Improved production of acetate and propionate by Propionibacterium freudenreichii

Propionibacterium freudenreichii is a commercially important bacterium that is well-known for its role as ripening starter in the cheese industry and its probiotic potential. These bacteria may beneficially modulate the intestinal ecosystem and can exert anti-neoplastic effects via the production of short chain fatty acids (SCFAs), acetate and propionate. Several studies have demonstrated that the SCFA production by P. freudenreichii is responsible for its probiotic abilities. The aim of this work was to optimize the acetate and propionate production by P. freudenreichii towards its future use as a nutraceutical agent. In order to optimize the production of the abovementioned SCFAs in a minimal synthetic media the different composition of the several components were evaluated. Characterization of the acetate and propionate production in a medium mimicking the content of the human colon (MCHC) and a medium used by colorectal carcinoma cell lines (DMEM) was performed. The basal medium (BM) was found to be the most promising regarding the production of the SCFAs, showing 0.530 ± 0.011 g L-1 of biomass; high acetate and propionate yields (0.216 ± 0.001 g g-1 and 0.572 ± 0.002 g g-1, respectively), as well as high productivities (0.031 ± 0.000 g L-1 h-1 and 0.010 ± 0.000 g L-1 h-1, respectively). In the MCHC and DMEM media, it was possible to observe microbial growth (0.234 ± 0.006 g L-1 and 1.54 ± 0.00 g L-1, respectively); however the amounts of acetate and propionate were lower than the ones produced in BM medium. The results suggest that acetate and propionate production depends not only on the substrate type, but also on the medium constituents, being the simplest medium the one that show higher productivities as P. freudenreichii show low SCFA production when grown in MCHC and DMEM media. Future work will be conducted in order the increase bacteria growth and SCFA production in those media as this represents an essential feature for its use as a nutraceutical

Chondrogenic differentiation induced by extracellular vesicles bound to a nanofibrous substrate

Extracellular vesicles (EVs) are being increasingly studied owing to its regenerative potential, namely EVs derived from human bone marrow mesenchymal stem cells (hBM-MSCs). Those can be used for controlling inflammation, repairing injury, and enhancing tissue regeneration. Differently, the potential of EVs derived from human articular chondrocytes (hACs) to promote cartilage regeneration has not been thoroughly investigated. This work aims to develop an EVs immobilization system capable of selectively bind EVs present in conditioned medium obtained from cultures of hACs or hBM-MSC. For that, an anti-CD63 antibody was immobilized at the surface of an activated and functionalized electrospun nanofibrous mesh. The chondrogenic potential of bound EVs was further assessed by culturing hBM-MSCs during 28 days under basal conditions. EVs derived from hACs cultured under differentiation medium or from chondrogenically committed hBM-MSCs induced a chondrogenic phenotype characterized by marked induction of SOX9, COMP, Aggrecan and Collagen type II, and matrix glycosaminoglycans synthesis. Indeed, both EVs immobilization systems outperformed the currently used chondroinductive strategies. These data show that naturally secreted EVs can guide the chondrogenic commitment of hBM-MSCs in the absence of any other chemical or genetic chondrogenic inductors based in medium supplementation.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD grant to M.R.C. (PD/BD/113797/2015) financed by the Doctoral Program on Advanced Therapies for Health (PATH) (FSE/POCH/PD/169/2013) and the project Cells4_IDs (PTDC/BTM-SAL/28882/2017). This work was also financed by the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125)

The biomimetic surface topography of Rubus fruticosus leaves stimulate the induction of osteogenic differentiation of rBMSCs

