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Este trabalho apresenta o desenvolvimento e a caracterização de biovidros e vitrocerâmicos bioativos do sistema 3CaO.P2O5-SiO2-MgO, com índices de cristalinidade variados, visando o uso destes materiais em implantes estruturais ósseos. Amostras de composição nominal (% em peso) 57,75% CaO.P2O5 - 30% SiO2 - 17,25% MgO foram obtidas por fusão, e posteriormente, tratamentos térmicos foram realizados para a cristalização parcial dos vitrocerâmicos em temperaturas entre 700 ºC e 1100 ºC, com tempos diversos de até 480 minutos. Esses materiais foram caracterizados quanto ao seu comportamento térmico, quanto às fases cristalinas presentes e à microestrutura. Os resultados mostram que o vidro base apresenta separação de fases amorfas e os vitrocerâmicos apresentam como fase cristalina majoritária a fase whitlockite, podendo ser encontrada também a fase diopsídio, em tratamentos térmicos acima de 1050 °C. As propriedades mecânicas de dureza, tenacidade à fratura, módulo de fratura por ensaio de flexão e módulo elástico dinâmico foram determinadas. Todas as propriedades foram avaliadas em função do grau de cristalinidade do material, mostrando que a microestrutura e a fração cristalizada, bem como as diferentes fases cristalinas presentes, influenciam os resultados obtidos. O ganho máximo nas propriedades mecânicas ocorre em tratamentos térmicos a 975 °C, sendo atingidos valores próximos a 6,2 GPa, 1,7 MPa.m1/2 e 120 MPa para a dureza, a tenacidade à fratura e o módulo de fratura, respectivamente, em comparação aos valores de 4,8 GPa, 1,1 MPa.m1/2, 71 MPa dos vidros nucleados, o que indica aumento da ordem de 30% na dureza, 55% na tenacidade e 70% na resistência à fratura. Para o teste in vitro de citotoxicidade foram utilizadas células CHO como meio de cultura. Para os testes de bioatividade, as trocas iônicas entre os materiais e a solução acelular SBF ressaltam as diferenças em termos de reatividade química, sendo o efeito da cristalização de biovidros na formação da HCA observado por FTIR. Todas as amostras não apresentaram caráter citotóxico e o vitrocerâmico tratado a 775 °C mostrou ser o material mais bioativo, com início de formação da HCA em período de 24 h. Assim, os resultados obtidos indicam que o vidro e os vitrocerâmicos do sistema 3CaO.P2O5-SiO2-MgO são promissores candidatos ao uso como implantes ósseos, com distintas características e propriedades. / The current work presents the development and characterization of bioglass and bioactive glass-ceramic based on 3CaO.P2O5-SiO2-MgO-system, with different crystallization degrees, in order to use these materials in bone restorations. Samples of nominal composition (wt%) 57.75% CaO.P2O5-30% SiO2-17.25% MgO were obtained by melting and after heat-treated to partial crystallization of glass-ceramic at temperatures between 700 ºC and 1100 °C , with times up to 480 minutes. These materials were characterized by thermal analysis, X-Ray diffraction and scanning electron microscopy. The results showed amorphous phase separation in glasses. In glass-ceramics, it was identified whitlockite as major crystal phase, and diopside phase above 1050 °C. Hardness, fracture toughness, bending strength and elastic modulii of the samples were evaluated. These mechanical properties were evaluated according to the matrix crystallinity degree. The microstructure and the crystallization degree must be influencing the mechanical properties results. The optimized properties were observed in samples crystallized at 975 °C, with values near to 6.2 GPa, 1.7 MPa.m1/2 and 120 MPa for hardness, fracture toughness and bending strength, respectively, in comparison to 4.8 GPa, 1.1 MPa.m1/2 and 71 MPa presented by nucleated glasses at 700 °C, which indicates increases of 30% in hardness, 55% in fracture toughness and 70% in bending strength. In the cytotoxicity test, the culture medium was CHO cells. Ionic exchanges with the SBF-K9 acellular solution during the in vitro bioactivity tests highlight the differences in terms of chemical reactivity between the glass and the glassceramics. The effect of crystallinity on the rates of hydroxycarbonate apatite (HCA) formation was followed by FTIR. All samples were considered not cytotoxic. The glassceramic obtained at 775 °C presented the most interesting result, because onset for HCA formation is about 24 hours.The results obtained indicate that glasses and glass-ceramics of the 3CaO.P2O5-SiO2-MgO system are promissory candidates to be used as bonegraft implants.

