Electroless nickel composite (Ni-P/SiC) coating - A study on interface and corrosion characteristic

Surface behaviour is of paramount importance as failure and degradation tend to initiate from the surface. Electroless composite coating (NiP/SiC) was developed using SiC as reinforcing particles. As heat treatment plays a vital role in electroless nickel coating owing to the changes in microstructure, phase structure and mechanical properties, an insight at the interface changes in chemistry and micromechanical behaviour was investigated using scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) and microindentation techniques. Corrosion performance was analysed using electrochemical impedance spectroscopy (EIS). Absence of zinc and migration of copper at the interface was detected. Brittleness and microcracks was seen long the interface when indenting at load of 500 gf (Vickers). Corrosion performance is weaker than particles free coating. However, a thin blanket of NiP could enhance the resistance to corrosive medium.

Dietary exposure to aflatoxin and micronutrient status among young children from Guinea

SCOPE: Aflatoxin exposure coincides with micronutrient deficiencies in developing countries. Animal feeding studies have postulated that aflatoxin exposure may be exacerbating micronutrient deficiencies. Evidence available in human subjects is limited and inconsistent. The aim of the study was to investigate the relationship between aflatoxin exposure and micronutrient status among young Guinean children.

METHOD AND RESULTS: A total of 305 children (28.8 ± 8.4 months) were recruited at groundnut harvest (rainy season), of which 288 were followed up 6 months later post-harvest (dry season). Blood samples were collected at each visit. Aflatoxin-albumin adduct levels were measured by ELISA. Vitamin A, vitamin E and β-carotene concentrations were measured using HPLC methods. Zinc was measured by atomic absorption spectroscopy. Aflatoxin exposure and micronutrient deficiencies were prevalent in this population and were influenced by season, with levels increasing between harvest and post-harvest. At harvest, children in the highest aflatoxin exposure group, compared to the lowest, were 1.98 (95%CI: 1.00, 3.92) and 3.56 (95%CI: 1.13, 11.15) times more likely to be zinc and vitamin A deficient.

CONCLUSION: Although children with high aflatoxin exposure levels were more likely to be zinc and vitamin A deficient, further research is necessary to determine a cause and effect relationship. 

Evolution, gene regulation and functional analysis of BMP2 in fish

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-related diseases. The objective of this work was to collect new functional data and determine spatiotemporal expression patterns in a fish system aiming towards a better understanding of BMP2 function and regulation. Transcriptional and post-transcriptional regulation of gilthead seabream BMP2 gene was inferred from luciferase reporter systems. Several bone- and cartilage-related transcription factors (e.g. RUNX3, MEF2c, SOX9 and ETS1) were found to regulate BMP2 transcription, while microRNA 20a was shown to affect stability of the BMP2 transcript and thus the mineralogenic capacity of fish bone-derived host cells. The regulation of BMP2 activity through an interaction with the matrix Gla protein (MGP) was investigated in vitro using BMP responsive elements (BRE) coupled to luciferase reporter gene. Although we demonstrated the functionality of the experimental system in a fish cell line and the activation of BMP signaling pathway by seabream BMP2, no conclusive evidence could be collected on a possible interaction beween MGP and BMP2. The evolutionary relationship among the members of BMP2/4/16 subfamily was inferred from taxonomic and phylogenetic analyses. BMP16 diverged prior to BMP2 and BMP4 and should be the result of an ancient genome duplication that occurred early in vertebrate evolution. Structural and functional data suggested that all three proteins are effectors of the BMP signaling pathway, but expression data revealed different spatiotemporal patterns in teleost fish suggesting distinct mechanisms of regulation. In this work, through the collection of novel data, we provide additional insight into the regulation, the structure and the phylogenetic relationship of BMP2 and its closely related family members.

Contamination of farmed gilthead sea bream (Sparus aurata, Linnaeus, 1758) by the parasite Amyloodinium ocellatum: a proteomic approach

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Uniersidade do Algarve, 2015

DRIFT and DRUV spectroscopy methods for studying the interaction of metal compounds with native cellulose

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

In-situ development of self-defensive antibacterial biomaterials: phenol-g-keratin-EC based bio-composites with characteristics for biomedical applications

