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Orientador: Claudia R. B. Haddad / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T21:05:30Z (GMT). No. of bitstreams: 1 Dalio_RonaldoJoseDurigan_M.pdf: 923106 bytes, checksum: 4cfd1d62015260abdbf750e8baf62aa6 (MD5) Previous issue date: 2007 / Resumo: Plantas de Cajanus cajan receberam aplicações de brassinosteróide ou de clotrimazol (inibidor de síntese de brassinosteróides) e foram submetidas à salinidade. O objetivo deste trabalho foi verificar o efeito de duas concentrações de brassinosteróide (1 x 10-7 e 0,5 x 10-9 M) e uma de clotrimazol (1 x 10-4 M), no crescimento, composição bioquímica e transporte de aminoácidos em plantas de C. cajan, submetidas à duas concentrações de NaCl (200 e 400 mM). A salinidade afetou os parâmetros de crescimento (massas frescas e secas, comprimento da parte aérea e da maior raiz, número de folhas, área e suculência foliar), os parâmetros bioquímicos (teores de nitrato, prolina, aminoácidos livres, proteínas totais, açúcares solúveis, sacarose, clorofilas e carotenóides) e o padrão dos aminoácidos transportados. A proporção da maioria dos aminoácidos transportados no xilema diminuiu sob salinidade, com exceção de alanina e serina. A concentração de NaCl a 400 mM provocou as maiores alterações. A aplicação de clotrimazol mostrou-se eficiente na inibição dos efeitos provocados por brassinosteróide na grande maioria dos parâmetros estudados, sob salinidade. Não houve diferença em relação à eficiência das duas concentrações de brassinosteróide utilizadas na maioria dos parâmetros avaliados. A aplicação de brassinosteróide amenizou o efeito do estresse salino na maioria dos parâmetros de crescimento, dos parâmetros bioquímicos e no padrão de aminoácidos transportados em plantas de C. cajan, submetidas à salinidade / Abstract: Plants of Cajanus cajan received applications of brassinosteroid or clotrimazol (brassinosteroids synthesis inhibitor), and were subjected to salt stress. The goal of this study was to verify the effect of two concentrations of brassinosteroid (1.0 x 10-7and 0.5 x 10-9M), and one concentration of clotrimazol (1.0 x 10-4 M) on growth, biochemical composition and transport of amino acids in plants of C. Cajan, cultivated under two concentrations of NaCl (200 and 400 mM). The salt affected growth (fresh and dry mass, shoot length and main root length, number of leaves, leaf area and succulence of leaves) and biochemical parameters (nitrate, proline, free aminoacids, total protein, soluble sugars, sucrose, chlorophylls and carotenoids concentrations), and changed the pattern of amino acids transported. The proportion of most of the amino acids transported in the xylem was reduced by salinity. Alanine and serine were exceptions. The largest alterations were caused by 400 mM of NaCl. Clotrimazol was effective in inhibiting the effects caused by brassinosteroid for the great majority of studied parameters. There was no difference between the two concentrations of brassinosteroid for most parameters evaluated, under salinity. The application of brassinosteroid was effective diminishing the effect of salt on most of the growth and biochemical parameters, and in the patterns of amino acids transported in plants of C. Cajan subjected to salt stress / Mestrado / Mestre em Biologia Vegetal

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Uveal melanoma (UM) is the most common primary cancer of the eye and its prognosis is strongly influenced by the occurrence of metastases, which are both rapidly developing and mostly fatal. The most frequent driver mutations occur in a small number of genes including GNAQ, GNA11, BAP1, CYSLTR2 and SF3B1. Due to a lack of suitable animal models, the mechanism through which mutations in these genes cause or cooperate in UM initiation and progression is still largely unknown. We aimed at generating transgenic strains expressing the human mutant proteins in zebrafish uveal melanocytes, using the kita promoter. We used the binary Gal4/UAS system to express the mutant genes mentioned above. Moreover, we performed xenotransplantation experiments with uveal melanoma human and zebrafish cell lines in optically-clear, immunocompromised, zebrafish larvae. Transplanted fish developed melanoma near the site of transplantation in two weeks and showed metastatic growth within one month of age. This approach could be used for short-term assays in larvae, and be further developed for long-term uveal melanoma studies. In parallel, we performed a chemical screen using a transgenic model previously generated in our laboratory, where oncogenic RAS is expressed under the kita promoter. As adults transgenic kita:RAS develop cutaneous melanoma with high frequency and uveal melanoma with a much lower percentage. Larvae showed an increased number of melanocytes already at 3 days post fertilization (dpf) as the earliest evidence of abnormal melanocyte growth. Using this model we performed a chemical screen based on automated detection of a reduction of melanocytes number caused by any of the 1280 FDA or EMA approved drugs of the Prestwick library. The analysis showed that 55 molecules were able to reduce by 60% or more the number of melanocytes per embryo. We identified