A poluição dos rios brasileiros é, atualmente, um grande problema para as autoridades governamentais já que a demanda dos recursos hídricos vem crescendo nos últimos anos. Os rios Tietê, Pinheiros e Tamanduateí foram de grande importância para a cidade de São Paulo em seu período de industrialização, contudo o próprio desenvolvimento industrial impactou permanentemente os mesmos. Preocupados com a saúde ambiental, órgãos fiscalizadores estaduais iniciaram forte fiscalização para coibir e responsabilizar os principais culpados. Esta política pública resultou em uma diminuição das concentrações de poluentes orgânicos e inorgânicos, porém muito pouco foi efetivamente feito com relação ao esgoto doméstico, e, como consequência, a toxicidade das águas dos rios permanece elevada. Os estudos de Avaliação e identificação da toxicidade englobam diversas metodologias reunidas em três documentos pela USEPA - Agência de Proteção Ambiental dos Estados Unidos, onde método físico-químico e testes toxicológicos são usados para identificar substâncias tóxicas, ou grupo de substâncias tóxicas que contribuem efetivamente para a toxicidade total de uma amostra. A abordagem rotineiramente utilizada baseia-se no fracionamento da amostra de acordo com suas propriedades físico-químicas: volatilidade, solubilidade e capacidade de formar complexos, ou de ser reduzida, oxidada, ionizada, entre outras. Em conjunto com os ensaios químicos, os ensaios toxicológicos, em especial o Sistema Microtox®, contribuem na investigação da toxicidade para os rios metropolitanos Tietê, Pinheiros e Tamanduateí, que se mostrou eficaz na determinação dos grupos de compostos, sendo os compostos orgânicos apolares, orgânicos voláteis, tensoativos e as partículas, as principais fontes da toxicidade para esses rios. Embora muitas informações sobre a toxicidade tenham sido descobertas, a identificação dos compostos só pode ser realizada por análises químicas mais robustas uma vez que um grande número de substâncias, metabólitos e subprodutos de degradação são facilmente encontrados neste tipo de amostra. Mesmo sofrendo algumas modificações, deve-se incentivar o uso desta metodologia para outros rios a fim de determinar os principais toxicantes responsáveis pela toxicidade. / Pollution of Brazilian rivers is currently a major problem for government authorities as demand for water resources has been increasing in recent years. Tietê, Pinheiros and Tamanduateí rivers were of great importance for the city of São Paulo during its industrialization period, however, the industrial development itself permanently impacted them. Concerned about environmental health, state supervisory bodies have begun strong enforcement to curb and hold the main culprits accountable. This public policy has resulted in a decrease in concentrations of organic and inorganic chemicals, but very little has been done in relation to domestic sewage, and as a consequence, the toxicity of the rivers remains high. The Toxicology Assessment and Identification studies encompass various methodologies gathered in three documents by the USEPA, where physico-chemical method and toxicological tests are used to identify toxic substances, or group of toxic substances that effectively contribute to the total toxicity of a sample. The routinely used approach is based on fractionation of the sample according to its physicochemical properties: volatility, solubility and chemical properties such as the ability to form complexes, or to be reduced, oxidized, ionized, among others. In conjunction with the chemical tests, the toxicological tests in particular the Microtox® System contribute to the investigation of the toxicity to the metropolitan rivers, Tietê, Pinheiros and Tamanduateí, which proved to be effective in the determination of the groups of compounds, the organic compounds being apolar, organic volatiles, surfactants and particulates, the main sources of toxicity for the studied rivers. Although much information on toxicity has been discovered, the identification of compounds can only be performed by more robust chemical analyzes since a large number of substances, metabolites, degradation byproducts are readily found in this type of sample. Even with some modifications, it is necessary to encourage the use of this methodology for other rivers in order to determine the main xenobiotics responsible for toxicity.

