Les matériaux au contact des aliments (MCDA) constituent un enjeu majeur pour les industriels européens de l’alimentation et de l’emballage d’autant que les MCDA doivent être produits selon les bonnes pratiques de fabrication et être en accord avec les règlementations, notamment le règlement cadre européen 1935/2004. Les MCDA constituent une source potentielle de contamination des aliments car ils peuvent transférer des constituants aux aliments par migration. Parmi les MCDA, les matériaux papiers-cartons sont les plus utilisés après les matériaux plastiques et leur taux de recyclage en Europe était de 72% en 2015 et il ne cesse d’augmenter. En plus des substances dites intentionnelles (SI), des substances non intentionnelles (SNI) peuvent migrer du MCDA vers l’aliment ; elles peuvent être des impuretés, des produits de dégradation, des contaminants provenant des matériaux recyclés etc. Actuellement, de nombreuses SNI ne sont pas identifiées. La présence potentielle de SNI et la nature complexe de ces matériaux rendent difficile l’étude des MCDA papiers-cartons recyclés. Ce projet de thèse a pour objectif d’évaluer le danger lié de ces matériaux. Pour cela, des papiers-cartons recyclés ont été prélevés le long d’une chaine de production (du papier-carton à recycler de début de chaine au papier-carton issu du processus de recyclage), extraits dans l’eau puis lyophilisés. Des analyses chimiques ont été réalisées par spectrophotométrie et par des méthodes chromatographiques (HPLC, GC-MS, LC-MS, ICP-MS). Ces analyses ont montré la présence d’un plus grand nombre de substances dans le papier-carton de début de chaine par rapport au papier-carton de fin de chaine de recyclage. Parmi les composants détectés, des substances naturelles ont été identifiées dans les deux extraits étudiés, provenant notamment du bois qui est la matière première utilisée pour la fabrication du papier (polymères de cellulose, d’hémicellulose et de lignine, acides gras, sucres, acides résiniques ou encore des phénols). D’autres substances identifiées comme additifs ou contaminants ont été caractérisées dans les extraits étudiés, dont bisphénols, des molécules appartenant à la famille des phthalates, des amines aromatiques ou encore des hydrocarbures saturés d’huiles minérales. Il est à souligner qu’une grande partie des substances identifiées dans le produit de début de chaine étaient absentes du produit de fin de chaine. En parallèle des analyses chimiques, des biotests ont été réalisés afin d’identifier des mécanismes d’action et des potentiels effets « cocktail ». Grâce à une batterie de biotests, les effets des extraits sur des cibles toxicologiques ont été étudiés sur différentes lignées cellulaires humaines (deux lignées hépatiques HEpG2 et HepaRG, la lignée hERα-HeLa-9903 et la lignée MDA-kb2): cytotoxicité, génotoxicité et potentiel perturbateur endocrinien. A la plus forte concentration testée, seul l’extrait de produit de fin de chaine a entrainé un effet cytotoxique sur la lignée MDA-kb2. Cependant, les mêmes conditions de test ont montré que les produits de départ et de fin de chaine pouvaient entrainer un stress oxydant dans les lignées hépatiques utilisées. L’évaluation de la génotoxicité a montré des effets génotoxiques des deux papiers-cartons étudiés. Cependant, contrairement aux lésions du matériel génétique induites par le produit de départ, les dommages de l’ADN dus au produit de de fin de chaine pourraient ne pas être réparables pas les systèmes de réparation cellulaires et entrainer des mutations chromosomiques. Enfin, l’étude du potentiel perturbateur endocrinien a mis en évidence des activités oestrogéniques et antiandrogéniques dose-dépendantes des extraits étudiés. Ainsi, la mise en corrélation des résultats d’analyses chimiques et toxicologiques permet d’offrir une stratégie pertinente pour l’évaluation du danger lié aux MCDA, afin d’aider les industriels dans l’évaluation du risque des potentiels NIAS présents dans ces matériaux. / Food contact materials (FCM) are a major issue of concern for European packaging and food companies as FCM must be produced in accordance with the good manufacturing practices and be in compliance with European Regulation 1935/2004/UE. FCM could be a potential source of food contamination because they could release chemicals by a phenomenon called migration. Among FCM, paperboards are the most common materials after plastics and the recycling rate of paperboards is high and was 72% in Europe in 2015. In addition to started substances also called Intentionally Added Substances (IAS), Non-Intentionally Added Substances (NIAS) such as impurities, degradation products, contaminants from recycled materials etc, are also able to migrate from the FCM to food and many of them are not always fully characterized. Assessing the risk of recycled paperboards FCM is difficult because of NIAS and as recycled paperboards can be complex mixtures of substances. This thesis project uses a methodology combining chemical analysis and in vitro bioassays to evaluate the potential hazard of recycled food contact paperboard extracts. For this study, recycled paperboards have been sampled at different steps a food packaging production chain (the starting paperboard of the beginning of the production chain and the end paperboard issued from the recycling process), extracted with water and the dry matter content was recovered by freeze-drying. Chemical analysis of the extracts was performed by spectrophotometry and by chromatographic methods (HPLC, GC-MS, LC-MS and ICP-MS). Chemical analysis showed the presence of a higher number of substances in the starting paperboard than the end paperboard issued from the recycling process. Among these substances, several natural substances were detected in both extracts such as wood extractives (polymers of cellulose, hemicellulose and lignin, fatty acids, sugars, resin acids and phenols). Other substances identified as additives and contaminants were characterized in both extracts such as bisphenols, phthalates, aromatic amins or mineral oil saturated hydrocarbons. It is important to precise that a large number of the substances of the starting paperboard were not detected in the end paperboard. In parallel to chemical characterization, bioassays were used as relevant tools to identify mechanisms