clotrimazole, as the best candidate. The molecule is an azole derivative acting on the energetic metabolism of melanoma cells. We further tested two compounds for each of the 5 pharmacological classes, and a farnesyltransferase inhibitor (lonafarnib), that inhibits an essential post-translational modification of HRAS and suppresses the hyperpigmentation phenotype. Combinations of clotrimazole and lonafarnib showed the most promising results in zebrafish embryos, allowing a dose reduction of both drugs. We performed validation of these observations in the metastatic human melanoma cell line A375M, and in normal human epithelial melanocytes (NHEM) as control cells, in order to investigate the mechanism of action of clotrimazole in blocking the proliferation of transformed melanocytes. Viability assay and analysis of energy metabolism in clotrimazole treated cells show that this drug specifically affects melanoma cells in vitro and transformed melanocytes in vivo, having no effects on NHEM or wild type larvae. Similar effects were observed with another hit of the antifungal class, miconazole. Furthermore, we show that the effects of clotrimazole are mediated by the inhibition of hexokinase activity and suggest further testing of clotrimazole in combinatorial treatments. In conclusion, this thesis investigated different possibilities of modeling the rare cancer uveal melanoma in zebrafish, using both transgenic and transplantation approaches, and developed a pipeline for a high-throughput, semi-automated chemical screen in a zebrafish melanoma that identified clotrimazole and miconazole as targeting a metabolic vulnerability in melanoma cells.

Fish may be exposed to an array of pharmaceuticals that are discharged into the aquatic environment, paralleling advances in medical knowledge, research and technology. Pharmaceuticals by their nature are designed to target specific receptors, transporters, or enzymes. Nuclear receptors (NRs) are often a key component of the therapeutic mechanism at play, and many of these are conserved among vertebrates. Consequently, fish may be affected by environmental pharmaceutical exposure, however there has been relatively little characterisation of NRs in fish compared with in mammals. In this thesis common carp (C. carpio) were exposed to selected pharmaceuticals in vitro and in vivo to investigate effects centred on the pregnane X receptor (PXR) and peroxisome proliferator-activated receptor alpha (PPARα), two key NRs involved in organism responses to pharmaceutical exposure. The PXR acts as a xenosensor, modulating expression of a number of xenobiotic metabolising enzymes (XMEs) in mammals. In a primary carp hepatocyte model it was shown that expression of a number of XMEs was altered on exposure to rifampicin (RIF), as occurs in mammals. This response was repressed by addition of ketoconaozle (KET; PXR-antagonist), indicating possible PXR involvement. The genes analysed showed up-regulation on exposure to ibuprofen (IBU) and clofibric acid (CFA), but not clotrimazole (CTZ) or propranolol (PRP). The lack of response to mammalian PXR-agonist CTZ was unexpected. In contrast, the same XME genes were found to be up-regulated in vivo after 10 days of exposure of carp to CTZ, although this response occurred only for a relatively high exposure concentration. CTZ was found to concentrate in the plasma (with levels up to 40 times higher than the water). Development and application of a reporter gene assay to measure PXR activation in carp (cPXR) and human PXR showed CTZ activation of cPXR, supporting data from the in vivo studies. Furthermore, activation was seen at concentrations as low as 0.01 μM. Interestingly RIF did not induce a response in the cPXR reporter gene assay, contrasting with the hepatocyte culture work. Taken together, the data presented here suggests divergence in the PXR pathway between mammals and fish in terms of ligand activation and downstream gene targets. PPARα was investigated in carp in vivo using CFA as a mammalian PPARα-agonist. Overall the resulting data suggested a broadly similar role for this NR in lipid homeostasis in fish as for mammals, with a number of PPARα-associated genes and acyl-coA oxidase (ACOX1) activity up-regulated in response to CFA exposure. A number of XMEs were also up-regulated by CFA (in vivo and in vitro), potentially extending the role of PPARα in fish (carp) to regulation of xenobiotic metabolism. The work presented has provided further characterisation of PXR and PPARα in fish. Elucidation of these pathways is vital to provide meaningful data in terms of establishing toxicity and mechanism-of-action data for pharmaceuticals and other compounds in fish, to allow validation of read-across approaches and ultimately aid in their environmental risk assessment. In vitro approaches are attractive ethically, financially and can provide useful mechanistic characterisation of compounds and the primary hepatocyte model and reporter gene assays used here show potential for the screening of pharmaceutical compounds in fish. However, further understanding of the metabolism of drugs and chemicals in fish is required to establish the true value of these methods for informing on possible effects in fish, in vivo.