O desenvolvimento de novos modelos de pele e novas metodologias in vitro segue uma tendência mundial na busca pela redução ou substituição de testes em animais. Nesse contexto, kits de epiderme humana reconstruída (RHE) apresentam-se como uma plataforma promissoras para essa proposta e, alguns modelos encontram-se validados para ensaios de irritação e corrosão cutânea in vitro. Entretanto, em países como o Brasil, enfrentam-se questões alfandegárias e perda do material por perecibilidade, dificultando e até impedindo, a importação desses kits para utilização por parte das indústrias e laboratórios nacionais. Em contrapartida, o desenvolvimento de um modelo de RHE apresenta-se como um avanço tecnológico e ganho de autonomia para esses países. Assim, no capítulo 1 explorou-se o desenvolvimento de um modelo nacional de RHE (USP-RHE) que atendesse às exigências internacionais descritas no guia OECD 439. O modelo desenvolvido apresentou uma epiderme bem diferenciada e atendeu aos parâmetros de qualidade (histologia, viabilidade e função barreira) bem como da funcionalidade, a qual é expressa na capacidade de distinção entre irritantes e não irritantes, apresentando 85,7% de especificidade, 100% sensibilidade e 92,3% de acurácia quando comparada com a classificação in vivo obtida pelo ensaio do linfonodo local (LLNA). No capítulo 2, células monocíticas THP-1 em monocamada foram capazes de distinguir entre agentes sensibilizantes e não sensibilizantes por meio da expressão de CD86, CD54 e liberação de IL-8. Após a obtenção de RHE e THP-1 funcionais, um cross-talking foi estabelecido gerando uma RHE imunocompetente. A RHEI distinguiu satisfatoriamente entre agentes sensibilizantes e não sensibilizantes por meio da expressão de CD86 e CD54 na membrana das células THP-1. A liberação de IL-8 também foi avaliada na RHEI, mas, não demonstrou ser um bom indicador para a avaliação de sensibilização, ao contrário de IL-1α, que distinguiu satisfatoriamente agentes sensibilizantes de não-sensibilizantes, mas não foi capaz de hierarquizá-los. No capítulo 3, avaliou-se o papel de interleucinas do tipo Th2 e da depleção de colesterol na membrana plasmática no desenvolvimento de características morfológicas e moleculares da dermatite atópica (DA) in vitro em um modelo de RHE. Os resultados demonstram que o uso de IL-4, IL-13 e IL-25 em combinação com a depleção de colesterol na membrana plasmática mimetiza in vitro, as principais características da DA. No capítulo 4, buscou-se avaliar os efeitos imunossupressores da radiação ultravioleta na RHEI. Os ensaios foram realizados em diferentes períodos de exposição, entretanto, não foi possível observar tais efeitos. Os resultados justificam-se pela ausência da liberação de IL-10 pelo RHE imunocompetente, por exemplo, e demonstram uma limitação do RHE imunocompetente para avaliações de inativação da reposta imune. Neste trabalho, concluímos que foi possível obter uma RHE competitiva, similar aos modelos internacionais validados e que pode ser utilizada como plataforma para ensaios de irritação e sensibilização cutânea, além de ser uma plataforma para estudos da dermatite atópica. No modelo é possível estudar a ativação do sistema imune, o que o torna promissor como uma plataforma para avaliação de resposta imunológica in vitro. Conclui-se, portanto, que os objetivos foram amplamente atendidos além de oferecermos um protocolo de livre acesso para reprodução por outros laboratórios e um modelo para validação futura. / The development of new in vitro skin models and new methodologies follows a global trend in search for reductions or replacement of animal testing. In this context, Reconstructed Human Epidermis kits (RHE) are presented as a promising platform in the search for alternative methods to animal use, and some models are validated for skin irritation and corrosion in vitro tests. However, in countries such as Brazil, who face customs issues and loss of material due to perishability, making it challenging and even compromising the importation of these kits for use by industries and laboratories. In contrast, the development of an RHE model is presented as a technological breakthrough and gain of autonomy for these countries. Thus, in Chapter 1 we explored the development of a national model of RHE (USP-RHE) that meet international requirements described in OECD TG 439. The developed model presented a well-differentiated epidermis and met the quality parameters, for instance, histology, viability, and barrier function as well as the functionality