The interaction between cells and biomaterials is essential for the success of biomedical applications in which the implantation of biomaterials in the human body is necessary. It has been demonstrated that material's chemical, mechanical, and structural properties can influence cell behaviour. The surface topography of biomaterials is a physical property that can have a major role in mediating cellâ material interactions. This interaction can lead to different cell responses regarding cell motility, proliferation, migration, and even differentiation. The combination of biomaterials with mesenchymal stem cells (MSCs) for bone regeneration is a promising strategy to avoid the need for autologous transplant of bone. Surface topography was also associated with the capacity to control MSCs differentiation. Most of the topographies studied so far involve machine-generated surface topographies. Herein, our strategy differentiates from the above mentioned since we selected natural surface topographies that can modulate cell functions for regenerative medicine strategies. Rubus fruticosus leaf was the selected topography to be replicated in polycaprolactone (PCL) membranes through polydimethylsiloxane moulding and using soft lithography. Afterwards, rat bone marrow stem cells (rBMSCs) were seeded at the surface of the imprinted PCL membranes to characterize the bioactive potential of our biomimetic surface topography to drive rBMSCs differentiation into the osteogenic lineage. The selected surface topography in combination with the osteogenic inductive medium reveals having a synergistic effect promoting osteogenic differentiation.This work is a result of the project FROnTHERA (NORTE-01-0145-FEDER-000023), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and Portuguese Foundation for Science and Technology under the doctoral programme in Tissue Engineering, Regenerative Medicine and Stem Cells (PD/59/2013), (PD/BD/128087/2016) (COVID/BD/151599/2020) and by the project Cells4_IDs (PTDC/BTM-SAL/28882/2017)

Biomimetic surface topography as a potential modulator of macrophages inflammatory response to biomaterials

The implantation of biomaterial devices can negatively impact the local microenvironment through several processes including the injury incurred during the implantation process and the associated host inflammatory response. Immune cell responses to implantable biomaterial devices mediate host-material interactions. Indeed, the immune system plays a central role in several biological processes required for the integration of biomaterials such as wound healing, tissue integration, inflammation, and foreign body reactions. The implant physicochemical properties such as size, shape, surface area, topography, and chemistry have been shown to provide cues to the immune system. Its induced immune-modulatory responses towards inflammatory or wound healing phenotypes can determine the success of the implant. In this work, we aim to evaluate the impact of some biomimetic surface topographies on macrophages' acute inflammatory response. For that, we selected 4 different biological surfaces to replicate through soft lithography on spin casting PCL membranes. Those topographies were: the surface of E. coli, S.eppidermidis and L929 cells cultured in polystyrene tissue culture disks, and an Eggshell membrane. We selected a model based on THP-1-derived macrophages to study the analysis of the expression of both pro-inflammatory and anti-inflammatory markers. Our results revealed that depending on the surface where these cells are seeded, they present different phenotypes. Macrophages present a M1-like phenotype when they are cultured on top of PCL membranes with the surface topography of E. coli and S. epidermidis. When cultured on membranes with L929 monolayers or Eggshell membrane surface topography, the macrophages present a M2-like phenotype. These results can be a significant advance in the development of new implantable biomaterial devices since they can help to modulate the inflammatory responses to implanted biomaterials by controlling their surface topography.FCT -Fundação para a Ciência e a Tecnologia(PD/59/2013

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Desenvolvimento de uma Propionibacterium melhorada para potencial uso como um nutracêutico para a prevenção - tratamento do cancro colorectal

