SILK/METHACRYLATED GELLAN GUM SCAFFOLDS FOR CONTROLLING SEGMENTAL VASCULARIZATION IN MENISCUS TISSUE REGENERATION

J. Silva-Correia1,2*, H. Pereira1,2,3,4, L-P. Yan1,2, V. Miranda-Gonçalves2,5, J. Espregueira-

Mendes1,2,3, A.L. Oliveira6, R.M. Reis2,5,7, J.M. Oliveira1,2, R.L. Reis1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- Saúde Atlântica Sports Center – F.C. Porto Stadium, Minho University and Porto University Research Center, Portugal 4- Orthopedic Department Centro Hospitalar Póvoa de Varzim, Vila do Conde, Portugal 5- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 6- Department of Health Sciences, Portuguese Catholic University, Viseu, Portugal 7- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil The human meniscus has a limited healing potential, partly due to a poor vasculature, and thus treatment of meniscus lesions using tissue engineering strategies has recently been investigated as a promising alternative to total/partial meniscectomy [1]. Meniscus tissue engineering can benefit from the use of highly developed scaffolds that are able to mimic the anatomy of this complex tissue, i.e. enable controlling the segmental vascularization during tissue regeneration. In this work, novel scaffolds were produced by combining a porous silk polymeric matrix at 12 wt% (silk-12) [2] and the methacrylated gellan gum hydrogel (iGG-MA), which was previously shown to be able to prevent the ingrowth of endothelial cells and blood vessels into the hydrogels [3,4]. A chorioallantoic membrane (CAM) assay was used to investigate in vivo the ability of acellular and cell-laden silk-12/iGG-MA scaffolds to control cell ingrowth, segmental vascularization, and innervations. Different sterile silk-based scaffolds were tested on the CAM, which were composed by silk-12 alone or combined with iGG-MA hydrogel and/or human meniscus cells. The angiogenic response was evaluated after 4 days of implantation by analysing the number of blood vessels converging toward the scaffolds, whereas possible inflammation and endothelial cells ingrowths’ was investigated by histological (haematoxylin and eosin - H&E - staining) and immunohistochemical methods (SNA-lectin staining). Results have shown that silk-12 is highly susceptible to cells ingrowths’ and blood vessel formation. However, when the hydrogel was combined with the silk-12, an inhibitory effect was observed as demonstrated by the low number of convergent blood vessels and less number of endothelial cells invading the silk-12/iGG-MA scaffolds. The iGG-MA hydrogel acted as a physical barrier for endothelial cells ingrowths’ and vascular invasion. This data showed that silk-12/iGG-MA scaffolds are not toxic, enabled controlling vascularisation and thus can be potentially applied in meniscus regeneration. [1] H. Pereira, et al., Arthroscopy 27, 1706 (2011) [2] L.-P. Yan, et al., Acta Biomater 8, 289 (2012) [3] J. Silva-Correia, et al., J Tissue Eng Regen Med 5, e97 (2011) [4] J. Silva-Correia, et al., Tissue Eng Part A 18,1203 (2012)


Poster Session 1

44

FABRICATION OF HIERARCHICAL POLYMERIC CARRIERS FOR DRUG DELIVERY AND

CELL ENCAPSULATION USING SUPERHYDROPHOBIC SURFACES

Ana M. S. Costa1, Manuel Alatorre-Meda1, C. Alvarez-Lorenzo2, and João F. Mano1

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal; ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 2- Departamento de Farmacia y Tecnología Farmacéutica, Universidad de Santiago de Compostela, Spain The use of superhydrophobic surfaces (SH) for the production of polymeric particles has been pointed as a promising strategy for drug and cell encapsulation purposes. Compared to other methodologies, this technique allows encapsulation efficiencies of ca. 100% and occurs at mild conditions [1]. However, the obtained particles are 1 mm in size, hampering their application in clinical trials. Improving on this technique, we recently replaced the pipetting step by spraying the polymeric solution directly onto a SH surface, yielding microparticles [2]. Further exploring this optimization, the present communication reports on the ability of the developed spray-based technology to produce hierarchical carriers for protein and cell encapsulation. To this end, both L929 cells and BSA protein were encapsulated in dextran methacrylate microparticles (ca. 226.8 ± 38.8 μm). After encapsulation, the cell viability proved to be maintained for at least 7 days in culture, as indicated by fluorescence microscopy (Live/Dead assay) and colorimetric quantification (MTS assay). On the other hand, BSA protein release was found to be almost complete (ca. 80%) after 1 h of incubation at physiological conditions. Interestingly, systems with a higher complexity, entailing the confinement of obtained microcarriers within an alginate matrix, exhibited a delayed and slower delivery of protein (c.a. 50% at 7 h), reflecting the possibility of modulating the release profile. In sum, these results demonstrate, for the first time, that SH surfaces can also be used to produce hierarchical systems, which are expected to contribute to the development of customizable systems to transport bioactive agents and cells in tissue engineering applications. [1] Song et al., Soft Matter 6, 5868 (2010) [2] Costa et al., Langmuir 30, 4535 (2014)


Poster Session 1

45

STROMAL VASCULAR FRACTION FROM ADIPOSE TISSUE AND CELL SHEET

ENGINEERING TO BUILD VASCULARIZATION UNITS FOR TISSUE ENGINEERING AND REGENERATIVE MEDICINE

M. Costa1,2, M. T. Cerqueira1,2, D. B. Rodrigues1,2, T. C. Santos1,2, A. P. Marques1,2, R. P. Pirraco1,2

and R. L. Reis1,2

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Vascularization holds the gold key for the effective survival and engraftment of complex engineered tissues and organs for Tissue Engineering and Regenerative Medicine. The lack of adequate vascularization post-transplantation often results in cell necrosis and ultimate failure and rejection of the engineered construct. Herein, we propose a strategy capable of surpassing this obstacle. Harnessing easy accessible adipose tissue stromal vascular fraction (SVF) as a source for cells with intrinsic angiogenic potential, and cell sheet technology we were able to engineer cell sheets with high angiogenic potential. SVF was isolated from the adipose tissue of healthy human subjects after enzymatic digestion and 2x105 nucleated cells/well were seeded on 24 well plates for cell sheet formation. To further boost cells’ angiogenic potential, hypoxic conditions of 5% of oxygen were provided to some of the cells while the rest was cultured in typical normoxia, for up to 8 days of culture, in basal medium. Flow cytometry analysis demonstrated the presence of a heterogeneous population of mesenchymal progenitors, endothelial and hematopoietic cells. Furthermore, the proliferation of SVF cells was evaluated through dsDNA quantification, which showed a trend for higher number of cells in normoxic conditions after 5 and 8 days of culture when comparing to hypoxic ones. Immunocytochemistry results before incubation with CD31 and CD146 antibodies highlighted the presence of several vessel-like structures self-assembled as a network, more intense after 5 days for cells cultured in hypoxia. Cobalt chloride supplementation in normoxia increased segment formation while HIF-1α inhibitor supplementation in hypoxia decreased network formation. Taken together, results show that the effects of hypoxia are at least partially related with HIF-1 α signalling. After 8 days of culture and in the absence of any specific media supplementation the extension of the network is quite similar for both conditions although the level of branching is different. In vivo testing using the cell sheets detached from the wells and HIF expression analysis are currently underway. Taken together, the great potentiality of cell sheet technology with SVF cells cultured in hypoxia opens new exciting perspectives and may represent tremendously valuable vascularization units for tissue engineering strategies.


Poster Session 1

46

CELL SELECTIVE CHITOSAN MICROPARTICLES AS INJECTABLE CELL CARRIERS FOR

TISSUE REGENERATION

C. A. Custódio 1,2, M. T. Cerqueira 1,2, A.P. Marques 1,2, R. L. Reis 1,2, J. F. Mano 1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal The detection, isolation and sorting of cells holds an important role in cell therapy and regenerative medicine. [1] Also, injectable systems have been explored for tissue regeneration in vivo, because it allows repairing complex shaped tissue defects through minimally invasive surgical procedures. The goal of this work was to explore the use of functional chitosan microparticles, as a strategy for cell separation and expansion that may be used as an injectable system to form tissue constructs at the lesion site for tissue regeneration purposes. Chitosan microparticles were firstly functionalized with biotin. Such modification allows engineering the surface of microparticles with a variety of biotinylated biomolecules via streptavidin (SaV), increasing its versatility and yield due to the multiple binding sites for biotinylated molecules. We tested the immobilization of biotinylated antibodies to target endothelial cells and human adipose stem cells (ASCs). Biotinylated antibody anti-CD31 was used to target human umbilical vein endothelial cells (HUVECs) cells while biotinylated antibody anti-CD90 was used to capture ASCs. Experimental results showed that biodegradable and biocompatible particles functionalized with antibodies presented selective affinity to cells, making them potentially suitable for separating subpopulations of cells from complex mixtures. Besides the ability for cell separation, the cultured particles proved to be also suitable for cell expansion. A versatile, cost effective, and easy-to-operate system with the capability to simultaneously separate and expand different cells subsets in vitro was developed. Moreover, the aggregation of the functionalized microparticles has been also shown to successfully form 3D robust structures upon injection into a mould. Thus the herein developed microparticles demonstrated that might be potentially used for further studies accomplishing the formation of a construct in situ upon implantation using minimally invasive procedures. 1 Abdallah, BM, Kassem, M. Gene Ther. 15, 109-16 (2008)


Poster Session 1

47

EXPLORING MULTIPLE APPLICATIONS OF CELLULOSE NANOCRYSTALS IN

BIONANOCOMPOSITE SCAFFOLDS DEVELOPMENT

R. M. A. Domingues1,2, S. Chiera3,4, S. Betta3,4, A. Motta3,4, R. L. Reis1,2, M. E. Gomes1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- Department of Industrial Engineering and Biotech Research Centre, University of Trento, Italy 4- European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Trento, Italy Cellulose nanocrystals (CNCs) are high strength, biocompatible and low toxic bionanoparticles seen as excellent load-bearing components for tissue engineering (TE) applications comparatively to other non-biocompatible reinforcing nanofillers [1]. We are currently exploring various concepts envisioning their application in the development of nanocomposite scaffolds aimed at tendon TE strategies. One of these concepts relies on the fabrication of reinforced nanofibrous scaffolds which can mimic tendon tissues architecture while balancing the biological performance with satisfactory mechanical properties. For this purpose, CNCs, as high strength nanofillers, were incorporated in electrospun polycaprolactone/chitosan (PCL/CH) nanofiber bundles, resembling the typical tendon structure. In another perspective, CNCs can be used as templates for the deposition of magnetic nanoparticles (MNP) resulting in hybrid magnetic nanomaterials with enhanced dispersion properties and superparamagnetic character. We prepared a range of CNCs decorated with magnetite (Fe3O4) MNP. When incorporated in tendon mimetic PCL/CH nanofiber bundles, these hybrid nanocomposites are envisioned to synergistically result in mechanically competent and magnetically responsive scaffolds which can ultimately be used to create a stimulating microenvironment capable of enhancing cell behaviors in tendon TE strategies. Proteins, such as e.g. collagen or fibrin, but also the polysaccharide chitosan are natural polymers traditionally used in scaffolds fabrication. However, they are generally low strength matrices what limit their range of applications. A key for their effective reinforcement can be the incorporation of CNCs with proper functionalization in order to establish chemical interactions between matrix and nanofiller. We prepared a range of functionalized CNCs with different degrees of carbonyl groups and thus aiming to instigate the formation of matrix-nanofiller imine linkages. With this strategy, CNCs will have the double role of load-bearing nanfiller but also functional cross-linker. These properties are envisioned to be particularly useful to reinforce soft material such as e.g. hydrogels based on those natural polymers. [1] R.M.A. Domingues, M.E. Gomes, R.L. Reis, Biomacromolecules 15, 2327 (2014)


Poster Session 1

48

PROTEOLYTIC SYSTEMS AND AMP-ACTIVATED PROTEIN KINASE AS IMPORTANT

TARGETS FOR ACUTE MYELOID LEUKEMIA THERAPEUTIC APPROACHES

Ângela Fernandes1,2, Maria M. Azevedo1,2, Olga Pereira1,2, Belém Sampaio-Marques1,2, Artur Paiva3, Margarida Correia-Neves1,2, Isabel Castro1,2 and Paula Ludovico1,2

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- Blood and Transplantation Center of Coimbra, Portuguese Institute of Blood and Transplantation, Coimbra, Portugal The therapeutic strategy against acute myeloid leukemia (AML) has hardly been modified over four decades [1]. Although resulting in a favorable outcome in young patients, older individuals, the most affected population, do not respond adequately to therapy [2]. Intriguingly, the mechanisms responsible for AML cells chemoresistance/susceptibility are still elusive. Mounting evidence has shed light on the relevance of proteolytic systems, as well as the AMPK pathway, in cancer biology and treatment, but their exact role is still unknown [3-5]. Specifically in AML, these processes have been suggested to act as pro-survival mechanisms, conferring chemoresistance but also as mechanisms of tumor cell death facilitating the eradication of malignant cells [5-7]. In this work, two AML cell lines (HL-60 and KG-1) were exposed to conventional chemotherapeutic agents (cytarabine and/or doxorubicin) to assess the relevance of autophagy and UPS on AML cells’ response to antileukemia drugs. Our results clearly showed the antileukemia agents target both proteolytic systems. Doxorubicin enhanced UPS activity and autophagy was blocked in cells exposed to the drugs’ combination. Importantly, these effects were specific of the HL-60 cells, which suggests a heterogeneous response of AML subtypes to conventional chemotherapy. Nevertheless, the data clearly demonstrate autophagy’s protective role on HL-60 and KG-1 cells response to treatment. Specific modulators of autophagy and UPS are, therefore, promising targets for therapeutic intervention in AML if used in association with standard chemotherapy. [1] Kantarjian H, O'Brien S, Cortes J, et al. Therapeutic advances in leukemia and myelodysplastic syndrome over the past 40 years. Cancer, 113:1933-1952 (2008) [2] Ravandi F, Burnett AK, Agura ED, Kantarjian HM: Progress in the treatment of acute myeloid leukemia. Cancer, 110(9):1900-1910 (2007) [3] Green AS, Chapuis N, Lacombe C, Mayeux P, Bouscary D, Tamburini J. LKB1/AMPK/mTOR signaling pathway in hematological malignancies: from metabolism to cancer cell biology. Cell Cycle, 10:2115-2120 (2011) [4] Voorhees PM, Dees EC, O’Neil B, Orlowski RZ. The Proteasome as a Target for Cancer Therapy. Clinical Cancer Research, 9:6316-6325 (2003) [5] Torgersen ML, Engedal N, Boe SO, Hokland P, Simonsen A. Targeting autophagy potentiates the apoptotic effect of histone deacetylase inhibitors in t(8;21) AML cells. Blood, 122:2467-2476 (2013) [6] Altman JK, Szilard A, Goussetis DJ, et al. Autophagy is a survival mechanism of acute myelogenous leukemia precursors during dual mTORC2/mTORC1 targeting. Clin Cancer Res, 20:2400-2409 (2014) [7] Willems L, Chapuis N, Puissant A, et al. The dual mTORC1 and mTORC2 inhibitor AZD8055 has anti-tumor activity in acute myeloid leukemia. Leukemia, 26:1195-1202 (2012) Acknowledgements to funding bodies: This work was supported by FCT - Fundação para a Ciência e Tecnologia (PTDC/BIA-MIC/114116/2009). AF has fellowship from FCT (SFRH/BD/51991/2012).