expressed in the capacity of screening between irritants and nonirritants, with 85.7 % of specificity, 100 % of sensitivity and 91.7% of accuracy in comparision to in vivo UN GHS classification from Local limph node assay (LLNA). In chapter 2, monocytic THP-1 cell line, as monolayers, were able to distinguish between sensitizers and non-sensitizers by expression of CD86, CD54, and IL-8 release. In this model, functional RHE and THP-1 were used in a cross-talking, and thus an immunocompetent RHE (RHEI) was generated. The RHEI has distinguished satisfactorily between sensitizers and non-sensitizers through CD86 and CD54 expression that was larger and more sensitive in this model. The release of IL-8 was also evaluated in RHEI, however, did not demonstrate to be a good parameter for this evaluation, unlike IL-1α, which satisfactorily distinguished sensitizers from non-sensitizers, but was not able to hierarchize them. In chapter 3, we evaluated the role of Th2-related cytokines and plasma membrane cholesterol depletion (CD) in the development of atopic dermatitis (AD) morphological and molecular characteristics in an in vitro model of RHE. The results showed that combination of IL-4, IL-13 and IL-25 in combination with CD can reproduce the major features of AD in vitro. In Chapter 4, we sought to evaluate the ultraviolet radiation-induced immunosuppressive effects in RHE. The tests were performed at different times. However, it was not possible to observe such effects. The results are justified by the absence of IL-10 release by RHEI, for example, and show a limitation of RHEI for rating inactivation of the immune response. In this work, we conclude that it was possible to obtain a competitive RHE similar to the validated international models that can be used as a platform for irritation and skin sensitization tests, besides being a platform for the study of atopic dermatitis. Using this model is possible to explore the activation of immune system, which makes it promising as a platform for the evaluation of immune response in vitro. We conclude, therefore, that the objectives have been met as well as it is offering an open source protocol for breeding by other laboratories, thus offering the RHE model developed here for future validation tests.

O desenvolvimento de novos modelos de pele e novas metodologias in vitro segue uma tendência mundial na busca pela redução ou substituição de testes em animais. Nesse contexto, kits de epiderme humana reconstruída (RHE) apresentam-se como uma plataforma promissoras para essa proposta e, alguns modelos encontram-se validados para ensaios de irritação e corrosão cutânea in vitro. Entretanto, em países como o Brasil, enfrentam-se questões alfandegárias e perda do material por perecibilidade, dificultando e até impedindo, a importação desses kits para utilização por parte das indústrias e laboratórios nacionais. Em contrapartida, o desenvolvimento de um modelo de RHE apresenta-se como um avanço tecnológico e ganho de autonomia para esses países. Assim, no capítulo 1 explorou-se o desenvolvimento de um modelo nacional de RHE (USP-RHE) que atendesse às exigências internacionais descritas no guia OECD 439. O modelo desenvolvido apresentou uma epiderme bem diferenciada e atendeu aos parâmetros de qualidade (histologia, viabilidade e função barreira) bem como da funcionalidade, a qual é expressa na capacidade de distinção entre irritantes e não irritantes, apresentando 85,7% de especificidade, 100% sensibilidade e 92,3% de acurácia quando comparada com a classificação in vivo obtida pelo ensaio do linfonodo local (LLNA). No capítulo 2, células monocíticas THP-1 em monocamada foram capazes de distinguir entre agentes sensibilizantes e não sensibilizantes por meio da expressão de CD86, CD54 e liberação de IL-8. Após a obtenção de RHE e THP-1 funcionais, um cross-talking foi estabelecido gerando uma RHE imunocompetente. A RHEI distinguiu satisfatoriamente entre agentes sensibilizantes e não sensibilizantes por meio da expressão de CD86 e CD54 na membrana das células THP-1. A liberação de IL-8 também foi avaliada na RHEI, mas, não demonstrou ser um bom indicador para a avaliação de sensibilização, ao contrário de IL-1α, que distinguiu satisfatoriamente agentes sensibilizantes de não-sensibilizantes, mas não foi capaz de hierarquizá-los. No capítulo 3, avaliou-se o papel de interleucinas do tipo Th2 e da depleção de colesterol na membrana plasmática no desenvolvimento de características morfológicas e moleculares da dermatite atópica (DA) in vitro em um