Recently, the development of highly inspired biomaterials with multi-functional characteristics has gained considerable attention, especially in biomedical, and other health-related areas of the modern world. It is well-known that the lack of antibacterial potential has significantly limited biomaterials for many challenging applications such as infection free wound healing and/or tissue engineering etc. In this perspective, herein, a series of novel bio-composites with natural phenols as functional entities and keratin-EC as a base material were synthesised by laccase-assisted grafting. Subsequently, the resulting composites were removed from their respective casting surfaces, critically evaluated for their antibacterial and biocompatibility features and information is also given on their soil burial degradation profile. In-situ synthesised phenol-g-keratin-EC bio-composites possess strong anti-bacterial activity against Gram-positive and Gram-negative bacterial strains i.e., B. subtilis NCTC 3610, P. aeruginosa NCTC 10662, E. coli NTCT 10418 and S. aureus NCTC 6571. More specifically, 10HBA-g-keratin-EC and 20T-g-keratin-EC composites were 100% resistant to colonisation against all of the aforementioned bacterial strains, whereas, 15CA-g-keratin-EC and 15GA-g-keratin-EC showed almost negligible colonisation up to a variable extent. Moreover, at various phenolic concentrations used, the newly synthesised composites remained cytocompatible with human keratinocyte-like HaCaT, as an obvious cell ingrowth tendency was observed and indicated by the neutral red dye uptake assay. From the degradation point of view, an increase in the degradation rate was recorded during their soil burial analyses. Our investigations could encourage greater utilisation of natural materials to develop bio-composites with novel and sophisticated characteristics for potential applications.

Obesidade e cancro da próstata: Avaliação do efeito dos adipócitos nas células RM1 de carcinoma da próstata

O cancro da próstata é o segundo cancro mais frequente e a sexta causa de morte mundial por cancro no sexo masculino. A obesidade tem sido associada ao aumento da incidência e mortalidade por cancro, com alguma controvérsia. As alterações nas expressões de adipocinas associadas à obesidade têm sido um dos diversos mecanismos propostos para explicar a associação entre a obesidade e o cancro da próstata, nomeadamente na promoção do desenvolvimento e progressão celular do tumor. O objetivo deste trabalho é avaliar o efeito dos fatores produzidos pelos pré-adipócitos e os adipócitos na proliferação, migração e invasão das células de carcinoma da próstata independentes dos androgénios. As células RM1 foram cultivadas na presença de diferentes concentrações de insulina e leptina, bem como em meio condicionado (MC) de pré-adipócitos e adipócitos e co-cultivadas em sistema de transwells, com as mesmas células. A proliferação celular das RM1 foi avaliada recorrendo a contagem celular em camara de Neubauer e em citometro de fluxo, e aos ensaios metabólicos alamar blue e XTT. Efetuou-se um ensaio de migração por dano nas células RM1 na presença dos meios condicionados. A invasão das células foi avaliada recorrendo a um sistema de transwells, com membrana de matrigel, quando cultivadas com pré-adipócitos e adipócitos. A insulina aumentou significativamente a proliferação celular, ao contrário da leptina que não teve efeito. O meio condicionado dos pré-adipócitos aumentou ligeiramente a proliferação, enquanto meio condicionado dos adipócitos de 1 e 2 dias aumentou significativamente a proliferação das células RM1 (p<0.01), quando avaliada por XTT. Na câmara de Neubauer não se verificaram diferenças significativas na proliferação celular. Relativamente à migração celular, observou-se um aumento significativo da migração das células RM1 cultivadas com meio condicionado de adipócitos (MCA) e pré-adipócitos (MCPA) em comparação com o controlo (p<0.01). Observou-se um aumento significativo da invasão de células RM1 cultivadas com adipócitos e pré-adipócitos (p <0.05). Os adipócitos aumentaram significativamente a proliferação das células RM1 em co-cultura (p<0.01). Em conclusão, as células RM1 parecem ser influenciadas por fatores secretados pelos adipócitos, capazes de aumentar a sua capacidade de proliferar, invadir e migrar.

Multiple linear and principal component regressions for modelling ecotoxicity bioassay response

The ecotoxicological response of the living organisms in an aquatic system depends on the physical, chemical and bacteriological variables, as well as the interactions between them. An important challenge to scientists is to understand the interaction and behaviour of factors involved in a multidimensional process such as the ecotoxicological response.With this aim, multiple linear regression (MLR) and principal component regression were applied to the ecotoxicity bioassay response of Chlorella vulgaris and Vibrio fischeri in water collected at seven sites of Leça river during five monitoring campaigns (February, May, June, August and September of 2006). The river water characterization included the analysis of 22 physicochemical and 3 microbiological parameters. The model that best fitted the data was MLR, which shows: (i) a negative correlation with dissolved organic carbon, zinc and manganese, and a positive one with turbidity and arsenic, regarding C. vulgaris toxic response; (ii) a negative correlation with conductivity and turbidity and a positive one with phosphorus, hardness, iron, mercury, arsenic and faecal coliforms, concerning V. fischeri toxic response. This integrated assessment may allow the evaluation of the effect of future pollution abatement measures over the water quality of Leça River.