Poster Session 1

49

DEVELOPMENT OF NANOPARTICLE CARRIERS FOR THERAPEUTIC, IMAGING AND

THERANOSTIC APPLICATIONS

Helena Ferreira1,2, Ana Cartaxo1,2, Albino Martins1,2, Rui L. Reis1,2, Nuno M. Neves1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal There is a worldwide increased interest in the development of innovative, more effective and specialized release dosage forms. A delivery device that could efficiently deliver therapeutic agents in a controlled manner at the anatomical target, in concentrations lying within the therapeutic range and without toxic side effects, would represent a huge step in the pharmaceutical and medical field. Nanoparticles with different compositions and biological properties have been extensively investigated for drug, peptides, protein and nucleic acids delivery. Synthetic or natural biodegradable polymers and lipids have been extensively proposed as building blocks for the nanoparticles development. Those materials reportedly non-cytotoxic can be used alone, combined or even chemically modified to develop highly innovative and effective delivery devices. In the present work, it is highlighted the development of liposomes and polymer-based or protein-based particles for different therapeutic strategies [1-3]. Liposomes were developed with the intent to treat psoriasis, inflammatory conditions and chronic wounds, as well as to induce the osteogenic commitment of bone marrow mesenchymal stem cells for bone tissue engineering strategies. Polymeric particles, made of synthetic and natural polymers, were also prepared in order to load and release prednisolone or piroxicam that could be used in wound healing or in inflammatory diseases treatments. Additionally, these vehicles can incorporate an imaging agent, such as gold nanoparticles, making them useful in the early diagnostic of specific diseases, monitoring of the site of drug delivery and theranostics. The diagnosis in an early phase and along treatment modalities represents an enormous advance in the medical field. Indeed, this monitoring capacity allows establishing individual therapeutic regimens, maximizing the safety and efficacy of personalized therapies. [1] H. Ferreira et al, React. Funct. Polym. 73, 1328 (2013) [2] N. Monteiro et al, Biomaterials Science 2, 1195 (2014) [3] R. Silva et al, Colloids Surf. B 92, 277 (2012)


Poster Session 1

50

SPONTANEOUS HEALING OF MYCOBACTERIUM ULCERANS INDUCED LESIONS IN THE

GUINEA PIG MODEL

Alexandra G. Fraga1,2, Ana Rita Gomes1,2, Elly Marcq3, Gabriela Trigo1,2, José B. Gama1,2, Adhemar Longatto-Filho1,2, António G. Castro1,2, Jorge Pedrosa1,2

1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal; 3- Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium Mycobacterium ulcerans is the etiological agent of a necrotizing skin disease known as Buruli ulcer (BU). BU is characterized by a wide range of clinical forms, including nonulcerative cutaneous lesions that can evolve into severe ulcers, if left untreated; nevertheless, spontaneous healing is known to frequently occur. The characterization of several aspects of the host immune response to M. ulcerans has been carried out by using the mouse; a model of progressive infection that ultimately results in tissue ulceration. Although the mouse is the most widely used animal model to study mycobacterial infections, the guinea-pig has proven to be useful for a more comprehensive analysis of other mycobacterial infections, due to its similarities in the progression of infection to human disease. In this study, we report the guinea pig as being capable of not only spontaneously healing M. ulcerans-induced ulcerative lesions, but also of controlling bacterial proliferation. Histologically, infected tissue showed early edema, focal necrotic areas harbouring intense bacterial loads, peripheral inflammatory infiltrate and compromised epidermal layers. With the decrease in the bacterial viability, the inflammatory infiltrate was gradually replaced by granulation tissue allowing reepithelialisation of the ulcerated epidermis. The final phase of the healing was characterized by a reduction in the cellular activity and tissue remodelling of the wounded area. This study allowed us to describe a model of resistance to M. ulcerans infection. We intend to further dissect the host immune response of the guinea pig in order to uncover correlates of protection against M. ulcerans infection, which will provide new insights for the development of effective therapeutic and preventive strategies.


Poster Session 1

51

ANTIBACTERIAL PROPERTIES OF BIOACTIVE STARCH-SCAFFOLDS FUNCTIONALIZED

WITH SILANOL GROUPS

Rodrigues A. I.1,2, Franco A.R.1,2, Leonor I. B. 1,2, Reis R. L. 1,2 1- 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Bacterial infections are a significant problem on post-transplant and surgery because they can colonize implants surface [1]. Silicon-based biomaterials offer a great potential for biomedical applications, namely in bone regeneration, due to their osteoconduction properties. The antibacterial potential of these materials has also been studied, suggesting that the presence of silicon (Si) can decrease bacterial activity [2]. The aim of this study was to evaluate the antibacterial properties of Si based scaffolds against Escherichia coli and Staphylococcus aureus. The incorporation of Si ions into a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds was confirmed by X-ray photoelectron spectroscopy. The antibacterial potential of the produced SPCL scaffolds was assessed through radial diffusion assay, and their effect on bacterial growth was also determined. The number of viable bacteria counts significantly decreased in the modified scaffolds 4h after incubation, but no bacteria adhesion was observed after 24h incubation. Unmodified scaffolds showed no effect on bacterial growth. By inductively coupled plasma optical emission spectroscopy, an increase of Si ions in the medium was observed 4h after incubation. No significant pH medium variation was detected. Results suggest that SPCL scaffolds incorporating Si had a bacteriostatic effect against the tested bacteria, although no bacteria growth inhibition was observed. The present study demonstrates the positive effect of the presence of Si on the antibacterial properties of the produced scaffolds as a new way to respond to biomaterial-associated infections on prosthetic joints. A.I. Rodrigues thanks the Portuguese Foundation for Science and Technology (FCT) for providing a PhD scholarship (SFRH/BD/69962/2010). This work was partially supported by national funds through the FCT under the scope of the project PTDC/CTM-BIO/0814/2012 and PTDC/BBB-BIO/0827/2012. [1] A. Hess, et al. Diagn Microbiol Infect Dis 79, 73 (2014) [2] V. Mortazavi, et al. J Biomed Mater Res A 94, 160 (2010)


Poster Session 1

52

MOLECULAR MACHINERY INVOLVED IN PLASMA MEMBRANE REPAIR DURING

MYCOBACTERIUM TUBERCULOSIS INFECTION

Joana Gaifem1,2,3, António Gil Castro2,3, Margarida Saraiva2,3, Rui Appelberg1

1- Institute for Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal 2- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Tuberculosis (TB) is the deadliest bacterial pathogenesis of human society, claiming almost two million lives each year. The disease is spread worldwide and this is mainly due to the efficacy of Mycobacterium tuberculosis (Mtb) in impairing the immune response, by inhibition of phagosome maturation and modulation of cytokine expression and immune signaling pathways. It is described that Mtb infection causes microdisruptions in macrophage plasma membrane (PM) and the resealing of these lesions, which is essential for preventing macrophage necrosis, is impaired by virulent strains. Virulent strains evade the antimicrobial action of the macrophage through the manipulation of the intracellular trafficking of vacuolar compartments and by inducing the necrosis of infected cells. In opposite, avirulent strains are able to eradicate the bacteria by undergoing apoptosis [1]. The eicosanoid pathways are also differentially triggered by Mtb avirulent/virulent strains [2]. The aim of this project is to unveil the mechanisms and the regulation of PM repair. For that, mice peritoneal macrophages were infected with Mtb avirulent strain H37Ra and PM repair was analyzed by measuring LAMP-1 (a protein specific from lysosomes) translocation to the cell surface. Preliminary results from the cytokine profile during infection suggest that IL-17 seems to be involved in PM repair. Concerning the molecular machinery, siRNA transfection of Rab3a (selected in a shRNA screening as a candidate involved in lysosomal translocation) is being performed in mice peritoneal macrophages subsequently infected with H37Ra to study molecular factors that can be involved in the regulation of PM repair in context of Mtb infection. [1] M. Divangahi, M. Chen, H. Gan, D. Desjardins, T. T. Hickman, D. M. Lee, S. Fortune, S. M. Behar, H. G. Remold. Nature Immunology 10, 8, 889-906 (2009) [2] M. Chen, M. Divangahi, H. Gan, D. S. J. Shin, S. Hong, D. M. Lee, C. N. Serhan, S. M. Behar, H. G. Remold. The Journal of Experimental Medicine 205, 12, 2791-2801 (2008)


Poster Session 1

53

THE IMPACT OF IMMUNE-RELATED GENE POLYMORPHISMS IN HUMAN MYCOBACTERIUM

ULCERANS INFECTION Ana Rita Gomes1,2, Carlos Capela1,2, João Menino1,2, Ange Dossou1,2,3, Ghislain Sopoh3, Alexandra G. Fraga1,2, Fernando Rodrigues1,2, Jorge Pedrosa1,2 1 - Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2 - ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3 - Centre de Dépistage et de Traitment de l`Ulcere de Buruli (CDTUB) d`Allada, Allada, Benin Differential susceptibility to infection is dependent on both environmental and host genetic factors. Regarding Buruli Ulcer (BU), a necrotizing skin disease caused by Mycobacterium ulcerans infection, there is evidence supporting the role of host genetics in disease susceptibility. It has been described that not every person living in BU endemic areas develop disease, and there is also a wide spectrum of clinical manifestations (e.g. nodules, papules, oedemas, plaques, ulcers and osteomyelitis) within the affected population. Moreover, epidemiological data indicates that family history of BU is a risk factor for developing disease. Yet, little is known about the role of host genetics in BU. The study of immune-related genetic variants may help predict susceptibility to M. ulcerans infection. Therefore, we used a population-based candidate-gene approach, selecting single nucleotide polymorphisms (SNPs) previously associated with other mycobacterioses. We genotyped 42 SNPs in 208 BU patients and 300 unrelated age/gender/water-contact matched controls from Benin, of which 34 were in Hardy-Weinberg equilibrium. We observed that SNPs in the TNFA and PACRG gene were associated to an increased risk of BU development [TNFA, rs1800629 OR=1.44(1.00-2.06); PACRG, rs1333955 OR=1.36(1.01-1.82)]. Moreover, we assessed the role of the SNPs in disease severity within the affected population. SNPs in the IL12RB1 and BCL2L11 gene were associated with non-ulcerative lesions [IL12RB1, rs375947 OR=0.48 (0.26-0.87); BCL2L11, rs17041883 OR=0.41(0.20-0.87), rs3761704 OR=0.42(0.20-0.89)]. A SNP in the VDR gene was related with less severe forms of disease [rs7975232 OR=0.61(0.38-0.97)], while SNPs in the TLR4, NOD2 and IL10 genes were associated with the most severe types of lesion [TLR4, rs5030719 OR=3.48(1.18-10.3); IL10, rs1800872 OR=1.63(1.05-2.51); NOD2, rs9302752 OR=2.23(1.14-4.37)], namely large ulcerative lesions, osteomyelitis and/or multiple lesions. Collectively, these results provide insights into mechanisms of pathogenesis and immune protection in BU, which will be further pursued.


Poster Session 1

54

ISOLATION AND CHARACTERIZATION OF DISTINCT SUB-POPULATIONS OF ADIPOSE

STEM CELLS PREDISPOSED TO TENOGENIC DIFFERENTIATION

Pavel M. Gershovich1,2, Ana I. Gonçalves1,2, Márcia T. Rodrigues1,2, Rui L. Reis1,2, Manuela E. Gomes1,2

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Cell therapies for tendon regeneration are of particular interest given the low regenerative potential of this tissue. Human adipose tissue is an abundant source of stem cells with multipotential capacities, which may be employed for tendon repair and regeneration. We hypothesized that the isolation of adipose stem cells (hASCs) subpopulations based on the use of immunomagnetic beads coated with specific antibodies can be an effective strategy for detection of a subset of cells that might be more prone to tenogenic differentiation [1,2]. Therefore, this study focus on the isolation of the subpopulations of hASCs positive for tenomodulin (TNMD), STRO-1, SSEA-4 and CD29 markers and characterization of their tenogenic differentiation potential. For this purpose, the SVF obtained by enzymatic digestion of adipose tissue samples was sorted using immunomagnetic beads coated with the selected specific antibodies. Distinct subpopulations of hASCs and unsorted population of hASCs were plated at the same density (2.000 cells/cm2) with subsequent induction to tenogenic differentiation for 28 days [3]. Differentiation potential was assessed by real time RT-PCR, immunocytochemistry and flow cytometry analysis. The results indicate markedly up-regulated gene expression of the tenocyte lineage markers scleraxis (SCX) and TNMD accompanied by increased number of cells expressing these protein markers in TNMD positive subpopulation. Also, sorted cells demonstrated more pronounced formation of extracellular matrix. Thus, TNMD positive subpopulation of hASCs is predisposed to tenogenic commitment and can be considered as a promising source of progenitor cells for connective tissue engineering. Other distinct subpopulations characterization is ongoing. [1] T. Rada et al. J Tissue Eng Regen Med, 6, 1 (2012) [2] S.M. Mihaila et al. Tissue Eng. Part A, 19, 235 (2013) [3] A.I. Gonçalves et al. PlosOne, 8, 1 (2013)


Poster Session 1

55

TARGETING CD147/BASIGIN SENSITIZES A549 LUNG CARCINOMA CELLS TO

PHENFORMIN

Sara Granja1,2, Fátima Baltazar1,2 1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal 2- ICVS/3B’sPT Government Associate Laboratory, Braga/Guimarães, Portugal Most cancer and particularly those programmed for rapid growth rely on aerobic glycolysis to generate the energy needed, a phenomenon termed “Warburg effect”(1). To maintain high glycolytic rates, cells must efficiently export lactic acid through members of a family of proton-coupled monocarboxylate transporters (MCT1/4) (2). These transports require a chaperone, Basigin (BSG), for trafficking to the plasma membrane and exert their activity (3). Firstly, we analysed the expression of MCT1, MCT4 and BSG in 50 cases of lung cancer and we observed that all proteins were upregulated in tumour tissues when compared to non-tumoural tissue. Targeting bsg in A549 cells, via ‘Zinc-Finger Nuclease’ (ZFNs), we successfully isolated cells knocked out for BSG, decreasing the expression and activity of both MCT1 and MCT4 by respectively 95 and 80%. We noticed that A549 cells grown in normoxia have a moderate rate of glycolysis, which is reduced by 2-fold in BSG-null cells. Both wild type and BSG-null are extremely sensitive to the mitochondria inhibitor phenformin in normoxia. However, only BSG-null cells remain sensitive to phenformin in hypoxia (1% O2) for cell proliferation in vitro, and tumour growth in nude mice. Our results demonstrated that inhibiting MCTs via BSG disruption sensitized A459 tumour cells to phenformin-induced cell death. [1].Warburg O., Science 1956:309–314 [2].Pouysségur J, Nature, 2006 441: 437–443 [3].Halestrap AP., IUBMB Life 2012 64(2): 109-19


Poster Session 1

56

THE ROLE OF TET ENZYMES AND DNA HYDROXYMETHYLATION IN NEURONAL

DIFFERENTIATION

Marta Guedes1,2, Claudia Antunes1,2, Mafalda Santiago1,2, Nuno Sousa1,2, Miguel Branco3, Joana Marques1,2

1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/ Guimarães, Portugal 3- Blizard Institute, Queen Mary University of London, United Kingdom In recent years, TET enzymes have been shown to convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), being implicated in the mechanism of active DNA demethylation. Interestingly, the highest levels of 5hmC were described in several brain regions and neural cell types, suggesting a role in neural differentiation and function. To investigate the role of TET enzymes and 5hmC in neural precursor cells (NPCs), we used a stable and inducible knockdown system [1, 2] and an in vitro neural differentiation protocol [3]. We show that Tet3 transcription dramatically increases from embryonic stem (ES) cells to NPCs whereas Tet1 levels remain similar. Knockdown of Tet3 in NPCs resulted in increased levels of Oct4 pluripotency marker, suggesting that Tet3-deficient NPCs revert to a more undifferentiated state. Genome-wide analysis of DNA methylation and hydroxymethylation, by RRBS and oxidative RRBS respectively, show a decrease in global 5hmC levels upon Tet3 knockdown, affecting genes involved in neuron differentiation and fate commitment. Additionally, increased levels of 5mC are observed in genes involved in neural precursor cell proliferation and also neuron fate commitment. In conclusion, our results suggest that TET3 is involved in maintaining methylation and hydroxymethylation levels in NPCs, mainly at genes regulating neuronal differentiation, pointing to a critical role in the nervous system. [1] M. Iacovino et al., Stem Cells 29, 1580 (2011) [2] G. Ficz et al., Nature 473, 398 (2011) [3] M. Bibel et al., Nat Protoc 2, 1034 (2007)


Poster Session 1

57

PERIPHERAL NERVE INJURY – IMPACT ON BEHAVIOR IN PAINFUL AND SILENT

CONDITIONS

Marco Rafael Guimarães*; Ana Margarida Cunha*; Ana Rosa Soares; Madalena Esteves; Sónia Borges; Ana João Rodrigues; Nuno Sousa; Armando Almeida; Hugo Leite-Almeida.