modelo de RHE. Os resultados demonstram que o uso de IL-4, IL-13 e IL-25 em combinação com a depleção de colesterol na membrana plasmática mimetiza in vitro, as principais características da DA. No capítulo 4, buscou-se avaliar os efeitos imunossupressores da radiação ultravioleta na RHEI. Os ensaios foram realizados em diferentes períodos de exposição, entretanto, não foi possível observar tais efeitos. Os resultados justificam-se pela ausência da liberação de IL-10 pelo RHE imunocompetente, por exemplo, e demonstram uma limitação do RHE imunocompetente para avaliações de inativação da reposta imune. Neste trabalho, concluímos que foi possível obter uma RHE competitiva, similar aos modelos internacionais validados e que pode ser utilizada como plataforma para ensaios de irritação e sensibilização cutânea, além de ser uma plataforma para estudos da dermatite atópica. No modelo é possível estudar a ativação do sistema imune, o que o torna promissor como uma plataforma para avaliação de resposta imunológica in vitro. Conclui-se, portanto, que os objetivos foram amplamente atendidos além de oferecermos um protocolo de livre acesso para reprodução por outros laboratórios e um modelo para validação futura. / The development of new in vitro skin models and new methodologies follows a global trend in search for reductions or replacement of animal testing. In this context, Reconstructed Human Epidermis kits (RHE) are presented as a promising platform in the search for alternative methods to animal use, and some models are validated for skin irritation and corrosion in vitro tests. However, in countries such as Brazil, who face customs issues and loss of material due to perishability, making it challenging and even compromising the importation of these kits for use by industries and laboratories. In contrast, the development of an RHE model is presented as a technological breakthrough and gain of autonomy for these countries. Thus, in Chapter 1 we explored the development of a national model of RHE (USP-RHE) that meet international requirements described in OECD TG 439. The developed model presented a well-differentiated epidermis and met the quality parameters, for instance, histology, viability, and barrier function as well as the functionality expressed in the capacity of screening between irritants and nonirritants, with 85.7 % of specificity, 100 % of sensitivity and 91.7% of accuracy in comparision to in vivo UN GHS classification from Local limph node assay (LLNA). In chapter 2, monocytic THP-1 cell line, as monolayers, were able to distinguish between sensitizers and non-sensitizers by expression of CD86, CD54, and IL-8 release. In this model, functional RHE and THP-1 were used in a cross-talking, and thus an immunocompetent RHE (RHEI) was generated. The RHEI has distinguished satisfactorily between sensitizers and non-sensitizers through CD86 and CD54 expression that was larger and more sensitive in this model. The release of IL-8 was also evaluated in RHEI, however, did not demonstrate to be a good parameter for this evaluation, unlike IL-1α, which satisfactorily distinguished sensitizers from non-sensitizers, but was not able to hierarchize them. In chapter 3, we evaluated the role of Th2-related cytokines and plasma membrane cholesterol depletion (CD) in the development of atopic dermatitis (AD) morphological and molecular characteristics in an in vitro model of RHE. The results showed that combination of IL-4, IL-13 and IL-25 in combination with CD can reproduce the major features of AD in vitro. In Chapter 4, we sought to evaluate the ultraviolet radiation-induced immunosuppressive effects in RHE. The tests were performed at different times. However, it was not possible to observe such effects. The results are justified by the absence of IL-10 release by RHEI, for example, and show a limitation of RHEI for rating inactivation of the immune response. In this work, we conclude that it was possible to obtain a competitive RHE similar to the validated international models that can be used as a platform for irritation and skin sensitization tests, besides being a platform for the study of atopic dermatitis. Using this model is possible to explore the activation of immune system, which makes it promising as a platform for the evaluation of immune response in vitro. We conclude, therefore, that the objectives have been met as well as it is offering an open source protocol for breeding by other laboratories, thus offering the RHE model developed here for future validation tests.