Selon les dernières estimations de l'OMS, on enregistre chaque année dans le monde 498.9 millions de nouveaux cas d'infections sexuellement transmissibles (IST) dont 276.4 millions sont dus au parasite Trichomonas vaginalis (T. vaginalis). Au niveau du tractus génital, la colonisation et l’irritation de la muqueuse vaginale par T. vaginalis favorisent la survenue de complications infectieuses. Ces infections associées peuvent conduire à des infections chroniques et avoir à terme des conséquences graves (stérilité, rupture prématuré du placenta, mort prématurée du nourrisson). De plus, ces infections vaginales représentent des facteurs qui favorisent les infections par le Virus d’Immunodéficience Humaine (VIH-1).A l’heure actuelle, la lutte contre ce type d’infections consiste à agir tant au niveau curatif, que préventif. Ainsi, l’objectif de ce projet est de développer de nouvelles formulations pour la prévention et le traitement des pathogènes qui colonisent les muqueuses vaginales. Dans ce contexte, la formulation que nous proposons est composée de metronidazole inclut dans un hydrogel thermogélifiant à base de pluronic® F127 et de chitosane. Il a été montré que cet hydrogel conserve ces propriétés physiques à une température physiologique même après dilution dans les fluides vaginaux. Ces hydrogels sont stables et permettent une libération prolongée du metronidazole. La formulation n’a montré aucune toxicité envers les cellules HeLa ni envers la muqueuse vaginale porcine. L’efficacité de cette formulation a été prouvée envers T. vaginalis et présente un effet protecteur envers les cellules HeLa en présence de T. vaginalis. L’ensemble des résultats suggère donc la capacité́ de cette formulation à constituer une double barrière, physique et pharmacologique, protectrice de la muqueuse vaginale vis-à-vis de T. vaginalis. / According to the latest WHO estimates, 498 millions of new cases of sexually transmitted infections (STIs) are recorded annually in the world, including 276.4 million due to the parasite Trichomonas vaginalis (T. vaginalis). In the genital tract colonization and irritation of the vaginal mucosa by T. vaginalis promote the occurrence of infectious complications. Associated infections can lead to chronic infections and eventually have serious consequences (infertility, premature placental abruption, premature death). In addition, these vaginal infections can promote infection by Human Immunodeficiency Virus (HIV-1).Currently, the fight against these infections is to act at curative and preventive level. Thus, the objective of this project is to develop new formulations for the prevention and treatment of pathogens that colonize the vaginal mucosa. In this context, we propose a formulation composed of metronidazole and chitosan include in a thermogelling hydrogel of pluronic F127®.It was shown that the hydrogel retains its physical properties even at a physiological temperature and after dilution in the vaginal fluids. These hydrogels are stable and allow a sustained release of the metronidazole. The formulation showed no toxicity against HeLa cells or porcine vaginal mucosa. The effectiveness of this formulation has been proven against T vaginalis and has a protective effect on HeLa cells in the presence of T. vaginalis. The overall results therefore suggest the ability of this formulation to form a double barrier, physical and pharmacological, than protect vaginal mucosa against T. vaginalis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work aimed the preparation of clotrimazole-loaded nanocapsule suspensions for vulvovaginal fungal infections treatment. For the first time, virgin coconut oil and medium chain triglycerides were used as an oil core of polymeric nanocapsules containing clotrimazole. The analytical method for quantification of this drug into nanoparticles was developed and validated. The method proved to be selective, linear, precise, accurate and robust. Using the method of interfacial deposition of preformed polymer, the nanoparticles were successfully prepared at three different concentrations of clotrimazole (1, 2 and 3 mg/mL). The physicochemical parameters measured were pH, particle diameter, polydispersity index, drug content, encapsulation efficiency, zeta potential and stability in storage during 60 days. The encapsulation efficiency was near to 100%, zeta potential was positive due to the cationic polymer employed, the