*Contributed equally Chronic pain is frequently accompanied by a deterioration of emotional behavior and cognitive function. Curiously, left- and right-sided nerve injuries impact differently on emotional and cognitive behaviors although having the same allogeneic potential. Also, while mechanical allodynia (a hallmark of neuropathic pain) appears soon after nerve injury, anxiety- and depression-like behaviors take 4-6 weeks to manifest. These observations suggest some degree of dissociation between pain and the behavioral impairments indicating that the nerve injury per se (in the absence of pain) might play a detrimental role.

To test this hypothesis, Sprague-Dawley rats, a strain in which 15-20% of neuropathic animals are resistant to the development of mechanical allodynia, were tested in a battery of behavioral paradigms – dark/light (anxiety), forced swimming (depression), spontaneous burrowing (general wellbeing), fear-conditioning and variable delay-to-signal (VDS; impulsivity) – after the induction of an unilateral spared nerve injury neuropathy in the left (SNI-L) or in the right (SNI-R) side. Results show that both allodynic and non-allodynic SNI-L animals presented an increased anxiety-like profile. On the contrary, both allodynic and non-allodynic SNI-R animals presented increased impulsivity in the VDS task. Our results support the hypothesis that the nerve injury alone, in the absence of pain, is sufficient to affect behavior; further the observed side-specific deficits obey to the same patters previously observed suggesting that the impact on brain function is anatomically restricted. The volumes of critical cortical and subcortical brain regions on both hemispheres are presently being evaluated.


Poster Session 1

58

SYNTHESIS AND CHARACTERIZATION OF BIOACTIVE BIODEGRADABLE CHITOSAN

COMPOSITE SPHERES WITH SHAPE MEMORY CAPABILITY

Álvaro J. Leite1,2 , Sofia G. Caridade1,2 , João F. Mano1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Chitosan spheres incorporating bioactive glass nanoparticles were prepared to obtain a composite system able to induce the deposition of an apatite layer upon immersion in a biological-like environment. Spheres were synthesized with different concentrations of bioactive glass nanoparticles obtained from a sol-gel route and genipin (the crosslinking agent). Biomimetic superhydrophobic surfaces were used to support droplets of chitosan-based solutions that after crosslinking enabled to produce well developed spherical particles with controlled sizes, between 1 mm and 7.5 mm. After SEM and EDS analysis, it was observed the successful formation of bone-like apatite on the surface when the spheres were immersed in a simulated body fluid. Lower genipin concentration promoted more apatite formation. The spheres presented shape memory behaviour triggered by hydration with high values of shape fixity and shape recovery. This effect could be used to introduce these spheres in a bone defect showing a good geometrical accommodation in the implanted site. The bioactive spheres allowed the incorporation of a drug model and its effective release. Overall the developed nanocomposite spheres showed great potential for bone tissue engineering in particular as a device to be implanted using minimal invasive procedures.


Poster Session 1

59

MOLECULAR MECHANISMS REGULATING IL-10 PRODUCTION IN INNATE IMMUNE CELLS

ACTIVATED VIA TLRS

D. Lobo-Silva1,2, L. Moreira-Teixeira1,2, F. Castro1,2, J. Pedrosa1,2, M. Correia-Neves1,2, A. G. Castro1,2, S. Roque1,2 and M. Saraiva1,2

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Activation of both the innate and adaptive immune systems is a tightly controlled process. This control is essential to prevent immunopathology and is achieved in most immune responses, for example, through the production of anti-inflammatory cytokines, such as interleukin (IL)-10. Production of IL-10 is tightly regulated, so that an efficient immune response is achieved in the absence of tissue damage. This project focuses on the molecular mechanisms regulating the expression of IL-10 by myeloid cells, namely macrophages, dendritic cells and microglial cells in response to toll-like receptor (TLR) activation. So far, our results suggest the existence of common and distinct mechanisms regulating TLR-induced IL-10 production in each cell type. Specifically, the kinetics of Il10 gene transcription appears to be cell-specific, with implications on the intensity of the pro-inflammatory responses driven upon cell activation. Acknowledgements to funding bodies: This work was supported by PTDC/BIA-BCM/102776/2008 and FCT-ANR/BIM-MEC/0007/2013. DS is supported by a FCT PhD grant: SFRH/BD/88081/2012. LMT is supported by an FCT Post-doc grant SFRH/BPD/77399/2011. MS is an Associate- FCT Investigator.


Poster Session 1

60

MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM TUBERCULOSIS PHYLOGENETIC

LINEAGES IN A GROUP OF LOW RISK TUBERCULOSIS PATIENTS FROM THE NORTH OF PORTUGAL

Henrique Machado1,2,*,Helder Novais Bastos1,2,3,*, Maria Isabel Veiga1,2, Jeremy Sousa1,2, Nuno S.

Osório1,2, Angélica Ramos4, Teresa Carvalho4, Jorge Pedrosa1,2, Fernando Rodrigues1,2, João Tiago Guimarães4,5, António Gil Castro1,2 and Margarida Saraiva1,2

1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of

Minho, 4710-057 Braga, Portugal. 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- Department of Pneumology, São João Hospital Center, Porto, Portugal. 4- Department of Clinical Pathology, São João Hospital Center & Institute of Public Health,

University of Porto, Porto, Portugal. 5- Department of Biochemistry, Faculty of Medicine, University of Porto, Porto, Portugal. *These authors contributed equally in this study. Background: Mycobacterium tuberculosis complex (MTBC) comprises seven genetic lineages of bacteria with distinct phylogeographic distribution, each of which can be divided into sublineages. Recent reports have suggested a change of MTBC genetic diversity in Western Europe due to new migration flows. The present study aims to investigate the phylogenetic distribution of MTBC isolates collected from a group of low risk tuberculosis (TB) patients in a main hospital in the Porto area. Methods: Socio-demographic, clinical and microbiological features of 681 patients attending the Centro Hospitalar São João, Porto, between 2007 and 2013, were collected. Known risk factors and comorbidities were excluded so that a group of 263 low risk TB patients was defined. MTBC strains from these patients were isolated and genotyped using single nucleotide polymorphisms (SNPs) as stable genetic markers to define the different lineages and sublineages. The presence of these genetic markers was determined by TaqMan SNP genotyping real-time PCR assays. Results: Of the 263 patients analysed, most cases (96.2%) were Portuguese-born Caucasians. Male gender was predominant (69.2%) and mean age (±SD) was 48.7±19.1 years. Drug resistance was present in 41 (15.9%) cases. SNP-typing revealed that 259 isolates (98.5%) belong to Lineage 4 (L4 or Euro-American). Within L4, the sublineage LAM (Latin American-Mediterranean) represented 62.7% of the isolates. Among non-L4 isolates, 2 were Portuguese-born Caucasians, 1 was African and 1 Asian. Conclusions: We show a highly homogeneous phylogenetic composition of MTBC in a population of north of Portugal, where L4 and LAM sublineage dominate. This predominance remained stable across the years analysed. The methods used in this study appear well suited to monitor the MTBC population structure in Portugal. Key-words: Tuberculosis, lineages, phylogeography, genotyping Acknowledgements: We acknowledge the Fundação Amélia de Mello/José de Mello Saúde and Sociedade Portuguesa de Pneumologia (SPP) for financial support. This work is co-funded by Programa Operacional Regional do Norte (ON.2 – O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER). MS is an Associate FCT-Investigator. MIV and NSO are postdoctoral fellows from FCT.


Poster Session 1

61

GENETIC DIVERSITY IN MYCOBACTERIUM TUBERCULOSIS AND ITS IMPACT ON

HOST/PATHOGEN CO-EVOLUTION

C. Magalhães1,2, F. Rodrigues1,2, E. Torrado1,2, J. Pedrosa1,2, A. G. Castro1,2, M. Saraiva1,2 and N. S. Osório1,2

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Tuberculosis (TB) is a global disease caused by infection with bacteria from the Mycobacterium tuberculosis complex (MTBC). There is compelling evidence that both pathogen and human genetic diversity influence TB outcome and that T-cell mediated immunity is critical for the control of M. tuberculosis infection. However, the full repertoire of MTBC peptides that bind in vivo to the human HLA class I and II molecules is yet to be determined. We have studied the diversity across the MTBC phylogeny of a previously defined in vivo expressed M. tuberculosis genomic island (iVEGI). We found that the 43 genes in this region were not equally diverse with 26 genes showing a level of nucleotide diversity above the whole-genome average. As expected, the majority of the SNPs were lineage-specific but homoplastic substitutions were also found suggesting that selection pressures acted differentially on individual sites. The use of codon-based substitution models and maximum-likelihood (ML) methods in these genes highlighted 21 amino acid residues from 13 genes as being under diversifying (positive) selection. The impact of these amino acid substitutions on the antigenicity of its encoding peptides was predicted for a set of 72 HLA class I and 42 HLA II human alleles. Of particular interest were the above 2.5 fold change in the predicted number of peptides and HLA I alleles strongly binding to amino acid positions 186 of Rv0962c/lprP, 191 of Rv0988 and 68 of Rv0990c and the predicted number peptides and HLA II alleles strongly binding to amino acid position 233 of Rv0974c/accD2. These results support the occurrence of targeted events of T-cell driven diversifying selection in 4 iVEGI genes. HLA-peptide binding in vitro assays to confirm the predictions are ongoing. Understanding the relationship between host-driven positive selection and antigenic variation in MTBC may allow the development of more effective vaccines.


Poster Session 1

62

THE CHOROID PLEXUS IS MODULATED BY VARIOUS STIMULI: IMPLICATIONS TO

DISEASES OF THE CENTRAL NERVOUS SYSTEM

Fernanda Marques1,2, Sandro D Mesquita1,2, Catarina Ferreira1,2, João C Sousa1,2, Margarida Correia-Neves1,2, Nuno Sousa1,2, Joana A Palha1,2

1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.

2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal. The brain barriers are essential because they protect the brain but also because they regulate the exchange of cells and molecules in and out of the brain; both functions are responsible for the homeostasis of the central nervous system (CNS). Together with the blood brain barrier (BBB), the blood cerebrospinal fluid barrier (BCSFB), composed by the choroid plexus (CP) epithelial cells and by the arachnoid membrane, form the barriers of the brain. Structurally, the CP can be divided in two independent compartments; one is formed by a unique and continuous line of epithelial cells which rests upon a basal lamina that contains the other compartment: a central core formed by connective and highly vascularised tissue populated by different cell types (fibroblasts, macrophages and dendritic cells). Importantly, depending on the disease type, the cells present in this second compartment may greatly vary. The CP epithelial cells produce most of the cerebrospinal fluid. It is now accepted that the “barriers” are more than just physical barriers, since they respond to various peripheral and CNS stimuli, therefore contributing to brain homeostasis in health and in disease. In fact, the CP transcriptome and secretome are diverse depending on the nature and duration of the stimuli. The versatility of the CP homeostasis should therefore be further studied towards a better understanding on how its modulation may contribute to treat diseases of the CNS, such as multiple sclerosis and Alzheimer’s disease.


Poster Session 2

63

Poster number

First Author Title

P2_.01 Ana Marques SHARK SKIN COLLAGEN MEMBRANES AS DRUG DELIVERY SYSTEMS

P2_.02 Olga Martinho HER FAMILY OF RECEPTORS ARE POTENTIAL TARGETS FOR THERAPY IN CERVICAL CANCER

P2_.03 Ana Catarina Martins DOES HYPERGLYCEMIA AFFECT ACUTE MYELOID LEUKEMIA CELL SURVIVAL AND CHEMORESISTANCE? – THE ROLE OF CXCL12 CHEMOATTRACTION AXIS

P2_.04 Vera Miranda-Gonçalves INVOLVEMENT OF MONOCRBOXYLATE TRANSPORTER 1 (MCT1) IN GLIOBLASTOMA AGGRESSIVENESS

P2_.05 Filipa Morais-Santos TARGETING LACTATE TRANSPORT SUPPRESSES IN VIVO BREAST TUMOR GROWTH

P2_.06 L. Moreira-Teixeira TYPE I INTERFERON REGULATES LUNG IMMUNOPATHOLOGY BY MODULATING THE INTENSITY AND PROFILE OF INFLAMMATORY RESPONSE TO MYCOBACTERIUM TUBERCULOSIS INFECTION

P2_.07 Liane Moura CORK EXTRACTS INDUCE A CYTOPROTECTIVE EFFECT AGAINST H2O2 OXIDATIVE DAMAGE IN SH-SY5Y CELLS

P2_.08 Catarina Oliveira NANOPARTICLES FOR SUSTAINED DRUG DELIVERY BASED IN MARINE POLYSACCHARIDES

P2_.09 Nuno Oliveira LIQUID MARBLES TO CULTURE ADHERENT CELLS FOR HIGH-THROUGHPUT DRUG SCREENING

P2_.10 Daniela P. Pacheco NEW MULTI-FUNCTIONAL THERAPEUTIC SYSTEM FOR BURULI ULCER

P2_.11 Daniela Pacheco INJECTABLE PIEZOELECTRIC DRUG DELIVERY SYSTEM: A NOVEL STRATEGY TO TARGET TISSUE REGENERATION

P2_.12 Patricia Patrício GENETIC AND EPIGENETIC REGULATION OF NEUROPLASTIC PHENOMENA IN THE ADULT HIPOCAMPAL NEUROGENIC NICHE - IMPLICATIONS FOR DEPRESSION

P2_.13 Olga Pereira ROLE OF AUTOPHAGY (MACROAUTOPHAGY) ON THE DIABETIC PATIENTS´ PRONENESS TO INFECTIOUS DISEASES AND CANCER

P2_.14 Sandra Pina OXIME CLICK CHEMISTRY TO MIMIC MULTIVALENT INTERACTIONS OF GLYCOSAMINOGLYCANS

P2_.15 Ricardo Pires CORK ANTI-OXIDANT COMPOUNDS AS A NOVEL THERAPEUTIC STRATEGY FOR MACHADO-JOSEPH DISEASE

P2_.16 João Pizarro SUBREGION-DEPENDENT SPECIFICITIES OF THE HIPPOCAMPAL FORMATION TO THE ACUTE APPLICATION OF AΒ OLIGOMERS

P2_.17 Sónia Puga PHARMACOLOGICAL SCREENING OF A NOVEL DRUG FOR THE CONTROL OF NEUROPATHIC PAIN AND ASSOCIATED DEPRESSION


Poster Session 2

64

P2_.18 Lara Reys METHACRYLATED FUCOIDAN AS PHOTOCROSSLINKABLE BIOPOLYMER FOR TISSUE ENGINEERING APPLICATIONS

P2_.19 Sofia Ribeiro THE EFFECTS OF COPPER-CONTAINING BIOACTIVE GLASSES ON THE IN VITRO BEHAVIOUR OF HUMAN ADIPOSE DERIVED STEM CELLS

P2_.20 Viviana Ribeiro SMART SILK FIBROIN HYDROGELS AND ITS POTENTIAL APPLICATION AS SUPPRESSOR OF ANGIOGENESIS AND TUMOR PROGRESSION

P2_.21 Daniel Rodrigues FUNCTIONALIZED DENDRONIZED POLYMERS FOR T-CELL THERAPY

P2_.22 Belem Sampaio-Marques INCREASED AUTOPHAGY IN AGING YEAST CELLS EXPRESSING ALPHA-SYNUCLEIN PROMOTES DNA REPLICATION STRESS

P2_.23 Lívia Santos DEVELOPMENT OF MAGNETIC MEMBRANES AND PRELIMINARY BIOLOGICAL ASSESSMENT OF MAGNETIC ACTUATION ON HUMAN ADIPOSE STEM CELLS

P2_.24 Tírcia Santos XENOGENEIC MODULATION OF MACROPHAGES AND DENDRITIC CELLS BY UNDIFFERENTIATED AND DIFFERENTIATED ADIPOSE TISSUE-DERIVED STEM CELLS

P2_.25 Carla Silva REAL TIME ANALYSIS OF THE ENZYMATIC DIGESTION OF CHONDROITIN SULFATE

P2_.26 João Silva MARINE SPONGES – AN ALTERNATIVE COLLAGEN/GELATIN SOURCE FOR BIOMEDICAL APPLICATIONS

P2_.27 Lucilia Silva HYALURONATE DEGRADATION FRAGMENTS FROM GGHA SPONGY-LIKE HYDROGELS

P2_.28 Marta Silva TEMPORARY AND REVERSIBLE INDUCTION OF COSTAL CARTILAGE PLASTICITY FOR THE MINIMALLY INVASIVE TREATMENT OF PECTUS CARINATUM

P2_.29 Gabriela Soares COLLAGEN/APATITE SCAFFOLDS OBTAINED FROM SHARK SKIN AND TEETH

P2_.30 Jeremy Sousa GENETIC DIVERSITY IN PHTHIOCEROL DIMYCOCEROSATE OF MYCOBACTERIUM TUBERCULOSIS: AN EVOLUTIONARY PERSPECTIVE

P2_.31 Pathomthat Srisuk COMBINING ELECTROACTIVE SPONGY LIKE HYDROGELS AND ELECTRICAL STIMULATION FOR SKELETAL MUSCLE TISSUE ENGINEERING.