of action and potential “cocktail effects”. A battery of bioassays has been performed using different human cell lines (two hepatic cell lines HepG2 and HepaRG, the hERα-HeLa-9903 cell line and the MDA-kb2 cell line) to check toxicological endpoints which are relevant to low exposure: cytotoxicity, genotoxicity and potential endocrine disruption. No cytototoxic effects of both extracts were observed on both human hepatic cell lines HepG2 and HepaRG neither on the HeLa 9903 cell line; whereas the end paperboard extract had cytotoxic effects on MDA-kb2 cells at the highest tested concentration. However, in the same conditions, the starting and the recycled end paperboard extracts were able to induce oxidative stress in the used cell lines. The results of the genotoxicity study showed genotoxic effects of the starting paperboard and the end paperboards. The DNA damage induced by the starting paperboard in the used cellular models might be repaired by cell repair systems. In contrast, data of this study suggest that the end paperboard was able to induce DNA damage that might turn into chromosomal mutations. Data of the endocrine disruption potential showed that the tested FCM paperboard extracts induced significant agonist and dose dependent estrogenic and antiandrogenic activities in the used cell lines. The correlation of the results of the in vitro toxicological study and chemical analysis offers a relevant strategy for hazard assessment of FCM in order to help manufacturers to manage the risk of NIAS.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

Made available in DSpace on 2016-03-15T14:17:06Z (GMT). No. of bitstreams: 2 3.pdf: 1873007 bytes, checksum: 8eae9ab2248d143593f9cbc784373efc (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / As vacinas são produtos imunobiológicos reconhecidamente seguros, eficazes e com grande participação no controle e/ou erradicação de doenças imunopreveníveis, comprovando uma grande relação custo-benefício. Atualmente, Bio-Manguinhos é o principal fornecedor de vacinas para o Ministério da Saúde e para suprir as demandas do Programa Nacional de Imunização utiliza como uma das estratégias de fornecimento, acordos de transferência de tecnologia com empresas mundiais, como a GlaxoSmithKline e a Sanofi Pasteur, com o objetivo de suprir demandas epidemiológicas brasileiras e adquirir desenvolvimento tecnológico para a instituição, o que caracteriza um processo inovador de grande relevância. Os produtos gerados através deste processo precisam ser submetidos a testes de Controle de Qualidade e pesquisa clínica para comprovar a qualidade e a confirmação terapêutica, através de metodologias analíticas e bioanalíticas (bioensaios) validadas. Este trabalho tem como objetivo avaliar a aplicabilidade da RDC 27 da ANVISA e analisar os demais documentos de validação de bioensaios preconizados pela OMS, FDA, USP e EMEA. Para isto, foi realizada uma análise desses documentos para a verificação de diferenças, semelhanças e aplicação. Um estudo de caso foi realizado através da validação do teste de neutralização por redução de placa de lise para sarampo em placas de 96 poços (MICRO-PRNT) que é considerado pela literatura o método mais sensível e específico para a qualificação e quantificação de anticorpos neutralizantes produzidos após a vacinação. Porém, A validação foi desenhada de maneira mais direcionada as características particulares da metodologia, utilizando parâmetros e critérios de aceitação pertinentes, e as diretrizes aplicáveis dos órgãos citados acima. A validação proposta neste estudo apresentou resultados satisfatórios onde todos os parâmetros estabelecidos foram validados. Pode-se concluir que a RDC 27 é direcionada a bioensaios com característica cromatográfica e que os documentos dos demais órgãos também não apresentam uma aplicabilidade total. Na realidade é necessário que a ANVISA elabore um guia ou legislação de validação de metodologia que atenda as particularidades dos bioensaios utilizados nas pesquisas clínicas de vacinas para comprovação de imunogenicidade. / Vaccines are immunobiological products and are known to be safe, effective and with great participation in the control and / or eradication of immunopreventable diseases, proving that is an excelent cost benefit relation. Currently, Bio-Manguinhos is the main provider of vaccines for the Ministry of Health and to meet the demands of the National Immunization Program and uses as a strategy of supply, technology transfer agreements with global companies such as GlaxoSmithKline and Sanofi Pasteur, aiming to meet brazilian epidemiological demands and acquire technological development for the institution, configuring an innovative process of great relevance. The products generated through this process need to undergo Quality Control testing and clinical research to demonstrate the quality and therapeutic effectiveness through analytical and bioanalytical methods (bioassays) validated. This study aims to evaluate the applicability of the RDC 27 ANVISA and analyze other documents validation of bioassays recommended by WHO, FDA, USP and EMEA. For this, an analysis of these documents for verification of differences, similarities and application was done. A case study was performed through validation by reduction neutralization test for lysis plate for measles in 96-well plates (MICRO-PRNT) which is considered by the literature the most sensitive and specific method for qualification and quantification of neutralizing antibodies produced after vaccination. However, validation was designed in a more direct way to particular characteristics of the methodology, using parameters and acceptance criteria that are relevant and also applicable directives of the organs mentioned above. The validation proposed in this study showed satisfactory results where all parameters set were validated. It can be concluded that the RDC 27 is directed to bioassays with chromatographic characteristics and also that the documents of the other organs did not show an overall applicability. In reality, it is necessary that ANVISA prepares a guide or legislation of methodology validation that meets the particularities of bioassays used in clinical research of vaccines to prove the immunogenicity and also be useful for other bioassays with biological targets.