Este trabalho apresenta o desenvolvimento e a caracterização de biovidros e vitrocerâmicos bioativos do sistema 3CaO.P2O5-SiO2-MgO, com índices de cristalinidade variados, visando o uso destes materiais em implantes estruturais ósseos. Amostras de composição nominal (% em peso) 57,75% CaO.P2O5 - 30% SiO2 - 17,25% MgO foram obtidas por fusão, e posteriormente, tratamentos térmicos foram realizados para a cristalização parcial dos vitrocerâmicos em temperaturas entre 700 ºC e 1100 ºC, com tempos diversos de até 480 minutos. Esses materiais foram caracterizados quanto ao seu comportamento térmico, quanto às fases cristalinas presentes e à microestrutura. Os resultados mostram que o vidro base apresenta separação de fases amorfas e os vitrocerâmicos apresentam como fase cristalina majoritária a fase whitlockite, podendo ser encontrada também a fase diopsídio, em tratamentos térmicos acima de 1050 °C. As propriedades mecânicas de dureza, tenacidade à fratura, módulo de fratura por ensaio de flexão e módulo elástico dinâmico foram determinadas. Todas as propriedades foram avaliadas em função do grau de cristalinidade do material, mostrando que a microestrutura e a fração cristalizada, bem como as diferentes fases cristalinas presentes, influenciam os resultados obtidos. O ganho máximo nas propriedades mecânicas ocorre em tratamentos térmicos a 975 °C, sendo atingidos valores próximos a 6,2 GPa, 1,7 MPa.m1/2 e 120 MPa para a dureza, a tenacidade à fratura e o módulo de fratura, respectivamente, em comparação aos valores de 4,8 GPa, 1,1 MPa.m1/2, 71 MPa dos vidros nucleados, o que indica aumento da ordem de 30% na dureza, 55% na tenacidade e 70% na resistência à fratura. Para o teste in vitro de citotoxicidade foram utilizadas células CHO como meio de cultura. Para os testes de bioatividade, as trocas iônicas entre os materiais e a solução acelular SBF ressaltam as diferenças em termos de reatividade química, sendo o efeito da cristalização de biovidros na formação da HCA observado por FTIR. Todas as amostras não apresentaram caráter citotóxico e o vitrocerâmico tratado a 775 °C mostrou ser o material mais bioativo, com início de formação da HCA em período de 24 h. Assim, os resultados obtidos indicam que o vidro e os vitrocerâmicos do sistema 3CaO.P2O5-SiO2-MgO são promissores candidatos ao uso como implantes ósseos, com distintas características e propriedades. / The current work presents the development and characterization of bioglass and bioactive glass-ceramic based on 3CaO.P2O5-SiO2-MgO-system, with different crystallization degrees, in order to use these materials in bone restorations. Samples of nominal composition (wt%) 57.75% CaO.P2O5-30% SiO2-17.25% MgO were obtained by melting and after heat-treated to partial crystallization of glass-ceramic at temperatures between 700 ºC and 1100 °C , with times up to 480 minutes. These materials were characterized by thermal analysis, X-Ray diffraction and scanning electron microscopy. The results showed amorphous phase separation in glasses. In glass-ceramics, it was identified whitlockite as major crystal phase, and diopside phase above 1050 °C. Hardness, fracture toughness, bending strength and elastic modulii of the samples were evaluated. These mechanical properties were evaluated according to the matrix crystallinity degree. The microstructure and the crystallization degree must be influencing the mechanical properties results. The optimized properties were observed in samples crystallized at 975 °C, with values near to 6.2 GPa, 1.7 MPa.m1/2 and 120 MPa for hardness, fracture toughness and bending strength, respectively, in comparison to 4.8 GPa, 1.1 MPa.m1/2 and 71 MPa presented by nucleated glasses at 700 °C, which indicates increases of 30% in hardness, 55% in fracture toughness and 70% in bending strength. In the cytotoxicity test, the culture medium was CHO cells. Ionic exchanges with the SBF-K9 acellular solution during the in vitro bioactivity tests highlight the differences in terms of chemical reactivity between the glass and the glassceramics. The effect of crystallinity on the rates of hydroxycarbonate apatite (HCA) formation was followed by FTIR. All samples were considered not cytotoxic. The glassceramic obtained at 775 °C presented the most interesting result, because onset for HCA formation is about 24 hours.The results obtained indicate that glasses and glass-ceramics of the 3CaO.P2O5-SiO2-MgO system are promissory candidates to be used as bonegraft implants.