Les particules de dioxyde de titane (TiO2) sont utilisées dans de nombreux secteurs industriels du fait de leurs propriétés physiques et chimiques intéressantes. Depuis une dizaine d’années, elles sont également utilisées sous forme nanoparticulaire car la taille nanométrique leur apporte de nouvelles propriétés, recherchées dans certaines applications industrielles. Elles sont par exemple utilisées comme colorant blanc dans le secteur de la cosmétologie, de la pharmacologie et dans les industries agroalimentaires. Dans ces dernières, l’utilisation de ces particules est autorisée car le TiO2 est un composé insoluble et relativement inerte. Le colorant alimentaire E171, autorisé depuis 1966, est ainsi constitué de particules de TiO2, initialement sous forme micrométrique, mais il s’avère que selon les procédés de fabrication, entre 10 et 43 % (selon les études) de ces particules présentent un diamètre inférieur à 100 nm, i.e. sont sous forme nanométrique. Ce n’est pas un nanomatériau du point de vue de la définition européenne, il n’est donc pas soumis à l’obligation d’étiquetage dans les produits alimentaires. Le E171 est présent dans de nombreux aliments sans que son impact sur la santé humaine, après ingestion, n’ait été clairement documenté. De plus en plus d’études s’intéressent à la toxicité des nanoparticules (NPs) après leur ingestion, mais peu d’entre elles ont été menées avec le E171 à proprement parler. Les études in vivo et in vitro publiées à ce jour démontrent que les NPs de TiO2 sont peu toxiques. Leur absorption intestinale et leur translocation vers le système sanguin puis des organes secondaires est faible. Les principaux effets décrits sont une augmentation des espèces réactives de l’oxygène associées à un stress oxydant, l’induction de marqueurs de l’inflammation, et plus récemment l’induction du stress du réticulum endoplasmique. Des effets sont également rapportés sur différents paramètres de la barrière intestinale, i.e. le microbiote, le mucus, les transporteurs membranaires, les jonctions cellulaires et l’immunité intestinale. Chez certaines personnes, cette barrière est compromise, elles sont donc potentiellement plus sensibles aux micros et nanos-particules contenues dans l’alimentation. Leur épithélium intestinal est enflammé, et à long terme, ces personnes peuvent développer des maladies inflammatoires chroniques de l’intestin et dans les cas les plus graves, des cancers.L’objectif de cette thèse est d’étudier la toxicité du colorant alimentaire E171 et d’approfondir les connaissances relatives à l’impact des NPs de TiO2 sur le système gastro-intestinal. Pour cela, nous avons travaillé avec différents modèles cellulaires d’épithélia intestinaux humain, un modèle d’épithélium jointif composé d’entérocytes Caco-2, un modèle d’épithélium sécrétant une couche de mucus, composé de cellules Caco-2 et HT29-MTX et enfin un modèle d’épithélium bordant les plaques de Peyer, composé des cellules Caco-2(C1) et RajiB. Ces modèles cellulaires ont été exposés de façon aigüe (6 h, 24 h et 48 h) ou chronique (21 jours), au colorant E171 ainsi qu’à deux NPs de TiO2 : A12, qui a la même structure cristalline que le E171 et P25, une NP très documentée dans la littérature. Nos résultats montrent que le E171 et les NPs de TiO2 sont modérément toxiques, ils n’engendrent pas de mortalité cellulaire ni de dommages à L’ADN. Néanmoins, ils provoquent une accumulation d’espèces réactives de l’oxygène intracellulaires et modulent certains marqueurs impliqués dans le stress oxydant, le stress du réticulum endoplasmique et l’inflammation. Ils impactent également la sécrétion et la composition de la couche de mucus, l’expression des transporteurs ABC, qui sont des paramètres impliqués dans la fonction de barrière de l’épithélium intestinal, le rendant possiblement plus vulnérable aux agressions extérieures. / Micro-sized titanium dioxide (TiO2) particles are used for years by industrials for their attractive physical and chemical properties. The use of TiO2 nanoparticles (NPs) is also constantly increasing, because the nanometric size gives new interesting properties to particles which industrials are looking for. In some daily-life products including paints, plastics, paper, medicines and food, micro-sized TiO2 particles are used as a pigment for their opacifying and whitening capacities. The use of TiO2 as a food additive, i.e. E171 in the EU, has been authorized in most countries since the 60ies, without any established acceptable daily intake, because of their low toxicity and intestinal absorption. However, it was recently shown that E171 can contain up to 43% of particles with diameter ranging from 1 to 100 nm, i.e. NPs. Still, E171 is not a nanomaterial as described in the European recommendation of definition because it contains less than 50% of NPs (in number). Food grade TiO2 is present in a wide range of food products while little is known about its toxicological impact to human health. The toxicity of ingested TiO2, either nano- or micro-sized, is increasingly documented, still E171 itself is rarely used in these studies.According to in vivo and in vitro studies, TiO2 particles were proven relatively safe for intestinal cells, no cytotoxicity neither genotoxicity were reported. Nevertheless, particles were often reported to increase reactive oxygen species (ROS) cell content, to impair autophagic processes and modulate gene expression and the content of proteins involved in oxidative stress, endoplasmic reticulum stress and inflammatory response regulation. Interestingly, their reported impact on intestinal cells suggests alteration of almost all the components of the intestinal barrier function, i.e. microbiota, mucus, cell junctions and transporters. This intestinal barrier function is altered in patients suffering from intestinal bowel diseases, these persons are thus possibly more sensitive to mineral particulate in food.The present study aimed at improving knowledge on the toxicity of food-grade TiO2. To this purpose, the impact of E171 was evaluated on in vitro cell models representative of the human intestinal epithelium, i.e. a model of differentiated Caco-2 enterocytes, a model of mucus-secreting epithelium obtained by coculture of Caco-2 and HT29-MTX mucus-secreting cells and a model of the follicle-associated epithelium, which lines Peyer patches, obtained by coculture of Caco-2(C1) and RajiB cells. These cell models were either acutely exposed for 6 h, 24 h and 48 h or chronically exposed for 21 days to E171. In parallel, they were exposed to two model TiO2-NPs, A12 which has the same crystalline structure as E171 and P25, a well-documented TiO2-NPs. Our results show that E171 and TiO2-NPs induced no overt cell mortality but significant oxidative stress, and that they oxidatively damage DNA. They modulate the expression of genes involved in oxidative stress and endoplasmic reticulum stress regulation. They also modulate the expression of genes, as well as the content of proteins from mucus, ABC transporters and inflammatory markers, which are the main players of the intestinal barrier function and presumably increase epithelium sensitivity to xenobiotics. These data suggest that they may be implicated in the development or aggravation of inflammatory bowel diseases.

