Thesis (Ph. D.)--University of Texas Health Science Center at Houston, School of Public Health, 1995. / Includes bibliographical references (leaves 100-107).

Made available in DSpace on 2015-03-03T11:52:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-26Bitstream added on 2015-03-03T12:07:07Z : No. of bitstreams: 1 000808319.pdf: 876369 bytes, checksum: 3244e6e3938737edaba8e2c8144fa57a (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Investigamos as repercussões do padrão alimentar ocidental, rico em gordura, (óleo de milho) (20%) e pobre em micronutrientes [ácido fólico (1 mg/g), colina (0,12%) e fibra (2%)], ocorrido durante a gestação, lactação e puberdade na susceptibilidade ao desenvolvimento de câncer do cólon induzido pela 1,2-dimetilhidrazina (DMH) no rato. Avaliamos se a reintrodução da dieta Ocidental durante a vida adulta em animais previamente expostos à dieta Ocidental durante a vida intrauterina, lactação e puberdade, pode modificar a susceptibilidade à oncogênese do cólon pela DMH. Ratas fêmeas SD prenhes receberam dieta semi-purificada controle (AIN-93) ou do tipo Ocidental (WD - Western Style Diet) a partir do dia gestacional 12 (DG 12). Após o desmame (dia 21), a prole de machos permaneceu com o mesmo tipo de dieta de suas mães até o dia pós-natal 50 (DPN 50). Nesse momento, receberam quatro injeções subcutâneas de DMH (40mg/kg) e foram divididos nos seguintes grupos/dietas: Grupo I (AIN-93/DMH/AIN-93) recebeu dieta controle durante todo o experimento (DG 12- DPN 370), o Grupo II (AIN-93/DMH/Ocidental) recebeu dieta Ocidental somente na vida adulta, a partir do DPN 260, Grupo III (Ocidental/DMH/AIN-93) recebeu dieta Ocidental desde a vida intrauterina até o início da puberdade (DG 12 - DPN 50), e o Grupo IV (Ocidental/DMH/Ocidental) recebeu dieta Ocidental desde a vida intrauterina até o início da puberdade (DG 12 - DPN 50), e foi reintroduzido à dieta Ocidental na vida adulta (DPN 260). Todos os animais foram eutanasiados ao final da 50ª semana de experimento (DPN 370). O cólon foi removido e submetido à análise estereoscópica e histológica para determinar o número, distribuição e multiplicidade dos focos de criptas aberrantes (FCAs). As características das lesões neoplásicas e os níveis séricos de insulina e leptina foram analisados à necropsia. A exposição à dieta Ocidental durante a gestação, ... / We examined the effects of a Western Style Diet (WD), high in fat (corn oil) (20%) and low in micronutrients [i.e.; folic acid (1 mg/g), choline (0.12%) and fiber (2%)] during pregnancy, lactation and in prepubertal stage to examine the susceptibility to colon carcinogenesis induced by dimethylhydrazine (DMH) in male Sprague-Dawley (SD) rats as later adults. We also evaluated whether WD reintroduction during adulthood can modify susceptibility to colon carcinogenesis. Pregnant SD rats received one of two diets AIN-93G or WD at gestational day 12 (GD 12). F1 male pups were weaned (21d) and fed the same AIN- 93G or WD diets from their dams until postnatal day 50 (PND 50). At this point, they were given four subcutaneous injections of carcinogen DMH (40 mg/body weight) and were shared the following diets/group: group I (AIN-93/DMH/AIN-93) a lifetime control diet-fed group (GD 12 until postnatal day - PND 370), group II (AIN-93/DMH/WD) received WD only in adulthood (at PND 260), group III (WD/DMH/AIN-93) received WD during pregnancy from dams until prepubertal stage (GD 12 - PND 50) and group IV (WD/DMH/WD), received WD since pregnancy from dams (GD 12) until prepubertal stage (PND 50) and was reintroduced to WD during adulthood (PND 260). All groups were euthanized at the end of 50th week (PND 370). The colon was removed and the aberrant crypt foci (ACF) were stereoscopically and histologically scored for number, distribution and multiplicity. Neoplastic lesions and serum insulin and leptin concentrations were analysed at necropsy. ACF multiplicity and dysplastic lesions were significantly higher in the group fed WD during pregnancy until prepubertal stage (group III) than control group (group I). In the group III, the number of adenocarcinomas were higher than control (group I). In the group fed WD only in adulthood (group II), ACF multiplicity was significantly higher than in the control (group I). The group ... / CNPq: 142195/2010-3 / FAPESP: 10/18827-0