Les écosystèmes aquatiques constituent la destination finale des contaminants. Les organismes aquatiques sont ainsi impactés par un cocktail de molécules, dont les produits phytosanitaires. Le caractère perturbateur endocrinien (PE) de certains d’entre eux est particulièrement préoccupant. La présente étude explore les effets de pesticides modèles à potentiel PE – la chlordécone (CLD) et le pyriproxyfène (PXF), seuls et en mélange – chez le copépode estuarien Eurytemora affinis par des approches écotoxicogénomiques innovantes. En réponse aux PE, l’accent a été porté sur les effets de ces composés sur la reproduction, la croissance ou le développement. En parallèle, l’affinité des pesticides aux récepteurs aux oestrogènes et aux androgènes humains d’une part, et aux ecdystéroïdes d’autre part, a été étudiée grâce à des outils bio analytiques afin d’évaluer la pertinence de l’utilisation du potentiel PE défini par les tests cellulaires comme critère d’exclusion dans la réglementation européenne des pesticides. Ces travaux de thèse présentent, pour la première fois, les transcriptomes et protéomes d’E. affinis après exposition aux PE. Un effet sexe-dépendant a été observé par les deux approches « omics » révélant un plus fort impact des composés sur les copépodes mâles. Des gènes et protéines, impliqués dans le processus de mue et dans la gamétogénèse ont été identifiés comme candidats pour le développement de biomarqueurs moléculaires de PE chez les crustacés. Les tests cellulaires ont permis de mettre en évidence des interactions entre les contaminants et les récepteurs hormonaux. Toutefois, le screening de molécules par les tests YES/YAS/anti-YES/anti-YAS s’est heurté à quelques limites d’interprétations. Enfin, les résultats du test S2 EcR, souligne la pertinence de l'utilisation couplée de tests spécifiques d’un système endocrinien de vertébrés et d’invertébrés dans l’évaluation du risque des composés PE. / Aquatic ecosystems constitute the chemicals’ final destination. Among the xenobiotics, endocrine disruptors (ED) are compounds of major concern. In this context, ED pesticides effects were investigated in the widespread copepod Eurytemora Affinis using ecotoxicogenomics technics. In response to PE pesticides, focus was made on reproduction, growth and development. In order to evaluate the endocrine activity, in vitro tests (YES/YAS/anti-YES/anti-YAS and S2 EcR) were used to screen the compounds alone and in mixtures. These results were discussed to evaluate the “cut off criterion” used in European assessment. This work presents for the first time, transcriptomes and proteomes of E. affinis after PXF and CLD –alone and in binary mixture- exposure. A sex dependent effect was observed by the two “omics” approaches. Male copepods were more impacted by contaminants than female copepods. Genes and proteins (e.g. chitin deacetylase, kelch protein) were identified as candidates for the development of ED molecular biomarkers. In vitro tests highlighted binding of pesticides with both vertebrate and invertebrate receptors. However, a toxicity for the highest concentrations tested and some limits for the interpretation of mixtures results were limiting in YES/YAS/anti-YES/anti-YAS assays. The last observation represents particularly a major concern for interpretation of the ED mode of action of environmental matrix. S2 EcR highlighted some complementary results about the mode of action of chemicals alone and in binary mixtures. These results accentuate the need to combine vertebrate specific test and invertebrate specific test in ED risk assessment.

This dissertation examines the ability of in vitro biotransformation assays to provide an indication of metabolic potential. The potential for xenobiotic compounds to bioconcentrate in aquatic organisms is expressed through the bioconcentration factor (BCF). The metabolic loss of ibuprofen, norethindrone and propranolol was measured using rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) gill and liver S9 fractions, microsomes and cell suspensions. Metabolic transformation rates (kM) were extrapolated from in vitro intrinsic clearance of parent compound (CLm) and integrated into a refined BCF model. In general, CLm of test compounds was greater in liver S9 fractions and hepatocytes. However, the influence of hepatic metabolism on kM and BCF was limited by hepatic blood flow (20-25%) compared to gill blood flow (~100%). A significant difference was noted between BCF solely based on KOW and BCF including kM. These studies indicate that the inclusion of kM in BCF models can bring predicted bioconcentration estimates closer to in vivo values. Primary cell suspensions are preferred over subcellular fractions as cell suspensions possess both phase I and phase II enzyme activity. Further study was conducted on ibuprofen biotransformation pathways. As fish do not contain the same cytochrome P450 (CYP) 2C homologs known to metabolize ibuprofen in mammals, it cannot be assumed that piscine biotransformation is similar. Metabolite analysis found 2-hydroxy-ibuprofen as the major metabolite in S9 and microsomal fractions. Additional assays involving the induction and inhibition of specific CYP isozymes support CYP1A2 as an alternative metabolic pathway.