O aumento da resistência microbiana devido a fatores como uso excessivo e ineficiente de antibióticos convencionais acarreta a necessidade da busca por novos compostos bioativos que atuem por mecanismos de ação diferentes aos fármacos já conhecidos. Na agricultura, o uso intensivo de pesticidas para o combate de microrganismos que comprometem principalmente a parte alimentícia também traz diversos problemas relacionados à resistência antimicrobiana e a riscos ambientais, oriundos do acúmulo dessas substâncias no solo. Dentro deste aspecto, o pseudofungo Pythium aphanidermatum, da classe dos oomicetos, destaca-se por ser uma espécie agressiva e altamente resistente a fungicidas comuns, apodrecendo raízes e frutos de cultivos de tomate, beterraba, pepino, pimentão, etc. A própolis verde, constituída em sua grande parte por material resinoso coletado e processado pela abelha da espécie Apis mellifera tem sido utilizada na medicina tradicional devido ao seu amplo espectro de ações preventivas e tratamentos de doenças, possuindo propriedades anti-inflamatórias, antimicrobianas, anticancerígenas e antioxidantes, tornando-se um produto de grande interesse na busca de novos compostos bioativos. Dentro destes aspectos apresentados, neste trabalho investigamos a ação da própolis verde contra o fitopatógeno P. aphanidermatum e identificamos através da técnica de cromatografia e bioensaios que a Artepillin C (3,5-diprenil-4-ácido-hidroxicinâmico), majoritária na própolis verde, foi o principal composto nesta ação. Os efeitos terapêuticos desta molécula tem sido foco de muitos estudos, porém ainda não há evidência em sua interação com agregados anfifílicos que mimetizam membranas celulares. O caráter anfifílico do composto, elevado pela presença dos grupos prenilados ligados ao ácido cinâmico, favoreceram a sua inserção nas membranas modelo, principalmente em seu estado agregado. Estas conclusões puderam ser inferidas devido às alterações nas propriedades das bicamadas lipídicas na presença da Artepillin C, podendo causar, especificamente para o caso de fitopatógenos como o P. aphanidermatum, perdas funcionais das proteínas de membranas, liberação de eletrólitos intracelulares e desintegração citoplasmática dos micélios e esporos. Ainda, as diferentes composições lipídicas nas vesículas influenciam no modo de interação do composto e consequentes alterações em suas estruturas, principalmente na presença do colesterol, que auxilia na manutenção da permeabilidade da bicamada lipídica, que pode contribuir para a integridade do conteúdo citoplasmático da célula. / The increase in the microbial resistance due to the excessive and inefficient use of conventional antibiotics brings the necessity to search new bioactive compounds which play their mechanism of action differently from the known drugs. In the agriculture, the intensive use of pesticide for the combat of microorganisms which undermine mainly the food portion also brings several issues related to the antimicrobial resistance and environment risks, originated from the high amount of these substances on the soil. In this aspect, the fungus-like Pythium aphanidermatum microorganism, from class Oomycete, stands out for being an aggressive species and highly resistant to common fungicides, rotting roots and fruits of tomato, beet, cucumber, pepper, etc. Green propolis, constituted by resinous material collected and processed by bees of the species Apis mellifera, has been used in the traditional medicine due its wide spectrum of preventive actions and diseases treatments, promoting anti-inflammatory, antimicrobial, anticancer and antioxidant properties, becoming a product of interest for investigation in the research of new bioactive compounds. Under all the aspects showed so far, in this work we investigated the action of the green propolis against the phytopathogen P. aphanidermatum and identified through chromatography and bioassays that Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid), majority in the green propolis, was the main compound in this action. The therapeutic effects of this molecule have been the focus of several studies, but, so far there is no evidence for its interaction with amphiphilic aggregates that mimic cell membranes. The amphiphilic character of the compound, enhanced by the presence of two prenylated groups bounded to the cinnamic acid, favors the insertion of the compound in the model membranes mainly in its aggregation state. These conclusions could be inferred due the alterations in the properties of the lipid bilayer in the presence of Artepillin C, that may cause, specifically in the case of phytopathogens like P. aphanidermatum, functional losses of membrane proteins, releasing of intracellular electrolytes and cytoplasmatic disintegration of mycelium and spores. Moreover, the difference of the lipid composition in the vesicles influence in the action of the compound and consequent alteration in their structures, mainly in the presence of cholesterol, that provides the maintenance of permeability of the lipid bilayer, contributing to the integrity of the cytoplasmic material of the cell.

