This thesis aimed to verify the causal association between "B. hampsonii" and the re-emergence of mucohaemorrhagic diarrhea in North American swine farms, to investigate the role of the intestinal microbiome as a predisposing factor for infection, to develop a porcine colon in vitro culture model and to apply this model in investigating early host-pathogen interactions. Two infection trials were conducted to determine the pathogenicity of "B. hampsonii" clade II and clade I. Weanling pigs were divided into control (n=6) and inoculated (n=12) groups. In each trial, pigs were inoculated with "B. hampsonii" clade II (tissue homogenate or pure culture) or clade I (pure culture) or sterile culture media. Animals were monitored for clinical signs of diarrhea and upon observation of bloody diarrhea they were necropsied for characterization of lesions. Fecal shedding of "B. hampsonii" was monitored throughout the trials using culture and quantitative real-time PCR. Pre and post-diarrhea fecal samples from the clade II infection trial were used to study the microbiome response to "B. hampsonii" infection and to determine if pre-inoculation microbiome composition differed between pigs that did or did not develop clinical disease. For in vitro model development, numerous factors associated with explant survivability in culture were investigated to develop a protocol for culture of porcine colon explants. The optimized model was used to study the first 12 hours of "B. hampsonii" clade II interaction with the host using a combination of histopathology and gene expression analysis. Pigs inoculated with "B. hampsonii" clade I (9/11) and clade II (9/12 and 8/12 in the tissue homogenate and pure culture experiments, respectively) developed mucohaemorrhagic diarrhea and colitis within 14 days of inoculation. In all trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of affected or unaffected clade II inoculated pigs. After the development of diarrhea, the fecal microbiome of diarrheic pigs was more dense and had a had a lower Bacteroidetes:Firmicutes ratio when compared to inoculated but unaffected or control pigs. Cultured porcine colon explants displayed differentiated epithelium and crypts after 5 days in culture, while expressing GAPDH at a constant rate. For explants to thrive in vitro our results suggested the use of distal spiral colon, processed immediately after euthanasia, and cultured in an oxygen-rich gas mix with air-liquid culture interface in media containing antibiotics and antifungals. Explants exposed to "B. hampsonii" for 12 hours had a greater number of necrotic cells and thicker catarrhal exudate than control explants. Interaction of spirochaetes with the epithelium, necrotic cells and crypts was visible under optical microscopy, and a trend of increased expression of IFN-γ and e-cadherin in inoculated explants relative to control explants was observed. Taken together, results of this thesis demonstrate that "B. hampsonii" causes mucohaemorrhagic diarrhea in pigs and modulates their intestinal microbiome. The development of an in vitro infection model that replicates in vivo features facilitated the observation of the initial events in "B. hampsonii" interaction with the colon. When explants were exposed to "B. hampsonii" similar histological lesions to in vivo were observed. This system provides a powerful model for future studies of the pathogenesis of "B. hampsonii" and other enteric pathogens of pigs.