Cell metabolism disorder is considered as both direct and indirect consequence of oncogenic mutations for tumoigenesis. Autophagy is a metabolic stress response and a mechanism of natural cellular degradation. It is believed that autophagy, as well as metabolism, is a crucial process for the adaptation of cancer cells to changes in nutrient availability. Understanding the relationship between metabolism and autophagy and targeting on the key steps are regarded as a promising strategy to treat cancer. Halofuginone (HF), a semisynthetic quinazolinone alkaloid originally derived from the plant Dichroa febrifuga Lour., has gained attention for its potential therapeutic effects in a variety of cancers. We hypothesize that HF may inhibit cancer cell proliferation by inducing changes in glucose metabolism and modulating related autophagy. A series of studies, from in vitro to in vivo, were designed to approve this concept in colorectal cancer (CRC);Firstly, we found that HF inhibited human CRC cell proliferation and induced the generation of reactive oxygen species (ROS) and apoptosis. As expected, a reduced level of NADPH was also observed, at least in part due to inactivation of glucose-6-phosphate dehydrogenase (G6PD) in pentose phosphate pathway (PPP) upon HF treatment. Given these findings, we further investigated metabolic regulation of HF through Akt/mTORC1-mediated aerobic glycolysis and found that HF downregulated the Akt/mTORC1 signaling pathway. Moreover, metabolomics found slower rates in both glycolytic flux and glucose-derived tricarboxylic acid cycle flux. Meanwhile, both glucose transporter GLUT1 and hexokinase-2 in glycolysis were suppressed in CRC cells by HF. These findings support our notion that HF regulates the Akt/mTORC1 signaling pathway to dampen glucose uptake and glycolysis in CRC cells. Furthermore, HF retarded tumor growth in nude mice inoculated with HCT116 cells and reduced the viability of primary cells from the tissues of CRC patients. This finding further confirmed our hypothesis that HF inhibits CRC cell growth through metabolic regulation of Akt/mTORC1. Because mTORC1 can inhibit autophagy through phosphorylation and inactivation of the initiating kinase ULK1 in cancer cells, we further studied the HF effects on CRC in different nutritional conditions. The results showed that HF in nutrient-rich conditions could reduce SQSTM1/p62 through mTORC1-mediated phosphorylation at Ser757 of ULK1. More interestingly, HF elevated SQSTM1 protein level in low nutrient condition through AMPK-mediated phosphorylation at Ser317/777 of ULK1. It showed that HF could regulate nutrient-sensing mTORC1-ULK1 or AMPK-ULK1 to dually modulate autophagy in CRC cells. Further study by using a variety of methods, including mRFP-GFP-LC3 puncta formation, transmission electron microscope (TEM) analysis and monodansylcadaverine (MDC) staining, found that HF could induce autophagosome formation and inhibit autophagosome membrane elongation, depending on nutrient-sensing pathways. Furthermore, we found HF pronouncedly enhanced expression level of Atg7 under nutrient-rich conditions while it decreased Atg7 in CRC cells under nutrient-poor conditions. These two findings imply that Atg7 is required in dual regulation of autophagic flux depending on nutrient conditions. This conclusion was then validated by comparing with autophagy-related proteins in Atg7 knockout (KO) MEFs and Wild-type (WT) MEFs upon HF treatment. Importantly, through analysis of metabolome and metabolic enzymes, we found that HF inhibited glycolysis under nutrient-rich conditions while it inhibited gluconeogenesis under nutrient-poor conditions in an Atg7-dependent manner. In subsequent studies, we found that caloric restriction (CR) in a xenograft mouse model, which mimics low nutrition in vitro, enhanced the anticancer activity of HF. Further analysis of the expression of TQTSM1 and LC3 in tumor tissues demonstrated that HF is an autophagic inducer in xenograft-bearing nude mice with ad libitum feeding, whereas it is an autophagic inhibitor when using CR.;In summary, this study explains how HF controls CRC cell growth through its influences on glucose metabolism and autophagy regulation. HF not only dually regulates autophagy in vitro and in vivo to inhibit cancer cell growth and proliferation through nutrient-sensing pathways under different conditions, but also modulates glycolysis/gluconeogenesis through an autophagic pathway. These results suggest that HF could turn out to be a potent therapeutic drug for treating CRC.