Abordagem e maneio médico-cirúrgico de feridas abertas em cães e gatos : caracterização etiológica e estudo de padrões traumáticos

Dissertação de Mestrado Integrado em Medicina Veterinária

Neurolysis using a carbohydrate polymer gel for the treatment of postoperative neuropathic pain.

Perineural and intraneural fibrosis is thought to be the main cause of failure of the many surgical treatments of neuropathic pain. We have used Adcon-T/N carbohydrate polymer gel for prevention of perineural fibrosis in several parts of the body. In this retrospective study, 54 patients who presented with postoperative neuropathic pain had microsurgical epineural neurolysis and relocation of a terminal neuroma. In 19 of them, the carbohydrate gel was applied at the same time. The mean follow-up was four years and the nerve distribution varied. Postoperative improvement in pain scores (visual analogue scale (VAS) and neuropathic pain scale inventory (NPSI)), sensitivity, overall improvement and satisfaction were equivalent in the two groups, with pain relief in about 80% of the patients. There was no significant beneficial effect in the carbohydrate gel group. Patients treated with this device had a higher infection rate (21 compared with 0, p = 0.01) and delayed wound healing (31.6 compared with 11.8, p = 0.2). We conclude that good long-term pain relief is obtained postoperatively independently of the addition of carbohydrate gel. There was a slight but not significant trend towards profound pain relief with the gel.

Role of intracellular signaling pathways activated in fibroblasts in response to lipoproteins

Summary The best described physiological function of low-density lipoproteins (LDL) is to transport cholesterol to target tissues. LDL deliver their cholesterol cargo to cells following their interaction with the LDL receptor. LDL, when their vascular concentrations increase, have also been implicated in pathologies such as atherosclerosis. Among the cell types that are found in blood vessels, endothelial and smooth muscle cells have dominated cellular research on atherosclerotic mechanisms and LDL activation of signaling pathways, while very little is known about adventitial fibroblast activation caused by elevated lipoprotein levels. Since fibroblasts participate in wound repair and since it has recently been recognized that fibroblasts may play pivotal roles in vascular remodeling and repair of injury, we assessed whether lipoproteins affect fibroblast function. We have found that LDL specifically mediate the activation of a class of mitogen-activated protein kinases (MAPKs): the p38 MAPKs. The activation of this pathway in turn modulates cell shape by promoting lamellipodia formation and extensive cell spreading. This is of particular interest because it provides a mechanism by which LDL can promote wound healing or vessel wall remodeling as observed during the development of atherosclerosis. In order to understand the molecular mechanisms by which LDL induce p38 activation we searched for the component in the LDL particle responsible for the induction of this pathway. We found that cholesterol is the major component of lipoprotein particles that mediates their ability to stimulate the p38 MAPK pathway. Furthermore, we investigated the cellular mechanisms underlying the ability of LDL to induce cell shape changes and whether this could participate in wound repair. Our recent data demonstrates that the capacity of LDL to induce fibroblast spreading relies on their ability to stimulate IL-8 secretion, which in turn leads to accelerated wound healing. LDL-induced IL-8 production and subsequent wound closure are impaired upon inhibition of the p38 MAPK pathway indicating that the LDL-induced spreading and accelerated wound sealing rely on the ability of LDL to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Therefore, regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL-cholesterol levels, IL-8 production and extensive remodeling of the vessel wall. Résumé: La fonction physiologique des lipoprotéines à faible densité (LDL) la mieux décrite est celle du transport du cholestérol aux tissus cibles. Les LDL livrent leur cargaison de cholestérol aux cellules après leur interaction avec le récepteur au LDL. Une concentration vasculaire des LDL augmenté est également impliquée dans le développement de l'athérosclérose. Parmi les types de cellule présents dans les vaisseaux sanguins, les cellules endothéliales et les cellules du muscle lisse ont dominé la recherche cellulaire sur les mécanismes athérosclérotiques et sur l'activation par les LDL des voies de signalisation intracellulaire. A l'inverse peu de choses sont connues sur l'activation des fibroblastes de l'adventice par les lipoprotéines. Puisqu'il a été récemment reconnu que les fibroblastes peuvent jouer un rôle central dans la remodélisation vasculaire et la réparation tissulaire, nous avons étudié si les lipoprotéines affectent la fonction des fibroblastes. Nous avons constaté que les LDL activent spécifiquement une classe de protéines kinases: les p38 MAPK (mitogen-activated protein kinases). L'activation de cette voie module à son tour la forme de la cellule en favorisant la formation de lamellipodes et l'agrandissement des cellules. Cela a un intérêt particulier car il fournit un mécanisme par lequel les LDL peuvent promouvoir la cicatrisation ou la remodélisation des parois vasculaires comme observés lors du développement de l'athérosclérose. Pour comprendre les mécanismes moléculaires par lesquels les LDL provoquent l'activation des p38 MAPK, nous avons cherché à identifier les composants dans la particule de LDL responsables de l'induction de cette voie. Nous avons constaté que le cholestérol est l'élément principal des particules de lipoprotéine qui contrôle leur capacité à stimuler la voie des p38 MAPK. En outre, nous avons examiné les mécanismes cellulaires responsables de la capacité des LDL à induire des changements dans la forme des cellules. Nos données récentes démontrent que la capacité des LDL à induire l'agrandissement des cellules, ainsi que leur aptitude à favoriser la cicatrisation, reposant sur leur capacité à stimuler la sécrétiond'IL-8. La production d'IL-8 induite par les LDL est bloquée par l'inhibition de la voie p38 MAPK, ce qui indique que l'étalement des cellules induit par les LDL ainsi que l'accélération de la cicatrisation sont liés à la capacité des LDL à stimuler la sécrétion d'IL8 via l'activation des p38 MAPK. La régulation de la forme et de la migration des fibroblastes par les lipoprotéines peuvent donc participer au développement de l'athérosclérose qui est caractérisée par l'augmentation des niveaux de production de LDL-cholestérol et d'IL-8 ainsi que par une remodélisation augmentée de la paroi du vaisseau.