Dissertação de mestrado em Genética MolecularPropionibacterium freudenreichii is a commercially important bacterium that is well-known for its role as ripening starter in the cheese industry and its probiotic potential. These bacteria may beneficially modulate the intestinal ecosystem and can exert anti-neoplastic effects, particularly against colorectal cancer (CRC), via the production of short chain fatty acids (SCFAs), namely acetate and propionate. Thus, propionibacteria can be envisaged as a potential nutraceutical towards the prevention/treatment of CRC. In that sense, the aim of this thesis was to develop strategies to enhance the production of SCFA by P. freudenreichii under the simulation of human colon environment, as well as to evaluate its effects on CRC cells. In order to optimize and characterize the production of SCFAs, acetate and propionate by P. freudenreichii, different culture media and different fermentation conditions were evaluated. Moreover, the SCFAs toxic concentrations for the bacterium were determined. Additionally, a digestive stress challenge and random mutagenesis of P. freudenreichii DSM 20271 were performed. Finally, the effect of the propionibacteria fermentation broth on CRC cells and the CRC cells conditioned medium on the growth and biotransformation performance of the bacteria were studied. The basal medium (BM) was found to be the best to produce SCFA by P. freudenreichii with high amounts of acetate and propionate being produced, mainly when supplemented with glycerol. However, the results obtained with the medium “mimicking the content of the human colon” (MCHC) were not favorable regarding SCFAs production. The adapted P. freudenreichii to digestive stress lost the ability to produce high amounts of SCFAs in yeast extract-lactate (YEL) and BM media, in particular propionate. Moreover, partial inhibition of the bacteria growth and SCFAs production occurred at the following concentrations of pure SCFAs: 4 g L-1 acetate; 3 g L-1 propionate; 6 g L-1 propionate; 1 g L-1 acetate and 3 g L-1 propionate. Pure acetate and propionate, as well as the bacterial fermentation broth inhibited the CRC cells RKO proliferation and promoted their accumulation in the sub-G1 phase of the cell cycle. In conclusion, the results gathered in this work suggest that the coculture of P. freudenreichii and CRC cells was found to be possible and favorable for the bacteria and that P. freudenreichii could potentially be used in the CRC prevention/treatment via their ability to produce SCFAs.A Propionibacterium freudenreichii é uma bactéria comercialmente importante, conhecida pela sua utilização como cultura de arranque na produção de queijo, bem como pelo seu potencial probiótico. Estas bactérias podem modular beneficamente o ecossistema intestinal e exercer os efeitos antineoplásicos, em particular contra o cancro colorectal (CRC), através da produção de ácidos gordos de cadeia curta (AGCC), nomeadamente acetato e propionato. Assim, as propionibactérias podem ser vistas como potenciais nutracêuticos para a prevenção/tratamento do CRC. Nesse sentido, o objectivo desta tese foi desenvolver estratégias para aumentar a produção de AGCC pela P. freudenreichii em condições que simulam o cólon humano, bem como avaliar o seu efeito nas células de CRC. A fim de otimizar e caracterizar a produção dos AGCC, acetato e propionato, diferentes meios de cultura e diferentes condições foram avaliados. Além disso, as concentrações de AGCC tóxicas para a bactéria foram determinadas. Adicionalmente, realizaram-se ensaios de adaptação ao stresse digestivo e de mutação aleatória na P. freudenreichii DSM 20271. Finalmente, foi estudado o efeito do meio de fermentação da bactéria nas células de CRC, bem como o efeito do meio condicionado das células de CRC no crescimento e produção de AGCC pela bactéria. O meio basal (BM) demonstrou ser o melhor para produzir AGCC pela P. freudenreichii, obtendo-se grandes quantidades de acetato e propionato, principalmente no meio BM suplementado com glicerol. No entanto, os resultados obtidos com o meio que "imita o conteúdo do cólon humano" (MCHC) não foram favoráveis relativamente à produção de AGCC. A P. freudenreichii adaptada ao stresse digestivo perdeu a capacidade de produzir grandes quantidades de AGCC, particularmente propionato, nos meios de extrato de levedura-lactato (YEL) e BM. Adicionalmente, observou-se uma inibição parcial do crescimento bacteriano e produção de AGCC para as seguintes concentrações de AGCC puros: 4 g L-1 acetato; 3 g L-1 propionato; 6 g L-1 propionato; 1 g L-1 acetato e 3 g L-1 propionato. O acetato e o propionato, bem como o meio de fermentação da bactéria inibiram a proliferação das células de CRC RKO e induziram um aumento de células na fase sub-G1 do ciclo celular. Em conclusão, os resultados deste trabalho sugerem que a co-cultura entre células de CRC e P. freudenreichii é possível e favorável para a bactéria e que a P. freudenreichii poderá potencialmente ser usada na prevenção/tratamento de CRC através da sua capacidade de produzir AGCC