P2_.32 Raquel Teixeira LAYER-BY-LAYER ASSEMBLY OF POLY-L-LYSINE/GLYCOSAMINOGLYCAN FILMS: A COMBINED ANALYSIS OF QCM-D AND ZETA POTENTIAL MEASUREMENTS

P2_.33 Catarina Vale MULTIFUNCTIONAL BIOMIMETIC LbL COATINGS THAT COMBINE BIOACTIVITY WITH SUPERIOR ADHESION FOR ORTHOPAEDIC APPLICATIONS

P2_.34 Maria Isabel Veiga COMPLEX POLYMORPHISMS IN THE PLASMODIUM FALCIPARUM MULTIDRUG RESISTANCE PROTEIN 2 GENE AND ITS CONTRIBUTION TO ANTIMALARIAL RESPONSE

P2_.35 Stephanie Vial GELA1:C74OLD NANORODS AS INTRACELLULAR DRUG RELEASE SYSTEMS FOR REGENERATIVE MEDICINE


Poster Session 2

65

SHARK SKIN COLLAGEN MEMBRANES AS DRUG DELIVERY SYSTEMS

Ana L. P. Marques1,2, Alberta Domingues1,2, Joana Moreira-Silva1,2, R.I. Perez-Martin3, C.G. Sotelo3,

Tiago H. Silva1,2, Rui L. Reis1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- CSIC, Inst Invest Marinas, Vigo 36208, Galicia, Spain Collagens are the most abundant proteins in mammals. Current industrial procedures extract collagen from bovine and porcine sources. However, religious constraints and transmission of diseases have empowered the search for new sources. Abundant and underexplored, marine byproducts are a good alternative, also giving a new end to fish processing products, with economic benefits. In this work, type I collagen was extracted from shark (Scyliorhinus canicula) skins and then processed into collagen membranes. Skins were treated with 0.1M NaOH to remove non-collagenous proteins, and plunged in 0.5M acetic acid to extract collagen. After centrifugation and dialysis, collagen is recovered by freeze-drying. Collagen was dissolved in 0.5M acetic acid to obtain 1%(w/v) solution. A 5%(v/v) hexamethylene diisocyanate(HMDI) was added to obtain a ratio of (0%) 1% and 5% with respect to collagen and let to react for 24h. The solution was then cast in a petri dish and left to dry at room temperature. Membranes loaded with a model drug were also prepared following the same procedure, but adding dexamethasone previously solubilized in acetone to the collagen solution. Collagen extraction was successfully achieved. SDS-PAGE analysis showed a type I collagen, due to molecular weight of its 3 polypeptide chains (α1, α2 and β). µ-DSC analysis returned a denaturation temperature of 33.15 °C, lower than mammalian collagen. The aminoacid analysis showed an existence of hydroxyproline (characteristic of collagen) and a high quantity of glycine (about 1/3 of total proteins), which is the aminoacid present in every sequence of three aminoacid residues (Gly-X-Y). Membranes with higher degree of crosslinking show higher Young’s modulus. The release profile of dexamethasone was studied, being progressively released, reaching a threshold at around 8h. Membranes’ properties can be tuned adjusting the crosslinking ratio, showing potential to be applied in tissue engineering, as dermal application or drug release.


Poster Session 2

66

HER FAMILY OF RECEPTORS ARE POTENTIAL TARGETS FOR THERAPY IN CERVICAL

CANCER

Olga Martinho1,2, Ana Martins-Barbosa1,2, Renato Silva-Oliveira1,2,3, Adhemar Longatto-Filho1,2,3, Rui Manuel Reis1,2,3

1-Life and Health Sciences Research Institute (ICVS), Health Sciences School, University of Minho, Braga, Portugal; 2-ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal; 3-Molecular Oncology Research Center, Barretos Cancer Hospital, São Paulo, Brazil; Despite technological advances, up to 35% of all cervical cancer patients will develop a metastatic and lethal disease. In the past two decades we have seen the successful development of Receptor Tyrosine Kinases (RTK) targeted drugs, expanding the treatment options for cancer patients. However, there is approved molecular targeted therapies in cervical cancer mainly because the preclinical studies are scarce. Thus, the major aim of this work was to screen for effective molecular targeted therapies in cervical cancer and to find out potential predictive factors for therapy response, with focus in the HER family of receptors. By screening for the activation of 42 different RTK in human cervical cancer cell lines we observed that exclusively HER family of receptors, such as EGFR, HER2 and HER4 were activated. In accordance, we found by in vitro assays that only HER inhibitors (such as lapatinib and AST1036, in contrast to cediranib, sunitinib and imatinib) were effective in the reduction of cell lines aggressiveness. Those results were validated by the in vivo Chick Chorioallantoic Membrane (CAM) Assay. Finally, by immunohistochemistry in a series of 231 adernocarcinoma cervical cancer tissues we found EGFR, HER2 , HER3 and HER4 positivity in both cytoplasm and membrane of the cells being HER3 the most expressed (74,7% of cases) and EGFR (18.8% of cases) the less frequent. Importantly, we observed a statistically significant association (p<0.05) between HER2 overexpression (in around 45% of cases) and poor prognosis of the patients. In conclusion, we performed the first comparative study of HER family receptors expression in a large series of cervical carcinoma cases. We found that the expression HER2 could constitute an independent prognostic marker for this patients. Importantly, we demonstrate for the first time that exclusively HER family of receptors are activated in cervical cancer cell lines, being HER targeted therapies effective in the blockage of cervical cancer cells aggressiveness. Thus, with this work we propose HER family inhibitors as potential anti-cancer agents in cervical cancer patients.


Poster Session 2

67

DOES HYPERGLYCEMIA AFFECT ACUTE MYELOID LEUKEMIA CELL SURVIVAL AND

CHEMORESISTANCE? – THE ROLE OF CXCL12 CHEMOATTRACTION AXIS

Ana Catarina Correia Martins1,2, Maria Azevedo1,2 and Paula Ludovico1,2

1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal The cross-talk between acute myeloid leukemia (AML) cells and their neighboring stromal cells is often the key for cancer cells survival, resistance to treatment and disease relapse [1]. Diabetes mellitus induces persistent changes in the bone marrow (BM) microenvironment [2] and is often associated with poorer AML outcomes [3]. However, the association between these two disorders has not been clarified. The CXCL12-CXCR4 chemoattraction axis is a possible mechanism by which diabetes affects AML progression. CXCL12 plays a role in diabetes and its altered function is known to lead to enhanced survival of the cancer cells and chemoresistance in AML [4]. The aim of this project is to study the CXCL12 chemoattraction axis in a diabetic context and evaluate its effect on AML cells tumorigenicity and chemoresistance, as well as, identify a possible intracellular signaling pathway responsible for driving the effect, using a diabetes in vitro model. [1] F. Ayala, R. Dewar, M. Kieran & R. Kalluri, Leukemia 23, 2233–41 (2009). [2] M. Fujimiya, K. Nagaishi, T. Yamashita & K. Ataka, Anatomical Record (Hoboken) 295, 917–21 (2012). [3] R. Gibson, S. Graham, A. Lilienfeld, L. Schuman, M. Levin, et al. Journal of the National Cancer Institute 56, 891–8 (1976). [4] P. S. Becker, Scientific World Journal 2012, 856467 (2012).


Poster Session 2

68

INVOLVEMENT OF MONOCRBOXYLATE TRANSPORTER 1 (MCT1) IN GLIOBLASTOMA

AGGRESSIVENESS

Vera Miranda-Gonçalves1,2, Rui M. Reis1,2,3, Fátima Baltazar1,2 1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; 2- ICVS/3B’s-PT Government Associate Laboratory, Braga/Guimarães, Portugal;; 3- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil Introduction: Tumor cells present a characteristic glycolytic metabolism with increased lactate production, which is transported to the microenvironment through monocarboxylate transporters (MCTs), to maintain the physiological intracellular pH. Glioblastomas present a high heterogeneity with high levels of hypoxia and glycolysis, which for instance confer resistance to therapy. Thus, it is important to understand the role of metabolism-related proteins, particularly MCTs, in the response to hypoxia on glioblastomas. Material and Methods: Evaluation of metabolic marker expressions (MCT1, MCT4, CD147 and GLUT-1) and hypoxic markers (CAIX and HIF-1α) expression was performed in a glioblastoma series and also in in vitro U251 and SW1088 glioma cell lines. Additionally, the effect of MCTs downregulation on cell proliferation, cell invasion and cellular metabolism was also evaluated. Results: In glioblastoma tissues, the expression of MCTs, CD147 and other metabolic markers increased significantly in hypoxic regions. The plasma membrane of GLUT-1 and CAIX, as well as, the nuclear HIF-1α expressions were restricted to areas adjacent to necrosis (hypoxia). Importantly, the plasma membrane expression of MCT1 and CD147, but not MCT4, increased in hypoxic regions. In glioma cell lines, hypoxia induced an increase in MCT1 and CD147, but not MCT4, plasma membrane expression in the most oxidative cell line (SW1088). Additionally, we observed that MCT1 downregulation, decreased lactate production, leading to a decrease in cell proliferation and invasion. Conclusion: MCT1 is responsible for lactate efflux in hypoxia-induced glycolytic phenotype, being an important mediator in cell survival and aggressiveness of glioblastomas, thereby representing a promising therapeutic target.


Poster Session 2

69

TARGETING LACTATE TRANSPORT SUPPRESSES IN VIVO BREAST TUMOR GROWTH

Filipa Morais-Santos1,2, Sara Granja1,2, Vera Miranda-Gonçalves1,2, António H.J. Moreira1,2, Sandro

Queirós1,2, João L. Vilaça1,2, Fernando C. Schmitt3,4,5, Joana Paredes3, Adhemar Longatto1,2,7, Fátima Baltazar1,2*, Céline Pinheiro1,2,6,7*

1- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus of Gualtar, Braga, Portugal

2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- IPATIMUP - Institute of Molecular Pathology and Immunology of University of Porto, Porto, Portugal; 4- Medical Faculty of the University of Porto, Porto, Portugal 5- Department of Pathology and Medicine, Laboratoire National de Sante, Luxembourg 6- Barretos School of Health Sciences, Dr. Paulo Prata - FACISB, Barretos, Sao Paulo, Brazil 7- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Sao Paulo, Brazil

* These authors contributed equally to the work. Acidification of tumour microenvironment by lactate extrusion, performed by up-regulation of lactate transporters (MCTs) has been associated with higher cell proliferation, migration, invasion, angiogenesis and increased cell survival. Previous results from our group showed up-regulation of MCT1 in breast carcinoma samples and demonstrated the importance of in vitro MCT inhibition, however additional studies are needed to validate the potential of lactate transport inhibition for cancer therapy. Therefore, this work aimed to evaluate the effect of MCT silencing in in vitro and in vivo breast cancer models. The effect of MCT1, MCT4 or MCT1 plus MCT4 silencing on lactate efflux, proliferation, cell biomass, migration and invasion was evaluated in MDA-MB-468, MDA-MB-231 and BT20 breast cancer cell lines, under normoxia and hypoxia conditions. Also, silenced cells were used to induce tumor xenografts in nude mice. Our results showed a decrease in in vitro tumour cell aggressiveness reducing lactate transport, cell proliferation, migration and invasion and importantly inhibited in vivo tumour formation and growth. This work demonstrates the contribution of MCTs for cancer cell aggressiveness and, more importantly, shows, for the first time, the disruption of in vivo breast tumour growth by targeting lactate transport. This work was supported by FCT fellowships (SFRH/BPD/69479/2010 and SFRH/BD/87139/2012) and FCT grant (PTDC/SAU-FCF/104347/2008, under the scope of “Programa Operacional Temático Factores de Competitividade” (COMPETE) of “Quadro Comunitário de Apoio III” and co-financed by Fundo Comunitário Europeu FEDER).


Poster Session 2

70

TYPE I INTERFERON REGULATES LUNG IMMUNOPATHOLOGY BY MODULATING THE

INTENSITY AND PROFILE OF INFLAMMATORY RESPONSE TO MYCOBACTERIUM TUBERCULOSIS INFECTION

L. Moreira-Teixeira1,2,3, J. Sousa1,2, F. McNab3, E. Torrado1,2, J. Gaifém1,2,4, F. Castro1,2, V.

Cardoso1,2, J. Pedrosa1,2, R. Appleberg4, A. G. Castro1,2, A. O’Garra3,5 and M. Saraiva1,2 1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- MRC National Institute for Medical Research, London, United Kingdom 4- IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal 5- NHLI, Faculty of Medicine, Imperial College, London, United Kingdom Mycobacterium tuberculosis (Mtb) is a successful pathogen that has an outstanding ability to spread and cause infections with multiple outcomes, depending on host and bacterial factors. Data from our laboratory shows that different strains were recognized by different Toll-like receptors (TLRs), with an impact in the outcome of infection. We also observed that in vitro TLR4-activating strain induces nitric oxide synthase 2 (Nos2) expression in macrophages, in a type I interferon dependent pathway. Since IFN-γ is a strong inducer of Nos2, to address the role of type I IFN in vivo, we compared the outcome of Mtb infection in type I IFN receptor deficient mice to that of mice deficient for both type I and type II IFN receptor. We found that double-deficient mice were more susceptible to infection, with higher bacterial burdens and enhanced lung pathology when compared to WT or type I IFN receptor deficient mice. However, while in vitro both type I and type II IFN induce the expression of Nos2, the enhanced susceptibility of double-deficient mice is not associated with reduced expression of Nos2. Instead, we found that underlying the enhanced susceptibility of type I and type II IFN receptor double-deficient mice was a higher recruitment of neutrophils to the lungs, likely as a result of higher IL-17, GM-CSF and chemokine gene expression. Importantly, we found that these neutrophils, despite expressing classical markers of the granulocyte lineage, display a hybrid neutrophil-dendritic phenotype, with the expression of CD11c and MHC-II. We are currently investigating the molecular mechanisms underlying the differentiation of this neutrophil population in the lungs of Mtb infected mice. Our data support a model whereby type I IFN prevents the accumulation of a specific population of neutrophils that may serve as host cells to Mtb, and prevent its efficient elimination by the acquired immune response.