<p>Humans are exposed to metals and metal-containing materials daily, either conscious, e.g. using metal tools or objects, or unconscious, e.g. during exposure to airborne metal-, and metal-containing particles. The diffuse dispersion of metals from different sources in the society, and the concern related to its potential risk for adverse effects on humans have gained an increased public and governmental attention both on a national and international level. In this context, the knowledge on metal release from metallic objects or metal-containing particles is essential for health risk assessment.</p><p>This thesis focuses on the study of metal release from powder particles of stainless steel and Cu-based materials exposed to synthetic body fluids mainly for simulating lung-like environments. The study comprises: i) development of a suitable experimental method for metal release studies of micron sized particles, ii) metal release data of individual alloy constituents from stainless steel powder particles of different particle sizes, and iii) Cu release from different Cu-based powder particles. In addition, the influence of chemical and physical properties of metallic particles and the test media are investigated. Selected results from Ni powder particles exposed to artificial sweat are presented for comparison. The outcome of this research is summarized through ten questions that are formulated to improve the general understanding of corrosion-induced metal release from metallic particles from a health risk perspective.</p><p>A robust, reproducible, fairly simple, and straightforward experimental procedure was elaborated for metal release studies on particles of micron or submicron size. Results in terms of metal release rates show, for stainless steel powder particles, generally very low metal release rates due to a protective surface oxide film, and Fe preferentially released compared to Cr and Ni. Metal release rates are time-dependent for both stainless steel powder particles and the different Cu-containing powders investigated. The release of Cu from the Cu-containing particles depends on the chemical and compositional properties of the Cu-based material, being either corrosion-induced or chemically dissolved. Moreover, the test medium also influences the metal release process. The metal release rate increases generally with decreasing pH of the test media. However, even at a comparable pH, the release rate may be different due to differences in the interaction between the particle surface and specific media.</p><p>The nature of particles is essentially different compared to massive sheet in terms of physical shape, surface composition and morphology. The surface area, and even the surface composition of metallic particles, depend on the particle size. The specific surface area of particles, area per mass, is intimately related to the particle size and has a large effect on the metal release process. Release rates increase with decreasing particle size due to a larger active surface area that takes part in the corrosion/dissolution process. The surface area that actually is active in the corrosion and metal release process (the effective area) governs the metal release process for both particles and massive sheet of metals or alloys. For particles, the effective surface area depends also on agglomeration conditions of particles during exposure.</p>

Les allergies représentent un problème majeur dans le domaine des maladies professionnelles et ont un impact sérieux sur la vie des travailleurs. Les allergies professionnelles sont principalement cutanées et respiratoires ; elles peuvent être causées par des produits chimiques de bas poids moléculaire. Dans le passé, les tests destinés à identifier les produits susceptibles d’entraîner des allergies étaient réalisés sur l’animal. Or, la législation européenne engage à limiter le recours à l’expérimentation animale pour évaluer le pouvoir sensibilisant des substances chimiques, incitant à développer des tests in vitro de substitution. C’est dans ce contexte que nous avons cherché à développer des modèles de cultures cellulaires destinés à identifier les substances sensibilisantes. Un premier modèle utilisant des cellules dendritiques dérivées de moelle osseuse (BMDC) de souris BALB/c a été développé et a donné des résultats prometteurs pour l’identification des produits sensibilisants et leur catégorisation selon leur puissance sensibilisante. De plus, la voie de signalisation Nrf2/Keap1 semble être impliquée dans la réponse de ce modèle cellulaire aux sensibilisants. Dans le but de compléter ce modèle et d’évaluer la capacité des BMDC à activer les lymphocytes T (LT), un modèle de coculture de BMDC et LT a été mis au point avec un sensibilisant de référence avant d’être testé sur un ensemble de produits de référence (sensibilisants cutanés et respiratoires, irritants et non sensibilisants). Les BMDC de notre modèle, exposées à des sensibilisants, se sont révélées capables d’activer les LT en coculture. Enfin, des essais préliminaires utilisant des cellules de souris de souche C57BL6/J dans notre modèle de coculture ont donné des résultats comparables à ceux obtenus avec des cellules issues de la souche BALB/c. Les modèles de cultures