Biofuncionalização da superfície de membranas fibrosas de policaprolactona para terapias avançadas de tecido esquelético e neural

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisDamage of the skeletal and neural tissues has a significant impact over the quality-of-life of patients and high socio-economical costs. Current treatment options are not effective in long term, due to the suboptimal integration with the host tissue and limited bioactivity of implantable biomaterials. The immobilization of biomolecules at the surface of biomedical devices has attracted increasing interest, allowing for their local bioavailability avoiding systemic side effects and longer half-life. Envisioning the development of advanced therapies, the electrospun nanofibrous meshes (NFMs) were used as a substrate due to their fibrous structure mimic the extracellular matrix (ECM) of many tissues, allowing cell-cell and cell-biomaterial interactions. For that, the surface of polycaprolactone NFMs was activated and functionalized with amine groups allow for covalent immobilization of defined antibodies, with the capacity to selectively bind autologous biomolecules. Different biofunctional substrates with chondrogenic inductive properties were developed through the surface biofunctionalization of NFM with endogenous human fibronectin, extracellular vesicles or the combination of endogenous Transforming Growth Factor-133 and Insulin-like Growth Factor-I. All these biofunctional substrates successfully induced the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) under basal culture conditions. Blood-derived Nerve Growth Factor bound to the surface of NFMs remains bioactive, being an effective inducer of the neurogenic differentiation of a relevant cell line. Additionally, we developed a biofunctional system able to mimic the vasculature of bone tissue, comprising Bone Morphogenetic Protein 2 and Vascular Endothelial Growth Factor in a parallel pattern design. This biofunctional system enabled a spatially defined osteogenic and angiogenic differentiation of hBM-MSCs. The surface biofunctionalization of biomaterial substrates enables developing biofunctional systems envisioning patient-specific devices promoting skeletal and neural tissue regeneration that can maximize and extend the local efficacy and minimize the side effects of the use of biologic based therapies in patients.A deterioração dos tecidos esquelético a neural têm um impacto significativo na qualidade de vida dos pacientes e um elevado custo socioeconómico. Os tratamentos atualmente disponíveis não são eficazes a longo termo, devido à inadequada integração com o tecido hospedeiro e à baixa bioatividade dos biomateriais implantados. A imobilização de biomoléculas constitui uma estratégia alternativa, permitindo a biodisponibilidade local das biomoléculas evitando efeitos colaterais sistêmicos. Ambicionando desenvolver terapias avançadas, malhas fibrosas produzidas por "electrospinning" (NFMs) foram usadas como substratos poliméricos devido à sua estrutura fibrosa similar a matriz extracelular (ECM) de muitos tecidos, promovendo as interações célula-célula e célula-biomaterial. Para isso, NFMs de policaprolactona foram ativadas e funcionalizadas com grupos amina, permitindo a imobilização covalente de anticorpos pré-definidos, com capacidade de ligar seletivamente biomoléculas autólogas. Foram desenvolvidos diferentes substratos biofuncionais, com propriedades indutoras de diferenciação condrogénica, mediante ligação de fibronectina humana, vesículas extracelulares ou a combinação do fator de transformação do crescimento beta 3 com o fator de crescimento semelhante à insulina tipo I. Todos estes substratos biofuncionalizados foram capazes de induzir a diferenciação condrogénica de células estaminais mesenquimais derivadas de medula óssea humana (hBM-MSCs) sendo cultivadas em condições basais. O fator de crescimento nervoso ligado à superfície das NFMs permanece bioativo, sendo um indutor eficaz da diferenciação neurogénica de uma linha celular relevante. Numa outra abordagem, foi desenvolvido um sistema biofuncional capaz de mimetizar a vasculatura de um tecido ósseo, ligando paralelamente a proteína morfogenética óssea 2 e o fator de crescimento do endotélio vascular sobre uma mesma NFM. Este sistema biofuncional permitiu a diferenciação osteogénica e angiogénica de hBM-MSCs espacialmente definida. Concluindo, a bioftmcionalização de substratos produzidos por "electrospinning" permite o desenvolvimento de dispositivos biomédicos personalizados, capazes de promover a regeneração do tecido esquelético e neural, maximizando a eficácia local e minimizando os efeitos colaterais do uso de terapias biológicas em pacientes.To the financial support of the Portuguese Fotmdatron for Science and Technology to maize possible this PhD by awarded me with a PhD scholarship (PD/BD/113797/2015) under the Doctoral Program on Advanced Therapies for Health (FSE/POCK/PD/169/2013). The experimental work was funded by the projects SPARTAN (PTDC/CTM-BIO/4388/2014) and FRONthera (NORTE-01-0145-FEDER-0000232)

