Cylindrospermopsin (CYN) is a toxic alkaloid produced by several freshwater cyanobacterial species, the most prevalent in Australian waters being Cylindrospermopsis raciborskii. The occurrence of CYN-producing cyanobacteria in drinking water sources worldwide poses a potential human health risk, with one well-documented case of human poisoning attributed to the toxin. While extensive characterisation of CYN-induced toxicity has been conducted in rodents both in vivo and in primary cell cultures, little is known about mechanisms of toxicity in human cell types. This thesis describes studies undertaken to further define the molecular mechanisms of CYN toxicity in human cells. Concentration-response relationships were determined in various cultured human cell types using standard toxicity assays. As expected, CYN caused dose-dependent decreases in the growth of three cell lines, HepG2, Caco-2 and HeLa, and one primary cell type, human dermal fibroblasts, according to tetrazolium reduction assays. CYN treatment did not disrupt cellular membranes according to the lactate dehydrogenase release assay in HepG2 or Caco-2 cells after 24, 48 or 72 h exposure, but did cause membrane disruption in fibroblasts after 72 h exposure to relatively high concentrations of the toxin. Apoptosis occurred more readily in HeLa cells than HepG2 cells or fibroblasts, with 72 h exposure to 1 &mug/mL required before statistically significant rates of apoptosis occurred in the latter cell types. CYN did not appear to directly affect the structure of actin filaments or microtubules under the conditions used in the present study. The major portion of the work presented in this thesis comprises a large-scale interrogation of changes in gene expression induced by the toxin in cultured cells. To assess the effects of CYN on global gene expression, relative messenger RNA (mRNA) levels in human dermal fibroblasts and HepG2 cells after 6 h and 24 h exposure to 1 &mug/mL CYN were determined using oligonucleotide microarrays representing approximately 19 000 genes. Overall, the number of transcripts significantly altered in abundance was greater in fibroblasts than in HepG2 cells. In both cell types, mRNA levels for genes related to amino acid biosynthesis, carbohydrate metabolism, and protein folding and transport were reduced after CYN treatment, while transcripts representing genes for apoptosis, RNA biosynthesis and RNA processing increased in abundance. More detailed data analyses revealed the modulation of a number of stress response pathways—genes regulated by NF-&kappaB were induced, DNA damage response pathways were up-regulated, and a large number of genes involved in endoplasmic reticulum stress were strongly down-regulated. Genes for the synthesis and processing of mRNA, tRNA and rRNA were strongly up-regulated, indicating that CYN treatment may increase the turnover of all forms of cellular RNA. A small group of genes were differentially expressed in HepG2 cells and fibroblasts, revealing cell-specific responses to the toxin. Selected changes in transcript level were validated using real-time quantitative reverse transcriptase PCR (qRT-PCR). The modulation of stress response pathways by CYN, indicated by microarray analysis, was further investigated using other methods. The role of tumour suppressor protein p53 in CYN-mediated gene expression was confirmed by measuring the expression of known p53-regulated genes following CYN treatment of HepG2 cells and human dermal fibroblasts using qRT-PCR. Western blotting of protein extracts from CYNtreated cells showed that p53 protein accumulation occurred in HepG2 cells, providing additional evidence of the activation of the p53 pathway by CYN in this cell line. The immediate-early genes JUN and FOS were found to be induced by CYN in a concentration-dependent manner, and MYC was induced to a lesser extent. The mitogen-activated protein kinase c-Jun NH2-terminal kinase, implicated in the ribotoxic stress response initiated by damage to ribosomal RNA, appeared to become phosphorylated in HeLa cells after CYN exposure, suggesting that ribotoxic stress may occur in response to CYN in at least some cell types. The expression of a reporter gene under the control of a response element specific for NF-&kappaB was induced at the mRNA level but inhibited at the protein level. This shows that while transcription factors such as p53 and NF-&kappaB are apparently activated in response to the toxin, transactivation of target genes may not necessarily manifest a corresponding increase at the protein level. The current work contributes significantly to the current understanding of cylindrospermopsin toxicity in human-derived cell types, and provides further insight into putative modes of action.