by Wai-Man Tong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 155-174). / Abstracts in English and Chinese. / ABSTRACT --- p.I-IV / Chapter 1. --- INTRODUCTION / Chapter 1.1. --- General / Chapter 1.1.1. --- Ribosome Inactivating Protein --- p.1 / Chapter 1.1.1.1. --- Ricin --- p.2 / Chapter 1.1.1.2. --- Trichosanthin --- p.5 / Chapter 1.1.2. --- In Vitro Study of RIP --- p.6 / Chapter 1.2. --- Uptake of Ribosome Inactivating Proteins / Chapter 1.2.1. --- Suicide Transport --- p.7 / Chapter 1.2.1.1 . --- Endocytic Uptake of Ricin --- p.8 / Chapter 1.2.1.2. --- Endocytic Uptake of Trichosanthin --- p.11 / Chapter 1.2.2. --- Pervious Studies in This Laboratory --- p.11 / Chapter 1.3. --- Apoptosis And Ribosome Inactivation / Chapter 1.3.1. --- Apoptosis / Chapter 1.3.1.1. --- Morphological Feature of Apoptosis --- p.14 / Chapter 1.3.1.2. --- Molecular Changes of Apoptosis --- p.15 / Chapter 1.3.2. --- Toxicity of Ribosome Inactivating Protein / Chapter 1.3.2.1. --- Toxicity of Ricin --- p.20 / Chapter 1.3.2.2. --- Toxicity of Trichosanthin --- p.21 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1. --- GENERAL / Chapter 2.1.1. --- Cell Culture / Chapter 2.1.1.1 . --- Schwann Cell Culture --- p.23 / Chapter 2.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.24 / Chapter 2.1.1.3. --- Identification of Schwann Cell and Dorsal Root Ganglion Neuron --- p.25 / Chapter 2.1.2. --- Labeling of Toxins --- p.26 / Chapter 2.1.3. --- Administration of Toxin --- p.27 / Chapter 2.2. --- UPTAKE OF RIBOSOME INACTIVATING PROTEINS / Chapter 2.2.1. --- Real-Time Observation of Toxin Uptake by Neurons --- p.27 / Chapter 2.3. --- APOPTOSIS STUDY OF RIBOSOME INACTIVATING PROTEINS' TOXICITY / Chapter 2.3.1. --- TUNEL Staining --- p.28 / Chapter 2.3.2. --- Annexin V Staining --- p.30 / Chapter 2.4. --- MOLECULAR STUDY OF THE DEATH MECHANISM OF RIBOSOME INACTIVATING PROTEINS / Chapter 2.4.1. --- NIH/3T3 Cell Line Culture --- p.33 / Chapter 2.4.2. --- Differential Display / Chapter 2.4.2.1. --- Differential Display --- p.34 / Chapter 2.4.2.2. --- Cloning and Sequencing --- p.38 / Chapter 2.4.2.3. --- RT-PCR --- p.42 / Chapter 2.4.3. --- Two Dimension Gel Electrophoresis --- p.43 / Chapter 2.4.4. --- Ribosomal RNA Analysis --- p.48 / Chapter 3. --- RESULTS / Chapter 3.1. --- General / Chapter 3.1.1. --- Cell Culture / Chapter 3.1.1.1 . --- Schwann Cell Culture --- p.50 / Chapter 3.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.51 / Chapter 3.1.1.3. --- Identification of Schwann Cell and Dorsal Root Ganglion Neuron --- p.51 / Chapter 3.1.2. --- RIPs Labeling --- p.52 / Chapter 3.2. --- Uptake of Ribosome Inactivating Protein / Chapter 3.2.1. --- Real-Time Observation of Toxin Uptake --- p.53 / Chapter 3.3. --- Apoptosis Study of Ribosome Inactivating Proteins' Toxicity / Chapter 3.3.1. --- TUNEL Staining --- p.55 / Chapter 3.3.2. --- Annexin V Assay / Chapter 3.3.2.1. --- Schwann Cell Culture --- p.57 / Chapter 3.3.2.2. --- Dorsal Root Ganglion Neuron Culture --- p.58 / Chapter 3.3.2.3. --- Unique Observable Pattern --- p.60 / Chapter 3.4. --- Molecular Study of the Death Mechanism of Ribosome Inactivating Proteins / Chapter 3.4.1. --- NIH/3T3 Cell Line Culture --- p.60 / Chapter 3.4.1.1. --- TUNEL Staining --- p.61 / Chapter 3.4.1.2. --- Annexin V Staining --- p.61 / Chapter 3.4.2. --- Differential Display / Chapter 3.4.2.1. --- Observation --- p.61 / Chapter 3.4.2.2. --- Primer Combination --- p.62 / Chapter 3.4.2.3. --- Differential Display --- p.62 / Chapter 3.4.3. --- Two-Dimensional Gel Electrophoresis / Chapter 3.4.3.1. --- Observation --- p.63 / Chapter 3.4.3.2. --- Comparison of Gels --- p.63 / Chapter 3.4.4. --- Ribosomal RNA Analysis --- p.63 / Chapter 4. --- DISCUSSION / Chapter 4.1. --- General / Chapter 4.1.1. --- The Selection of In Vitro Model / Chapter 4.1.1.1. --- Schwann Cell Culture --- p.65 / Chapter 4.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.66 / Chapter 4.1.2. --- Labeling of Toxins with Fluorochromes --- p.67 / Chapter 4.1.3. --- Dosage Used in In Vitro Study --- p.68 / Chapter 4.2. --- Uptake of Ribosome Inactivating Proteins / Chapter 4.2.1. --- Real-Time Examination of Toxin Uptake --- p.69 / Chapter 4.3. --- Involvement of Apoptosis in Ribosome Inactivating Proteins' Intoxication / Chapter 4.3.1. --- TUNEL Staining --- p.75 / Chapter 4.3.2. --- Annexin V and Propidium Iodide Staining --- p.77 / Chapter 4.3.3. --- Special Pattern of Fluorescence Signal in Neuronal Cell Bodies --- p.82 / Chapter 4.4. --- Molecular Study of Death Mechanism of Ribosome Inactivating Proteins / Chapter 4.4.1. --- NIH/3T3 Cell Line Culture --- p.84 / Chapter 4.4.2. --- Differential Display --- p.84 / Chapter 4.4.3. --- Two Dimensional Polyacrylamide Gel Electrophoresis --- p.86 / Chapter 4.4.4. --- Ribosomal RNA Alternation --- p.88 / Chapter 5. --- CONCLUSIONS --- p.89 / Chapter 6. --- "FIGURES, GRAPHS AND LEGENDS" --- p.91 / Chapter 7. --- REFERENCES --- p.155 / APPENDIX / Appendix A Materials --- p.175 / Appendix B Source of Chemicals and Equipments --- p.184 / ACKNOWLEDGEMENTS --- p.186