The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes.

Cellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely blocks only galvanotaxis and does not decrease migration speed. Overexpression of ENaC is sufficient to drive galvanotaxis in otherwise unresponsive cells. Pharmacologic blockade or maintenance of the open state of ENaC also decreases or increases, respectively, galvanotaxis, suggesting that the channel open state is responsible for the response. Stable lamellipodial extensions formed at the cathodal sides of wild-type cells at the start of galvanotaxis; these were absent in the ENaC knockout keratinocytes, suggesting that ENaC mediates galvanotaxis by generating stable lamellipodia that steer cell migration. We provide evidence that ENaC is required for directional migration of keratinocytes in an electric field, supporting a role for ENaC in skin wound healing.

Demonstration of the effectiveness of zinc in diarrhoea of children living in Switzerland.

We designed a double-blinded randomized clinical trial of zinc (10 or 20 mg of zinc sulphate for 2-5 month-old or 6-59 month-old children, respectively, during 10 days) vs. placebo in otherwise healthy children aged 2 months to 5 years who presented with acute diarrhoea (i.e. ≥3 stools/day for less than 72 h). Eighty-seven patients (median age 14 months; range 3.1-58.3) were analysed in an intention-to-treat approach. Forty-two patients took zinc and 45 placebo. There was no difference in the duration nor in the frequency of diarrhoea, but only 5% of the zinc group still had diarrhoea at 120 h of treatment compared to 20% in the placebo group (P = 0.05). Thirty-one patients (13 zinc and 18 placebo) were available for per-protocol analyses. The median (IQR) duration of diarrhoea in zinc-treated patients was 47.5 h (18.3-72) and differed significantly from the placebo group (median 76.3; IQR 52.8-137) (P = 0.03). The frequency of diarrhoea was also lower in the zinc group (P = 0.02). CONCLUSION: zinc treatment decreases the frequency and severity of diarrhoea in children aged 2 months to 5 years living in Switzerland. However, the intention-to-treat analysis reveals compliance issues that question the proper duration of treatment and the choice of optimal pharmaceutical formulation.

Les cellules stromales multipotentes accélèrent la guérison de plaies cutanées chez les souris irradiées via la sécrétion de la chimiokine SDF-1α

