It has been suggested that plasma membranes play a role in tumor production. Changes in plasma membrane lipid composition may change membrane fluidity and disrupt cellular communication. These changes in membrane lipid composition appear to be related to the fatty acid content of the animal's diet. The purpose of this investigation was to compare the effect of dietary linoleic and stearic acids and tumor size on cholesterol/phospholipid ratios and fatty acids in membranes of mammary adenocarcinomas.Plasma membranes of mammary tumors from Strain A/St mice were isolated by centrifugation and the lipids extracted. Phospholipid content was assayed by the method of Bartlett (102); cholesterol and fatty acids by gas liquid chromatography.Plasma membranes of tumors from mice fed the high linoleic diet (SAFF) were found to have the highest cholesterol/phospholipid ratios (Mean=0.396); mice fed high stearic acid diets produced tumors with the lowest ratios (0.280). Membranes of tumors from mice fed SA-4 and Stock diets had intermediate ratios (0.0.341 and 0.0.346, respectively). / Department of Biology

The role of dietary fatty acids and their subsequent effects on metabolism has received considerable attention in mammalian species. It is becoming increasingly clear that fatty acids have metabolic roles over and above their influence on energy density of the diet. Recent studies have linked changes in the fatty acyl composition of the plasma membrane, induced by the dietary fat profile, to alterations in both lipid and glucose metabolism. These dietary induced changes have profound effects on insulin action, glucose transport and enzyme activity that regulate triglyceride and fatty acid synthesis, factors that ultimately influence protein and lipid deposition of animals. Because of their high growth rate, broiler chickens have a high requirement for energy and the use of triglycerides as a major energy source has resulted in a fat carcass. A change in the glucose-insulin balance has been suggested as being the main reason for differences in adiposity between broilers selected for fatness or leanness. The hypotheses of this thesis is based on the finding that dietary polyunsaturated fatty acids (PUFA�s) increase the sensitivity of muscle tissue to insulin and this would presumably augment insulin-stimulated glucose uptake into muscle cells. Therefore, increasing the capacity of broiler muscle tissue to utilise glucose as its principal energy substrate would reduce the bird�s reliance on triglycerides and this inturn would result in a leaner carcass. The aims of this study are firstly to explore the role that dietary PUFA�s from the n-3 and n-6 series have on the growth and body composition of broiler chickens and secondly to determine the relationships between dietary fatty acid profile, tissue insulin sensitivity and lipid deposition. Because dietary fatty acids have been implicated in the modulation of hormones important for the growth and development of animals, a third aim of this thesis is to determine the effects of dietary n-3 and n-6 polyunsaturated fatty acids on pituitary and adrenal sensitivity. Since the modulation of metabolism by dietary fatty acids has been attributed to changes in the fatty acyl composition of the plasma membrane, the final aim of this study will be to investigate and characterise the molecular species of the breast muscle choline and ethanolamine phospholipids. Feeding either fish oil a source of n-3 PUFA�s or sunflower oil a source of n-6 PUFA�s fatty acids to broiler chickens resulted in a significant (P<0.01) reduction in the abdominal fat pad mass and a modest increase in breast muscle mass when compared to broilers fed edible tallow. Associated with the changes in carcass composition was an alteration in energy substrate utilisation. This was reflected by lower respiratory quotients and reduced triglyceride and insulin concentrations for the chickens fed the two PUFA diets. Coupled to the shift in energy metabolism was a significant (P<0.05) increase in the proportion of PUFA�s incorporated into the abdominal fat pad and breast muscle. The dietary fat supplements resulted in the incorporation of specific fatty acid subtypes. Feeding fish oil significantly increased the proportion of long-chain n-3 PUFA�s whereas feeding sunflower oil significantly increased the proportion of long-chain n-6 PUFA�s compared to tallow feeding whose tissues were dominated by a higher proportion of saturated fatty acids. It was further shown that dietary n-3 and n-6 PUFA�s enhanced glucose /insulin action. Feeding either fish oil or sunflower oil to broiler chickens increased insulin action when examined by an intravenous glucose tolerance test. The maximal insulin release in response to the glucose infusion was higher in the tallow fed group compared to either the sunflower oil or fish oil groups. To estimate the disappearance rate of glucose from the plasma and its incorporation into tissues, 2-deoxy-D-3H glucose was infused into each chicken. There were no significant differences in the clearance rate of 2-deoxy-D-3H glucose from the plasma. However, when measured under steady state conditions, the labelled glucose incorporation into the breast muscle was greater in birds fed fish oil compared to either tallow or sunflower oil feeding. The dietary fatty acid induced increase in insulin action suggests that the sensitivity of muscle cells to insulin was enhanced. This modulation of tissue sensitivity by dietary fatty acids was also shown to occur at the level of the pituitary. To provide an estimate of pituitary sensitivity, bolus GnRH and GHRH infusions were given on different days to chickens fed the three dietary treatments. Feeding sunflower oil (n-6 PUFA�s) increased the level of GH