pH was around 5.6 and drug contents were close to the theoretical values. The size distribution was nanometer (140-200 nm) with polydispersity index lower than 0.2. The formulations had adequate physicochemical characteristics and were stable during storage. Photodegradation studies have shown that the nanoencapsulation improved the stability of clotrimazole against UVC radiation compared to free drug solution after 14 hours of experiment. The in vitro drug release using the dialysis bag technique was characterized by a prolonged release. No burst effect was observed. Profilesare based on an anomalous transport and first order kinetics, regardless of the oil used. The in vitro microbiological test of nanocapsules suspensions was performed against Candida albicans and Candida glabrata susceptible and resistant to fluconazole by microdilution method. In combination with clotrimazole into nanoparticles, medium chain triglycerides was reported to have similar MICs of methanolic solution containing the oil and the drug. In addition to the antifungal activity in solution, coconut oil did not lose its activity after incorporation into the nanostructures and, in combination with drug, showed greater inhibition of microbial growth than the nanocapsules of medium chain triglycerides with clotrimazole. Finally nanoparticle suspensions were incorporated into hydrogels containing polymers with mucoadhesive properties, Pemulen® and Pullulan, which presented appropriate drug content, pH and spreadability. The formulations developed in this study represent promising alternatives for treatment of vulvovaginal candidiasis. / Este trabalho objetivou a preparação de suspensões de nanocápsulas contendo clotrimazol para o tratamento de infecções fúngicas vulvovaginais. Pela primeira vez, o óleo de coco virgem e os triglicerídeos de cadeia média foram empregados como núcleo oleoso de nanocápsulas poliméricas contendo clotrimazol. A metodologia analítica para a quantificação do fármaco nas nanopartículas foi desenvolvida e validada. O método mostrou-se seletivo, linear, preciso, exato e robusto. A partir do método de deposição interfacial do polímero pré-formado, as nanopartículas foram preparadas com sucesso em três concentrações diferentes de clotrimazol (1, 2 e 3 mg/mL). Os parâmetros físico-químicos avaliados foram pH, diâmetro de partícula, índice de polidispersão, teor, eficiência de encapsulamento, potencial zeta e estabilidade frente ao armazenamento por 60 dias. A eficiência de encapsulamento foi próxima a 100%, o valor do potencial zeta foi positivo devido ao polímero catiônico empregado, valor de pH em torno de 5,6 e teores de fármaco próximos aos teóricos. A distribuição de tamanho foi nanométrica (140-200 nm), com índice de polidispersão menor que 0,2. As formulações apresentaram características físico-químicas adequadas e foram estáveis durante o armazenamento. Estudos de fotodegradação mostraram que o nanoencapsulamento melhorou a estabilidade do clotrimazol sob radiação UVC, em comparação com a solução do fármaco livre, após 14 horas de experimento. A liberação do fármaco in vitro, a partir da técnica de sacos de diálise, foi caracterizada como uma liberação prolongada sem efeito burst, que foi mediada por transporte anômalo e seguiu cinética de primeira ordem, independente do óleo utilizado. As suspensões foram capazes de diminuir a velocidade de liberação do clotrimazol nas 24 horas de experimento. A partir do método de microdiluição, procedeu-se a avaliação microbiológica in vitro das suspensões de nanocápsulas contra Candida albicans e Candida glabrata sensíveis e resistentes ao fluconazol. Em combinação com o clotrimazol nas nanopartículas, os triglicerídeos de cadeia média apresentaram valores de CIMs semelhantes à solução metanólica contendo o óleo e o fármaco. Além de apresentar atividade antifúngica em solução, o óleo de coco não perdeu sua atividade após a incorporação na nanoestrutura e, em associação com o fármaco, apresentou maior inibição do crescimento microbiano do que as nanocápsulas de triglicerídeos de cadeia média com clotrimazol. Por fim, as suspensões nanoparticuladas foram incorporadas em hidrogéis contendo polímeros com propriedades mucoadesivas, Pemulen® e Pullulan, que apresentaram teor, pH e espalhabilidade adequados. As formulações desenvolvidas neste estudo representam alternativas promissoras para o tratamento da candidíase vulvovaginal.