Poster Session 2

71

CORK EXTRACTS INDUCE A CYTOPROTECTIVE EFFECT AGAINST H2O2 OXIDATIVE

DAMAGE IN SH-SY5Y CELLS

Liane I. F. Moura1,2, Ivo M. Aroso1,2, Rui L. Reis 1,2, Ricardo A. Pires1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Alzheimer disease (AD) is a prevalent neurological disorder in the World, characterized by aggregation/accumulation of amyloid-β peptide (Aβ) in the brain, which induces neuronal cell death. Aβ-peptide induces hydrogen peroxide (H2O2) and reactive oxygen species (ROS) formation, which contributes to oxidative damage/pathogenicity of AD[1]. To control AD progression, it is important to search for anti-oxidant (AO) compounds that inhibit Aβ aggregation. Cork is the outer bark of Quercus suber. Its chemical composition includes polyphenols (that are readily extracted) which contain bioactive compounds that present AO activity[2]. In this context, we analyzed the cytoprotective effects of cork compounds, towards the human neuroblastoma cell line (SH-SY5Y) cultured in the absence/presence of H2O2 (to mimic the oxidative environment in the brain of AD patients). The cork polyphenolic raw extract (WE) was obtained by standard water extraction and fractionated by HPLC. MALDI-TOF was used to characterize two of the most relevant compounds isolated from WE: F6 and F8 (castalagin and vescalagin, respectively). The human neuroblastoma SH-SY5Y cells were pre-treated during 24h with FT, F6, F8 (10-200μg/mL) and/or treated with 800μM H2O2 and their cellular viability was evaluated by MTS. Initial screening demonstrated that FT, F6, F8 did not present cytotoxicity towards SH-SY5Y cells, up to a concentration of 200μg/mL, while the treatment with 800μM H2O2 alone reduced significantly the viable cells. Moreover, the SH-SY5Y cells pre-treated with <100μg/mL of cork compounds (FT, F6, F8) did not present any cytoprotective capacity after incubation with 800μM H2O2. However, cells pre-treated with the same cork compounds (at concentrations ≥100μg/mL) presented the capacity to retain the metabolic activity of the SH-SY5Y cells. These results indicate that the cork extractives (including castalagin and vescalagin) are able to reduce the cytotoxic effect of H2O2 usually present in the biological milieu of neurons of AD patients. Acknowledgements: FP7 project POLARIS (REGPOT-2012-2013-1-316331). [1] Xie H., Wang J., Jiang Z. Molecules 19, 5119-5134 (2014). [2] Santos A.O, Pinto P.C.R.O., Silvestre A.J.D., Neto C.P. Industrial Crops and Products, 31 521–526 (2010).


Poster Session 2

72

NANOPARTICLES FOR SUSTAINED DRUG DELIVERY BASED IN MARINE

POLYSACCHARIDES

C. Oliveira 1, 2, T. H. Silva 1,2, R. L. Reis 1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Naturally derived polymers are of special interest due to their biological and chemical similarities to natural tissues. Additionally, the vast range of properties and characteristics of marine organisms and their materials may be of great interest when concerning a potential use in biomedical applications, such as biomaterials for tissue regeneration and drug delivery. In particular, nanoparticles are ideal for use as drug delivery systems since they can improve macromolecules stability, protect them from enzymatic degradation and control their release profile. Moreover, they can penetrate cells and tissue gap to arrive at target organs. In this study, nanoparticles were prepared based on the electrostatic interactions (polyelectrolyte complexation) between two marine origin materials with opposite charge. Chitosan is positively charged and was combined with different sulfated polyanions: chondroitin sulfate or fucoidan. Sulfated polyanions were selected due to their similarities to glycosaminoglycans. The polyanion is added, drop-by-drop, to the solution of chitosan and a spontaneously formation of the nanoparticles occurs. Different conditions were assessed to optimize parameters such as polymers concentration, pH, polymer ratio in final solution, time of stirring and ionic strength. The effects of these five factors were studied on the average nanoparticles size, polydispersity and zeta potential. The size of the nanoparticles ranged from 150 to 700 nm, depending on the concentrations, ratio and polymer used. The selection of production conditions can thus be used to render particles with the desired size and surface charge. For instance, decreases in polymers concentration lead to smaller nanoparticles and the increase in polymer ratio from 1:1 to 1:3 (excess of polyanion) turn the zeta potential from positive to negative values. The final goal is to develop an optimized system for sustained drug delivery, targeted therapeutic and signaling.


Poster Session 2

73

LIQUID MARBLES TO CULTURE ADHERENT CELLS FOR HIGH-THROUGHPUT DRUG

SCREENING

Nuno M. Oliveira1,2, Clara R. Correia1,2, Rui L. Reis1,2, João F. Mano1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Liquid marbles (LM) were defined as liquid droplets covered with a hydrophobic powder (HP). To manufacture the LM is only to roll a liquid droplet over HP. The hydrophobic shell avoids any direct contact between the liquid and the external environment. LM make possible an easily manipulation of liquids, being good candidates to be used as micro-bioreactors. LM were employed to produce cancer cell spheroids and embryoid bodies [1,2]. We propose a strategy to culture anchorage-dependent cells using the LM. The rational is based on the possibility of fast producing well designed LM in which high-throughput drug screening studies can be performed. The HP is hydrophobized Diatomaceous Earth. The poly(L-lactic acid) microparticles (PLLAmp) were used to provide the anchorage points for cells. The L929 cell line was used as anchorage-dependent cell model. Iron chloride was used as cytotoxic agent and was injected into the LM. The metabolic activity and proliferation of cells were accessed by MTS/AlamarBlue assay and DNA quantification. The AlamarBlue measurement was performed by taking a picture of LM and measure the red color intensity by ImageJ. The metabolic activity decrease around 50% in LM cultured without PLLAmp comparing with LM with PLLAmp. Taking advantage of self-healing LM ability, by injecting a cytotoxic agent was shown the applicability of the system to test drug effects over cells on a liquid environment. A systematic decrease in cellular metabolic activity with increasing iron content was detected. It was showed that it is possible to culture anchorage-depend cells inside of LM using PLLAmp as cell adhesion supports, and analyze cell response to an injected agent without destroy the LM. [1] T. Arbatan, A. Al-Abboodi, F. Sarvi, P.P.Y. Chan, W. Shen, Adv. Healthcare Mater. 1, 467 (2012) [2] F. Sarvi, T. Arbatan, P.P.Y. Chan, W. Shen, RSC Adv. 3, 14501 (2013)


Poster Session 2

74

NEW MULTI-FUNCTIONAL THERAPEUTIC SYSTEM FOR BURULI ULCER

Daniela P. Pacheco1,3, Alexandra G. Fraga2,3, Vítor M. Correlo1,3, Carine M. Gonçalves2,3 Alexandra

P. Marques1,3, Rui L. Reis1,3, Jorge Pedrosa2,3 1- 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal; 2- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Buruli Ulcer (BU) is an emerging necrotizing skin disease caused by Mycobacterium ulcerans. The pathogenesis of BU is associated with the cytotoxic toxin mycolactone. Initially, BU appears as a painless nodule in the skin. Subsequent necrosis of the nonulcerative forms expands to the epidermis, leading to ulceration. The ulcer base and surrounding dermal areas show extensive coagulative necrosis with clumps of extracellular bacilli. Although antibiotherapy is effective for small lesions, extensive ulcers are generally treated by surgical removal of affected skin, followed by skin grafting. Even after clearance of infection, patients are often left with disfiguring scars and reduced mobility. Gellan gum-based hydrogel has been previously proposed for skin tissue engineering purposes. Specifically, this hydrogel significantly contributed to improve skin healing by maintaining wounds moist and allowing for facilitated nutrients, cells and metabolites diffusion due to high water content. Taking this into consideration, we are interested in testing gellan gum-based hydrogel to promote skin regeneration and improve functionality/esthetic appearance of chronic BU lesions. For that, experimentally induced M. ulcerans lesions will be treated with the hydrogel and wound healing will be screened by macroscopic analysis of the wound area and by histological analysis through (ex: tissue stratification, collagen synthesis, and epithelialisation). In addition to being an intrinsic healing promoter, gellan gum-based hydrogel can have a drug delivery role. Microparticles can be randomly dispersed within the hydrogel or disposed along a gradient adjusted according to the required delivery of the antibiotics in order to attain optimal concentrations, dosage and time of administration. In that sense, the antimycobacterial effect of this drug delivery system will be evaluated by performing bacterial counts of the experimentally induced M. ulcerans lesion. This new multi-functional therapeutic system will improve not only the efficacy antibiotherapy but will also improve the functionality/esthetic appearance of the healed tissue.


Poster Session 2

75

INJECTABLE PIEZOELECTRIC DRUG DELIVERY SYSTEM: A NOVEL STRATEGY TO TARGET

TISSUE REGENERATION

Daniela P. Pacheco1,3, Alexandra G. Fraga2,3, Vítor M. Correlo1,3, Alexandra P. Marques1,3, Rui L. Reis1,3, Jorge Pedrosa2,3

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Therapeutic induction of tissue repair or function restoration requires recapitulation of the spatial and temporal native microenvironments, in many cases by providing exogenous instructive bioactive signals using drug delivery systems (DDS). Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a natural and biodegradable polymer with piezoelectric properties, i.e. capable of suffering electric polarization due to mechanical stress, and vice-versa. Naturally occurring electric currents are an intrinsic property of human tissues, likely to act as an integrator of cellular organization, development as well as tissue regeneration. Under this context, it is herein reported the development of an injectable piezoelectric DDS system incorporating hydrophilic and hydrophobic model active agents with targeted and stimuli-sensitive features aiming to modulate the defined biological functions and tackle tissue regeneration. Microparticles of PHBV incorporating a model protein, Bovine Serum Albumin (BSA), and glucocorticoid, Dexamethasone (Dex), were produced by a double emulsification-solvent evaporation method with modifications. Variations of the composition of the organic phase during processing allowed tuning surface topography, size distribution and core porosity of the PHBV microparticles and, thus, the in vitro release profile of Dex, but not of BSA, which followed typical first order release kinetics independently of the conditions and the incorporated agent. Uncontrollable displacements that greatly affect the concentration of active agents at the target tissues are among a major limitation of the use of microparticulate DDS. A DDS responsive to electrical stimulation that combines PHBV microparticles with injectable gellan gum, which tackle the reduced residence time of microparticles at the injection site, and takes advantage of its piezoelectric character to release the loaded active agents, was successfully achieved. By varying the properties of the hydrogel and the intensity of the provided electric signal, we were able to design systems with different release profiles, which can be then tuned according to tissue and pathology/injury specific requirements.


Poster Session 2

76

GENETIC AND EPIGENETIC REGULATION OF NEUROPLASTIC PHENOMENA IN THE ADULT

HIPOCAMPAL NEUROGENIC NICHE - IMPLICATIONS FOR DEPRESSION P. Patrício1,2, A. Mateus-Pinheiro1,2, M. Santiago1,2, N.D. Alves1,2, J. Marinho1,2, M. Morais1,2, A.R. Machado-Santos1,2, J. Correia1,2,C. Antunes1,2, M. Branco3, J.M. Bessa1,2, W. Reik4, N. Sousa1,2, J. Marques1,2, L. Pinto1,2. 1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3 - Blizard Institute, Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom 4 - Epigenetics Programme, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK, and Centre for Trophoblast Research, University of Cambridge Depression is a complex and multidimensional disorder affecting around 20% of the world population. Alterations in adult hippocampal cell genesis and dendritic morphology are known to be involved in the pathophysiology of the disorder and in the actions of antidepressants (ADs). Stress-exposure is a common environmental etiological factor leading to various pathological marks in neurochemical and neuroplastic phenomena, many of them underlying the so-called stress-related disorders, such as depression. To better understand the molecular regulation of these phenomena, we analyzed both genetic and epigenetic mechanisms occurring in the hippocampal dentate gyrus of a chronic mild stress (CMS) rat model of depression, both untreated and treated with ADs. For the gene expression studies we performed a genome-wide study using microarray analysis of the macrodissected hippocampal dentate gyrus (DG). RNA profiling disclosed the differential molecular regulation in control, CMS-exposed and AD-treated rats. These results were further correlated with relevant behavioral and physiological parameters to unveil an integrated network that maintain homeostasis in the hippocampal niche [1]. Several studies have shed light on how epigenetic mechanisms serve as mediators of the pathological effects of stress on brain homeostasis. In this way, we aimed also to study the effects of stress on the epigenetic landscapes of the hippocampal DG. For that, we have conducted genome-wide analysis of methylation and hydroxymethylation in these areas. So far, hydroxymethylation pathways are likely to be involved in the pathological effects of stress exposure, as they are differentially regulated in control and stress-exposed animals. [1] P. Patrício, A. Mateus-Pinheiro, M. Irmler, N.D Alves, A.R. Machado-Santos, M. Morais, J.S. Correia, M. Korostynski, M. Piechota, R. Stoffel, J. Beckers, J.M. Bessa, O.F. Almeida, N. Sousa, and L. Pinto, Neuropsychopharmacology, e-pub ahead of print (2014)


Poster Session 2

77

ROLE OF AUTOPHAGY (MACROAUTOPHAGY) ON THE DIABETIC PATIENTS´ PRONENESS

TO INFECTIOUS DISEASES AND CANCER

Olga Pereira1,2, Margarida Correia-Neves1,2, Henrique Girão3 and Paula Ludovico1,2 1 - Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2 - ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3 - Institute for Biomedical Imaging and Life Science (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal Diabetes mellitus comprises a group of metabolic disorders characterized by chronic hyperglycemia that has gained global epidemic proportions [1]. Epidemiological studies have pointed diabetes as a risk factor for the development and treatment failure of tuberculosis [2,3], an infectious disease caused by Mycobacterium tuberculosis, and acute myeloid leukemia (AML) [4], a group of hematological malignancies characterized by accumulation of immature myeloid cells in bone marrow. Despite the accumulating epidemiological evidence, the biological mechanisms underlying the diabetic patients´ proneness to tuberculosis and AML development/treatment failure were not yet identified. Recent studies have implicated the dysregulation of autophagy (macroautophagy) in the diabetes pathophysiology [5]. Interestingly, studies have also suggested the autophagy dysregulation as player in the tuberculosis [6] and AML [7] development. Plus, autophagy dysregulation has also been implicated in the AML chemoresistance [8]. Accordingly, the main objective of this project is to elucidate whether dysregulated autophagy contributes to the diabetic patients´ proneness to tuberculosis and AML development/treatment failure. To achieve this goal, we intend to: (Task 1) assess autophagy on human blood and bone marrow cells from diabetic individuals; (Task 2) evaluate autophagy and its role on the cell death type determination on human blood cells from diabetic individuals in response to M. tuberculosis; (Task 3) evaluate autophagy and its role on the tumorigenicity, chemoresistance and cell death type modulation on AML cell lines subjected to hyperglycemia and on AML cells from diabetic individuals. Obtained results will clarify whether dysregulated autophagy plays a role on the diabetic patients´ proneness to tuberculosis and AML. [1] Diagnosis and classification of diabetes mellitus. Diabetes Care, 36 Suppl 1: p. S67-74 (2013). [2] K.E. Dooley and R.E. Chaisson, Tuberculosis and diabetes mellitus: convergence of two epidemics. Lancet Infect Dis, 9(12): p. 737-46 (2009). [3] M.A. Baker et al., The impact of diabetes on tuberculosis treatment outcomes: A systematic review. BMC Medicine, 9(81): p. 1-15 (2011). [4] J.J. Castillo et al., Increased incidence of non-Hodgkin lymphoma, leukemia, and myeloma in patients with diabetes mellitus type 2: a meta-analysis of observational studies. Blood, 119(21): p. 4845-50 (2012). [5] C.D. Gonzalez et al., The emerging role of autophagy in the pathophysiology of diabetes mellitus. Autophagy, 7(1): p. 2-11 (2011). [6] V. Deretic et al., Autophagy in immunity against mycobacterium tuberculosis: a model system to dissect immunological roles of autophagy. Curr Top Microbiol Immunol, 335: p. 169-88 (2009). [7] A. Puissant, G. Robert, and P. Auberger, Targeting autophagy to fight hematopoietic malignancies. Cell Cycle, 9(17): p. 3470-8 (2010). [8] J.K. Altman et al., Autophagy is a survival mechanism of acute myelogenous leukemia precursors during dual mTORC2/mTORC1 targeting. Clin Cancer Res., 20 (9): p. 2400-2409 (2014).