that was released in response to the GHRH infusion when compared to birds fed either tallow or fish oil (n-3 PUFA�s). This dietary fatty acid modulation appears to be specific to certain pituitary cell types as there was no effect on LH secretion following the GnRH infusion. Dietary fatty acid modulation of endocrine gland sensitivity is particular to the gland type. Although the dietary treatments mediated a distinct pattern in pituitary sensitivity to GHRH infusion, these same three diets did not influence adrenal sensitivity, as there was no difference in the corticosterone profile following either ACTH or CRF infusion. The previously observed physiological changes for the three dietary groups was expected to be positively correlated to an alteration of the plasma membrane phospholipids induced by the dietary fatty acids. Supplementation with fish oil (n-3 PUFA�s) significantly increased levels of both eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) into the choline (PC) and ethanolamine (PE) breast muscle phospholipids compared to either sunflower oil (n-6 PUFA�s) or tallow supplementation. The increase in n-3 PUFA incorporation was associated with a corresponding decrease in the proportion of arachidonic acid (AA; 20:4n-6) an event that would presumably alter substrate availability for the 1- and 2-series eicosanoids. However, feeding sunflower oil or tallow gave a molecular species profile that was remarkably similar in both fatty acid subtype and proportion. This suggests that the plasma membrane dynamics would be similar for these two dietary groups. Therefore, it is appears that factors other than a change in the fatty acyl- composition of the plasma membrane may be responsible for modifying the physiology of the broiler.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Nanoemulsões catiônicas têm sido recentemente propostas como sistemas carreadores de DNA. O presente trabalho teve por objetivo avaliar a influência de diferentes fosfolipídeos sobre propriedades dos complexos formados entre nanoemulsões e pDNA (pTracerTMCMV2). Em uma primeira etapa, nanoemulsões catiônicas constituídas de triglicerídeos de cadeia média, estearilamina, lecitina de gema de ovo ou fosfolipídeos isolados (DSPC, DOPC, DSPE ou DOPE), glicerol e água foram preparadas através do procedimento de emulsificação espontânea. Independente do tipo de fosfolipídeo empregado, esse procedimento conduziu à obtenção de nanoemulsões catiônicas monodispersas com diâmetro de gotícula e potencial zeta de cerca de 250 nm e +50 mV, respectivamente. A complexação do pDNA com as nanoemulsões catiônicas, avaliada através do retardamento de migração do pDNA em gel de agarose por eletroforese, foi total quando o complexo apresenta uma relação de cargas [+/-] ≥ 1,0. Nestas condições, os complexos formados foram protegidos da degradação pela enzima DNase I. Em uma segunda etapa, a citotoxicidade das nanoemulsões e dos complexos com o pDNA sobre células Hep G2 foi avaliada, através do ensaio de MTT. Os resultados obtidos demonstraram que a adição de quantidades crescentes das nanoemulsões, conduz a uma toxicidade progressiva sobre as células, independente do pH do meio. Dentre as formulações estudadas, aquelas estabilizadas pelos fosfolipídeos DSPC e DSPE, de elevada temperatura de transição de fases, foram marcadamente menos tóxicas, em comparação com as formulações obtidas com lecitina, DOPC e DOPE. Essa mesma tendência foi detectada para os complexos formados com o pDNA. Em uma última etapa, foi realizado um estudo preliminar de transferência gênica em células Hep G2, utilizando a técnica de PCR em tempo real. Dentre as diferentes formulações testadas, a maior quantidade de DNA de GFP detectada parece ser para a formulação obtida com o fosfolipídeo DSPC, ilustrando as potencialidades de uso das nanoemulsões desenvolvidas como reagentes de transfecção de pDNA em células Hep G2. Em conclusão, o conjunto dos resultados obtidos demonstra o efeito dos fosfolipídeos empregados sobre propriedades físico-químicas, complexação, estabilidade, citotoxicidade e transfecção de nanoemulsões catiônicas como sistemas carreadores de pDNA. / Cationic nanoemulsions have been recently considered as potential delivery systems for DNA. The aim of the present work was to evaluate the influence of different phospholipids on the properties of complexes formed between nanoemulsions and pDNA (pTracerTM-CMV2). First, cationic nanoemulsions composed of medium chain triglycerides, stearlyamine, egg lecithin or isolated phospholipids (DSPC, DOPC, DSPE or DOPE), glycerol and water were prepared through spontaneous emulsification process. Independently of the type of phospholipid used this procedure results in monodisperses cationic nanoemulsions with droplet size and zeta potential of about 250 nm and +50 mV, respectively. The complexation of pDNA with cationic nanoemulsions, analyzed by agarose gel retardation assay was total when the complex possesses a charge relation [+/-] ≥ 1.0. In these conditions the complexes were protected from enzymatic degradation by DNase I. After that, the cytotoxicity of the nanoemulsion and the complexes with pDNA in Hep G2 cells was evaluated through MTT assay. The results showed that the addition of increasing amount of nanoemulsion leads to a progressive toxicity on the cells independently of the media’s pH. Among the studied formulations the ones stabilized with the phospholipids DSPC and DSPE, that have elevated phase transition temperatures, were much less cytotoxic in comparison with the formulations obtained with lecithin, DOPC and DOPE. This same trend was detected for the complexes formed with pDNA. Finally a preliminary study of gene transfer to Hep G2 cells was performed using real-time PCR technique. Among the different formulations tested, the major quantity of reporter DNA detected seems to be for the formulation obtained with the DSPC phospholipid. This shows the potentialities of the use of nanoemulsions as transfection reagents of pDNA in Hep G2 cells. In conclusion, the overall results show the effect of the phospholipids on physicochemical properties, complexation, stability, cytotoxicity and transfection of cationic nanoemulsions as delivery systems for pDNA.