Made available in DSpace on 2015-12-10T14:23:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-08-28. Added 1 bitstream(s) on 2015-12-10T14:30:04Z : No. of bitstreams: 1 000853903.pdf: 918508 bytes, checksum: d82c80d39357f403a66df5582254910d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O interferon alfa (IFN-α), um IFN do tipo I, se apresenta como uma citocina com grande potencial terapêutico, pois atua no combate direto às células tumorais, além de agir sobre a maturação de células dendríticas (DCs), que são células apresentadoras de antígenos profissionais e peças chave na elaboração da uma resposta antitumoral. Entretanto, a administração sistêmica de citocinas pode produzir toxicidade importante nos pacientes, de modo que a indução de sua produção in situ poderia representar uma forma de imunomodulação mais adequada. Assim, o objetivo deste estudo é verificar a ação de vetores lentivirais carregando o gene do IFN-α para transdução de células tumorais, permitindo assim a produção localizada de IFN-α, a fim de explorar, in vitro, seu potencial lítico e imunomodulatório sobre DCs. Vetores lentivirais carregando o gene do IFN-α humano (Lego-IFN) ou GFP (Lego-GFP) foram utilizados para a transdução in vitro de células de câncer colorretal. A transdução foi feita com diferentes multiplicidades de infecção (MOIs - 0.3, 1.0, 2.0, 4.0) para avaliarmos o efeito dose-dependente, seguido de co-cultura com DCs derivadas de monócitos de doadores saudáveis (DC-0.3, DC-1.0, DC-2.0, DC-4.0). Após 48h de co-cultura, as DCs foram avaliadas fenotípica e funcionalmente, através da análise dos marcadores de membrana por citometria de fluxo, capacidade de aloestimulação e de indução de linfócitos T citotóxicos (CTLs). Nós observamos que a transdução com Lego-GFP, mas não com Lego-IFN, aumentou a imunogenicidade das células tumorais, com aumento de expressão de CD54 e HLA-DR. A co-cultura de DCs com células tumorais transduzidas com Lego-IFN aumentou discretamente seu perfil de ativação, mas não seu potencial aloestimulatório in vitro. Observamos que linfócitos cultivados com DC-2.0 produziram níveis mais altos de IFN-γ, sugerindo a indução de um perfil Th1, enquanto que... / Interferon alpha (IFN-α) is a type I IFN with great therapeutic potential, since it is able to directly fight tumor cells and enhance the maturation of dendritic cells (DCs), the main antigen-presenting cells, required for an effective antitumor response. However, the systemic administration of cytokines can induce severe collateral effects. Therefore, the induction of cytokine secretion in situ should represent a more adequate approach for cytokine-based immunotherapy. Thus, the goal of this study was to induce IFN-α secretion by colon cancer cells by transduction with a lentivirus vector carrying the human IFN-α gene, followed by analysis of its immunomodulatory potential over DCs. Transduction was made with different multiplicities of infection (MOIs - 0.3, 1.0, 2.0 and 4.0) to evaluate the dose-dependent effects. Such cells were co-cultured with monocyte-derived DCs from healthy donors (DC-0.3, DC-1.0, DC-2.0 and DC-4.0). Forty-eight hours later, DCs were evaluated for their phenotype (surface activation/maturation markers) by flow cytometry, their ability to induce allogeneic response in a mixed leukocyte reaction (MLR) and effectiveness to induce cytotoxic T cells. We observed that transduction with Lego-GFP, but not Lego-IFN, increased tumor cells' immunogenicity with up-regulation of the markers CD54 and HLA-DR. Co-culture of Lego-IFN-transduced tumor cells with DCs slightly enhanced their activation phenotype but not their potential to stimulate T cell proliferation in vitro. Furthermore, we observed that lymphocytes cultured with DC-2.0 produced higher levels of IFN-γ, suggesting an induction of Th1 profile on T cells, while DC-GFP induced more IL-10 and IL-4. Additionally, DC-4.0 was more efficient in generating cytotoxic T lymphocytes (CTLs) that the control DC, however DC-GFP induced even more CD8+T cell proliferation. The enhancement of tumor cell immunogenicity and the superior induction of CLTs ...