Crescente ênfase vem sendo dada ao estudo e desenvolvimento de alimentos ricos em carboidratos não-disponíveis em virtude de seus benefícios já comprovados. O presente trabalho visou caracterizar carboidratos da banana verde e avaliar efeitos fisiológicos de seus carboidratos não-disponíveis sobre a tolerância à glicose, entre outros parâmetros relacionados a ela ou à saúde em geral. A massa de banana verde (MBV) (banana verde cozida com casca) e o amido de banana verde (ABV) (amido isolado) foram os principais produtos estudados. Foram realizados ensaios de curta duração em humanos e em ratos, e de média duração somente em ratos. Para a caracterização dos produtos e rações utilizados, foram avaliados principalmente os teores de amido resistente (AR), fibra alimentar (FA), fração indigerível (FI) e o perfil da fermentação in vitro. Os efeitos fisiológicos dos produtos e rações foram avaliados por curvas glicêmicas, teste de tolerância à glicose (TTG), perfil lipídico e de fermentabilidade in vivo. Os produtos de banana verde e as rações elaboradas com os mesmos apresentaram alto teor de carboidratos não-disponíveis, diferentes em quantidade e qualidade, mas com alta fermentabilidade, sinalizada pelo aumento da produção de ácidos graxos de cadeia curta (AGCC) e diminuição de pH na fermentação colônica in vitro ou in vivo. A ingestão de MBV ou ABV como primeira dieta, em ensaios de curta duração com ratos, afetou a glicemia pós-prandial de uma segunda dieta padrão, o que não foi observado com rações contendo tais produtos em menor proporção. Por outro lado, em 28 dias as rações contendo MBV ou ABV causaram menor secreção de insulina em ilhotas pancreáticas, o que possibilita poupar as células ß. Os resultados indicam que os produtos estudados têm grande potencial para serem utilizados na elaboração de alimentos destinados à prevenção de determinadas doenças crônicas não-transmissíveis, como diabetes tipo 2. / The study of unavailable carbohydrates has been of great concern due to their proven benefits to health. This work aimed to characterize carbohydrates of unripe bananas, as well as to evaluate physiological effects of their unavailable carbohydrates over glucose tolerance, among other parameters related to it or to individuals\' health in general. The unripe banana mass (MBV) (unripe banana cooked with peel) and the unripe banana starch (ABV) (isolated starch from the fruit in natura) were the main studied products. Short term assays were carried out with humans and rats, while medium term ones were realized with rats. In order to characterize the products and diets used in this work, several parameters were evaluated, such as quantifications of resistant starch (RS), dietary fiber (DF), indigestible fraction (IF) and the study of the in vitro fermentation profile. The physiological effects of the products and diets were evaluated through glycemic curves, glucose tolerance test (GTT), and lipidic and fermentative characteristics. The unripe banana products, as well as the diets elaborated with them, presented high amount of unavailable carbohydrates, in different quantity and quality, but all of them with high fermentative rates, signalized buy an increase in short chain fatty acids (SFCA) production and decrease in pH on colonic fermentation carried out in vivo or in vitro. In the short term assays, the MBV and ABV intakes affected the postprandial glycemic response to a following diet in rats. Similar effect was not noticed in medium term assays after the intake of diets containing unripe banana products in lower proportions for 28 days. On the other hand, in this medium term assays, rats diets containing MBV or ABV lead to lower insulin secretion in isolated pancreatic islets, which may preserve ß cells function. The results of this work point that the unripe banana products have great potential to be applied in special food elaboration aiming the prevention of some non-transmissible chronic diseases.