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T12:57:27Z No. of bitstreams: 1 Iukary Takenami Avaliação de biomarcadores....2015.pdf: 2420884 bytes, checksum: b10235421512081a9897b20afdf98354 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T12:57:43Z (GMT) No. of bitstreams: 1 Iukary Takenami Avaliação de biomarcadores....2015.pdf: 2420884 bytes, checksum: b10235421512081a9897b20afdf98354 (MD5) / Made available in DSpace on 2016-02-04T12:57:43Z (GMT). No. of bitstreams: 1 Iukary Takenami Avaliação de biomarcadores....2015.pdf: 2420884 bytes, checksum: b10235421512081a9897b20afdf98354 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO: A tuberculose (TB), doença crônica infecciosa causada por Mycobacterium tuberculosis, é considerada um grave problema de saúde pública no país. A caracterização de antígenos protéicos e/ou lipídios que induzem uma resposta imunológica no hospedeiro, torna-se um importante passo para o desenvolvimento de novas ferramentas de diagnóstico e resposta terapêutica. Dentre os diferentes antígenos, em especial a mammalian cell entry protein 1A (proteína Mce1A), e os fosfolipídios da parede celular do bacilo como a cardiolipina (CL), os fosfatidilinositol (FI), fosfatidilcolina (FC), fosfatidiletanolamina (FE) e o sulfatide (SL), são, em sua maioria altamente imunogênicos, podendo então ser úteis no sorodiagnóstico. Portanto, o objetivo do estudo é avaliar a produção de anticorpos anti- Mce1A e anti-fosfolipídios como biomarcadores no diagnóstico e no monitoramento do tratamento da TB pulmonar. Além disso, o estudo também objetivou avaliar o perfil de citocinas e quimiocinas produzidas em sobrenadantes de cultura após estímulo in vitro com a proteína Mce1A. PACIENTES E MÉTODOS: O estudo foi conduzido no 6º Centro de Saúde Rodrigo Argolo e no Instituto Brasileiro para Investigação da Tuberculose (IBIT). A população de estudo foi composta por pacientes recém diagnosticados com TB pulmonar, seus respectivos comunicantes domiciliares (infectados por M. tuberculosis e saudáveis) e pacientes diagnosticados com outras doenças pulmonares. RESULTADOS: Pacientes com TB produzem uma forte e consistente resposta de anticorpos anti-Mce1A e anti-fosfolipídios (anti-CL, anti-FE, anti-FI e anti-FC) quando comparados com os indivíduos do grupo controle. Além disso, após início do tratamento os níveis de anti-Mce1A e anti-fosfolipídios diminuem significativamente. O sobrenadante de culturas dos pacientes TB, após cultura com Mce1A, induzem uma acentuada produção de TNF, o que não se observa nas demais citocinas e quimiocinas avaliadas. CONCLUSÃO: Estes resultados sugerem que os anticorpos anti- Mce1A e anti-fosfolipídios desempenham potencial papel como biomarcadores sorológicos no diagnóstico da TB pulmonar. Além disso, a proteína Mce1A parece desempenhar um papel importante na produção de TNF, que pode contribuir com a indução de necrose pelo bacilo, permitindo sua evasão das respostas imunes e favorecendo a dispersão do bacilo para outras células não infectadas. / INTRODUCTION: Tuberculosis (TB), chronic infectious disease caused by Mycobacterium tuberculosis, is still a serious public health problem in the country. The characterization of protein and/or lipids antigens that induce an immune response in the host, it is an important step in the development of new diagnostic tools and monitoring TB treatment response. Among the different antigens, particularly mammalian cell entry protein 1A (Mce1A protein), and phospholipids from the cell wall of bacillus such as cardiolipin (CL), phosphatidylinositol (PI), phosphatidylcholine (PTC), phosphatidylethanolamine (PE) and sulfatide (SL), are highly immunogenic and can be used for improvement of the serodiagnosis. Therefore, the aim of the study is to evaluate the production of anti-Mce1A and anti-phospholipids as biomarkers for diagnosis and monitoring of TB treatment response. In addition, the study also aimed to evaluate the profile of cytokines and chemokines produced in vitro after stimulation with Mce1A protein in culture supernatants. PATIENTS AND METHODS: The study was conducted on the 6º Centro de Saúde Rodrigo Argolo and the Instituto Brasileiro para Investigação da Tuberculose (IBIT). The study population consisted of newly diagnosed pulmonary TB patients, their household contacts (infected by M. tuberculosis and healthy) and patients diagnosed with other lung diseases. RESULTS: Patients with TB produce a strong and consistent response to anti-Mce1A and anti-phospholipids (anti-CL, anti-PE, anti- PI and anti-PTC) than those in the control groups. Furthermore, after the beginning of the treatment, anti-Mce1A and anti-phospholipid levels were significantly decreased compared with TB patient at baseline. Culture supernatants of TB patients after stimulation with Mce1A induce a strong TNF production, which is not observed in the other evaluated cytokines and chemokines. CONCLUSION: These results suggest that anti-Mce1A and anti-phospholipids play role as potential serum biomarker in the diagnosis of pulmonary TB. Furthermore, Mce1A protein appears to play an important role in TNF production, which may contribute to the induction of necrosis by M. tuberculosis, allowing for avoidance of immune responses and facilitating the dispersion of bacillus to other uninfected cells.