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2017-06-08T12:36:15Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese Débora Oliveira da Silva.pdf: 11845528 bytes, checksum: 2ed9c55ca22d8379a068646ccd7d870a (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-06-08T13:14:51Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese Débora Oliveira da Silva.pdf: 11845528 bytes, checksum: 2ed9c55ca22d8379a068646ccd7d870a (MD5) / Made available in DSpace on 2017-06-08T13:14:51Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese Débora Oliveira da Silva.pdf: 11845528 bytes, checksum: 2ed9c55ca22d8379a068646ccd7d870a (MD5) Previous issue date: 2017-02-17 / Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGS / A planta Pereskia aculeata é uma hortaliça não convencional que possui grande potencial alimentar devido aos seus elevados teores de minerais, aminoácidos, vitaminas, fibras e antioxidantes. Entretanto, informações sobre características fenológicas, químicas e toxicológicas da planta, especialmente quando cultivada em região subtropical de clima temperado, são limitadas ou inexistentes. Assim, a planta P. aculeata foi cultivada sob clima temperado, em Pelotas, RS, Brasil, e avaliada quanto aos seguintes aspectos: (a) toxicidade aguda, (b) citotoxicidade, (c) características fenológicas e (d) características físico-químicas. A análise da toxicidade aguda foi realizada por meio de ensaio biológico, que utilizou 24 ratas, adultas, da linhagem Wistar. Os animais foram distribuídos em quatro grupos, de acordo com as doses administradas de extrato etanólico da planta P. aculeata: 0 mg/kg, 1250 mg/kg, 2500 mg/kg e 5000 mg/kg de massa corporal. O experimento teve duração de 15 dias e após a eutanásia foram realizadas avaliações histopatológicas em 8 órgãos. A citotoxicidade da P. aculeata foi avaliada utilizando-se alface (L. sativa) como modelo experimental. Sementes de alface foram tratadas com diferentes doses de extrato de P. aculeata (0,13 mg/ml; 1,30 mg/ml; 3,25 mg/ml; 6,50 mg/ml; 13,00 mg/ml) e avaliadas quanto ao índice de germinação, crescimento de raiz, crescimento de parte aérea, e índice mitótico. Durante 12 meses a planta foi avaliada periodicamente quanto a características físico-química (teor de umidade, área foliar, proteína, cor, fenóis totais e atividade antioxidante) e fenológicas (mudança foliar, floração e frutificação). Observou-se que P. aculeata não possui efeito tóxico em ratos, na dose de até 5000 mg/kg de massa corporal. A planta apresentou reduzido efeito citotóxico sobre alface, consistindo em alteração no crescimento de raízes e partes aéreas, porém sem comprometimento da germinação e sem efeito genotóxico na semente. No estudo fenológico observou-se boa adaptação da planta ao clima temperado, caracterizado pelo pleno crescimento e desenvolvimento. Entretanto, destacase um período de quiescência durante o inverno, no qual a planta não produziu folhas. Concluiu-se que o cultivo de P. aculeata é viável em clima temperado e que seu consumo alimentar é seguro. / The Pereskia aculeata plant has valuable nutritional properties and could be a supplementary food source, because it contains high amounts of minerals, amino acids, vitamins, fibers and antioxidants. However, we have limited information about toxicity, cytotoxicity, phenological and physicochemical characteristics of this plant, particularly when cultivated in subtropical areas with temperate climate. Thus, we analyzed Pereskia aculeata plants cultivated in Pelotas, RS, Brazil, under temperate climate, for: (a) acute toxicity, (b) cytotoxicity, (c) phenological characteristics and (d) physicochemical properties. The acute toxicity experiment was performed with 24 female Wistar rats. The animals were divided into four groups, according to the single dose of ethanolic plant extract they received: 0 mg/kg, 1250 mg/kg, 2500 mg/kg e 5000 mg/kg of body weight. After a 15 days period of observation the euthanasia was performed and 8 different animal tissues were sampled for histopathological analysis. The cytotoxicity was evaluated using lettuce (L. sativa) as an experimental model. The lettuce seeds were treated with different doses of P. aculeata extract (0.13 mg/ml; 1.30 mg/ml; 3.25 mg/ml; 6.50 mg/ml; 13.00 mg/ml) and analyzed for germination and mitotic indexes, and also for roots and shoots length. During 12 months cultivation period the plant was analyzed for physicochemical properties (humidity, color, leaf area, protein content, antioxidant activity, total phenolic content) and phenological aspects (leaf changes, flowering and fructification). Our results for acute toxicity of Pereskia aculeata show that it has non-toxic effect to rats at concentrations of 5000 mg/kg of body weight. We also found this plant to have minimal deleterious cytotoxicity effects on lettuce, consisting of abnormal growth of roots and shoots, but without affecting germination or causing genotoxic effect on lettuce seeds. Furthermore, Pereskia aculeata showed a good adaptation under temperate climate, with normal growth and development. Though, a quiescent state happened in the winter and the plant did not produce leaves. We conclude that cultivation of Pereskia aculeata under temperate climate is feasible and also, the leaves of these plants are safe for consumption as food.