Made available in DSpace on 2014-06-11T19:27:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-21Bitstream added on 2014-06-13T20:17:31Z : No. of bitstreams: 1 fragoso_mf_me_botfm.pdf: 663141 bytes, checksum: 3faf3707c9cd0b2583b7df103e5d313a (MD5) / Açaí, fruto da Euterpe oleraceae Martius, é consumido in natura e em uma variedade de bebidas e alimentos. Esse fruto tem sido bastante consumido e produzido no Brazil, devido à alta popularidade como alimento funcional com capacidade antioxidante e antiinflamatória. Este trabalho investigou o efeito protetor potencial do consumo da polpa de açaí (PA) seca por pulverização na carcinogênese de cólon induzida pela 1,2-dimetilhidrazina (DMH) em ratos Wistar adultos (peso médio de 200g). Após 4 semanas de administração de DMH, os grupos passaram a receber dieta padrão, dieta contendo 2.5% ou 5.0% de PA e 0.2% de N-acetilcisteína (NAC) durante 10 semanas de tratamento para análise de focos de criptas aberrantes (FCA). Adicionalmente, dois grupos receberam dieta padrão e dieta contendo 5.0% de PA durante 20 semanas de tratamento para análise de tumores de cólon. No experimento de 14 semanas, foi observada redução significante no número de criptas aberrantes (CA) e em FCA (1-3 CA) nos grupos que receberam 5.0% de PA e 0.2% de NAC (37% e 47% de inibição, p = 0.036; 37% e 41% de inibição, p = 0.042), respectivamente, quando comparados ao grupo não tratado. No experimento de 24 semanas, foi observada redução no número de adenocarcinomas invasivos e multiplicidade de tumores no grupo que recebeu 5.0% de PA na dieta (p < 0.005 e p = 0.001, respectivamente) quando comparados ao grupo não tratado. Além disso, redução significante na proliferação celular de tumores marcados com Ki-67 e na taxa de crescimento tumoral foi observada no grupo que recebeu 5.0% de PA (p = 0.003 e p = 0.001). Portanto, os resultados desse trabalho indicam que uma dieta acrescida de 5.0% da polpa de açaí seca por pulverização pode reduzir o desenvolvimento de FCA e de tumores de colón induzidos em ratos, indicando seu uso como um potencial alimento funcional / Acai, fruit from Euterpe oleraceae Martius, is consumed in natura and in a variety of beverages and food preparations. This fruit has been widely consumed and produced in Brazil, due to the high popularity as a functional food with antioxidant and anti-inflammatory capacities. This study investigated the potential protective effect of spray-dried açai pulp (AP) intake on colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in male adult Wistar rats (mean weight of 200g). After 4 weeks of DMH administration, groups were fed standard diet, diet containing AP at 2.5% or 5.0% or 0.2% N-acetylcysteine (NAC) during 10 weeks for analysis of aberrant crypt foci (ACF) as endpoint. Additionally, two groups were fed standard diet or diet containing AP at 5.0% during 20 weeks for analysis of colon tumor as endpoint. In 14-week ACF assay, a significant reduction in number of aberrant crypt (AC) and in ACF (1-3 AC) were observed in groups fed 5.0% AP and 0.2% NAC (37% and 47% inhibition, p = 0.036; 37% and 41% inhibition, p = 0.042), respectively, when compared to the untreated group. In 24-week tumor assay, a significant reduction in number of invasive adenocarcinomas and tumor multiplicity were observed in group fed 5.0% AP (p < 0.005 and p = 0.001, respectively) when compared to untreated group. Also, significant reduction in tumor Ki-67 cell proliferation and growth index was observed in group fed 5.0% AP (p = 0.003 and p = 0.001). Therefore, the findings of this study indicate that spray-dried açai pulp feeding at 5.0% may reduce the development of chemically-induced ACF and colon tumor in male rats, indicating their use as a potential functional food

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-28Bitstream added on 2014-06-13T20:10:15Z : No. of bitstreams: 1 moura_na_me_botib.pdf: 645966 bytes, checksum: 470176f1b4d022fe7693f78122d3d799 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Yacon (Smallanthus sonchifolius) é uma raiz originária da região dos Andes que tem se destacado pelos seus compostos bioativos principalmente frutanos como futooligossacarídeos e inulina. O presente projeto teve como objetivo determinar a atividade quimioprotetora da ingestão de Yacon sobre o desenvolvimento de lesões pré-neoplásicas (focos de criptas aberrantes-FCA) induzidas pela dimetilhidrazina (DMH) em ratos Wistar machos. Os animais foram divididos em seis grupos com 5 a 12 animais cada. Os