A colonoscopia com magnificação de imagem (CMI), associada à cromoendoscopia, tem mostrado a possibilidade de se diferenciar lesões neoplásicas e não-neoplásicas. Duzentas e treze lesões colorretais foram magnificadas e analisadas, através da classificação de Kudo, por 3 observadores distintos e também comparadas com os resultados histopatológicos. Houve bom grau de concordância entre os observadores (Kappa = 0,561) para os diversos padrões de criptas. Não houve diferenças entre eles com relação à chance de acerto (p = 0,121; 0,500; 0,405). A acurácia do método foi de 84%, sensibilidade de 91,4%, especificidade de 67,2%, valores preditivos positivo e negativo de 86,6% e 79,3%, índice de Kappa de 0,61. Esses resultados mostram que a CMI não deve ser utilizada na definição de conduta frente a lesões polipóides / Magnifying colonoscopy (MC) associated with chromoendoscopy has showed the possibility of differentiating neoplastic from non-neoplastic lesions. Two hundred and thirteen lesions were magnifyied and analysed, according to Kudo's classification, by 3 observers and compared with histopathological results. There was a good agreement index among them (Kappa = 0.561) with respect to the aspects of the pits. No differences were found in relation to the possibility of being correct (p = 0.121; 0.500; 0.405). Accuracy of method was 84%, sensitivity of 91.4%, specificity of 67.2%, positive and negative predictive values of 86.6% and 79.3%; Kappa index of 0.61. These results show that MC must not be used to define what should be done with polypoid lesions

強心苷是一類多年普遍用於心力衰竭治療的化合物,包括蟾蜍靈和地高辛。鈉泵（也可稱為鈉鉀ATP酶）是強心苷的受體。最近流行病學研究，體外實驗，動物實驗和臨床試驗表明，強心苷具有癌症治療的強大潛力。 / 大腸癌是全球第三大殺手，約有一半的大腸癌患者需要手術切除後的輔助治療。因此，通過化療殺死腫瘤細胞，是一個可行的辦法來治療大腸癌患者。在本課題的研究中，強心苷抗人結腸癌的作用在HT-29和Caco-2細胞上進行了評價與闡釋。在結腸癌細胞研究模型中，蟾蜍靈誘導caspase非依賴性的細胞死亡，伴隨沒有早期凋亡，沒有聚（ADP-核糖）聚合酶(PARP)與caspase-3裂解，這些發現與強心苷誘發其它類腫瘤細胞凋亡的機製完全不同。相反，蟾蜍靈激活自噬途徑，促進LC3-II積累和自噬流動。此外，其它強心苷如地高辛與烏本苷也促使LC3-II在HT-29細胞內聚集。沉默ATG5和Beclin-1顯著降低蟾蜍靈誘導的LC3- II積累和細胞死亡。蟾蜍靈誘導的自噬與活性氧（ROS）產生和JNK活化相關。我們的研究結果揭示了蟾蜍靈藥物對抗結腸癌細胞的一種新的機制，開闢了強心苷通過自噬途徑來治療大腸癌的可能性。 / 最近的研究表明，強心苷誘導多種癌細胞系的細胞包括促使凋亡與自噬的細胞週期阻滯在G2／M期。然而，沒有詳細的信息闡述強心苷如何阻滯細胞週期進展。在本課題研究中，我們研究了強心苷介導的細胞週期阻滯的分子機制。蟾蜍靈處理的HeLa H2B-YFP細胞被阻滯在前中期，伴隨姐妹染色單體凝聚，染色體未排列在赤道板，未退出有絲分裂期。這一結果被蟾蜍靈誘導的四倍DNA含量細胞既不在四倍體G1期也不在胞質分裂期進一步證明。此後，我們檢測了紡錘體組裝和染色體分離所需的Aurora激酶和Polo-like kinase 1 (Plk1)。結果發現，在HT-29和HeLa細胞上，蟾蜍靈和其它強心苷能顯著降低總蛋白質和磷酸化的Aurora激酶與Plk1。此外，我們還發現，蟾蜍靈通過PI3K下調有絲分裂酶的活性。這些結果已經通過沉默鈉泵α做了驗證。總之，我們的結果表明, 蟾蜍靈和其它強心苷鈉鉀泵抑製劑強有力的抑制細胞在前中期是通過PI3K/HIF-1α/NF-κB途徑下調Aurora激酶的蛋白質和磷酸化水平和Plk1的蛋白質水平。我們的研究發現在了解如何利用強心苷的潛能治療癌症以及認知鈉泵在細胞週期中的功能方面提供了有用的信息。 / The sodium pump (also known as Na+/K+-ATPase) is the receptor for cardiac glycosides, a group of compounds including bufalin and digoxin which have been commonly used for heart failure treatment for many years. Recent epidemiological studies, in vitro studies, animal studies and clinical trials have shown that cardiac glycosides have potential applications for cancer treatment. / Colorectal cancer is the third leading cause of cancer death worldwide and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treat patients with colorectal cancer. In this study, the effects of cardiac glycosides were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to their well documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis, as well as poly(ADP-ribose) polymerase (PARP) and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. Moreover, other cardiac glycosides digoxin and ouabain could also induce the accumulation of LC3-II in HT-29 cells. The silencing of ATG5 and Beclin-1 significantly reduced bufalin-induced LC3-II accumulation and cell death. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS) and JNK activation. My findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer by cardiac glycosides through an autophagy pathway. / Recent studies have revealed that cardiac glycosides induce G2/M phase arrest in many cancer cells, which include apoptosis- and autophagy-promoting cells. However, no detailed information is available on how cardiac glycosides arrest cell cycle progression. In this study, I studied the molecular mechanisms of cell cycle arrest mediated by cardiac glycosides. Bufalin-treated HeLa H2B-YFP cells were arrested at prometaphase, as characterized by the presence of sister chromatid cohesion, absence of chromosomes alignment on the metaphase plate, and failure to exit mitosis. This result was further confirmed by bufalin-induced cells with 4N DNA content in neither tetraploid G1 phase nor cytokinesis. Thereafter, I detected the Aurora kinases and Polo-like kinase 1 (Plk1), which are required for both spindle assembly and chromosome segregation. It was found that bufalin and other cardiac glycosides could significantly reduce the total protein and phosphorylation of Aurora kinases and Plk1 in HT-29 and HeLa cells. In addition, I found that PI3K was responsible for the bufalin-induced downregulation of the activities of mitotic kinases. This result was validated by silencing of sodium pump alpha. Taken together, my results demonstrate that bufalin and other cardiac glycoside inhibitors of the sodium pump potently arrest cancer cells at prometaphase by downregulating the total protein and phosphorylation of Aurora kinases and the total protein of Plk1 through the PI3K/HIF-1α/NF-κB pathway. My findings provide useful information in understanding how cardiac glycosides could be exploited for their potentials in treating cancer and in identifying the function of sodium pump in cell cycle progression. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xie, Chuanming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 133-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.i / Acknowledgements --- p.iii / Abstract --- p.vi / Abstract (in Chinese) --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvi / List of Tables --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.2 --- The chemical structure of cardiac glycosides --- p.2 / Chapter 1.3 --- The traditional use of cardiac glycosides in cardiology --- p.4 / Chapter 1.4 --- The role of cardiac glycosides in cancer treatment --- p.4 / Chapter 1.5 --- The mechanisms of action by cardiac glycosides in cancer --- p.5 / Chapter 1.5.1 --- The structure and functions of cardiac glycosides receptor sodium pump --- p.5 / Chapter 1.5.2 --- Sodium pump as anticancer target --- p.6 / Chapter 1.5.3 --- The signal pathways involved in anticancer effect of cardiac glycosides --- p.7 / Chapter 1.6 --- The role of cardiac glycosides in apoptosis and autophagy --- p.8 / Chapter 1.7 --- Objectives of this project --- p.12 / Chapter Chapter 2 --- Bufalin induces autophagy but not apoptosis in human colon cancer cells --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.19 / Chapter 2.2.1 --- Reagents and antibodies --- p.19 / Chapter 2.2.2 --- Cell culture --- p.19 / Chapter 2.2.3 --- Cell viability and cell death assay --- p.20 / Chapter 2.2.4 --- Annexin V and PI staining --- p.20 / Chapter 2.2.5 --- Cell cycle analysis --- p.21 / Chapter 2.2.6 --- Analysis of cleaved caspase-3-positive cells by flow cytometry --- p.21 / Chapter 2.2.7 --- Western blot analysis --- p.21 / Chapter 2.2.8 --- Immunofluorescence analysis of LC3 distribution --- p.22 / Chapter 2.2.9 --- RNA isolation and RT-PCR --- p.22 / Chapter 2.2.10 --- siRNAs transfection and treatments --- p.23 / Chapter 2.2.11 --- Transmission electron microscopy --- p.23 / Chapter 2.2.12 --- Statistical analysis --- p.24 / Chapter 2.3 --- Results --- p.24 / Chapter 2.3.1 --- Bufalin induces cell death and cell cycle arrest at G2/M phase in colon cancer cells --- p.24 / Chapter 2.3.2 --- Bufalin induces caspase-independent