Quando se trata de efluentes muito complexos e de amostras de água ou de sedimento de locais poluídos, é inviável ou até mesmo impossível detectar a presença de todas as substâncias presentes. Os principais contaminantes associados à poluição das águas naturais, tem-se os metais pesados, uma classe de compostos de toxicidade elevada e que são bioacumulados nos seres vivos. Desses metais, o cobre e o níquel se destacam, tanto por seu amplo uso em processos industriais, o que acarreta sua presença em diversos tipos de despejos, quanto por sua toxicidade elevada. O objetivo deste trabalho foi determinar o potencial tóxico do sulfato de cobre e sulfato de níquel, de sua mistura e seus complexos com EDTA frente a Daphnia similis para utilização na avaliação do desempenho do processo de separação com membranas no tratamento de efluentes. Em um estudo de toxicidade, as interações do metal com o organismo-teste são influenciadas pela espécie testada, pela combinação dos metais ou pela composição do meio aquoso. O íon Cu2+ apresentou toxicidade superior ao Ni2+, sendo que na mistura desses dois íons, prevaleceu o resultado obtido para o Cu2+, decorrente de sua maior toxicidade. A complexação dos metais reduz significativamente o potencial tóxico dos metais / When it comes to very complex effluents and water samples or sediment contaminated sites, it is impractical or even impossible to detect the presence of all substances. The main contaminants associated with the pollution of natural waters, has the heavy metals, a class of compounds of high toxicity and are bioaccumulated in living organisms. Of these metals, copper and nickel stand out, both for its wide use in industrial processes, leading to their presence in several kinds of evictions, and by their high toxicity. The objective of this study was to determine the potential toxic effects of copper salts II and nickel II of its moisture and its complexes with EDTA compared to Daphnia similis for use in assessing the performance of the separation membrane in the treatment of effluents. In a study of toxicity, the interactions of metal with the test organisms are influenced by the species tested, the combination of metals or the composition of the aqueous medium. The ion Cu 2+ toxic than the Ni2+, and the mixture of these two ions, prevailed the result obtained for the Cu2+, due to its greater toxicity. The complexation of metals greatly reduces the potential toxic metals

Microalgas e cianobactérias têm sido amplamente recomendadas para biomonitoração de metais pesados e outros poluentes, sendo considerados indicadores sensíveis às alterações ambientais e utilizados como organismos testes na regulamentação dos níveis de metal. Estes micro-organismos fotossintetizantes são produtores primários da base da cadeia alimentar aquática e são os primeiros a serem afetados pela poluição por metais pesados. O cobre é um metal normalmente considerado como nutriente essencial para a vida aquática mas pode ser tóxico para algumas espécies. Portanto, neste estudo foram avaliados o efeito tóxico e a bioacumulação de cobre (II) em quatro espécies de micro-organismos fotoautotróficos componentes do fitoplâncton dulcícola, duas cianobactérias filamentosas (Anabaena sp. e Oscillatoria sp) e duas microalgas da classe das clorofíceas (Monorraphidium sp. e Scenedesmus sp.). O meio de cultivo utilizado nos ensaios foi o ASM-1 com e sem a presença de cobre (0,6 mg/L a 12 mg Cu2+/L) onde, o efeito tóxico do metal foi monitorado por contagem celular para as microalgas e por peso seco para as cianobactérias. A bioacumulação do metal foi avaliada da mesma forma para todos os micro-organismos, através de coletas de amostras no decorrer do experimento e determinação da concentração de cobre em solução por espectrometria de absorção atômica com chama. Os resultados obtidos mostram que o efeito tóxico do metal é diretamente proporcional à concentração inicial para os micro-organismos estudados, mas que o cobre (II) foi mais tóxico para as cianobactérias que para as microalgas verdes. A bioacumulação teve uma relação direta com o efeito tóxico do metal sobre os micro-organismos. Os resultados obtidos permitem sugerir que cobre (II) tem efeito negativo no fitoplâncton, inibindo o crescimento e alterando parâmetros metabólicos como a fotossíntese. A bioacumulação do metal pode comprometer os níveis tróficos da cadeia alimentar, afetando seu transporte para seres superiores / Microalgal and cyanobacterial cells are widely used in the biomonitoring of metal contaminated areas as well as other pollutant agents, being considered sensitive microorganisms in relation to environmental changes and also used as test-organisms in the regulation of low levels of metals. These photosynthesizing microorganisms are primary producers in the aquatic food chain, and are the first ones to be affected by heavy