Nanoemulsões catiônicas têm sido recentemente propostas como sistemas carreadores de DNA. O presente trabalho teve por objetivo avaliar a influência de diferentes fosfolipídeos sobre propriedades dos complexos formados entre nanoemulsões e pDNA (pTracerTMCMV2). Em uma primeira etapa, nanoemulsões catiônicas constituídas de triglicerídeos de cadeia média, estearilamina, lecitina de gema de ovo ou fosfolipídeos isolados (DSPC, DOPC, DSPE ou DOPE), glicerol e água foram preparadas através do procedimento de emulsificação espontânea. Independente do tipo de fosfolipídeo empregado, esse procedimento conduziu à obtenção de nanoemulsões catiônicas monodispersas com diâmetro de gotícula e potencial zeta de cerca de 250 nm e +50 mV, respectivamente. A complexação do pDNA com as nanoemulsões catiônicas, avaliada através do retardamento de migração do pDNA em gel de agarose por eletroforese, foi total quando o complexo apresenta uma relação de cargas [+/-] ≥ 1,0. Nestas condições, os complexos formados foram protegidos da degradação pela enzima DNase I. Em uma segunda etapa, a citotoxicidade das nanoemulsões e dos complexos com o pDNA sobre células Hep G2 foi avaliada, através do ensaio de MTT. Os resultados obtidos demonstraram que a adição de quantidades crescentes das nanoemulsões, conduz a uma toxicidade progressiva sobre as células, independente do pH do meio. Dentre as formulações estudadas, aquelas estabilizadas pelos fosfolipídeos DSPC e DSPE, de elevada temperatura de transição de fases, foram marcadamente menos tóxicas, em comparação com as formulações obtidas com lecitina, DOPC e DOPE. Essa mesma tendência foi detectada para os complexos formados com o pDNA. Em uma última etapa, foi realizado um estudo preliminar de transferência gênica em células Hep G2, utilizando a técnica de PCR em tempo real. Dentre as diferentes formulações testadas, a maior quantidade de DNA de GFP detectada parece ser para a formulação obtida com o fosfolipídeo DSPC, ilustrando as potencialidades de uso das nanoemulsões desenvolvidas como reagentes de transfecção de pDNA em células Hep G2. Em conclusão, o conjunto dos resultados obtidos demonstra o efeito dos fosfolipídeos empregados sobre propriedades físico-químicas, complexação, estabilidade, citotoxicidade e transfecção de nanoemulsões catiônicas como sistemas carreadores de pDNA. / Cationic nanoemulsions have been recently considered as potential delivery systems for DNA. The aim of the present work was to evaluate the influence of different phospholipids on the properties of complexes formed between nanoemulsions and pDNA (pTracerTM-CMV2). First, cationic nanoemulsions composed of medium chain triglycerides, stearlyamine, egg lecithin or isolated phospholipids (DSPC, DOPC, DSPE or DOPE), glycerol and water were prepared through spontaneous emulsification process. Independently of the type of phospholipid used this procedure results in monodisperses cationic nanoemulsions with droplet size and zeta potential of about 250 nm and +50 mV, respectively. The complexation of pDNA with cationic nanoemulsions, analyzed by agarose gel retardation assay was total when the complex possesses a charge relation [+/-] ≥ 1.0. In these conditions the complexes were protected from enzymatic degradation by DNase I. After that, the cytotoxicity of the nanoemulsion and the complexes with pDNA in Hep G2 cells was evaluated through MTT assay. The results showed that the addition of increasing amount of nanoemulsion leads to a progressive toxicity on the cells independently of the media’s pH. Among the studied formulations the ones stabilized with the phospholipids DSPC and DSPE, that have elevated phase transition temperatures, were much less cytotoxic in comparison with the formulations obtained with lecithin, DOPC and DOPE. This same trend was detected for the complexes formed with pDNA. Finally a preliminary study of gene transfer to Hep G2 cells was performed using real-time PCR technique. Among the different formulations tested, the major quantity of reporter DNA detected seems to be for the formulation obtained with the DSPC phospholipid. This shows the potentialities of the use of nanoemulsions as transfection reagents of pDNA in Hep G2 cells. In conclusion, the overall results show the effect of the phospholipids on physicochemical properties, complexation, stability, cytotoxicity and transfection of cationic nanoemulsions as delivery systems for pDNA.