animais dos Grupos 1 a 4 e Grupos 5 e 6 receberam respectivamente, quatro injeções subcutâneas de DMH (40 mg/Kg) e solução de EDTA (veículo da DMH) nas duas semanas iniciais do experimento respectivamente. Os animais receberam ração basal até a sexta semana do experimento e a partir desta os animais dos grupos 2, 3, 4, 5 receberam ração acrescida de extrato de Yacon a 0,5%, 1%, 1% e 1%, respectivamente. Os animais do grupo 4 receberam Lactobacilus casei (2,5 x 1010 de UFC por Kg de ração). O sacrifício ocorreu na vigésima semana de experimento para análise de focos de criptas aberrantes (FCA) e tumores. Nossos resultados mostraram uma redução no número, multiplicidade de FCA e no número de adenocarcinomas invasivos nos grupos tratados com 1% yacon (G3) e na combinação simbiótica (G4), (0,05 < p < 0, 001). A multiplicidade de tumores (invasivos e não invasivos) foi significativamente menor no grupo tratado com a combinação simbiótica (p < 0,02). Observou-se também uma redução significativa nas taxas de proliferação celular tanto em criptas colônicas como em tumores nos grupos tratados com 1% yacon (G3) e na combinação simbiótica (G4), p < 0.001. Os resultados sugerem que a ingestão de extrato de yacon exerce atividade quimiopreventiva contra carcinogênese de cólon / Yacon (Smallanthus sonchifolius) is a tuberous root native to the Andean region of South America which contains high amounts of inulin-type fructans. The present study investigated the beneficial potential of yacon root intake on development of colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in male Wistar rats. Six groups were studied: Groups 1–4 were given four s.c. injections of DMH (40 mg/kg b.w.) twice a week, during two weeks, whereas Groups 4 and 5 received similar injections of EDTA solution (DMH vehicle). After 6 weeks of DMH-initiation, groups were fed with basal diet (G1 and G6) or basal diet containing dried extract of yacon root at 0.5% (G2), 1.0% (G3 and G5) or a synbiotic formulation (G4) (1.0% yacon root plus Lactobacillus casei at 2.5 x 1010 CFU per g diet) for 13 weeks. At 20 week, all animals were killed and the colons were analyzed for development of aberrant crypt foci (ACF) and tumor. A significant reduction in number and multiplicity of ACF and in number of invasive adenocarcinomas was observed in the groups orally treated with 1.0% yacon root (G3) or their synbiotic formulation (G4) (0.05 < p < 0.001). Tumor multiplicity (noninvasive plus invasive) was significantly lower solely in group fed with symbiotic formulation (p < 0.02). Also, a reduction in cell proliferation indexes in colonic crypt and tumor were observed in groups orally treated with 1.0% yacon root (G3) or their synbiotic formulation (G4) (p < 0.001). The findings this study suggest that yacon root intake may have potential as chemopreventive agent against colon carcinogenesis

Orientador: Luis Fernando Barbisan / Banca: Camila Renata Correa / Banca: Sérgio Britto Garcia / Resumo: Açaí, fruto da Euterpe oleraceae Martius, é consumido in natura e em uma variedade de bebidas e alimentos. Esse fruto tem sido bastante consumido e produzido no Brazil, devido à alta popularidade como alimento funcional com capacidade antioxidante e antiinflamatória. Este trabalho investigou o efeito protetor potencial do consumo da polpa de açaí (PA) seca por pulverização na carcinogênese de cólon induzida pela 1,2-dimetilhidrazina (DMH) em ratos Wistar adultos (peso médio de 200g). Após 4 semanas de administração de DMH, os grupos passaram a receber dieta padrão, dieta contendo 2.5% ou 5.0% de PA e 0.2% de N-acetilcisteína (NAC) durante 10 semanas de tratamento para análise de focos de criptas aberrantes (FCA). Adicionalmente, dois grupos receberam dieta padrão e dieta contendo 5.0% de PA durante 20 semanas de tratamento para análise de tumores de cólon. No experimento de 14 semanas, foi observada redução significante no número de criptas aberrantes (CA) e em FCA (1-3 CA) nos grupos que receberam 5.0% de PA e 0.2% de NAC (37% e 47% de inibição, p = 0.036; 37% e 41% de inibição, p = 0.042), respectivamente, quando comparados ao grupo não tratado. No experimento de 24 semanas, foi observada redução no número de adenocarcinomas invasivos e multiplicidade de tumores no grupo que recebeu 5.0% de PA na dieta (p < 0.005 e p = 0.001, respectivamente) quando comparados ao grupo não tratado. Além disso, redução significante na proliferação celular de tumores marcados com Ki-67 e na taxa de crescimento tumoral foi observada no grupo que