metal pollution. Copper is a metal usually considered as an essential nutrient for aquatic forms of life, however toxic for some species. In this way, in the present study, the toxic effect of copper ion and its bioaccumulation by photoautotrophic microbes from freshwater phytoplankton: two filamentous cyanobacteria (Anabaena sp. and Oscillatoria sp) and two chlorophyte microalgae Monorraphidium sp. and Scenedesmus sp.). The culture medium used in the tests was the ASM-a medium in absence and contaminated with copper ion (0,6 mg/L to 12 mg Cu2+/L). The toxic effect of copper was monitored through cell count (microalgal cells) and dry weight measurements (cyanobacterial cells), and copper bioaccumulation was evaluated for all the species tested, through determination of the residual metal concentration in solution, by flame atomic absorption spectrometry. Results obtained indicated that the toxic effect of the metal was directly proportional to the initial concentration of the ion for all the species tested; however, copper ion was more toxic to cyanobacterial than to microalgal cells. The bioaccumulation of copper was directly related to the toxic effect of the ion over the microorganisms. Results obtained suggest that copper ion has a negative effect on the phytoplankton, inhibiting cell growth and also affecting metabolic parameters such as photosynthesis. The bioaccumulation of the metal can markedly affect the trophic levels in the food chain, thus affecting its transport to higher species

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Os recifes de corais são ecossistemas diversos com alta densidade de biodiversidade, o que leva a intensa competição entre as espécies. Estas espécies podem produzir substâncias desconhecidas, muitas com valor farmacológico. Chromonephthea braziliensis é um coral mole invasor, originário do Oceano Indo-Pacífico, que foi possivelmente transportado por plataformas de petróleo e cuja presença é uma ameaça para a biodiversidade da região de Arraial do Cabo (RJ). Esta espécie produz metabólitos secundários que são responsáveis pela indução de danos para o ecossistema local. A finalidade deste estudo é a busca de novas substâncias para quimioterapia geral com base na estrutura de compostos bioativos. Extratos desse coral foram preparados a partir de colônias liofilizadas (solventes: hexano, diclorometano, acetato de etila e metanol). Realizaram-se análises químicas para a caracterização dos extratos e avaliaram-se as atividades: mutagênicas e citotóxicas, usando o ensaio de mutação reversa bacteriana (Salmonella/microssoma) com as linhagens TA97, TA98, TA100 e TA102; genotóxicas, utilizando análise da quebra do DNA e formação de micronúcleos na linhagem RAW 264,7 de macrófagos e; tóxicas para náuplios de microcrustáceos Artemia salina. Observou-se citotoxicidade, na presença de S9 mix, dos extratos diclorometano para a linhagem TA102 na concentração de 20 g/100 L/placa e metanol para a TA97 com 5 e 20 g/100 L/placa. Os extratos diclorometano, acetato de etila e metanol apresentaram genotoxicidade no DNA plasmidial em concentrações elevadas (250 g/mL), mas nenhum dano ao DNA foi observado no ensaio de micronúcleo. Todos os extratos foram tóxicos para os náuplios de microcrustáceos em pelo menos uma das concentrações usadas (0,01 1000 g/mL), e a LC50 pode ser determinada apenas para os extratos hexano, diclorometano e acetato de etila. / Coral reefs are diverse ecosystems that have a high density of biodiversity leading to intense competition among species. These species may produce unknown substances, many with pharmacological value. Chromonephthea braziliensis is an invasive soft coral from the Indo-Pacific Ocean that has been possibly transported by oil platforms and whose presence can be a threat to Arraial do Cabo regions biodiversity. This species produces secondary metabolites that are responsible for inducing damage to the local ecosystem. The purpose of this study is the search of new drugs for generally chemotherapy based on the structure of bioactive compounds. Coral extracts were prepared from dried colonies (solvents: hexane, dichloromethane, ethyl acetate, and methanol). Chemical analyses were performed to characterize the extracts and evaluated their cytotoxic and mutagenic activities, using the bacterial reverse mutation assay (Salmonella/microsome) with TA97 strains, TA98, TA100 and TA102; genotoxic activity, using DNA breakage analysis and micronucleus formation and; toxic effects for nauplii microcrustaceans. Cytotoxicity was observed in the presence of S9 mix for dichloromethane extract for strain TA102 at the 20 g/plate concentration and methanol extract for TA97 at 5 and 20 g/plate. The dichloromethane, ethyl acetate and methanol extracts present genotoxicity for plasmidial DNA at high concentrations (250 g/mL), but no DNA damage was observed in micronucleus assay. All extracts were toxic for nauplii microcrustaceans at least one of tested concentrations (0.01 1000 g/mL), but LC50 was calculated only for the hexane, dichloromethane and ethyl acetate extracts.