Stimulation of neurite outgrowth using autologous NGF bound at the surface of a fibrous substrate

Peripheral nerve injury still remains a major clinical challenge, since the available solutions lead to dysfunctional nerve regeneration. Even though autologous nerve grafts are the gold standard, tissue engineered nerve guidance grafts are valid alternatives. Nerve growth factor (NGF) is the most potent neurotrophic factor. The development of a nerve guidance graft able to locally potentiate the interaction between injured neurons and autologous NGF would be a safer and more effective alternative to grafts that just release NGF. Herein, a biofunctional electrospun fibrous mesh (eFM) was developed through the selective retrieval of NGF from rat blood plasma. The neurite outgrowth induced by the eFM-NGF systems was assessed by culturing rat pheochromocytoma (PC12) cells for 7 days, without medium supplementation. The biological results showed that this NGF delivery system stimulates neuronal differentiation, enhancing the neurite growth more than the control condition.Portuguese Foundation for Science and Technology (FCT), grant numbers PTDC/BTM-SAL/28882/2017 (Cells4_Ids) and PTDC/CTM-BIO/4388/2014 (SPARTAN). The PhD grant of M.R.C. (PD/BD/113797/2015) was funded by the FCT Doctoral Program on Advanced Therapies for Health (PATH) (FSE/POCH/PD/169/2013) and the A.M. was financed by the IF grant IF/00376/201

Fibronectin bound to a fibrous substrate has chondrogenic induction properties

Articular cartilage is an avascular tissue characterized by a dense and specific extracellular matrix (ECM). Fibronectin (FN) is a key constituent of the pericellular ECM, assembled into a fibrillar matrix through a cell-mediated process, being implicated in chondrogenic events. In this study, we evaluate the chondrogenic potential of FN bound to the surface of an electrospun nanofibrous mesh (NFM). For that, an anti-FN antibody was immobilized at the surface of NFMs, rendering them capable of selectively binding endogenous FN (eFN) from blood plasma. The chondrogenic potential of bound eFN was further assessed by culturing human bone marrow-derived mesenchymal stem cells (hBM-MSCs) for 28 days, in a basal growth medium. The biological results indicate that NFMs functionalized with eFN were able to successfully induce the chondrogenesis of hBM-MSCs, as demonstrated by the high expression of SOX9, Aggrecan, and Collagen type II. Therefore, biofunctionalized nanofibrous substrates comprising eFN significantly enhance the efficacy of a cartilage tissue-engineering strategy.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the Ph.D. grant of M.R.C. (PD/BD/113797/2015) financed by the FCT Doctoral Program on Advanced Therapies for Health (PATH) (FSE/POCH/PD/169/2013), the IF grant of A.M. (IF/00376/2014), and the project SPARTAN (PTDC/CTMBIO/4388/2014