Poster Session 2

78

HIERARCHICAL IONIC-DOPED COMPOSITE SCAFFOLDS FOR OSTEOCHONDRAL TISSUE

ENGINEERING

S. Pina1,2, J.M. Oliveira1,2, R.L. Reis1,2

1- 3B´s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Osteochondral (OC) tissue engineering have the potential of producing sufficient numbers of grafts for the repair/regeneration of OC defects [1]. OC defects are lesions of the articular cartilage and underlying bone often derived from trauma related injuries or osteoarthritis, causing joint pain and deformity. Currently, OC lesions treatments are well established and effective for reducing pain improving the patient’s quality of life [2]. However, the cartilage has a limited regeneration capacity demanding new therapeutic options for complete OC lesions healing. Therefore, the main purpose for OC tissue engineering is to recreate a biomimetic scaffold combining different materials with cell-recognition sites. In addition, looking to the architectures of bone and cartilage, specific structured scaffolds are demanding for OC defect treatment. Thus, an OC scaffold ought to consist of a cartilaginous layer and an underlying osseous layer for regeneration of cartilage and subchondral bone respectively, involving different materials, morphologies and properties [3]. Composites involving biopolymeric matrices and bioresorbable fillers have been seen as a promising approach for bone/cartilage repair and regeneration. Biopolymers have great similarities with the extracellular matrix, chemical versatility, and good biological performance [4]. On the other hand, bioresorbable fillers as calcium phosphates have favourable osteoconductivity, resorbability and biocompatibility [5]. Benefits have been observed from doping calcium phosphates with different ions known to improve cell-material interactions and the mechanical properties of the biomimetic materials [6]. The objective of the present study is to develop composite scaffolds made of silk and Sr-doping calcium phosphates for the simultaneous repair and regeneration of bone and cartilage tissues, as well as their interface. [1] S. Nukavarapu, D. Dorcemus, Biotech Adv 31, 706 (2013) [2] O. Schindler, Acta Orthop Belg 77, 709 (2011) [3] J. M. Oliveira, M. T. Rodrigues, S. S. Silva, P. B. Malafaya, M. E. Gomes, C. A. Viegas, I. R. Dias, J. T. Azevedo, J. F. Mano, R. L. Reis, Biomaterials 27, 6123 (2006) [4] J. Yang, A. Liu, Y. Han, Q. Li, J. Tian, C. Zhou, J Biomed Mater Res A (2013) [5] A. J. Ambard, L. Mueninghoff, J Prosthod 15, 321 (2006) [6] S. Pina, S. I. Vieira, P. Rego, P. M. C. Torres, F. Goetz-Neunhoeffer, J. Neubauer, O. A. B. da Cruz e Silva, E. F. da Cruz e Silva, J. M. F. Ferreira, Eur Cells Mater 20, 162 (2010)


Poster Session 2

79

CORK ANTI-OXIDANT COMPOUNDS AS A NOVEL THERAPEUTIC STRATEGY FOR

MACHADO-JOSEPH DISEASE

Ana R. Araújo,1,2 Ana Jalles,2,3 Patrícia Maciel,2,3 Rui L. Reis,1,3 Andreia Teixeira-Castro,2,3 Ricardo A. Pires1,3

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Machado-Joseph disease (MJD) is a neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) tract within the C-terminal of the ataxin-3 (ATXN3) protein. Mutant ATXN3 acquires the ability to self-associate and enter an aggregation process, which is associated with several pathophysiological consequences for neurons. An imbalance in the oxidative state of the neuronal cells has been linked to the onset and progression of MJD [1]. In order to control disease progression, polyphenols have been tested as compounds that combine anti-oxidant (AO) properties with the capacity to inhibit protein aggregation. Cork polyphenolic extracts usually present AO activity [2], however, there is no report on their capacity to inhibit ATXN3 aggregation, nor to minimize the MJD symptoms. In this context, we collected the cork water extract (WE) and fractionated it (by HPLC) in order to test their activity towards MJD. We tested these compounds in the C. elegans MJD animal model due to the fact that this nematode presents several of the MJD characteristic symptoms in humans, namely, motor neuron dysfunction and the accumulation of ATXN3 aggregates in specific neurons. The toxic evaluation of the cork compounds (food clearance assay, 7 days) leads us to conclude that they are not toxic towards MJD and wild-type (WT) animals, at concentrations up to 1mg/mL (WE) or 500μg/mL (HPLC fractions). Next, we evaluated the impact of WE on the motility of MJD animals, and we observed a 63% improvement on the mutant ATXN3-mediated neurological dysfunction. In addition, WE-treated WT animals did not present any improvement in locomotion, suggesting that the WE action may be specific to mutant ATXN3-mediated pathogenesis. Treatment with WE was also able to suppress mutant ATXN3 aggregation in vivo. These results suggest that WE show neuroprotective properties probably through the enhancement of cell proteostasis. Acknowledgements: We acknowledge FP7-REGPOT-CT2012-316331-POLARIS for funding. 1. Butterfield, D.A. and J. Kanski, Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins. Mechanisms of Ageing and Development, 2001. 122(9): p. 945-962. 2. Santos, S.A.O., et al., Chemical composition and antioxidant activity of phenolic extracts of cork from Quercus suber L. Industrial Crops and Products, 2010. 31(3): p. 521-526.


Poster Session 2

80

SUBREGION-DEPENDENT SPECIFICITIES OF THE HIPPOCAMPAL FORMATION TO THE

ACUTE APPLICATION OF AΒ OLIGOMERS

João Pizarro, Nuno Sousa, Vítor Pinto

Alzheimer’s disease (AD) is a neurodegenerative disorder and the main cause of dementia worldwide. It is characterized by the presence of extracellular plaques of Amyloid-β (Aβ) and intracellular neurofibrillary tangles of Tau protein. Although AD has been intensively studied either by using transgenic animal models or by the application of Aβ peptides in the hippocampus, in vivo and in vitro, the exact mechanisms leading to neurodegeneration still remain unclear. Probably due to methodological variations, but also because of different intrinsic properties of the different hippocampal subregions, several contradictions are reported in the literature regarding the susceptibility of the CA1 and the dentate gyrus (DG) to Aβ peptides. Indeed, the application of different neurotoxic concentrations of Aβ (200 nM, 500 nM and 1 µM) in CA1, only resulted in increased paired pulse facilitation in the presence of 1 µM of Aβ. Conversely, in the DG, there were no differences in any of the three measures assessed (input-output relationship, paired pulse facilitation and LTP), in the presence of 200 nM of Aβ. These results showed that, despite the marked intrinsic differences between CA1 and the DG, these two subregions were not differently affected by the application of Aβ oligomers. Possible methodological variations are discussed that could explain the observed results.


Poster Session 2

81

PHARMACOLOGICAL SCREENING OF A NOVEL DRUG FOR THE CONTROL OF

NEUROPATHIC PAIN AND ASSOCIATED DEPRESSION

S. Puga1,2, M. Guimarães1,2, A. David-Pereira1,2, H. Leite-Almeida1,2, F. Pinto-Ribeiro1,2, A. Almeida1,2

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Neuropathic pain afflicts millions worldwide, is very debilitating and represents a major clinical challenge since it is often refractory to conventional therapies and is associated with mood comorbidities. In this work, we aimed at validating a novel class of therapeutical agents as analgesics in the rat spared nerve injury (SNI) model of neuropathic pain. The chronic administration of this drug significantly reduced mechanical allodynia, and improved the depressive phenotype associated to SNI animals. Overall, this drug appears to be a potential candidate for the control of pain and associated depression in novel pain management therapies.


Poster Session 2

82

METHACRYLATED FUCOIDAN AS PHOTOCROSSLINKABLE BIOPOLYMER FOR TISSUE

ENGINEERING APPLICATIONS Lara L.Reys1,2,, Simone S. Silva1,2, Nuno M. Oliveira1,2, João F. Mano 1,2, Tiago H. Silva 1,2 and Rui

L. Reis 1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal In the last decades, marine organisms have been the focus of considerable attention as potential source of valuable materials. One important example is fucoidan, a sulfated polysaccharide extracted from the cell wall of brown seaweeds[1] . Fucoidan is composed by L-fucose, sulfate groups and glucuronic acid and have important bioactive properties such as anti-oxidative, anticoagulant, anticancer and reducing the blood glucose [1]. In this work, the biomedical potential of fucoidan was assessed by processing modified fucoidan (MFu) into particles by photopolymerization using superamphiphobic surfaces and visible light. For that, fucoidan was modified by methacrylation reaction using different concentrations (8% and 12% v/v) of methacrylate anhydride. Subsequently, MFu particles were produced by pipetting a solution of 5% of MFu with triethanolamine and eosin-y onto a superamphiphobic surface and then photocrosslinked using visible light [2]. The developed particles were characterized by FTIR, 1HNMR, assessment of sulfate degree and swelling behavior, as well as morphological evaluation. To examine cell behavior in developed structures, 3.5x105 cells/ml of L929-mouse fibroblast like cells were seeded in the MFu particles. Preliminary results after 14 days of culture present good cell viability and proliferation. These findings indicated that modified fucoidan particles could be a good candidate for biomedical applications. [1] T.H.Silva, A. Alves, E.G.Popa, L.L.Reys, M.E.Gomes, R.A.Sousa, S.S.Silva, J.F.Mano, R.L.Reis, Biomatter, 2:4.278-289,(2012). [2] S.M.Mihaila, A.K. Gaharwar, R.L.Reis, A.P.Marques, M.E.Gomes, A.Khademhosseini, doi:10.1002/adhm.201300317, (2012)


Poster Session 2

83

THE EFFECTS OF COPPER-CONTAINING BIOACTIVE GLASSES ON THE IN VITRO

BEHAVIOUR OF HUMAN ADIPOSE DERIVED STEM CELLS

S. C. Ribeiro1,2, A. Hoppe3, A. I. Gonçalves1,2, M. T. Rodrigues1,2, M. E. Gomes1,2, I. B. Leonor1,2, A. R. Boccaccini3, R. L. Reis1,2

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, 91058 Erlangen, Germany. Bioactive glasses (BGs) are inorganic and bioactive biomaterials that can be used to fabricate scaffolds for tissue engineering. The ionic dissolution products from these inorganic materials have notable effects on the behaviour of cells. Therefore, incorporating specific metallic ions in the base BG composition will enhance the biological activity of BGs concerning osteogenesis and angiogenesis [1]. Copper (Cu), an essential ion in the human body, has been reported to have osteogenic potential and to improve angiogenesis [2, 3]. Thus, Cu has been combined with 45S5 Bioglass® in order to enhance the biological activity of this material to produce improved bone tissue scaffolds [4]. This study investigates the effect of ionic dissolution products of Cu-doped BG and 45S5 Bioglass® (used as control), on the proliferation and osteogenic differentiation of human adipose stem cells (hASCs). Cu-doped BG powders of three compositions (0.1, 1 and 2.5 wt.% CuO) were cultured with hASCs for 3, 7, 14 and 21 days in α-MEM medium. Characterization was performed in terms of cell viability, cell proliferation, alkaline phosphatase (ALP) activity quantification and immunocytochemistry analysis for osteocalcin (OCN), osteopontin (OPN) and collagen I (Col I) proteins detection. Cell culture experiments revealed good in vitro biocompatibility of the Cu-BGs (0.1 wt.%) towards hASCs cells. Immunofluorescence revealed that hASCs expressed higher levels of OPN and Col I for 0.1wt% Cu-BGs formulation. Furthermore, ALP activity results suggest an osteogenic potential of the Cu-BGs (0.1 wt.%) formulation. In summary, preliminary results show the potential effects of the ionic dissolution products of Cu-BGs powder inducing osteogenesis of hASCs and warrant further in vitro and in vivo investigations. The authors gratefully acknowledge financial support for AH via a STSM fellowship from EU COST Action NAMABIO and to the national funds through the FCT under the scope of the project PTDC/CTM-BIO/0814/2012. [1] A. Hoppe et al. Biomaterials 32: 2757 (2011) [2] G. F. Hu et al. J Cell Biochem 69(3): 326 (1998) [3] A. Ewald et al. J Biomed Mater Res A 100(9): 2392(2012) [4] A. Hoppe et al. J. Mater. Chem. B 1: 5659 (2013)


Poster Session 2

84

SMART SILK FIBROIN HYDROGELS AND ITS POTENTIAL APPLICATION AS SUPPRESSOR

OF ANGIOGENESIS AND TUMOR PROGRESSION

Viviana P. Ribeiro1,2, Joana Silva-Correia1,2, Vera Miranda-Gonçalves2,3, Leping Yan1,2, Ana L. Oliveira4, Rui M. Reis2,3,5, Joaquim M. Oliveira1,2 and Rui L. Reis1,2

1- 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal ICVS - Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 3- CBQF–Center for Biotechnology and Fine Chemistry, Portuguese Catholic University, Porto, Portugal 4- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil Common methods used to prepare silk fibroin(SF) hydrogels are based on SF conformation transition from amorphous to β-sheet in aqueous status, which implies longer gelation time and harsh preparation conditions [1]. These requirements hinder the final application of SF as injectable system for cell encapsulation/drug delivery. Recently, enzyme-mediated in situ hydrogels attracted great attention because of their specificity and non-toxic nature [2]. Proteins with tyrosine groups can be used to prepare hydrogels in situ using horseradish peroxidase(HRP) and hydrogen peroxide(H2O2) [3]. SF demonstrated to be a promising biomaterial since it contains 5 mol% tyrosine groups. In this study, enzymatically cross-linked SF hydrogels were prepared using high concentrate aqueous SF solution (16 wt%) mediated by a HRP/H2O2 system [4]. The aim of this work is assess the conformation transition in SF hydrogels and evaluate if this transition is allied to potential anti-tumoral applications. The β-sheet conformation transition was analyzed by means of TEM and ThioflavinT staining. Human neuronal glioblastoma (U251) and human cervical adenocarcinoma (HeLa) cells were incorporated into the hydrogels and cultured for 10days under standard culture conditions. Cell viability and proliferation were evaluated through Live/Dead staining and DNA quantification. The fast-formed hydrogels presented mainly amorphous conformation during the first week, but a conversion to a dominant β-sheet conformation was verified from day7. SF hydrogels were capable of incorporating cells and support their viability during the first 24hours of culturing. Nonetheless, the β-sheet conformation of SF induced hydrogels autofluorescence, hindering the cell viability/death evaluation from day7. From DNA quantification it was possible conclude that no cell proliferation occurred from day1 until day10. The obtained results reveal that the conformation transition is responsible for inhibiting cell growth and thus the developed SF hydrogels can be a very useful system for different biomedical applications, including suppressing angiogenesis and tumors progression in vivo. [1] U-J. Kim, et al. Biomacromolecules 5:786-792 (2004) [2] D. Gan, et al. J Am Chem Soc 123:7511-7517 (2001) [3] S. Sofia, et al. J Macromol Sci A 39:1151-1181(2002) [4] L. Yan, et al. National Patent Nr. 106041, priority date: 06-12 (2011)


Poster Session 2

85

FUNCTIONALIZED DENDRONIZED POLYMERS FOR T-CELL THERAPY

D.B. Rodrigues1,2; M.R.Carvalho1,2; J. M. Oliveira1,2;E. Torrado2,3; A.G.Castro2,3;T.C. Santos1,2;R.L.