Los campos de cultivo agrícola se ven afectados en la producción por diversos factores, entre los cuales se encuentra el ataque de plagas de insectos, siendo una medida para su control y exterminio el uso de insecticidas sintéticos, sin embargo, la mayoría genera una alta contaminación ambiental. Debido a esto, los bioinsecticidas de origen vegetal surgieron como una alternativa menos contaminante por su rápida degradación en el ambiente y baja peligrosidad para el hombre. El presente trabajo evaluó la toxicidad de los extractos acuosos, etanólicos y hexánicos de las hojas de Minthostachys mollis (Lamiaceae) “muña” y semillas de Annona muricata (Annonaceae) “guanábana”, Lupinus mutabilis (Fabaceae) “tarwi” y Chenopodium quinoa (Chenopodiaceae) “quinua” sobre hembras adultas del ácaro Tetranychus urticae (Trombidiformes: Tetranychidae) “arañita roja” y larvas del primer instar de Chrysoperla externa (Neuroptera: Chrysopidae) “león de áfidos”. Se emplearon dos concentraciones para todos los extractos: 10% y 20% (p/v), en un periodo de exposición entre las 24 y 72h. Los datos obtenidos fueron sometidos al análisis de varianza (ANDEVA) y la prueba post hoc de Duncan con un nivel de significancia de p≤0,05. Los parámetros de toxicidad se observaron en los valores NOEC (Concentración sin efecto observado) y LOEC (Concentración más baja con efecto observado). Se realizó el screening fitoquímico de los extractos botánicos empleados en los bioensayos. El extracto acuoso de M. mollis y el extracto etanólico de C. quinoa, ambos al 20% de concentración, causaron mortalidades en T. urticae del 28,98% y 29,63%, respectivamente. Los extractos hexánicos de A. muricata y M. mollis no presentaron actividad acaricida. El extracto etanólico de M. mollis registró la mayor toxicidad de todos los extractos evaluados a las 72h de exposición en C. externa, con una mortalidad del 75,76%. El extracto hexánico de A. muricata no produjo mortalidad en este insecto a las 24, 48 y 72h de exposición. La diferenciación en toxicidad (mayor a menor) de los extractos vegetales para C. externa fue: etanólico> acuoso> hexánico. Según el CR (cociente de riesgo) obtenido a las 48h de exposición, los extractos acuosos no representaron un riesgo en la mortalidad de C. externa. La secuencia de mayor a menor toxicidad del CR de los extractos acuosos fue: L. mutabilis˃ A. muricata˃ C. quinoa˃ M. mollis.Agricultural crops have been affected by several factors among which are plague of insects attack, being an effective measure for its control and extermination: synthetic insecticides use, however the majority of them produce high levels of environmental pollution. Due to this, bioinsecticides of plant origin emerging as alternative less polluting because of rapid degradation in the environment and low danger to man. This work analizes the toxicity of hexanic, ethanolic and aqueous extracts of leaves of Minthostachys mollis (Lamiaceae) “muña” and seeds of Annona muricata “guanábana” (Annonaceae), Lupinus mutabilis “tarwi” (Fabaceae) and Chenopodium quinoa “quinua” (Chenopodiaceae) on adult female mite of Tetranychus urticae (Trombidiformes: Tetranychidae): "red spider mite" and the first instar stage larva of Chrysoperla externa (Neuroptera: Chrysopidae): "lacewings". Two concentrations were used for all extract: 10% and 20% (w/v) in an exposure period between 24 and 72h. Analysis of variance (ANOVA) and post-hoc test of Duncan were performed with a significant level p ≤ 0.05. Toxicity endpoints were observed with values in NOEC (No Observed Effect Concentration) and LOEC (Lowest Observed Effect Concentration). Phytochemical screening of botanical extracts used in bioassays was performed. Aqueous extract of M. mollis and ethanolic extract of C. quinoa both at concentration of 20% causing losses in T. urticae of 28,98% and 29,63% respectively. Hexanic extracts of A. muricata and M. mollis did not show acaricide activity. Ethanolic extract of M. mollis registered the greater toxicity of all evaluated extracts at 72 hours in exposure of C. externa with mortality 75,76%. Hexanic extract of A. muricata had no significant effect on mortality in this insect at 24, 48 and 72 hours of exposure. Differentiation in toxicity (to a greater up lesser) of vegetal extracts for C. externa was: ethanol> aqueous> hexane. According to obtained quotient risk (CR) at 48 hours of exposure, aqueous extracts did not represent an increased risk of mortality of C. externa. The descending sequence of toxicity of CR of aqueous extracts was: L. mutabilis˃ A. muricata˃ C. quinoa˃ M. mollis.