Made available in DSpace on 2014-06-11T19:30:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-15Bitstream added on 2014-06-13T20:00:21Z : No. of bitstreams: 1 aoki_phb_me_bauru.pdf: 3726182 bytes, checksum: c582cd9cf1e3d2f3af8f65685764355e (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A presente dissertação de mestrado trata da fabricação de filmes ultrafinos (nanômetros de espessura) de fosfolipídios aniônicos (DPPG e CLP) e zwiteriônicos (DPPC e DOPC) utilizando as técnicas layer-by-layer (LbL) e Langmuir-Blodgett (LB), possibilitando assim a obtenção de filmes com diferentes arquiteturas moleculares. O objetivo principal foi explorar a aplicação destas diferentes arquiteturas moleculares na detecção do fármaco fenotiazínico azul de metileno (AM) em soluções diluídas. O crescimento linear, camada por camada, desses filmes foi verificado e monitorado por espectroscopia de absorção no UV-Vis. Verificou-se por FTIR que o crescimento dos filmes LbL e LB é determinado por interações eletrostáticas entre os fosfolipídios e o polieletrólito catiônico PAH utilizado como camada de suporte, favorecendo o crescimento dos filmes com fosfolipídios aniônicos. A morfologia foi analisada através de microscopias ópticas, AFM e MEV, revelando que os fosfolipídios estruturam-se como vesículas nos filmes LbL e monocamadas nos filmes LB. Filmes LbL e LB de fosfolipídios com diferentes espessuras foram então depositados sobre eletrodos interdigitados de Pt formando unidades sensoriais que foram aplicadas na detecção de AM via espectroscopia de impedância. Os resultados mostraram que as diferentes arquiteturas moleculares dos filmes LbL e LB levam a respostas elétricas distintas e permitem a detecção de AM em concentrações de nanomolar. A técnica SERS foi utilizada para obter informações estruturais do sistema AM/fosfolipídios nos níveis de diluição alcançados via espectroscopia de impedância. Para tanto, monocamadas LB foram depositados sobre filmes evaporados com 6 nm de Ag e nanopartículas de Ag foram dispersas no interior dos filmes LbL. A presença da Ag na forma de nanopartículas em ambos os casos permitiu a obtenção... / Thin films of anionic (DPPG and CLP) and zwiterionic (DPPC and DOPC) phospholipids with different molecular architectures were fabricated using the layer-by-layer (LbL) and Langmuir-Blodgett (LB) techniques. The main goal of this work was to explore the Application of these distinct molecular architectures in the detection of high diluted solutions of methylene blue (MB), a phenothiazine derivative drug. The linear growth, layer by layer, of these films was verified and monitored by UV-Vis absorption spectroscopy. It was found by FTIR that electrostatic interactions among the phosholipids and the supporting layer of PAH (cationic electrolyte) are the main driving force allowing the growth of the LbL and LB films, which favors the growth of the cationic phospholipids. The morphology was analyzed through optical microscopy, AFM and SEM, revealing that the phospholipids are structured as vesicles in the LbL films and as monolayers in the LB films. Different thickness of LbL and as monolayers in the LB filma containing phospholipids were deposited onto Pt interdigitated electrodes forming sensingn units applied in the detection of MB using impedance spectroscopy. The results showed that the different molecular architectures of the LbL and LB films take to different eletrical responses and allow the MP detection in concentrations of nanomolar. The SERS technique was applied to obtain structural information of the system MP/phospholipids at the dilution levels reached via impedance spectroscopy at the dilution levels reached via impedance spectroscopy. Then, LB monolayers were deposited onto 6nm of Ag evaporated films with and Ag evaporated films with and Ag nanoparticles were dispersed within the LbL films. The presence of Ag forming nanoparticles in both cases played a key role in achieving the MB SERRS signal allowing obtaining the detection in high dilution levels, approaching... (Complete abstract click electronic access below)

A fosfoetanolamina sintética é uma molécula fosforilada artificialmente, com síntese inédita realizada pela primeira vez pelo nosso grupo, diferindo-se das moléculas atuais pelo seu nível de absorção de aproximadamente 90%, com diversas propriedades antiinflamatórias e apoptóticas. O objetivo principal desse estudo e avaliar os efeitos antitumorais \"in vitro\" e \"in vivo\" da fosfoetanolamina sintética em células de melanoma B16F10 implantados em camundongos Balb-c. Foram utilizados grupos de 60 camundongos Balb-c, fêmeas com aproximadamente 20 g, tratados com água e ração \"ad libidum\". A atividade citotóxica do composto foi testada em linhagens tumorais pelo método colorimétrico MTT, e determinada à concentração inibitória (IC50%), sua toxicidade foi também testada em linfócitos T normais, em ensaios de proliferação celular, estimulados por mitógeno. Os animais portadores de tumores foram tratados após o 14º dia do implante tumoral com solução aquosa (i.p) de fosfoetanolamina sintética e o grupo controle recebeu solução salina, e foram avaliados os seguintes parâmetros: volume tumoral, área e número de metástases em órgãos internos. Foi também realizada a comparação da fosfoetanolamina sintética em relação aos quimioterápicos comerciais Taxol e Etoposideo separados nas diferentes fases do ciclo celular. Os resultados do tratamento com a fosfoetanolamina sintética \"in vitro\" mostraram que o composto induz citotoxicidade seletiva para as células tumorais com IC50% de 1.69 ug/ml sem afetar a capacidade proliferativa de células normais. Os animais portadores de tumores dorsais de melanoma B16F10 apresentaram