Reis1,2

1- 3B’s Research Group—Biomaterials, Biodegradables, and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal 2-ICVS-3B’s — PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- Institute of Life and Health Sciences, Health Sciences School, University of Minho, Braga, Portugal Over the past decade, medicine has focused much of its attention in immunotherapy, thus novel approaches evolving dendritic cells (DCs) in cancer immunotherapy have been developed, however not free of notorious drawbacks. Several studies [1, 2] have shown that antigen-specific lymphocytes can be primed to amplify in vivo or previously cultured in vitro to expand under desired conditions and to express specific mediators, in order to be administered afterwards. The main objective of this work was the development of functionalized artificial antigen presenting cells (aAPCs) using dendronized polymers to trigger mycobacterial specific T cells. A dendronized polymer, combining glycodendrimers in the inner core with natural-origin polymers in the external shell was created. Generation 5,5 poly(amidoamine) (PAMAM) dendrimers were grafted with carboxymethlychitosan (CMCht) creating a generation 7 dendrimer. Then the dendronized polymer was functionalized with anti-CD3 and anti-CD28 antibodies which augmented the particle size preventing its internalization. The modified dendronized polymer was characterized by NMR and Zetasizer, and internalization assays were also performed with a murine fibroblast-like cell line (L929). The grafting with CMCht was confirmed by the respective NMR spectra, where the correspondent peaks of CMCht were present in the modified dendronized polymer. By zetasizer analysis a dispersion of sizes was verified, were the PAMAM-CMCht modified dendrimer presented a size around ±50nm. The fluorescent microscopy images showed that the modified polymers with around 50nm diameter were easily uptake by L929 cells. The functionalization with the antibodies generated larger structures that macrophages cannot phagocytise but still capable of priming T-cell responses. These findings suggest that it is possible to create functional aAPCs based on natural-origin dendronized polymers, with the possibility to modulate the immune responses in primary infections and vaccination, and also to simplify the means necessary to prompt the correct activation and proliferation of naive T-cells in the lymph nodes. [1].. Fadel, T.R., et al.. Nature nanotechnology, 2014. 9(8): p. 639-647. [2]. Schilbach, K., et al.. Blood, 2005. 106(1): p. 144-149. Acknowledgments: Projecto Estratégico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013) Keywords (max.4): Dendronized polymers; artificial antigen presenting cells; immune modulation; T-cell activation


Poster Session 2

86

INCREASED AUTOPHAGY IN AGING YEAST CELLS EXPRESSING ALPHA-SYNUCLEIN

PROMOTES DNA REPLICATION STRESS

B. Sampaio-Marques1,2, A. Guedes1,2, W. C. Burhans3 and P. Ludovico1,2 1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- Dept. of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA Aging is a complex and multi-factorial process that results in the progressive accumulation of molecular alterations and disruption of different cellular functions. Genome instability and loss of proteostasis are among the proposed hallmarks of aging although the relationships between these two phenomena have been largely unexplored. Evidence exists that autophagy, a conserved proteolytic mechanism essential for the maintenance of metabolic and proteome homeostasis, is induced by DNA replication stress/DNA damage responses, suggesting a coordinated regulation of both phenomena. Nevertheless, the signaling/molecules underlying the interconnection between these two processes are still unclear. In the present work, we aimed to obtain new insights into the crosstalk between autophagy and DNA replication stress, during aging, in a proteotoxic stress context elicited by the heterologous expression of human α-synuclein, a protein associated with Parkinson’s disease. The data obtained showed that the increase in autophagy and shortened chronological lifespan (CLS) promoted by α-synuclein expression was associated with growth arrest of stationary phase cells in S-phase. Deletion of the RAS2 gene abrogates α-synuclein toxicity by forcing cells to arrest in G0/G1, suggesting that α-synuclein toxicity is associated with DNA replication stress. DNA replication stress was found to be related to lower levels of ATP and autophagy dependent-ribonucleotide reductase Rnr1 degration, which consequently is associated with the reduction of the dNTP pools. Our findings highlight that autophagy and replication stress are intricately coordinated, and that disruption of this relationship leads to genome instability and premature aging. This work was supported by Fundação para a Ciência e Tecnologia (FCT) and COMPETE/QREN/EU (PTDC/BIA-MIC/114116/2009). B.S.M. and A.S. have fellowships from FCT (SFRH/BPD/90533/2012 and UMINHO/BI/221/2013, respectively).


Poster Session 2

87

DEVELOPMENT OF MAGNETIC MEMBRANES AND PRELIMINARY BIOLOGICAL ASSESSMENT OF MAGNETIC ACTUATION ON HUMAN ADIPOSE STEM CELLS

L. J. Santos1,2, A. I. Gonçalves1,2, M. A. Silva1,2, M. T. Rodrigues1,2, R.L. Reis1,2 and M.E. Gomes1,2

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Tendons are poorly cellularised and vascularised tissues (1) and thus in face of an injury they present limited regeneration potential. The aim of this study is to develop novel starch poly-caprolactone (SPCL) and magnetic SPCL membranes and show preliminary evidences that magnetic actuation can be a successful strategy to elicit tenogenesis and hence contribute for tendon repair. In this work we prepared SPCL and magSPCL membranes according to the solvent casting method. The magSPCL membranes were prepared by doping iron oxide particles (Micromod) into the SPCL at a concentration of 1.8 (magSPCL-1.8) and 3.6 wt% (magSPCL-3.6). Casted membranes were characterised by superconducting quantum interference device (SQUID). Surface morphology and particle incorporation into the bulk material was probed by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Human adipose stem cells (hASCs) were seeded at a density of 2.000 cells/cm2 and cultured under static or magnetic stimulation conditions provided by Nanotherics (2Hz, 350 mT) in α-MEM medium (plus 1% antibiotic, 10% FBS). Degradation profile was analysed by mass loss and hASCs proliferation estimated through the PicoGreen® dsDNA Assay Kit. SEM and EDS confirmed that iron oxide particles were effectively incorporated into the bulk material and SQUID analysis demonstrated that magnetic SPCL membranes respond satisfactory to an external magnetic field with values above 4 emu/g. Mass loss profiles were similar among materials. Preliminary assessment of biological behaviour suggests that magnetic materials stimulate proliferation of hASCs and that magnetic actuation elicits stem cell differentiation as evidenced by the presence of collagen I. In conclusion, our results show that iron-doped SPCL exhibits adequate magnetic saturation values and that magnetic materials have a positive effect on the proliferation of hASCs. In the future we expect to further develop magnetic-based strategies that fine tune stem cell commitment into the tenogenic lineage. [1] Liu et al. TE:PartB 2011, 17(3):165


Poster Session 2

88

XENOGENEIC MODULATION OF MACROPHAGES AND DENDRITIC CELLS BY

UNDIFFERENTIATED AND DIFFERENTIATED ADIPOSE TISSUE-DERIVED STEM CELLS

T.C. Santos1,2; D.B. Rodrigues1,2, M.T. Cerqueira1,2; R.P. Pirraco1,2; A.P. Marques1,2; R.L. Reis1,2

1- 3B’s Research Group—Biomaterials, Biodegradables, and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal 2- ICVS-3B’s — PT Government Associate Laboratory, Braga/Guimarães, Portugal

Adult mesenchymal stem cells (MSCs) are considered to be "immunologically privileged" and have been widely studied as tools in the field of tissue engineering and regenerative medicine (TERM) as well as in immunotherapeutic approaches. In a previous work when human adipose tissue-derived stem cells (hASCs) subcutaneously implanted in mice we did not identify an adverse host response1. In this study we hypothesised that undifferentiated hASCs and derived osteoblasts and chondrocytes are unable to activate murine bone marrow-derived macrophages (mBMMØs) and dendritic cells (mBMDCs) into M2 phenotype, aiding tissue regeneration. Murine BMMØs or mBMDCs were plated in direct contact with undifferentiated and osteo or chondro-differentiated hASCs for 4h, 10h, 24h and 72h. The cytokine profile was analysed by qRT-PCR and the surface markers were detected by flow cytometry. The direct interaction of both cell types was observed by time lapse microscopy. The results showed that murine BMMØs and BMDCs activate in similarly after contacting tissue culture polystyrene or after contacting both undifferentiated and osteo or chondro-differentiated hASCs. This was confirmed by the expression of IL-1, IL-10, IL-4, TNF-α and IFN-γ (genetic profile) and surface markers (CD206++, CD336++, MHC II+ and CD86++) detection. These data suggest immunotolerance exerted by osteo- and chondro-differentiated hASCs in xenogeneic approaches. It is therefore possible to consider the potential of hASCs in contemporary xenogenic tissue engineering and regenerative medicine strategies, as well as host immune system modulation in autoimmune diseases. Acknowledgments: RL3-TECT-NORTE-01-0124-FEDER-000020, co-financed by North Portugal Regional Operational Program (ON.2-O Novo Norte), under the National Strategic Reference Framework, through the European Regional Development Fund

1-Santos, TC et al., Tissue Eng A, 19(7-8), 83, 2013


Poster Session 2

89

REAL TIME ANALYSIS OF THE ENZYMATIC DIGESTION OF CHONDROITIN SULFATE

Carla Silva1,2, Ramon Novoa-Carballal1,2, Rui L. Reis1,2 and Iva Pashkuleva1,2

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal We describe the use of gel permeation chromatography (GPC) setup with four size exclusion columns for real time analysis of enzymatically digested glycosaminoglycans (GAGs). This setup provides information about Mw and concentration of all species (low and high Mw) present in the digests in a single measurement. The data about high molecular weight fraction (often omitted in the analysis of GAG digests) provide direct evidence about the mechanisms of action of the enzymes [1, 2]. We proved the feasibility of this methodology by applying it to chondroitin sulfate (CS) substrates with different molecular weight and sulfation pattern and using different enzymes (hyaluronidase and chondroitinase). The results were validated by alternative methodologies usually used for characterization of GAG digests. MALDI-Tof, NMR and fractionalization of the obtained digests by ultrafiltration confirmed the results obtained by GPC setup and reveal further details about the degradation mechanisms: (i) both enzymes preferentially attack 4-sulfated chondroitin sulfates (CS) and (ii) additionally to the well documented endolytic activity of hyaluronidase [3] we also observed a low lyase activity for this enzyme reflected in the detected minor exolytic breakage. Finally, we demonstrate that 6-sulfated CS with medium molecular weight (12 to 60kDa) can be obtained in good yields by ultrafiltration, i.e. by easily up scalable process. [1] V. H. Pomin, Y. Park, R. Huang, C. Heiss, J. S. Sharp, P. Azadi, J. H. Prestegard, Glycobiology 22, 826-838 (2012). [2] K. Murata, Y. Yokoyama, Anal Biochem 149, 261-268 (1985). [3] K. S. Girish, K. Kemparaju, Life sciences 80, 1921-1943 (2007).


Poster Session 2

90

MARINE SPONGES – AN ALTERNATIVE COLLAGEN/GELATIN SOURCE FOR BIOMEDICAL

APPLICATIONS

J. C. Silva 1,2, A. A. Barros1,2, I. M. Aroso1,2,T. H. Silva 1,2, J.F. Mano1,2 A. R. C. Duarte1, 2 and R. L. Reis1, 2

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Marine sponges can be regarded as a promising alternative source for collagen, with interesting prospective for biomedical applications. Although, and despite of its unique physico-chemical properties, sponge-origin collagen is not available in large quantities, mainly due to the lack of efficient extraction methodologies [1]. The traditional procedures are very laborious, time consuming and present several drawbacks that are hampering the process scale up, such as the requirement of several operational steps, energy and high volumes of water and other reagents [2, 3]. In this sense, there is a necessity for an alternative methodology comprising fewer steps and, ideally, more environmentally sustainable. We have proposed a new technology for the extraction of marine sponge collagen based on water pressurized with carbon dioxide. This new methodology is comprised by one single extraction step, under mild operating conditions. The objective of this work is to optimize the operating conditions to obtain the highest yields and collagen quantity and quality. Pressure and time were the parameters studied through a two-level factorial design performed by statistical analysis using the software Design-Expert (Stat - Ease®). The reactor was loaded with the marine sponges and distilled water, heated to 37ºC and pressurized with CO2. The effects of pressures ranging 10-50 bars and extraction times between 3-24 hours were studied. In this work we demonstrate that the acidification of the aqueous solution promoted by CO2 induces collagen solubilisation in the aqueous media and the final extracts can be easily recovered after freeze-drying. The collagen content of each extract was determined using a dye-binding method (Sircol Assay). Additionally, circular dichroism analysis and Infra-red spectroscopy spectra are similar to the controls, suggesting that the original chemical structure from collagen is maintained. In overall, the results obtained have demonstrated the effectiveness and potential of this new green technology to obtain sponge-origin collagen with great applicability in the biomedical field. [1] S. Heinemann, H. Ehrlich, Biomacromolecules 8, 3452-3457 (2007) [2] D. Swatschek, W. Schatton, European Journal of Pharmaceutics and Biopharmaceutics 53, 107-113 (2002) [3] C. A. Pacak, J. M. Powers, Tissue Engineering Part C Methods 17, 879-885 (2011)


Poster Session 2

91

HYALURONATE DEGRADATION FRAGMENTS FROM GGHA SPONGY-LIKE HYDROGELS

L. P. da Silva1,2, M. T. Cerqueira1,2, R. P. Pirraco1,2, T. C. Santos1,2, R.L. Reis1,2, V. M. Correlo1,2, A.

P. Marques1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Spongy-like hydrogels are highly attractive materials for Tissue Engineering (TE) applications due to their biological and mechanical properties. In relation to precursor hydrogels, these materials depict features between sponges and hydrogels. Moreover, are structurally more stable, flexible, retain a high water amount and, more importantly, promote cell adhesion and proliferation. Bearing in mind one of the challenges of TE materials, which is the inefficient vascularization within thick 3D constructs, we hypothesised that spongy-like hydrogels containing hyaluronate (HA) would promote the migration and proliferation of endothelial cells upon degradation, due to the angiogenic nature of the released hyaluronate fragments [1]. GGHA spongy-like hydrogels (1 % and 2 %, w/v) were prepared as previously described [2–4]. The susceptibility of the GGHA spongy-like hydrogels to hyaluronidase degradation was demonstrated in vitro by the increased amount of reducing sugars and low molecular weight hyaluronate fragments in solution up to 21 days, as determined by the DNS assay and Gel Permeation Chromatograpphy (GPC), respectively. Moreover, HA fragments were able to bind to CD44 and RHAMM receptors of human dermal microvascular endothelial cells (hDMECs) as determined by flow cytometry, although a significant effect over hDMECS proliferation and migration was not observed. Nonetheless, after implanting the GGHA spongy-like hydrogels in ischemia hind limb mice model, increased blood perfusion in comparison to the control, was observed in the presence of GGHA 2% spongy-like hydrogels. GGHA spongy-like hydrogels, comprising the unique characteristics of GG spongy-like hydrogels and the bioactivity of hyaluronate, are potential materials to be used in acellular and cellular approaches to improve neotissue vascularization. [1] S. Ibrahim, A. Ramamurthi, J Tissue Eng Regen Med 2, 22–32 (2008). [2] M.T. Cerqueira, L.P. da Silva, T.C. Santos, R.P. Pirraco, V.M. Correlo, A.P. Marques, et al. Tissue Eng Part A 20,1369–78 (2014). [3] L.P. da Silva, M.T. Cerqueira, A.P. Marques, V.M. Correlo, R.A. Sousa, R.L. Reis, WO2014167513 (2013). [4] L.P. da Silva LP, M.T. Cerqueira, R.A. Sousa, R.L. Reis, V.M. Correlo, A.P. Marques, Acta Biomater 10, 4787-97 (2014).