Le cancer du sein est un problème de santé public majeur. Avec 500 000 décès dans le monde chaque année, il concentre de nombreux efforts dans la recherche de nouvelles thérapies et de biomarqueurs aussi bien diagnostiques, pronostiques que de suivi thérapeutique. D'un point de vue diagnostique, la mammographie est actuellement la méthode de dépistage de référence. Cependant, elle présente de nombreuses limites, notamment chez les femmes avec une densité mammaire élevée. L'identification de marqueurs permettant de mettre en évidence de petites tumeurs ou pronostiquer l'évolution de ces tumeurs serait une aide précieuse dans la prise en charge de la maladie. D'un point de vue thérapeutique, la radiothérapie est l'un des traitements principaux contre le cancer du sein. Cependant, certaines patientes peuvent développer d'importants effets secondaires tardifs sévères. La prédiction de cette radiotoxicité est un enjeu majeur car elle permettrait d'identifier les patients à risque, de leur proposer un traitement personnalisé et au final d'optimiser leur prise en charge thérapeutique. Malgré de nombreux efforts, il n'existe à l'heure actuelle aucun marqueur prédictif de la radiotoxicité tardive utilisé en routine clinique.Ce travail de thèse a pour objectif d'identifier des marqueurs protéiques, d'une part pour le diagnostic et le pronostic des formes précoces du cancer du sein, et d'autre part pour la prédiction de la réponse à la radiothérapie. Grâce à différentes approches de protéomique, j'ai pu identifier trois signatures protéiques : (1) une signature d'autoanticorps sériques (GAL3, RACK1, PAK2, PHB2 et RUVBL1) pour le diagnostic des carcinomes canalaires in situ (CCIS) et des cancers précoces, (2) une signature d'autoanticorps sériques (CIRBP, ECHDC1, HMGN1, PSRC1 et RBP-Jκ) pronostic de la progression de CCIS en cancer invasif et (3) cinq protéines lymphocytaires (AK2, ANX1, APEX1, HSPA8 et IDH2) prédictives d'une toxicité radio-induite tardive. Au delà de leur intérêt diagnostic, pronostic ou prédictif, ces signatures ont également permis d'identifier de nouveaux partenaires moléculaires susceptibles d'être impliqués dans les mécanismes de carcinogénèse mammaire et de radiotoxicité tardive. / Breast cancer is a major public health problem. With 500,000 deaths every year around the world, it focuses on many efforts in the search for new therapies and for therapeutic monitoring, diagnosis or prognosis biomarkers. From a diagnostic point of view, mammography remains gold standard for breast cancer detection. However, it has many limitations, especially in women with significant breast density. The identification of biomarkers for early diagnosis or for prognosis of in situ tumors in this population at risk would be a valuable improvement in the breast cancer management. Considering treatment strategies, radiotherapy has become a major treatment against breast cancer. However, some patients can develop severe radiation-induced late side effects. Their prediction is a main challenge as clinicians will be able to identify patients at risk and develop individualized treatment. Despite all efforts, no biomarkers are validated and used in clinical routine for normal tissues outcomes after irradiation. The aim of my PhD work was to identify protein markers for the diagnosis and prognosis of early-stage breast cancers, and also for the prediction of radiation-induced severe late effects. With different proteomic approaches, I was able to identify three protein signatures: (1) a serum autoantibody signature for the diagnosis of DCIS and node negative early-stage breast cancers (GAL3, RACK1, PAK2, and PHB2 RUVBL1), (2) a serum autoantibody signature of ductal carcinoma in situ progression to invasive breast cancer (CIRBP, ECHDC1, HMGN1, PSRC1 and RBP-Jκ) and (3) five lymphocyte proteins (AK2, ANX1, APEX1, HSPA8 and IDH2 ) that could predict late radiation-induced toxicity. In addition, these signatures allowed to identify new molecular partners likely to be involved in the mechanisms of mammary carcinogenesis and in the late radio-induced toxicity.

This thesis deals with forms of aluminium occurring in acidified surface waters. Special attention is paid to inorganic monomeric aluminium, fraction, which has toxic effect on some species of aquatic and terrestrial biota. The thesis primarily focuses on the analysis of surface water, groundwater and soil water chemistry in the year-round and long-term monitoring. The practical took place on an experimental mountain basin Uhlířská, which is drained by river Černá Nisa. Uhlířská basin is located in the Jizera Mountains and represents the area which was significantly exposed to the acid atmospheric deposition in the second half of the 20th century. Long-term exposure to acid precipitation caused the acidification of soil and watercourses, forest degradation and deforestation of large parts of the basin. Decrease in pH and an increase in concentration of toxic aluminium also caused species depletion of aquatic ecosystems. Monomeric forms of aluminium were determined spectrophotometrically, the concentration of total aluminium was determined by the method of flame atomic absorption spectrometry (FAAS), in case of low concentration electrothermal atomic absorption spectrometry (ETAAS) was used. Five forms of aluminium were obtained - Altot, AlAS, Alm, Alo and potentially toxic Ali. Appart from...