significativa redução carga tumoral, mostrando inibição da capacidade de crescimento e a metastatização. A avaliação hematológica não demonstrou alterações relevantes após a administração da fosfoetanolamina sintética pela via intraperitoneal nos animais portadores de melanoma. Conclui-se que a fosfoetanolamina sintética diminuiu significativamente o tamanho de tumores de forma seletiva, sem alterações em células normais, com vantagem em relação aos quimioterápicos comerciais, pois a mesma não apresentou os terríveis efeitos colaterias dos mesmos. Neste trabalho ficou evidente a capacidade inibitória da fosfoetanolamina sintética na inibição da progressão e disseminação das células tumorais. / The synthetic phosphoethanolamine is a phosphorilad artificially molecule, with unknown synthesis carried through for the first time for our group, differing itself from current molecules for its level of absorption of approximately 90%, with diverse anti-inflammatory and apoptotic as properties. The main objective of this study and to evaluate the anti tumor effect \"in vitro\" and \"in vivo\" of the synthetic phosphoethanolamine in cells of melanoma B16F10 implanted in mice Balb-c. Groups of 60 Balb-c mice had been used, females with approximately 20 g, treated with water and ration \"ad libidum\". The cytotoxic activity of the composition was tested in lives tumor or normal cells for colorimetric method MTT, and determined to the inhibitory concentration (IC50%) its toxicid also it was tested in normal linphocytes T, in assays of cellular proliferation, stimulated for mitogen. The bearing animals of tumors had been dealt with after 14º day the tumor inoculation with watery solution (i.p) of synthetic phosphoethanolamine and the group control received solution saline, and had been evaluated the following parameters: tumor volume, area and number of metastases in internal agencies. Also the comparison of the synthetic phosphoethanolamine in relation to the convencionaly treatment with quimioterapics separate Taxol and Etoposideo in the different phases of the cellular cycle was carried through. The results of the treatment with the synthetic phosphoethanolamine \"in vitro\" had shows that the composition induces selective citotoxicity for the tumor cells with IC50% of 1.69 ug/ml without affecting the proliferative capacity of normal cells. The bearing animals of dorsal tumors of melanoma B16F10 had presented significant reduction tumor load, showing to inhibition of the capacity of growth and the metastatization. The hematological evaluation did not show alterations the administration of the synthetic phosphoethanolamine by the intraperitoneal way in the bearing animals of melanoma. The synthetic phosphoethanolamine significantly reduced the size of tumors of selective form, without alterations in normal cells; with advantage in relation to the commercial quimioterapics therefore the same one did not present the terrible collaterals effect of the same ones. In this work it was evident the inhibitory capacity of the synthetic phosphoethanolamine in the inhibition of the progression and dissemination of the tumor cells.

A fosfoetanolamina sintética (FO-S), um fosfomonoéster, apresenta relevante atividade antitumoral. Contudo, a utilização de um carreador para encapsular a FO-S em lipossomas poderia favorecer a sua disponibilidade no microambiente tumoral, possibilitando o aumento da sua eficácia. Desta forma, o presente estudo avaliou a eficiência de encapsulamento da FO-S em lipossomas de DODAC e o seu potencial antitumoral. Os lipossomas foram preparados por ultrasonicação e caracterizados físicoquimicamente. A citotoxidade foi avaliada nas linhagens tumorais B16F10 (melanoma murino), Hepa1c1c7 (hepatocarcinoma murino) e Skmel-28 (melanoma humano) e nas células normais HUVEC, após o tratamento com diferentes concentrações dos lipossomas DODAC/FO-S, no tempo de 24 horas. A internalização dos lipossomas e o potencial elétrico mitocondrial foram analisados por microscopia confocal a laser. Adicionalmente, a expressão das proteínas caspases 3 e 8 ativas, receptor DR4, citocromo c, p53, p21, Bax, p27, CD44, CD90, Bcl-2 e ciclina D1 foi quantificada por citometria de fluxo. Para os estudos in vivo, os camundongos C57BL/6J portadores de hepatocarcinoma foram tratados com FO-S, DODAC/FO-S e DODAC, pelas vias intraperitoneal (IP) e intrahepática (IH), durante 20 dias. Os resultados demonstraram que os lipossomas apresentaram aspecto esférico e alta eficiência de encapsulação da FO-S, como também promoveram maior citotoxicidade nas linhagens tumorais estudadas, em comparação com FO-S. Além disto, nas células B16F10 e Hepa1c1c7, ocasionou parada nas fases S e G2/M do ciclo celular. A linhagem Hepa1c1c7 foi a mais sensível ao tratamento com os lipossomas DODAC/FO-S, os quais foram internalizados em até 6 horas e promoveram a diminuição de CD90, CD44, ciclina D1 e Bcl-2, o aumento de p53, p21, p27, Bax e caspases 8 e 3 ativas e a liberação do citocromo c. O aumento significativo das caspases 8 e 3 ativas, expressão do receptor DR4 e a liberação do citocromo c também ocorreu nas linhagens B16F10 e Skmel-28. Os resultados in vivo mostraram que os lipossomas DODAC/FO-S e a FO-S não induziram hepatotoxicidade, nefrotoxicidade e caquexia. Os lipossomas DODAC/FO-S não ocasionaram mielossupressão e hemólise, apresentando menor toxicidade em relação a FO-S, administrada pelas vias IP e IH. Além disto, os tratamentos com DODAC/FO-S (IH) e FO-S (IH e IP) foram efetivos em diminuir o número de células na fase S. Contudo, apenas os lipossomas DODAC/FO-S (IH) reduziram significamente os focos tumorais, aumentando as áreas de necrose, promovendo também o aumento da expressão gênica da p53, ciclina