Poster Session 2

92

TEMPORARY AND REVERSIBLE INDUCTION OF COSTAL CARTILAGE PLASTICITY FOR THE

MINIMALLY INVASIVE TREATMENT OF PECTUS CARINATUM

Marta Alves da Silva1,3, Francesca Mitzman2,3, Emanuel M. Fernandes1,3, Ruben Pinheiro2,3, Jorge Correia-Pinto2,3, Nuno Neves1,3

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 3- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Pectus carinatum (PC) is defined as the outward displacement of the sternum and/or abnormal protrusion of the ribs. The anomaly is thought to be due to abnormal cartilage growth possibly secondary to pathologic patterning associated with skeletal tissue metabolism. The pathology may involve symmetrical or asymmetrical PC, according to the bilateral or unilateral development of the protrusion. The most common treatments are the external bracing technique, applying direct pressure on the protruding area of the chest during long periods. The treatment involves considerable pain and discomfort to the patient and even permanent skin lesions, leading to patient non-compliance with the treatment. The surgical approach is also possible in patients with severe PC but involves complete removal of the costal cartilage and leads frequently to inconsistent results. Therefore, it is of the utmost importance to find more consistent, effective and also less painful or radical treatments for this condition. The correction of asymmetrical PC with unilateral cartilage protrusion is expected to greatly benefit from strategies that promote temporary and reversible cartilage weakening/softening. The herein proposed concept is to find a percutaneous method/ technique enabling temporary weakening of the costal cartilage, reducing the time and pressure needed to be applied to the patients in bracing treatment for correcting PC. Based on the clinical scenario, the strategy used should involve a minimum number of perforations of the cartilage tissue and the delivery of an enzymatic cocktail solution to perform tissue digestion. We were able to successfully define an enzymatic cocktail and the optimal concentration levels that significantly reduce the cartilage mechanical properties. Histological analysis showed that enzymes effectively cleave the cartilage ECM components leading to the desired tissue weakening. The biomechanical properties of the cartilage under flexural stress revealed that the presence of the drilled holes is the main factor controlling the reduction of cartilage stiffness. Moreover, the flexural strength was further reduced by the enzymatic treatment over time. Further studies are required to confirm the safety and efficacy of this alternative clinical strategy.


Poster Session 2

93

COLLAGEN/APATITE SCAFFOLDS OBTAINED FROM SHARK SKIN AND TEETH

G. S. Diogo1,2, E. López3, R. Canadas1,2, E. Balboa3, C. Rodríguez-Valencia3, M. López-Álvarez3, P. Coladas3, P. González3, J. Serra3, R.I. Pérez-Martín4, C.G. Sotelo4, J. M. Oliveira1,2, T.H. Silva1,2, J.

Moreira-Silva1,2, R.L. Reis1,2

1- 3B's Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal. 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal. 3- New Materials Group, Applied Physics Dpt, Institute of Biomedical Research of Vigo (IBIV), University of Vigo, Campus Lagoas-Marcosende, 36310 Vigo, Spain. 4- Instituto de Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain Introduction. Bone defects may compromises the mechanical and biological functions of bone tissue, becoming a worldwide problem. Collagen/apatite composites can act as a temporary templates that can mimic the bone tissue matrix since collagen confers elasticity and tensile strength, while apatite granules can provides stiffness and compressive strength. In this work, scaffolds of marine origin were developed, combining collagen extracted from shark skin and biological apatites derived from shark teeth, for applications in bone tissue regeneration. Experimental. Collagen type I was obtained from P. glauca skin by acid solubilisation. Apatite granules were obtained by subjecting shark teeth (I. oxyrinchus, P. glauca) to pyrolysis (950ºC, 12h), followed by a ball milling and sieving processes. Collagen and apatite granules were mixed and crosslinked in situ, frozen in molds and freeze-dried to produce porous structures. Two different crosslinkers (HMDI and EDC/NHS) in different concentrations, 1%,5% and 12.5%,25%, respectively, apatite grain size (20-63µm) and three different collagen:apatite ratios were tested (70:30, 50:50, 30:70). To evaluate scaffolds stability, structures of each condition were incubated in culture medium for up to 15 days. Scaffolds produced with different formulations were analysed by micro-CT and SEM for morphological assessment and mechanical properties were evaluated by compression tests. Preliminar cytotoxicity tests, consisting in metabolic activity and cell proliferation, were performed for the crosslinkers with better results in morphological and mechanical characterization. Results and Discussion. micro-CT analysis confirmed that the apatite particles are homogeneously distributed into the scaffold structures. In general the results were better for, EDC/NHS 12.5% and HMDI 5% chemical crosslinker. The structures were stable in culture medium by up to 15 days except for the formulations crosslinked with HMDI 1%. Depending on the crosslinking conditions, different structural integrity, morphology and mechanical properties can be achieved. Conclusions. The ability to obtain collagen/apatite scaffolds from shark skin and teeth was demonstrated.


Poster Session 2

94

GENETIC DIVERSITY IN PHTHIOCEROL DIMYCOCEROSATE OF MYCOBACTERIUM

TUBERCULOSIS: AN EVOLUTIONARY PERSPECTIVE

J. Sousa1,2, C. Magalhães1,2, E. Torrado1,2, J. Pedrosa1,2, A. G. Castro1,2, M. Saraiva1,2 and N. S. Osório1,2

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Mycobacterium tuberculosis is the etiologic agent of tuberculosis (TB), a disease that remains a major cause of mortality worldwide. The M. tuberculosis cell envelope is a complex structure with a major role in the bacterial resistance to the hostile environments encountered during the infection. Recently, it has been proposed that phthiocerol dimycocerosate (PDIM), a cell wall lipid, is involved in mechanisms of M. tuberculosis evasion to the immune system by masking the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) such as toll-like receptors (TLRs). Data from our laboratory showed that different clinical isolates of M. tuberculosis can be recognized by different TLRs with an impact in the immune response. Based on these results and on the fact that TB-causing bacteria can be divided into seven distinct phylogeographic lineages, we investigated the genetic diversity across the different lineages of M. tuberculosis in the genes involved in the synthesis of this lipid. The results based on the analysis of 300 genome sequences from M. tuberculosis clinical isolates showed that the genes that codify polyketide synthases PpsA-E are highly conserved, with the exception of a cluster of homoplasic variability in the PpsA gene. Analysis of the amino acid sequence suggests that this variability may have functional impact. This work highlights sites of variability that might impact the synthesis of PDIM in different M. tuberculosis strains.


Poster Session 2

95

COMBINING ELECTROACTIVE SPONGY LIKE HYDROGELS AND ELECTRICAL

STIMULATION FOR SKELETAL MUSCLE TISSUE ENGINEERING. P.Srisuk1,2,3, F.V.Berti1,2, L. P. da Silva1,2, A. Zonari1,2, A. P. Marques1,2, V.M.Correlo1,2, R.L.Reis1,2*

1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Ave Park, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- Faculty of Pharmaceutical Sciences, Khon Kaen University, 40002, Khon Kaen, Thailand Despite, topography, material stiffness and mechanical force, electrical stimulation is an emergent factor affecting cellular behaviours and activity [1]. Thus, electroactive hydrogels have a great potential as bioactive materials for tissue engineering applications [2]. PPy and PANi are reported to be biocompatible materials able to promote cell growth, adhesion and proliferation at the polymer-tissue interface through electrical stimulation [3]. Nevertheless, both conductive polymers tend to easily lose their mechanical stability. To avoid these problem, PPy and PANi are usually combined with other polymers to improve their mechanical properties. The goal of this study was to combine gellan gum (GG) with PPy or PANi to obtain electroactive sponge like hydrogels (SLH) that were further used to study the effect of electrical stimulation (direct current) on myoblasts. After fabrication, GG-SLH, PPy.GG-SLH and PANi.GG-SLH were physico-chemically characterized by FTIR, DSC, SEM and Universal mechanical test. L929 metabolic activity and proliferation were evaluated by MTS and DNA assays, respectively. Confocal microscopy and SEM were used to analyse the morphology and adhesion of myoblast after electrical stimulation (20mV/mm). The results revealed that it was possible to prepare electroactive PPy.GG-SLH and PANi.GG-SLH by in situ polymerization with improved mechanical properties when compared with GG-SLH. According to in vitro biological assays, PPy.GG-SLH and PANi.GG-SLH did not shown any toxicity for L929 cells. Additionally, the adhesion and spreading of myoblasts were greater on PPy.GG-SLH and PANi.GG-SLH than GG-SLH. The electrical stimulus with 20mV increased myoblasts spreading on both materials. Finally, we concluded that PPy and PANi.GG-SLH have high potential to be used on novel skeletal muscle tissue engineering. [1] Tandon N, Cannizzaro C, Chao P-HG, Maidhof R, Marsano A, Au HTH, et al., Nat Protocols 4(2), 155-73. (2009) [2] Li L, Ge J, Guo B, Ma PX, Polymer Chemistry 5(8), 2880-90 (2014) [3] Bendrea A.D., Cianga L., Cianga I., J.Biomater Appl 25, 3-84 (2011)


Poster Session 2

96

LAYER-BY-LAYER ASSEMBLY OF POLY-L-LYSINE/GLYCOSAMINOGLYCAN FILMS: A

COMBINED ANALYSIS OF QCM-D AND ZETA POTENTIAL MEASUREMENTS

Teixeira R.1,2, Reis R.L.1,2, Pashkuleva I.1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal Layer-by-layer (LbL) films made of natural biopolymers have received considerable attention in biomedical fields because of their unique properties. Assemblies incorporating proteins and/or glycosaminoglycans (GAGs) are of particular interest since these are main components of the extracellular matrix (ECM) and therefore, they are expected to promote cell adhesion and proliferation [1]. GAGs have a natural tendency to swell in water, imparting to the films a viscoelastic behavior. The build-up is affected by the entrapped water, which contributes to the exponential growth of the LbL construct. It is generally accepted that the exponential growth of multilayers is a consequence of the “in-and-out” diffusion of shorter polymer chains. In this work, we have investigated the role of water and charge in the exponential build-up. Our initial hypothesis was that higher electrostatic interactions between the poyions will reduce the water content in each bilayer but will increase it between bilayers. We used therefore poly-L-lysine (PLL) as polycation, and the GAGs hyaluronan (HA), chondroitin sulfate (ChS) or heparin (Hep) (increasing negative charge) as polyanions. The build-up process was followed in situ by Quartz crystal microbalance with dissipation (QCM- We found that HA multilayers can grow either linearly or exponentially depending on the MW of the polyanion. Among the sulfated polyanions, only ChS multilayers grow exponentially. Hep, which behaves as a strong polyelectrolyte, produces thinner films that grow in a linear fashion. Surface zeta potentials showed that PLL-ended films become more basic as the films grow (isoelectric point, IEP≈8), and their titration profiles tend to overlap, regardless of the used polyanion. Among polyanion-ended films, remar [1] I. Pashkuleva, R.L. Reis, J. Mater. Chem. 20, 8803 (2010)


Poster Session 2

97

MULTIFUNCTIONAL BIOMIMETIC LbL COATINGS THAT COMBINE BIOACTIVITY WITH

SUPERIOR ADHESION FOR ORTHOPAEDIC APPLICATIONS

S. Rego,1,2 A. C. Vale,1,2 G. M. Luz,1,2 S.G. Caridade,1,2 J. F. Mano,1,2 N. M. Alves1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal In a marine environment, specific proteins are secreted by mussels and used as a bioglue to stick to a surface allowing generate irreversible bonding. Adhesive secreted proteins of mussels present an unusual amino acid 3,4-dihydroxyphenylalanine, DOPA [1]. The outstanding adhesive properties of these materials in the harsh conditions of the sea, impossible to mimic by human efforts until now, have been attributed to the presence of the catechol groups present in DOPA [2]. So, inspired by nature and in particular by the mussel adhesive proteins (MAPs), layer-by-layer films (LbL) with superior adhesive properties in wet environments were developed in this work. These LbL films were based on natural polymers combined with catechol groups, in particular hyaluronic acid and chitosan that have demonstrated adequate properties such as biocompatibility for several biomedical applications, in order to obtain films with enhanced tissue-adhesive properties and improved cell behaviour. Moreover, in order to develop truly multifunctional mussel inspired LbL films, bioactive nanoparticles were incorporated in their composition, besides the presence of the catechol groups. The final goal is to specifically use these films as coatings of a variety of implants for orthopaedic applications in order to improve both cell response and osteoconductivity in a simple and versatile way. Several techniques were used to characterize the properties of the developed films, such as UV spectrophotometry, quartz-crystal microbalance with dissipation monitoring (QCM-D), mechanical tests and SEM. [1] T.H. Anderson, J. Yu, A. Estrada, M.U. Hammer, J.H. Waite and J.N. Israelachvili, Adv. Funct. Mater. 20, 4196-4205 (2010). [2] H. Lee, Y. Lee, A.R. Statz, J. Rho, T.G. Park and P.B. Messersmith, Adv. Mater. 20, 1619-1623 (2008).


Poster Session 2

98

COMPLEX POLYMORPHISMS IN THE PLASMODIUM FALCIPARUM MULTIDRUG

RESISTANCE PROTEIN 2 GENE AND ITS CONTRIBUTION TO ANTIMALARIAL RESPONSE

M. Isabel Veiga1,2,3, Nuno S. Osório1,2, Pedro E. Ferreira3, Oscar Franzén3, Sabina Dahlstrom3, J. Koji Lum4, Francois Nosten5,6 and J. Pedro Gil3,7

1- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- Department of Medicine, Karolinska Institute, Stockholm, Sweden 4- The Harpur College of Arts and Sciences, Binghamton University, NY, USA 5- Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 6- Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK 7- Center for Biodiversity, Functional and Integrative Genomics, Universidade de Lisboa, Portugal; Plasmodium falciparum has the capacity to escape essentially all antimalarial drugs action. ATP–binding cassette (ABC) transporter proteins are known to be causative of multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for Multidrug Resistance Protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 Thai isolates. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs) /Kb was identified in this gene, with some SNPs being novel. Additionally, we also report that Pfmrp2 harbours a significant number of micro-indels, some previously not reported. We then investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in the artemisinin based combination therapy and with the parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms to modulate the parasite in vitro response to quinoline antimalarials, including chloroquine, piperaquine and mefloquine and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.


Poster Session 2

99

GELLAN-GUM COATED GOLD NANORODS AS INTRACELLULAR DRUG RELEASE SYSTEMS

FOR REGENERATIVE MEDICINE

S. Vial1,2, S. Vieira1,2, M. Carvalho1,2, R. L. Reis1,2, P. L. Granja3, and J. M. Oliveira1,2 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 3- INEB – Instituto de Engenharia Biomedica, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal

Due to their unique physicochemical properties, gold nanorods (AuNR) have been widely studied for biomedical applications including diagnosis, cell tracking, thermal therapy and drug delivery system (DDS) [1]. Their optical properties arising from Surface Plasmon Resonance, their easy of synthesis, their ability to be functionalized with a wide variety of biomolecules (peptides, DNA, proteins) and small molecules and their high surface area make them interesting as drug delivery carrier for tissue engineering and cancer therapy. However, to improve their biocompatibility and stability under biological conditions, surface modification needs to be envisaged, while enable the controlled release of drugs/bioactive agents. In this purpose, gold nanorods were coated with Gellan Gum (GG), a biodegradable and biocompatible natural-based polymers [2, 3]. The hybrid materials AuNR-GG have shown to be stable in a wide range of pH and ionic strength conditions. Moreover, no cytotoxicity has been detected in vitro after 14 days of incubation in two different cell lines: SaOS-2, human osteoblast-like cell line, and rabbit adipose stem cells. Dark-field microscopy and Transmission electron microscopy have confirmed the internalization of the nanoparticles inside cells, aggregated in vesicles. Subsequently, BSA-FTIC, as model drug, have been entrapped in the AuNR-GG. Using GG allowed to significantly improve the drug loading and the stability of the carrier. The proposed system has shown interesting features, in agreement with DDS requirements and further will be extended for cancer therapy and osteogenesis studies.

[1] X. Huang, S. Neretina, M. A. El-Sayed, Advanced Materials 21, 4880 (2009) [2] J. Silva-Correia, J. M. Oliveira, S. G. Caridade, J. T. Oliveira, R. A. Sousa, J. F. Mano, R. L. Reis, Journal of Tissue Engineering and Regenerative Medicine 5, 97 (2011) [3] S. Dhar, E. M. Reddy, A. Prabhune, V. Pokharkar, A. Shiras, B. L. V Prasad, Nanoscale 3, 575 (2011)

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