Le traitement du cancer à l’aide d’une exposition aux radiations ionisantes (RI) peut mener au développement de plusieurs effets secondaires importants, dont un retard de réparation et de régénération des tissus. Les mécanismes responsables de ces effets demeurent largement inconnus encore aujourd’hui, ce qui a pour effet de limiter le développement d’approches thérapeutiques. À l’aide d’un modèle de guérison de plaie cutanée chez la souris, nous avons cherché à déterminer les mécanismes par lesquels l’exposition aux RI limite la régénération de la peau. Nos résultats démontrent que l’induction de la "stromal-derived growth factor 1α" (SDF-1α), une cytokine normalement surexprimée dans les tissus hypoxiques, est sévèrement diminuée dans les plaies de souris irradiées versus non-irradiées. Ce défaut corrèle avec un retard de guérison des plaies et est encore évident plusieurs mois suivant l’exposition aux RI, suggérant qu’il y a une altération permanente de la capacité de la peau à se réparer. Parce que SDF-1α est secrété principalement par les fibroblastes du derme, nous avons évalué le potentiel des cellules stromales multipotentes (MSCs), qui sont reconnues pour secréter des niveaux élevés de SDF-1α, à accélérer la régénération de la peau chez les souris irradiées. L’injection de MSCs en périphéries des plaies a mené à une accélération remarquable de la guérison de la peau chez les souris exposées aux RI. Les actions des MSCs étaient principalement paracrines, dû au fait que les cellules n’ont pas migré à l’extérieur de leur site d’injection et ne se sont pas différentiées en kératinocytes. L’inhibition spécifique de l’expression de SDF-1α a mené à une réduction drastique de l’efficacité des MSCs à accélérer la fermeture de plaie indiquant que la sécrétion de SDF-1α par les MSCs est largement responsable de leur effet bénéfique. Nous avons découvert aussi qu’un des mécanismes par lequel SDF-1α accélère la guérison de plaie implique l’augmentation de la vascularisation au niveau de la peau blessée. Les résultats présentés dans ce mémoire démontrent collectivement que SDF-1α est une importante cytokine dérégulée au niveau des plaies cutanées irradiées, et que le déclin du potentiel de régénération des tissus qui est observé suivant une exposition au RI peut être renversé, s’il est possible de restaurer le microenvironnement de la blessure avec un support stromal adéquat.

Étude du rôle de l’auto-antigène nucléaire centromérique B (CENP-B) et des auto-anticorps anti-CENP-B dans l’activation des cellules musculaires lisses vasculaires : implication potentielle dans la pathophysiologie de la sclérose systémique

La sclérose systémique (ScS) est une maladie auto-immune dont l’un des principaux auto-anticorps, dirigé contre la protéine centromérique B (CENP-B), est fortement associé à l’hypertension artérielle pulmonaire, l’une des causes majeures de décès dû à la ScS. L’hypertension résulte de l’occlusion progressive des vaisseaux suite à une hyperactivation des cellules musculaires lisses (CML) de la paroi vasculaire. Cependant, les facteurs responsables de ce remodelage vasculaire restent inconnus. Plusieurs études récentes ont démontré que certains auto-antigènes possèdent des fonctions biologiques additionnelles lorsqu'ils se retrouvent dans le milieu extracellulaire. En effet, une fois libérés par nécrose ou apoptose, ces auto-antigènes adoptent une activité biologique qui s'apparente à celles des cytokines et peuvent ainsi participer aux processus normaux de réparation de blessure et/ou acquérir une activité pathogène qui contribue au développement de certaines maladies auto-immunes. Nos résultats suggèrent que la CENP-B peut être ajoutée à cette liste de molécules bifonctionnelles. À l'aide des techniques d'immunofluorescence, d'ELISA cellulaire et de cytométrie en flux, nous avons démontré que la CENP-B se liait spécifiquement à la surface des CML vasculaire de l’artère pulmonaire avec une plus grande affinité pour le phénotype contractile que synthétique. Cette liaison provoquait la migration des cellules ainsi que la sécrétion de cytokines pro-inflammatoires telles que l’interleukine 6 et 8. Les mécanismes par lesquels la protéine exerçait ces effets impliquaient la phosphorylation de FAK et Src ainsi que la voie des MAP kinases, avec ERK1/2 et p38. Des études de signalisation intracellulaire effectuées à l’aide de plusieurs inhibiteurs spécifiques ainsi que des études de désensibilisation nous ont permis d’identifier le récepteur de la CENP-B en plus d’identifier les mécanismes complets de sa signalisation membranaire. Nous avons démontré que la CENP-B se liait de manière spécifique aux CML vasculaire via le récepteur de chémokine 3 (CCR3) pour ensuite transactiver le récepteur EGF, selon un mécanisme métalloprotéase-dépendant qui implique le relargage du HB-EGF. Cette transactivation est un processus important dans l’activation de la voie des MAP kinases ainsi que dans la sécrétion d’IL-8 induite par la CENP-B. Finalement, nous avons démontré que les auto-anticorps anti-CENP-B pouvaient abolir cette cascade de signalisation, empêchant ainsi la CENP-B d’exercer son rôle de cytokine. L’identification de la CENP-B comme ligand du CCR3 ouvre donc plusieurs perspectives quant à l’étude du rôle pathogène des auto-anticorps anti-CENP-B dans la ScS.