B1 e caspases 8 e 3. O conjunto dos resultados in vivo e in vitro demonstraram que a formulação lipossomal DODAC/FO-S foi capaz de maximizar os efeitos antitumorais da FO-S, ativando as vias intrínsecas e extrínsecas da apoptose / Synthetic phosphoethanolamine (PHO-S) - a phosphomonoester - has shown relevant anticancer effects. However, the utilization of a carrier to encapsulate the PHOS in liposomes can maximize its availability in the tumor microenvironment, allowing an increase in its effectiveness. Thus, the present study has evaluated efficiency of PHO-S encapsulation in DODAC liposomes and its antitumor potential. The liposomes were prepared by ultrasonication and physico-chemically characterized. The cytotoxic effects were evaluated on B16F10 cells (murine melanoma), Hepa1c1c7 cells (murine hepatocellular carcinoma), Skmel-28 (human melanoma) and in endothelial cells HUVEC, after treatment with DODAC/PHO-S liposomes at different concentrations for 24 hours. The internalization of the liposomes and mitochondrial electrical potential were analyzed by confocal laser microscopy. Additionally, the expression of active caspases 3 and 8, receptor DR4, cytochrome c, p53 p53, p21, Bax, p27, CD44, CD90, Bcl-2 and cyclin D1 proteins was quantified by flow cytometry. For in vivo studies, C57BL/6J mice with hepatocellular carcinoma were treated with PHO-S, DODAC/PHO-S and DODAC, by intraperitoneal (IP) and intratumoral (IT) routes for 20 days. The results demonstrated that liposomes presented spherical aspect and high PHO-S encapsulation efficiency, as also promoted high cytotoxic effect - compared with PHO-S. Furthermore, in B16F10 and Hepa1c1c7 cells, the liposomes induced S and G2/M cell cycle arrest. Hepa1c1c7 cells showed greater sensitivity to the DODAC/PHO-S formulation, which were internalized until 6 hours and promoted a decrease in the expression of CD90, CD44, cyclin D1 and Bcl-2, an increase of de p53, p21, p27, Bax and active caspases 8 and 3 and the liberation of cytochrome c. The significant increase in the expression of active caspases 3 and 8, DR4 receptor and liberation of cytochrome c also occurred in B16F10 and Skmel-28 cells. In vivo results showed that DODAC/PHO-S liposomes and PHO-S did not induce nephrotoxicity, hepatotoxicity and cachexia. DODAC/PHO-S liposomes did not cause myelosuppression and hemolysis, presenting lower toxicity in relation to PHO-S - when administered by IP and IT routes. Moreover, treatment with DODAC/PHO-S (IT) and PHO-S (IT and IP) effectively decreased the number of cells in S phase. However, only DODAC/PHO-S liposomes significantly reduced the number of tumor foci, increasing area of necrosis, and also promoting an increase in gene expression of p53, cyclin B1 and caspases 8 and 3. The set of in vitro and in vivo results demonstrated that DODAC/PHO-S liposomal formulation was capable of maximizing the PHO-S antitumor effects, activating the intrinsic and extrinsic pathways of the apoptosis

Submitted by Raquel Vergara Gondran (raquelvergara38@yahoo.com.br) on 2016-04-11T17:39:03Z No. of bitstreams: 1 tese melissa.pdf: 963723 bytes, checksum: 38c0d180ac706f670cc2675cd21665c5 (MD5) / Approved for entry into archive by cleuza maria medina dos santos (cleuzamai@yahoo.com.br) on 2016-04-23T14:02:41Z (GMT) No. of bitstreams: 1 tese melissa.pdf: 963723 bytes, checksum: 38c0d180ac706f670cc2675cd21665c5 (MD5) / Made available in DSpace on 2016-04-23T14:02:41Z (GMT). No. of bitstreams: 1 tese melissa.pdf: 963723 bytes, checksum: 38c0d180ac706f670cc2675cd21665c5 (MD5) Previous issue date: 2009 / A possibilidade de valoração de co-produtos e reciclagem de resíduos de processos agroindustriais utilizados em diferentes processos, inclusive os bioprocessos, está sendo amplamente investigada pelo mundo científico, que procura fontes alternativas para diversos produtos e suas aplicações. No alvo destas pesquisas está o farelo de arroz que, atualmente, é considerado um co-produto do beneficiamento do arroz que corresponde de 8 % a 10 % deste grão abundante no Rio Grande do Sul e constitui-se promissora fonte de proteínas, fibra dietética e compostos funcionais como antioxidantes, fosfolipídios, fenóis e ácido fítico. Este trabalho teve o objetivo de estudar a disponibilização de compostos funcionais em farelo de arroz, através da fermentação em estado sólido com Rhizopus oryzae. Durante o desenvolvimento do trabalho foi avaliado, no farelo de arroz fermentado, o efeito do processo fermentativo sobre a composição físico-química, o perfil de ácidos graxos, o conteúdo de fosfolipídios e a presença de compostos antioxidantes, bem como a atividade de enzimas durante o acompanhamento do processo fermentativo. O farelo de arroz utilizado foi fornecido pelo IRGA. O fungo agente fermentador foi Rhizopus oryzae e seus esporos foram propagados em agar batata dextrose, utilizando umar suspensão de Tween 80 (0,2 %), incubados durante 7 dias a 30 °C, até nova e completa esporulação do fungo. A fermentação foi realizada em biorreatores de bandejas onde o substrato (farelo de arroz) foi disposto e homogeneizado com a suspensão de esporos perfazendo a concentração inicial de 4,0x106 esporos.g-1 meio. A umidade do meio foi ajustada para 50 % e o processo ocorreu em estufa a 30 °C por 120 h. As amostras necessárias para as determinações analíticas foram coletadas no início do processo e a cada 24 h. Foram determinados os teores de umidade, cinzas, lipídios, proteína, fibra, açúcares redutores, aminoácidos digeríveis, ácido fítico, perfil de ácidos graxos com identificação e quantificação por cromatografia gasosa e fosfolipídios. Os compostos fenólicos dos fermentados foram extraídos com metanol a frio e quantificados por método espectrofotométrico