INTRODUÇÃO: Estudos indicam uma prevalência de 20 a 30% de hiperprolactinemia discreta em pacientes com lúpus eritematoso sistêmico (LES), sugerindo um possível papel da prolactina (PRL) na sua etiopatogenia. Como a expressão do gene da PRL é encontrada na maioria das células do sistema imunológico, onde atua como citocina, de forma parácrina e autócrina, a origem linfocitária desta PRL tem sido aventada. OBJETIVOS: estudar a expressão do gene da PRL linfocitária de pacientes com LES em atividade e inatividade de doença e de controles normais, e sua atividade biológica em bioensaios com células Nb2 e Ba/F-LLP; Determinar o nível sérico de PRL e a prevalência de macroprolactinemia numa população nossa com LES. MÉTODOS: grupo 1, composto de 73 pacientes (66 mulheres e 7 homens), sendo 28 pacientes com LES em atividade e 45 em inatividade de doença, onde foi avaliado o nível de PRL sérica e a prevalência da macroprolactinemia; grupo 2, derivado do grupo 1, com 30 pacientes: 18 com LES em atividade e 12 em inatividade e um grupo controle com 10 indivíduos normais, dos quais foram extraídos linfócitos do sangue periférico e colocados em cultura por 72 horas. Em seguida, o sobrenadante da cultura foi utilizado como amostra de PRL linfocitária em ensaios com células Nb2 (heterólogo) e Ba/F-LLP (homólogo) para avaliação da bioatividade. Os RNAs totais destes linfócitos foram extraídos e usados na RT-PCR em tempo real (método quantitativo), para comparar a expressão do gene da PRL em linfócitos de pacientes com LES em atividade e inatividade, utilizando pool de indivíduos normais como calibrador. RESULTADOS: hiperprolactinemia discreta foi encontrada em 21,9% (16 de 73 pacientes do grupo 1): 7 de 28 pacientes com LES em atividade (25%), e 9 de 45 em inatividade (20%). A presença de macroprolactinemia foi encontrada em 3 pacientes, todos com LES em inatividade. O nível de PRL sérica: grupo 1 (LES em atividade) teve mediana de 10,8 (4,9 38,9) ng/mL e o grupo 1 (LES em inatividade) mediana de 7,6 (1,9 49,6) ng/mL, não havendo diferença significante entre os dois subgrupos (p=0,123). No entanto, quando consideramos apenas o nível sérico da PRL monomérica, a mediana da PRL do grupo 1 (LES em inatividade) caiu para 7,3 ng/mL (1,9 20,6) ng/mL e assim, quando comparado novamente ao grupo 1 (LES em atividade), observamos que além de uma porcentagem maior dos casos em atividade apresentarem hiperprolactinemia, a mediana da PRL monomérica nesses pacientes é significantemente maior que nos pacientes em inatividade de doença (p= 0,016). Os bioensaios foram realizados com as amostras do grupo 2 (subgrupo do grupo 1): no ensaio com células Nb2, a bioatividade da PRL linfocitária foi semelhante, não havendo diferença significante entre os pacientes com LES em atividade e inatividade e desses com o grupo controle. Já o bioensaio com a Ba/F-LLP não mostrou sensibilidade adequada, portanto não sendo confiável para avaliação da PRL linfocitária. O RT-PCR em tempo real apresentou expressão gênica também semelhante entre os pacientes avaliados (grupo 2). CONCLUSÕES: A expressão do gene da PRL linfocitária e a sua bioatividade foram semelhantes nos pacientes com LES em atividade e inatividade. A prevalência de hiperprolactinemia nos nossos pacientes com LES foi de 21,9%, sendo maior nos pacientes com atividade de doença. A macroprolactinemia só foi encontrada em pacientes com LES inativo, sugerindo um possível efeito protetor deste achado. / INTRODUCTION: Studies point to a prevalence of 20-30% of discrete hyperprolactinemia in patients with systemic lupus erythematosus (SLE), suggesting a possible implication of prolactin (PRL) in the pathogenesis of this disorder. As the lymphocytic PRL gene expression is found in the majority of the immune cells, where it acts as citokine, by paracrine and autocrine regulation, the lymphocytic source of this PRL has been suggested. OBJECTIVES: 1) to study the lymphocytic PRL gene expression of patients with active and inactive SLE and normal controls, as well as its biological activity in bioassays using Nb2 (heterologous) and Ba/F-LLP cells (homologous); 2) To assess serum PRL level and the prevalence of macroprolactinemia in our population with SLE. METHODS: group 1, composed of 73 patients (66 women and 7 men), 28 patients with active and 45 with inactive SLE, where the serum PRL level and prevalence of macroprolactinemia were evaluated; group 2, a subset of group 1, with 30 patients: 18 with active and 12 with inactive SLE and 10 normal individuals as control group, from whom lymphocytes were extracted from peripheral blood and were set on culture for 72 hours. After that, the supernatant was taken as lymphocytic PRL samples in bioassays with Nb2 cells (heterologous) and Ba/FLLP (homologous) in order to assess its bioactivity. Total RNA from these lymphocytes was extracted and a comparison was made between lymphocytic PRL gene expression of patients with active and inactive SLE by real time RT-PCR, using normal pool as calibrator. RESULTS: mild hyperprolactinemia was found in 21.9% (16 of 73 patients of group 1), 7 of 28 patients in activity (25%), and 9 of 45 in inactivity (20%). Macroprolactinemia was found in 3 patients, all with inactive SLE. Regarding serum PRL levels group 1 (active SLE) had median of 10.8 (4.9 38.9) ng/mL and the group 1 (inactive SLE) median of 7.6 (1.9 49.6) ng/mL, without significant difference between the two sub-groups (p=0.123). However, when only monomeric PRL level was considered, the median of group 1 (inactive SLE) dropped to 7.3 ng/mL (1.9 20.6) ng/mL and, when compared again with group 1 (active SLE), we observed that beyond a bigger percentage of the cases in activity to present hyperprolactinemia, the medium of serum PRL level in patients with active SLE is significantly greater of that in the ones in inactivity (p= 0.016). The bioassays with the samples of group 2 (sub-group of group 1): The assays with Nb2 cells showed similar lymphocytic PRL bioactivity. They did not have significant difference between patients with SLE active and inactive and these with normal control group. The assays with Ba/F-LLP cells did not show adequate sensitivity, so not trustworthy for lymphocytic PRL evaluation. The real time RT-PCR also presented similar gene expression between patients (group 2). CONCLUSIONS: The gene lymphocytic PRL expression and its bioactivity were similar in patients with SLE in activity and inactivity of illness and the normal controls. The prevalence of hyperprolactinemia in our population of patients with SLE was of 21.9%, being greater in patients with active SLE. The macroprolactinemia was found only in patients with inactive SLE, suggesting a possible protective effect of this finding.

A água é um importante recurso natural que contribui para melhorias no bem-estar social e desenvolvimento inclusivo. A presença de produtos químicos, rejeitos radioativos e agentes infecciosos pode comprometer a qualidade desse recurso, afetando a biodiversidade e a subsistência de milhões de pessoas. A Ecotoxicologia Aquática é uma ciência que surgiu para dar suporte no enfrentamento dos problemas de contaminação dos corpos d\'água por compostos tóxicos. Seus instrumentos de análise são capazes de avaliar a toxicidade de compostos químicos, sinalizando os potenciais efeitos ecotoxicológicos e seus mecanismos de ação em organismos vivos em ambientes impactados. A Bacia Hidrográfica do Rio Pardo (UGRHI 04) abrange 27 municípios com um importante contingente populacional (1.092.477 habitantes), inserido em uma região cuja prática agrícola baseia-se na cultura de cana-de-açúcar para a produção de etanol e açúcar. Dentro desse contexto, o objetivo do estudo foi avaliar a toxicidade para organismos bioindicadores de amostras de água superficial do Rio Pardo, principal afluente da Bacia Hidrográfica do Rio Pardo. Foram realizados ensaios de toxicidade aguda com Daphnia similis e Vibrio fischeri, e toxicidade crônica com Ceriodaphnia dubia. Adicionalmente, analisaram-se Oxigênio Dissolvido (OD), pH e Temperatura da água. Os resultados obtidos mostraram que, os parâmetros físico-químicos analisados estão de acordo com os limites estabelecidos pela Resolução CONAMA 357/2005 [OD (>= 5 mgO2/L); pH (6 a 9)]. Nos ensaios de toxicidade aguda realizados com Daphnia similis, tanto na estação chuvosa quanto na estação seca, não observou-se toxicidade sobre os organismos testados. Todas as amostras foram classificadas como Não Tóxicas (NT). Nos ensaios de toxicidade crônica com Ceriodaphnia dubia, na estação chuvosa, nenhuma das amostras analisadas apresentou toxicidade. Das amostras analisadas na estação seca, 50% apresentaram Efeito Crônico (EC); os resultados mostraram diferença estatisticamente significante entre o número médio de neonatos produzidos por adulta no grupo controle (19,2) e nas amostras dos pontos de coleta 2 (10,7), 3 (10,5) e 5 (8,1). No entanto, não houve diferença estatisticamente significante nas amostras dos pontos de coleta 1 (13,7), 4 (12,3) e 6 (14,3), classificadas como NT. A comparação entre os resultados obtidos no presente estudo, nos ensaios de toxicidade crônica com Ceriodaphnia dubia, mostrou similaridade com a série histórica de dados (2010-2015) fornecida pela Agência Ambiental do Governo do Estado de São Paulo (CETESB). Nos ensaios de toxicidade aguda realizados com Vibrio fischeri, nas estações chuvosa e seca do ano de 2016, não observou-se toxicidade sobre a bactéria após os períodos de 5 e 15 minutos de exposição; todas as amostras foram classificadas como NT. Apesar do Rio Pardo estar inserido em área com reconhecido uso de produtos agrícolas e pouca proteção de Áreas de Preservação Permanente (APP), os resultados mostraram toxicidade apenas para o organismo Ceriodaphnia dubia, na estação seca do ano de 2016, possivelmente relacionada com a diminuição da vazão média e a concentração de poluentes na água. Cabe destacar a importância da manutenção do monitoramento periódico pela CETESB, considerando o Art. 14 da Resolução CONAMA 357/2005, que prevê a \"não verificação de efeito tóxico crônico a organismos\" para águas doces de Classe 1 e 2 no contexto brasileiro. / Water is an important natural resource that contributes to improvements in social well-being and inclusive development. The presence of chemicals, radioactive waste and infectious agents can compromise the quality of this resource, affecting the biodiversity and livelihoods of millions of people. Aquatic Ecotoxicology is a science that has emerged to support the problems of contamination of water bodies by toxic