cellulaires BMDC et de coculture BMDC-LT sont prometteurs dans le cadre du développement de méthodes de substitution à l’expérimentation animale pour l’évaluation du pouvoir sensibilisant de substances chimiques / Allergies constitute an important issue in the field of occupational health and have a serious impact on the lives of workers. Occupational allergies are mainly contact and respiratory allergies and can be caused by low molecular weight chemicals. In the past, the tests that were used to identify the potential allergens were carried out on animals. However, European legislation has provided the impetus for reducing the use of animal testing to assess the sensitization potential of chemicals and promoted the development of alternative in vitro tests. In this context, we aimed to develop cell culture models to identify sensitizers. A first model using bone marrow derived dendritic cells (BMDC) from BALB/c mice was developed and showed promising results for identifying sensitizers and classify them according to their allergenic potency. Moreover, the Nrf2/Keap1 pathway seems to be involved in the response of this cell model to sensitizers. In order to supplement this model and to assess the functionality of BMDC, a BMDC-T cell (TC) coculture model was developed with a reference sensitizer before being tested on a range of reference sensitizers (cutaneous and respiratory sensitizers, irritants and non-sensitizers). The BMDC of our model, while exposed to sensitizers, were able to activate TC in coculture. Finally, preliminary tests using the cells of C57BL6/J mice in our coculture model showed that similar results to those obtained with cells from the BALB/c strain. The models of BMDC cultures and BMDC-TC coculture are promising for the development of alternative methods to animal experimentation assessing the sensitizing potential of chemicals

Humans are exposed to metals and metal-containing materials daily, either conscious, e.g. using metal tools or objects, or unconscious, e.g. during exposure to airborne metal-, and metal-containing particles. The diffuse dispersion of metals from different sources in the society, and the concern related to its potential risk for adverse effects on humans have gained an increased public and governmental attention both on a national and international level. In this context, the knowledge on metal release from metallic objects or metal-containing particles is essential for health risk assessment. This thesis focuses on the study of metal release from powder particles of stainless steel and Cu-based materials exposed to synthetic body fluids mainly for simulating lung-like environments. The study comprises: i) development of a suitable experimental method for metal release studies of micron sized particles, ii) metal release data of individual alloy constituents from stainless steel powder particles of different particle sizes, and iii) Cu release from different Cu-based powder particles. In addition, the influence of chemical and physical properties of metallic particles and the test media are investigated. Selected results from Ni powder particles exposed to artificial sweat are presented for comparison. The outcome of this research is summarized through ten questions that are formulated to improve the general understanding of corrosion-induced metal release from metallic particles from a health risk perspective. A robust, reproducible, fairly simple, and straightforward experimental procedure was elaborated for metal release studies on particles of micron or submicron size. Results in terms of metal release rates show, for stainless steel powder particles, generally very low metal release rates due to a protective surface oxide film, and Fe preferentially released compared to Cr and Ni. Metal release rates are time-dependent for both stainless steel powder particles and the different Cu-containing powders investigated. The release of Cu from the Cu-containing particles depends on the chemical and compositional properties of the Cu-based material, being either corrosion-induced or chemically dissolved. Moreover, the test medium also influences the metal release process. The metal release rate increases generally with decreasing pH of the test media. However, even at a comparable pH, the release rate may be different due to differences in the interaction between the particle surface and specific media. The nature of particles is essentially different compared to massive sheet in terms of physical shape, surface composition and morphology. The surface area, and even the surface composition of metallic particles, depend on the particle size. The specific surface area of particles, area per mass, is intimately related to the particle size and has a large effect on the metal release process. Release rates increase with decreasing particle size due to a larger active surface area that takes part in the corrosion/dissolution process. The surface area that actually is active in the corrosion and metal release process (the effective area) governs the metal release process for both particles and massive sheet of metals or alloys. For particles, the effective surface area depends also on agglomeration conditions of particles during exposure. / QC 20101119