recebeu 5.0% de PA (p = 0.003 e p = 0.001). Portanto, os resultados desse trabalho indicam que uma dieta acrescida de 5.0% da polpa de açaí seca por pulverização pode reduzir o desenvolvimento de FCA e de tumores de colón induzidos em ratos, indicando seu uso como um potencial alimento funcional / Abstract: Acai, fruit from Euterpe oleraceae Martius, is consumed in natura and in a variety of beverages and food preparations. This fruit has been widely consumed and produced in Brazil, due to the high popularity as a functional food with antioxidant and anti-inflammatory capacities. This study investigated the potential protective effect of spray-dried açai pulp (AP) intake on colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in male adult Wistar rats (mean weight of 200g). After 4 weeks of DMH administration, groups were fed standard diet, diet containing AP at 2.5% or 5.0% or 0.2% N-acetylcysteine (NAC) during 10 weeks for analysis of aberrant crypt foci (ACF) as endpoint. Additionally, two groups were fed standard diet or diet containing AP at 5.0% during 20 weeks for analysis of colon tumor as endpoint. In 14-week ACF assay, a significant reduction in number of aberrant crypt (AC) and in ACF (1-3 AC) were observed in groups fed 5.0% AP and 0.2% NAC (37% and 47% inhibition, p = 0.036; 37% and 41% inhibition, p = 0.042), respectively, when compared to the untreated group. In 24-week tumor assay, a significant reduction in number of invasive adenocarcinomas and tumor multiplicity were observed in group fed 5.0% AP (p < 0.005 and p = 0.001, respectively) when compared to untreated group. Also, significant reduction in tumor Ki-67 cell proliferation and growth index was observed in group fed 5.0% AP (p = 0.003 and p = 0.001). Therefore, the findings of this study indicate that spray-dried açai pulp feeding at 5.0% may reduce the development of chemically-induced ACF and colon tumor in male rats, indicating their use as a potential functional food / Mestre

Orientador: Ramon Kaneno / Banca: Elaine Guadelupe Rodrigues / Banca: Daniela B. Kaneno / Resumo: O interferon alfa (IFN-α), um IFN do tipo I, se apresenta como uma citocina com grande potencial terapêutico, pois atua no combate direto às células tumorais, além de agir sobre a maturação de células dendríticas (DCs), que são células apresentadoras de antígenos profissionais e peças chave na elaboração da uma resposta antitumoral. Entretanto, a administração sistêmica de citocinas pode produzir toxicidade importante nos pacientes, de modo que a indução de sua produção in situ poderia representar uma forma de imunomodulação mais adequada. Assim, o objetivo deste estudo é verificar a ação de vetores lentivirais carregando o gene do IFN-α para transdução de células tumorais, permitindo assim a produção localizada de IFN-α, a fim de explorar, in vitro, seu potencial lítico e imunomodulatório sobre DCs. Vetores lentivirais carregando o gene do IFN-α humano (Lego-IFN) ou GFP (Lego-GFP) foram utilizados para a transdução in vitro de células de câncer colorretal. A transdução foi feita com diferentes multiplicidades de infecção (MOIs - 0.3, 1.0, 2.0, 4.0) para avaliarmos o efeito dose-dependente, seguido de co-cultura com DCs derivadas de monócitos de doadores saudáveis (DC-0.3, DC-1.0, DC-2.0, DC-4.0). Após 48h de co-cultura, as DCs foram avaliadas fenotípica e funcionalmente, através da análise dos marcadores de membrana por citometria de fluxo, capacidade de aloestimulação e de indução de linfócitos T citotóxicos (CTLs). Nós observamos que a transdução com Lego-GFP, mas não com Lego-IFN, aumentou a imunogenicidade das células tumorais, com aumento de expressão de CD54 e HLA-DR. A co-cultura de DCs com células tumorais transduzidas com Lego-IFN aumentou discretamente seu perfil de ativação, mas não seu potencial aloestimulatório in vitro. Observamos que linfócitos cultivados com DC-2.0 produziram níveis mais altos de IFN-γ, sugerindo a indução de um perfil Th1, enquanto que... / Abstract: Interferon alpha (IFN-α) is a type I IFN with great therapeutic potential, since it is able to directly fight tumor cells and enhance the maturation of dendritic cells (DCs), the main antigen-presenting cells, required for an effective antitumor response. However, the systemic administration of cytokines can induce severe collateral effects. Therefore, the induction of cytokine secretion in situ should represent a more adequate approach for cytokine-based immunotherapy. Thus, the goal of this study was to induce IFN-α secretion