compounds. Its analytical instruments are able to evaluate the toxicity of chemical compounds, signaling the potential ecotoxicological effects and their mechanisms of action in living organisms in impacted environments. The Pardo River Hydrographic Basin (UGRHI 04) covers 27 municipalities with a significant population (1.092.477 inhabitants), inserted in a region whose agricultural practice is based on the cultivation of sugarcane for the production of ethanol and sugar. In this context, the objective of the study was to evaluate the toxicity to bioindicators organisms of surface water samples of the Pardo River, main tributary of the the Pardo River Hydrographic Basin. Acute toxicity tests were performed with Daphnia similis and Vibrio fischeri, and chronic toxicity with Ceriodaphnia dubia. In addition, dissolved oxygen (DO), pH and water temperature were analyzed. The obtained results showed that the physical-chemical parameters analyzed are in accordance with the limits established by CONAMA Resolution 357/2005 [DO (>= 5 mgO2 / L); pH (6 to 9)]. In the acute toxicity tests performed with Daphnia similis, both in rainy season and in dry season, no toxicity was observed on the organisms tested. All samples were classified as Non-Toxic (NT). In the chronic toxicity tests with Ceriodaphnia dubia in the rainy season none of the analyzed samples presented toxicity. Of the samples analyzed in the dry season, 50% presented Chronic Effect (CE); The results showed a statistically significant difference between the mean number of neonates produced by the adult in the control group (19,2) and the collection points 2 (10,7), 3 (10,5) and 5 (8,1). However, there was no statistically significant difference in the samples from collection points 1 (13,7), 4 (12,3) and 6 (14,3), classified as NT. The comparison between the results obtained in the present study in the chronic toxicity tests with Ceriodaphnia dubia showed similarity with the historical data series (2010-2015) provided by the Environmental Agency of the State of São Paulo (CETESB). In the acute toxicity tests performed with Vibrio fischeri in the rainy and dry seasons of 2016, no toxicity was observed on the bacteria after periods of 5 and 15 minutes of exposure; All samples were classified as NT. Although Rio Pardo is inserted in an area with recognized use of agricultural products and little protection from Permanent Preservation Areas (PPA), the results showed toxicity only for the Ceriodaphnia dubia organismo, in the dry season of 2016, possibly related to the decrease in average flow and the concentration of pollutants in the water. It is important to highlight the importance of Maintenance of periodic monitoring by CETESB, considering Art. 14 of CONAMA Resolution 357/2005, which provides for \"non-verification of chronic toxic effect to organisms\" for Class 1 and 2 fresh waters in the Brazilian context

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No. of bitstreams: 1 Tese - Claudia Dantas Comandolli Wyrepkowski.pdf: 5493553 bytes, checksum: 2facc8046c9c0fe6bc72cf077d977196 (MD5) Previous issue date: 2016-09-24 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / The American Cutaneous Leishmaniasis (ACL) is a protozoonosis caused by parasites of the genus Leishmania, which is transmitted by the bite of the sand fly insect. The LTA, patients can present cutaneous form, with localized skin lesions that may progress to chronic lesions. The treatment of choice is pentavalent antimony and drugs of second choice are pentamidine and amphotericin B, but all have side effects and require parenteral administration. Natural products are presented as an alternative for the treatment of many diseases, and there are studies that demonstrate potential against Leishmania. In the Amazon region, Libidibia ferrea (Mart. Ex Tul) LP Queiroz stands out for its economic and pharmacological importance, due to anti-inflammatory and antimicrobial reported. This study aimed to evaluate the activity of extracts and fractions of L. ferrea against promastigotes and amastigotes of L. (L.) amazonensis and L. (V.) guyanensis also conducting its chemical characterization, and study the effect in topical formulations containing pentamidine, extracts and most promising fractions. And hexane extracts were prepared methanolic the epicarp, leaves and branches of L. ferrea, who underwent in vitro bioassays against promastigotes and amastigotes of L. (L.) amazonensis and L. (V) guyanensis, the counting method direct. The most active extracts were evaluated in vitro for cytotoxic activity in J774 macrophage lineage. The extracts and active fractions were analyzed by TLC assay, the quantized flavonoid and phenolic content, and analyzed by NMR and HPLC. The in vivo assays employed as an animal model hamsters infected with L. (L.) amazonensis. They were prepared and evaluated by topical application on lesions initially hydrating and anhydrous emulsions containing pentamidine isethionate (10%). The results of the bioassays showed that the methanolic extract from epicarp (EpMeOH) showed a higher activity against species of Leishmania, with EC50 15.01 µg/ml against L (L.) amazonensis. This extract had the highest concentration of phenolic (5.14%). A 2-DCM fraction also demonstrated activity with EC50 12.77 