com o reagente de Folin-Ciocalteau. A capacidade dos antioxidantes dos extratos do farelo fermentado foi estimada considerando o potencial em capturar os radicais 2,2-difenil-1-picrilidrazil (DPPH), inibir a peroxidação lipídica e a reação de escurecimento do guaiacol catalisada pela peroxidase. A fermentação em estado sólido reduziu em 40 %, 50 % e 60 %, significativamente (p<0,05), os teores lipídios, ácido fítico e açúcares redutores do farelo de arroz, respectivamente. O farelo fermentado apresentou teores aumentados de 30 % em cinzas, 50 % em fibras e 40 % em proteínas. A determinação de aminoácidos digeríveis indicou aumento de 27,6 % na digestibilidade das proteínas produzidas. O farelo de arroz fermentado por 24 h apresentou o maior conteúdo de compostos fenólicos totais (2200 µg ác.ferúlico/gfarelo), no entanto o extrato metanólico do farelo de arroz fermentado por 96 h inativou 50 % do DPPH reativo em 15 min (CE50 de 4,3 µg ác.ferúlico/mL). Este mesmo extrato reduziu em 57 % o valor do índice de peróxido no óleo de oliva após 30 dias de armazenamento. O extrato aquoso do farelo de arroz fermentado por 120 h foi o mais eficiente inibidor da reação de escurecimento catalisada pela peroxidase. Os teores de fosfolipídios foram aumentados em 1,8 mg P/g lipídio. No farelo fermentado os ácidos oléico, palmítico e linoléico foram os predominantes, ocorrendo ao longo da fermentação a redução dos ácidos graxos saturados (20 %) e o aumento dos ácidos graxos insaturados (5 %) Estes resultados indicam que a fermentação em estado sólido é uma poderosa ferramenta para agregar valor a este co-produto modificando a sua composição química e disponibilizando compostos de maior interesse, que podem ser aplicados em outros processos, subsidiando o agronegócio com alternativas para à sua sustentabilidade. O presente trabalho contribui com informações importantes para as etapas de investigação sobre a disponibilização destes biocompostos e suas futuras aplicações. / The possibility of co-product valuation and residue reuse of agroindustrial processes may be used in different processes, besides bioprocess is being investigated thoroughly by the scientific world which looks for alternative sources for several products and its applications. In the goal of these researches is rice bran, which is nowadays being considered a co-product of rice milling and corresponds from 8% to 10% of this abundant grain in Rio Grande do Sul. Thus, it is a promising source of proteins, dietary fiber and functional compounds like antioxidants, phospholipids, phenols and phytic acid. This work aimed to study the availability of functional compounds in rice bran through solid-state fermentation with Rhizopus oryzae. During the development of this work, the effect of the fermentative process was evaluated on fermented rice bran, that is, the physico-chemical composition, the fatty acid profile, phospholipids content and the presence of antioxidant compounds, as well as the enzyme activity during the fermentative process. Rice bran was supplied by IRGA (Instituto Rio Grandense de Arroz). The strain used for fermentation was from Rhizopus oryzae fungus and the spores were scraped from the slopes into aqueous emulsion of Tween 80 (0.2 %) incubated during 7 days at 30 °C until new and complete fungi sporulation. Fermentation was carried out in tray bioreactors, in which the substratum, rice bran, was placed and homogenized with spores suspension totalizing the initial concentration of 4.0x106 spores/g. The medium moisture was adjusted to 50 % and the process was carried out in a chamber at 30 ºC for 120 h. For analytical determination, the samples were withdrawn at the beginning of the process and each 24 h. The contents of moisture, ash, lipid, protein, fiber, reducing sugars, digestible aminoacids and phytic acid were determined, as well as the fatty acid profile, following identification and quantification by gaseous chromatography. The phenolic compounds were cold-extracted with methanol and quantified by spectrophotometer using the FolinCiocalteau reagent. The fermented bran extract antioxidant ability was estimated considering its potential in capturing DPPH radicals, inhibiting lipid peroxidation and guaiacol darkening reaction catalyzed by peroxidase enzyme. Solid-state fermentation reduced moisture, lipid, phytic acid and reducing sugars content of rice bran in, respectively, 24.6 %, 40 %, 50 % and 60 %. The fermented bran presented an increase of 30 % in ashes content, 50 % in fibers and 40 % in proteins. The digestible aminoacid determination indicated increase of 27.6 % in the digestibility of produced proteins. Rice bran fermented for 24 h showed the highest phenolic compound content (2200 µg ferulic acid/g bran), however the fermented rice bran methanolic extract at 96 h inactivated 50 % of the DPPH reagent in 15 min (CE50 of 4.3 µg ferulic acid/mL). This same extract reduced in 57% the peroxide index in olive oil after 30 days of storage. The fermented rice bran aqueous extract at 120 h was the most efficient inhibitor of the darkening reaction catalyzed by peroxidase. Phospholipids content were increased to 1.8 mgP/g lipid. Oleic, palmitic and linoleic fatty acids predominated in fermented bran, being observed a reduction in saturated fatty acids (20 %) and an increase (5 %) in unsaturated ones along the fermentation. These results point out that solid-state fermentation is a powerful tool to add value to rice bran, modifying its chemical composition in which components of major interest become available and can be applied to other processes, subsidizing agribusiness with important information for investigating steps related to these compounds, purifying method and application.