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-04-09T18:16:27Z No. of bitstreams: 1 SANDRO MARTINS DOLGHI - DISSERTAÇÃO PPG-CEMat 2014..pdf: 1349252 bytes, checksum: 7e20e2840dc2a5d2e873cd9b77ed0100 (MD5) / Made available in DSpace on 2018-04-09T18:16:27Z (GMT). No. of bitstreams: 1 SANDRO MARTINS DOLGHI - DISSERTAÇÃO PPG-CEMat 2014..pdf: 1349252 bytes, checksum: 7e20e2840dc2a5d2e873cd9b77ed0100 (MD5) Previous issue date: 2014-12-19 / O culto à beleza está em evidência no Brasil e no mundo, levando ao crescente aumento de técnicas e procedimentos para corrigir defeitos estéticos e minimizar os efeitos do envelhecimento. Diversas substâncias preenchedoras com fluidos de alta viscosidade ou partículas de polímeros em suspensão têm sido utilizadas em larga escala em procedimentos cosméticos e correção de lipodistrofia de pacientes HIV positivos, submetidos à terapia antirretroviral de alta atividade. O uso de polimetilmetacrilato (PMMA) como substância de preenchimento dérmico tem crescido vertiginosamente, assim como o número de reações adversas decorrentes de seu uso. O PMMA vem sendo utilizado, amplamente como matéria prima na fabricação de diversos dispositivos e produtos médicos desde a década de 40, especialmente em função de sua biocompatibilidade, entretanto, ainda há carência de informações e estudos sobre migração e resposta inflamatória das substâncias comercializadas no Brasil para fins de preenchimento dérmico. Este trabalho teve como objetivo avaliar as características físico-químicas de implantes de PMMA comercializados no Brasil para procedimentos de bioplastia, em três apresentações comerciais do produto com diferentes concentrações, visando delimitar limites aceitáveis para fins de registro destes produtos na Agência Nacional de Vigilância Sanitária (Anvisa), com foco na segurança sanitária. As amostras foram caracterizadas por Difração de Raios X (DRX), Espectroscopia na região do infravermelho com transformada de Fourier (FTIR), Microscopia Eletrônica de Varredura (MEV), Espectroscopia por Energia Dispersiva de Raios X (EDS), análise de Calorimetria Exploratória Diferencial (DSC), caracterização por Microscopia Óptica (MO). A técnica de DRX mostrou comportamento semicristalino do PMMA. Com a técnica de FTIR constatou-se os grupos funcionais presentes nos implantes. Por meio das técnicas de MO e MEV foi possível perceber a não homogeneidade em relação ao tamanho das microesferas de PMMA. Pôde-se concluir com os resultados de DSC que o pico endotérmico aumentou de acordo com as variações de concentração do PMMA. A partir dos dados obtidos neste trabalho constata-se que o consumidor está sujeito a riscos, visto que a irregularidade das superfícies e variações nos tamanhos das microesferas pode ocasionar reações adversas, em função da fagocitose das microesferas com tamanho inferior a 20 μm. O crescente uso das técnicas de preenchimento com implantes de PMMA, bem como sua indicação para uso em demais especialidades da medicina, o coloca como material a ser ainda amplamente estudado nas mais diversas aplicações do produto na área médica. / The cult of beauty is in evidence in Brazil and in the world, leading to increasing techniques and procedures to correct aesthetic defects and minimize the effects of aging. Several dermal fillers with high viscosity or polymer particles in suspension fluids have been used in large scale in cosmetic procedures and lipodystrophy correction of HIV-positive patients undergoing HAART activity. The use of polymethylmethacrylate (PMMA) as dermal filler substance has grown dramatically, and the number of adverse reactions arising from its use. PMMA has been used widely as a raw material in manufacturing various medical devices and products since the 40s, especially due to its biocompatibility, however, there is still a lack of information and studies on migration and inflammatory response of substances marketed in Brazil dermal filler purposes. This study aimed to evaluate the physical and chemical characteristics of PMMA implants sold in Brazil for bioplasty procedures in three commercial presentations of the product with different concentrations, in order to delimit acceptable limits for registration of these products at ANVISA, with focus on security health. The samples were characterized by X-ray Diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Spectroscopy Energy Dispersive X-ray (EDS) analysis of Differential Scanning Calorimetry (DSC), characterized by Optical Microscopy (OM). The XRD technique showed semi-crystalline behavior of PMMA. FTIR technique found the functional groups present on the implants. Through OM and SEM techniques we saw the inhomogeneity relative to the size of the PMMA microspheres. One can conclude from the results of the DSC endothermic peak increased in accordance with changes in the concentration of PMMA. From the data obtained in this study it appears that the consumer is subject to risks as uneven surfaces and variations in size of the microspheres can cause adverse reactions, depending on the phagocytosis of microspheres with size less than 20 microns. The increasing use of technical fill with PMMA implants, as well as indications for use in other specialties of medicine, places it as material to be still widely studied in various product applications in the medical field.

Do metal particles (including particles of pure metals, alloys, metal oxides and compounds) pose a hazard or risk to human health? In the light of this question, this thesis summarizes results from research conducted on metal particles, and describes the elaboration and implementation of an in vitro test methodology to study metal release from particles through corrosion and dissolution processes in synthetic biological media relevant for human exposure through inhalation/ingestion and dermal contact. Bioaccessible metals are defined as the pool of released metals from particles that potentially could be made available for absorption by humans or other organisms. Studies of bioaccessible metals from different metal particles within this thesis have shown that the metal release process is influenced by material properties, particle specific properties, size distribution, surface area and morphology, as well as the chemistry of synthetic biological test media simulating various human exposure scenarios. The presence of metal particles in proximity to humans and the fact that metals can be released from particles to a varying extent is the hazard referred to in the title. The bioavailable metal fraction of the released metals (the fraction available for uptake/absorption by humans through different exposure routes) is usually significantly smaller than the bioaccessible pool of released metals, and is largely related to the chemical form and state of oxidation of the released metals. Chemical speciation measurements of released chromium for instance revealed chromium to be complexed to its non-available form in simulated lung fluids. Such measurements provide an indirect measure of the potential risk for adverse health effects, when performed at relevant experimental conditions. A more direct way to assess risks is to conduct toxicological in-vitro testing of metal particles, for instance on lung cell cultures relevant for human inhalation. Induced toxicity of metal particles on lung cells includes both the effect of the particles themselves and of the released metal fraction (including bioaccessible and bioavailable metals), the latter shown to be less predominant. The toxic response was clearly influenced by various experimental conditions such as sonication treatment of particles and the presence of serum proteins. Thorough characterization of metal particles assessing parameters including chemical surface composition, degree of agglomeration in solution, size distribution, surface area and morphology was performed and discussed in relation to generated results of bioaccessibility, bioavailability and induced toxicity. One important conclusion was that neither the surface composition nor the bulk composition can be used to assess the extent of metals released from chromium-based alloy particles. These findings emphasize that information on physical-chemical properties and surface characteristics of particles is essential for an in-depth understanding of metal release processes and for further use and interpretation of bioaccessibility data to assess hazard and reduce any risks induced by human exposure to metal particles. / QC 20100803