µg/mL against L. (L.) amazonensis. No cytotoxic activity was observed in macrophages for both samples. The moisturizing emulsions containing pentamidine were most effective, although viable parasites were detected and all groups after eight days of treatment. These moisturizing emulsions were used as a base for creams with addition of 2.5% of EpMeOH extract and evaluated in two areas with skin lesions - snout and paw. The groups treated with cream EpMeOH extract (2.5%) and placebo showed differences in lesion volume compared to the control group after 20 days of treatment, but showed the presence of parasites, with no statistical difference between the control group. These results suggest that the treatment time and extract concentration to be increased beyond the proportion of use of EpMeOH extract in a more hydrophilic formulation. The hydrogel EpMeOH was then evaluated for lesions on the snout of hamsters as well as cream Fr2. After 40 days of treatment, the groups treated with these topical formulations showed significant differences between the control group and between their respective placebos, as volume, clinical aspect of the lesion, and as the parasitic quantification. Minor inflammatory infiltrate was observed in the hydrogel EpMeOH. This study identified extracts promising in the treatment of experimental cutaneous leishmaniasis and supports pointing new opportunities and innovation serving as a basis for herbal formulations to treat this neglected disease. / A Leishmaniose Tegumentar Americana (LTA) é uma protozoonose causada por parasitas do gênero Leishmania, que são transmitidos ao homem pela picada do inseto flebotomíneo. Na LTA, os pacientes podem apresentar a forma cutânea, com lesões localizadas na pele que podem evoluir para lesões crônicas. O tratamento de primeira escolha são antimoniais pentavalentes e os fármacos de segunda escolha são pentamidina e anfotericina B, porém todos apresentam efeitos colaterais e requerem administração parenteral. Os produtos naturais apresentam-se como uma alternativa para o tratamento de inúmeras enfermidades, e há trabalhos que demonstram potencial contra Leishmania. Na região Amazônica, a espécie Libidibia ferrea (Mart. ex Tul) LP Queiroz destaca-se pela sua importância econômica e farmacológica, devido à atividade anti-inflamatória e antimicrobiana relatada. O presente trabalho teve por objetivo avaliar a atividade de extratos e frações de L. ferrea contra promastigotas e amastigotas de L. (L.) amazonensis e L. (V.) guyanensis, realizando também a sua caracterização química, e estudar o efeito in vivo de formulações tópicas contendo pentamidina, os extratos e frações mais promissoras. Foram preparados extratos hexânicos e metanólicos do epicarpo, folhas e galhos de L. ferrea, os quais foram submetidos a bioensaios in vitro contra promastigotas e amastigotas de L. (L.) amazonensis e L. (V.) guyanensis, pelo método da contagem direta. Os extratos mais ativos foram avaliados in vitro quanto a atividade citotóxica em macrófagos de linhagem J774. Os extratos e frações ativos foram analisados por CCDC, quantificados o teor de fenólicos e flavonoides, e analisados por RMN e CLAE. Os ensaios in vivo empregaram hamsters como modelo animal, infectados com L. (L.) amazonensis. Foram preparadas e avaliadas por aplicação tópica nas lesões, inicialmente, emulsões hidratantes e anidras contendo isetionato de pentamidina (10%). Os resultados dos bioensaios demostraram que o extrato metanólico de epicarpo (EpMeOH) apresentou maior atividade frente as espécies de Leishmania, com EC50 de 15,01 µg/mL contra L (L.) amazonensis. Este extrato apresentou a maior concentração de fenólicos (5,14%). A fração 2-DCM demonstrou também atividade, com EC50 12,77 µg/mL contra L. (L.) amazonensis. Não foi observada atividade citotóxica em macrófagos para ambas as amostras. As emulsões hidratantes contendo pentamidina foram mais eficientes, embora fossem detectados parasitas viáveis em todos os grupos após oito dias de tratamento. Estas emulsões hidratantes foram utilizadas como base para cremes com adição de 2,5% do extrato EpMeOH e avaliados em duas áreas com lesões cutâneas - focinho e pata. Os grupos tratados com creme com extrato EpMeOH (2,5%) e o placebo mostraram diferenças quanto ao volume da lesão quando comparados ao grupo controle após 20 dias de tratamento, mas mostraram a presença de parasitas, sem diferença estatística entre o grupo controle. Estes resultados sugerem que o tempo de tratamento e a concentração do extrato devem ser aumentadas, além da proporção do uso do extrato EpMeOH em uma formulação mais hidrofílica. O hidrogel EpMeOH foi avaliado em lesões no focinho de hamsters, assim como emulsão Fr2. Após 40 dias de tratamento, os grupos tratados com estas formulações tópicas mostraram diferenças estatísticas entre o grupo controle e também entre seus respectivos placebos, quanto ao volume, aspecto clínico da lesão, e quanto à quantificação parasitária. Menor infiltrado inflamatório foi observado no grupo hidrogel EpMeOH. Este estudo permitiu identificar o extratos promissores no tratamento da leishmaniose tegumentar experimental e corrobora apontando novas oportunidades e inovação servindo como base para formulações de fitoterápicos para o tratamento desta doença negligenciada.