by colon cancer cells by transduction with a lentivirus vector carrying the human IFN-α gene, followed by analysis of its immunomodulatory potential over DCs. Transduction was made with different multiplicities of infection (MOIs - 0.3, 1.0, 2.0 and 4.0) to evaluate the dose-dependent effects. Such cells were co-cultured with monocyte-derived DCs from healthy donors (DC-0.3, DC-1.0, DC-2.0 and DC-4.0). Forty-eight hours later, DCs were evaluated for their phenotype (surface activation/maturation markers) by flow cytometry, their ability to induce allogeneic response in a mixed leukocyte reaction (MLR) and effectiveness to induce cytotoxic T cells. We observed that transduction with Lego-GFP, but not Lego-IFN, increased tumor cells' immunogenicity with up-regulation of the markers CD54 and HLA-DR. Co-culture of Lego-IFN-transduced tumor cells with DCs slightly enhanced their activation phenotype but not their potential to stimulate T cell proliferation in vitro. Furthermore, we observed that lymphocytes cultured with DC-2.0 produced higher levels of IFN-γ, suggesting an induction of Th1 profile on T cells, while DC-GFP induced more IL-10 and IL-4. Additionally, DC-4.0 was more efficient in generating cytotoxic T lymphocytes (CTLs) that the control DC, however DC-GFP induced even more CD8+T cell proliferation. The enhancement of tumor cell immunogenicity and the superior induction of CLTs ... / Mestre

Orientador: Maria Aparecida Marchesan Rodrigues / Coorientador: Luís Fernando Barbisan / Banca: Carla Adriene da Silva Franchi / Banca: Fernando Silva Ramalho / Banca: Juliana Elaine Perobelli / Banca: Luis Antonio Justilin Junior / Resumo: Investigamos as repercussões do padrão alimentar ocidental, rico em gordura, (óleo de milho) (20%) e pobre em micronutrientes [ácido fólico (1 mg/g), colina (0,12%) e fibra (2%)], ocorrido durante a gestação, lactação e puberdade na susceptibilidade ao desenvolvimento de câncer do cólon induzido pela 1,2-dimetilhidrazina (DMH) no rato. Avaliamos se a reintrodução da dieta Ocidental durante a vida adulta em animais previamente expostos à dieta Ocidental durante a vida intrauterina, lactação e puberdade, pode modificar a susceptibilidade à oncogênese do cólon pela DMH. Ratas fêmeas SD prenhes receberam dieta semi-purificada controle (AIN-93) ou do tipo Ocidental (WD - Western Style Diet) a partir do dia gestacional 12 (DG 12). Após o desmame (dia 21), a prole de machos permaneceu com o mesmo tipo de dieta de suas mães até o dia pós-natal 50 (DPN 50). Nesse momento, receberam quatro injeções subcutâneas de DMH (40mg/kg) e foram divididos nos seguintes grupos/dietas: Grupo I (AIN-93/DMH/AIN-93) recebeu dieta controle durante todo o experimento (DG 12- DPN 370), o Grupo II (AIN-93/DMH/Ocidental) recebeu dieta Ocidental somente na vida adulta, a partir do DPN 260, Grupo III (Ocidental/DMH/AIN-93) recebeu dieta Ocidental desde a vida intrauterina até o início da puberdade (DG 12 - DPN 50), e o Grupo IV (Ocidental/DMH/Ocidental) recebeu dieta Ocidental desde a vida intrauterina até o início da puberdade (DG 12 - DPN 50), e foi reintroduzido à dieta Ocidental na vida adulta (DPN 260). Todos os animais foram eutanasiados ao final da 50ª semana de experimento (DPN 370). O cólon foi removido e submetido à análise estereoscópica e histológica para determinar o número, distribuição e multiplicidade dos focos de criptas aberrantes (FCAs). As características das lesões neoplásicas e os níveis séricos de insulina e leptina foram analisados à necropsia. A exposição à dieta Ocidental durante a gestação,... / Abstract: We examined the effects of a Western Style Diet (WD), high in fat (corn oil) (20%) and low in micronutrients [i.e.; folic acid (1 mg/g), choline (0.12%) and fiber (2%)] during pregnancy, lactation and in prepubertal stage to examine the susceptibility to colon carcinogenesis induced by dimethylhydrazine (DMH) in male Sprague-Dawley (SD) rats as later adults. We also evaluated whether WD reintroduction during adulthood can modify susceptibility to colon carcinogenesis. Pregnant SD rats received one of two diets AIN-93G or WD at gestational day 12 (GD 12). F1 male pups were weaned (21d) and fed the same AIN- 93G or WD diets from their dams until postnatal day 50 (PND 50). At this point, they were given four subcutaneous injections of carcinogen DMH (40 mg/body weight) and were shared the following diets/group: group I (AIN-93/DMH/AIN-93) a lifetime control diet-fed group (GD 12 until postnatal day - PND 370), group II (AIN-93/DMH/WD) received WD only in adulthood (at PND 260), group III (WD/DMH/AIN-93) received WD during pregnancy from dams until prepubertal stage (GD 12 - PND 50) and group IV (WD/DMH/WD), received WD since pregnancy from dams (GD 12) until prepubertal stage (PND 50) and was reintroduced to WD during adulthood (PND 260). All groups were euthanized at the end of 50th week (PND 370). The colon was removed and the aberrant crypt foci (ACF) were stereoscopically and histologically scored for number, distribution and multiplicity. Neoplastic lesions and serum insulin and leptin concentrations were analysed at necropsy. ACF multiplicity and dysplastic lesions were significantly higher in the group fed WD during pregnancy until prepubertal stage (group III) than control group (group I). In the group III, the number of adenocarcinomas were higher than control (group I). In the group fed WD only in adulthood (group II), ACF multiplicity was significantly higher than in the control (group I). The group ... / Doutor

Orientador: Luís Fernando Barbisan / Banca: Luís Antônio Justulim Júnior / Banca: Carlos Eduardo Andrade Chagas / Resumo: Yacon (Smallanthus sonchifolius) é uma raiz originária da região dos Andes que tem se destacado pelos seus compostos bioativos principalmente frutanos como futooligossacarídeos e inulina. O presente projeto teve como objetivo determinar a atividade quimioprotetora da ingestão de Yacon sobre o desenvolvimento de lesões pré-neoplásicas (focos de criptas aberrantes-FCA) induzidas pela dimetilhidrazina (DMH) em ratos Wistar machos. Os animais foram divididos em seis grupos com 5 a 12 animais cada. Os animais dos Grupos 1 a 4 e Grupos 5 e 6 receberam respectivamente, quatro injeções subcutâneas de DMH (40 mg/Kg) e solução de EDTA (veículo da DMH) nas duas semanas iniciais do experimento respectivamente. Os animais receberam ração basal até a sexta semana do experimento e a partir desta os animais dos grupos 2, 3, 4, 5 receberam ração acrescida de extrato de Yacon a 0,5%, 1%, 1% e 1%, respectivamente. Os animais do grupo 4 receberam Lactobacilus casei (2,5 x 1010 de UFC por Kg de ração). O sacrifício ocorreu na vigésima semana de experimento para análise de focos de criptas aberrantes (FCA) e tumores. Nossos resultados mostraram uma redução no número, multiplicidade de FCA e no número de adenocarcinomas invasivos nos grupos tratados com 1% yacon (G3) e na combinação simbiótica (G4), (0,05 < p < 0, 001). A multiplicidade de tumores (invasivos e não invasivos) foi significativamente menor no grupo tratado com a combinação simbiótica (p < 0,02). Observou-se também uma redução significativa nas taxas de proliferação celular tanto em criptas colônicas como em tumores nos grupos tratados com 1% yacon (G3) e na combinação simbiótica (G4), p < 0.001. Os resultados sugerem que a ingestão de extrato de yacon exerce atividade quimiopreventiva contra carcinogênese de cólon / Abstract: Yacon (Smallanthus sonchifolius) is a tuberous root native to the Andean region of South America which contains high amounts of inulin-type fructans. The present study investigated the beneficial potential of yacon root intake on development of colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in male Wistar rats. Six groups were studied: Groups 1-4 were given four s.c. injections of DMH (40 mg/kg b.w.) twice a week, during two weeks, whereas Groups 4 and 5 received similar injections of EDTA solution (DMH vehicle). After 6 weeks of DMH-initiation, groups were fed with basal diet (G1 and G6) or basal diet containing dried extract of yacon root at 0.5% (G2), 1.0% (G3 and G5) or a synbiotic formulation (G4) (1.0% yacon root plus Lactobacillus casei at 2.5 x 1010 CFU per g diet) for 13 weeks. At 20 week, all animals were killed and the colons were analyzed for development of aberrant crypt foci (ACF) and tumor. A significant reduction in number and multiplicity of ACF and in number of invasive adenocarcinomas was observed in the groups orally treated with 1.0% yacon root (G3) or their synbiotic formulation (G4) (0.05 < p < 0.001). Tumor multiplicity (noninvasive plus invasive) was significantly lower solely in group fed with symbiotic formulation (p < 0.02). Also, a reduction in cell proliferation indexes in colonic crypt and tumor were observed in groups orally treated with 1.0% yacon root (G3) or their synbiotic formulation (G4) (p < 0.001). The findings this study suggest that yacon root intake may have potential as chemopreventive agent against colon carcinogenesis / Mestre