Para o sucesso das estratégias de manejo de Rhipicephalus (Boophilus) microplus (carrapato bovino) são necessários testes práticos, econômicos e confiáveis que possam detectar a presença de fenótipos resistentes a drogas em suas populações. O fipronil é um acaricida de uso relativamente recente não havendo testes padronizados para o diagnóstico de resistência do carrapato à molécula. No presente trabalho, foram padronizados bioensaios in vitro para esta finalidade: Teste de Imersão de Adultas, Teste de Imersão de Larvas e Teste de Pacote com Larvas. Os testes foram aplicados e, de forma inédita, populações resistentes foram diagnosticadas tanto no Brasil quanto no Uruguai. Ensaios com inibidores enzimáticos não evidenciaram participação importante de enzimas detoxificadoras no mecanismo de resistência. Foi demonstrada reação cruzada entre fipronil e lindano, não verificada para ivermectina. Em algumas situações, foi observado interferência do controle químico de pragas agrícolas no desenvolvimento de resistência dos carrapatos. / For the success of the strategies for the management of Rhipicephalus (Boophilus) microplus (cattle tick), practical, economical and reliable tests are needed to detect the presence of drug-resistant phenotypes in their populations. Fipronil is a relatively new acaricide with no standardized tests for the diagnosis of tick resistance to this molecule. In this study, were standardized in vitro bioassays for this purpose: Adult Immersion Test, Larval Immersion Test and Larval Packet Test. The tests were applied and for the first time, resistant populations were diagnosed in Brazil and Uruguay. Tests with enzymatic inhibitors showed no significant involvement of detoxification enzymes in the mechanism of resistance. Cross-resistance was demonstrated between lindane and fipronil but not with ivermectin. In some situations, it was observed interference of the chemical control of agricultural pests in the development of resistance in ticks.

Os fungos são microrganismos amplamente dispersos, podendo ser encontrados em vegetais, animais, solo e ambientes aquáticos, participando do ciclo de elementos na natureza. Embora muitos papéis ecológicos tenham sido estudados e descritos para os fungos terrestres, a ecologia de fungos marinhos ainda é pouco conhecida. Assim, os oceanos, que representam aproximadamente metade da biodiversidade global, são uma fonte enorme e virtualmente inexplorada de microrganismos produtores de novos produtos naturais. O objetivo deste trabalho foi isolar linhagens de fungos derivados de uma espécie de alga marinha do gênero Sargassum, visando à avaliação do seu potencial para a produção de metabólitos secundários bioativos. Ao todo foram isoladas 58 linhagens, das quais 52 foram crescidas em meio de cultura líquido e, após a extração com solventes orgânicos, deram origem a 99 extratos. Tais extratos foram avaliados por ensaios de atividade biológica, cromatografia em camada delgada (CCD), ressonância magnética nuclear (RMN) e cromatografia líquida acoplada a detectores de arranjo de diodos, espalhamento de luz evaporativo e espectrômetro de massas (LC - PDA - ELSD - MS). A avaliação pelo ensaio antibiótico foi o que resultou no maior número de extratos ativos (n = 13), seguido dos ensaios enzimático (n = 8), citotóxico (n = 3) e anti-tuberculose (n = 1). O extrato AS Fub 39, que apresentou atividade antibiótica, foi selecionado para estudos adicionais. Este extrato foi purificado por HPLC, e o seu composto majoritário identificado como sendo o 8-metóxi-3,5-dimetilisocroman-6-ol. Posteriormente, a linhagem AS Fub 39 foi taxonomicamente identificada como pertencendo à espécie Penicillium steckii. / Fungi are widely disperse microorganisms, typically associated with plants, animals, soil and aquatic environments (fresh and sea water), participating in the elements cycling. Although many ecological roles have been described for terrestrial fungi, ecological studies of marine derived fungi are still scarce. Therefore, oceans, which represent approximately half of the global biodiversity, are a huge and virtually unexplored source of microorganisms producers of interesting metabolites. The aim of this study was to isolate fungal strains derived from a marine algae of the Sargassum genus, and the evaluation of their metabolical potential for the production of secondary metabolites. Overall, 58 strains were isolated, of which 52 were grown in liquid culture media and extracted with organic solvents, originating 99 crude extracts. These extracts were analyzed by bioassays, thin layer chromatography (TLC), nuclear magnetic resonance (NMR) and liquid chromatography coupled with a photo diode array, an evaporative light scattering, and a mass spectrometry detectors (LC - PDA - ELSD - MS). The evaluation with the antibiotic assay resulted in the largest number of active extracts (n = 13), followed by the enzymatic (n = 8), the cytotoxic (n = 3) and the antituberculosis (n = 1) assays. The crude extract AS Fub 39, which presented antibiotic activity, was selected for additional studies. This extract was purified by HPLC, and its major compound identified as the 8-methoxy-3,5-dimethylisocroman-6-ol. Later, the AS Fub 39 strain was taxonomically identified as Penicillium steckii.