cell death in colon cancer cells --- p.28 / Chapter 2.3.3 --- Bufalin induces autophagy in colon cancer cells --- p.30 / Chapter 2.3.4 --- Bufalin-induced autophagy is dependent on ATG5 and Beclin-1 --- p.37 / Chapter 2.3.5 --- Increased autophagy is responsible for bufalin-induced cell death --- p.40 / Chapter 2.4 --- Discussion --- p.42 / Chapter Chapter 3 --- Bufalin mediates autophagic cell death through ROS generation and JNK activation --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Reagents and antibodies --- p.46 / Chapter 3.2.2 --- Cell culture --- p.47 / Chapter 3.2.3 --- Cell viability and cell death assay --- p.47 / Chapter 3.2.4 --- Western blot analysis --- p.47 / Chapter 3.2.5 --- Quantification of cells with > 5 LC3 punctate staining --- p.47 / Chapter 3.2.6 --- siRNAs transfection and treatments --- p.48 / Chapter 3.2.7 --- RNA isolation and RT-PCR --- p.48 / Chapter 3.2.8 --- ROS analysis --- p.48 / Chapter 3.2.9 --- JC-1 staining --- p.49 / Chapter 3.2.10 --- Statistical analysis --- p.49 / Chapter 3.3 --- Results --- p.50 / Chapter 3.3.1 --- Bufalin induces autophagy-mediated cell death via ROS generation --- p.50 / Chapter 3.3.2 --- Activation of JNK is required for the upregulation of ATG5 and Beclin-1, and subsequent autophagy-mediated cell death in response to bufalin --- p.54 / Chapter 3.3.3 --- ROS generation is upstream of JNK activation in bufalin-induced cell death --- p.59 / Chapter 3.3.4 --- Bufalin-induced ROS generation is derived from mitochondria --- p.62 / Chapter 3.4 --- Discussion --- p.66 / Chapter Chapter 4 --- Bufalin arrests cells at prometaphase --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Reagents and antibodies --- p.70 / Chapter 4.2.2 --- Cell synchronization --- p.70 / Chapter 4.2.3 --- Mitotic index analysis of phosphorylation of MPM2 --- p.71 / Chapter 4.2.4 --- Cell cycle analysis --- p.71 / Chapter 4.2.5 --- Time-lapse experiments --- p.71 / Chapter 4.2.6 --- Immunofluorescence analysis of phospho-histone H3 (Ser10) --- p.72 / Chapter 4.2.7 --- Western blot analysis --- p.73 / Chapter 4.3 --- Results --- p.73 / Chapter 4.3.1 --- Bufalin reduces mitotic marker phosphorylation of histone H3 and MPM2 and increases cells with 4N DNA content --- p.73 / Chapter 4.3.2 --- Increased cells with 4N DNA content after bufalin treatment are in neither a tetraploid G1 phase nor a cytokinesis arrest --- p.77 / Chapter 4.3.3 --- Bufalin-treated cells can enter prophase, but fail to pass through metaphase --- p.80 / Chapter 4.4 --- Discussion --- p.83 / Chapter Chapter 5 --- Bufalin induces prometaphase arrest through downregulating mitotic kinases --- p.87 / Chapter 5.1 --- Introduction --- p.87 / Chapter 5.2 --- Materials and Methods --- p.89 / Chapter 5.2.1 --- Reagents and antibodies --- p.89 / Chapter 5.2.2 --- Cell synchronization --- p.90 / Chapter 5.2.3 --- Immunofluorescence staining --- p.90 / Chapter 5.2.4 --- siRNAs transfection and treatments --- p.91 / Chapter 5.2.5 --- Western blot analysis --- p.91 / Chapter 5.2.6 --- Statistic analysis --- p.91 / Chapter 5.3 --- Results --- p.92 / Chapter 5.3.1 --- Bufalin downregulates Aurora A and B in protein and phosphorylation levels --- p.92 / Chapter 5.3.2 --- Bufalin prevents Aurora A recruitment to mitotic centrosomes and Aurora B recruitment to unattached kinetochores --- p.97 / Chapter 5.3.3 --- Bufalin prevents Plk1 recruitment to mitotic centrosomes and unattached kinetochores through downregulation of protein levels of Plk1 --- p.101 / Chapter 5.3.4 --- Bufalin decreases the activities of Aurora A, Aurora B and Plk1 through PI3K pathway --- p.105 / Chapter 5.3.5 --- HIF-1α and NF-κB pathways are involved in sodium pump-mediated the regulation of mitotic kinases --- p.109 / Chapter 5.4 --- Discussion --- p.112 / Chapter Chapter 6 --- General discussion --- p.115 / Chapter 6.1 --- Potential toxicity of bufalin --- p.115 / Chapter 6.2 --- Cardiac glycosides induced programmed cell death --- p.115 / Chapter 6.3 --- Signal pathways involved in cardiac glycosides-mediated autophagy --- p.117 / Chapter 6.4 --- The relationship between ROS and JNK in cardiac glycosides-induced autophagy --- p.120 / Chapter 6.5 --- The role of ROS in apoptosis and autophagy --- p.121 / Chapter 6.6 --- The role of cardiac glycosides in cell cycle arrest --- p.122 / Chapter 6.7 --- Application of cardiac glycosides in combination with chemotherapy and radiotherapy --- p.125 / Chapter Chapter 7 --- Conclusions and future perspectives --- p.127 / References --- p.133 / Appendices --- p.153 / Publication --- p.153

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 156-176). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

A colonoscopia com magnificação de imagem (CMI), associada à cromoendoscopia, tem mostrado a possibilidade de se diferenciar lesões neoplásicas e não-neoplásicas. Duzentas e treze lesões colorretais foram magnificadas e analisadas, através da classificação de Kudo, por 3 observadores distintos e também comparadas com os resultados histopatológicos. Houve bom grau de concordância entre os observadores (Kappa = 0,561) para os diversos padrões de criptas. Não houve diferenças entre eles com relação à chance de acerto (p = 0,121; 0,500; 0,405). A acurácia do método foi de 84%, sensibilidade de 91,4%, especificidade de 67,2%, valores preditivos positivo e negativo de 86,6% e 79,3%, índice de Kappa de 0,61. Esses resultados mostram que a CMI não deve ser utilizada na definição de conduta frente a lesões polipóides / Magnifying colonoscopy (MC) associated with chromoendoscopy has showed the possibility of differentiating neoplastic from non-neoplastic lesions. Two hundred and thirteen lesions were magnifyied and analysed, according to Kudo's classification, by 3 observers and compared with histopathological results. There was a good agreement index among them (Kappa = 0.561) with respect to the aspects of the pits. No differences were found in relation to the possibility of being correct (p = 0.121; 0.500; 0.405). Accuracy of method was 84%, sensitivity of 91.4%, specificity of 67.2%, positive and negative predictive values of 86.6% and 79.3%; Kappa index of 0.61. These results show that MC must not be used to define what should be done with polypoid lesions

Crescente ênfase vem sendo dada ao estudo e desenvolvimento de alimentos ricos em carboidratos não-disponíveis em virtude de seus benefícios já comprovados. O presente trabalho visou caracterizar carboidratos da banana verde e avaliar efeitos fisiológicos de seus carboidratos não-disponíveis sobre a tolerância à glicose, entre outros parâmetros relacionados a ela ou à saúde em geral. A massa de banana verde (MBV) (banana verde cozida com casca) e o amido de banana verde (ABV) (amido isolado) foram os principais produtos estudados. Foram realizados ensaios de curta duração em humanos e em ratos, e de média duração somente em ratos. Para a caracterização dos produtos e rações utilizados, foram avaliados principalmente os teores de amido resistente (AR), fibra alimentar (FA), fração indigerível (FI) e o perfil da fermentação in vitro. Os efeitos fisiológicos dos produtos e rações foram avaliados por curvas glicêmicas, teste de tolerância à glicose (TTG), perfil lipídico e de fermentabilidade in vivo. Os produtos de banana verde e as rações elaboradas com os mesmos apresentaram alto teor de carboidratos não-disponíveis, diferentes em quantidade e qualidade, mas com alta fermentabilidade, sinalizada pelo aumento da produção de ácidos graxos de cadeia curta (AGCC) e diminuição de pH na fermentação colônica in vitro ou in vivo. A ingestão de MBV ou ABV como primeira dieta, em ensaios de curta duração com ratos, afetou a glicemia pós-prandial de uma segunda dieta padrão, o que não foi observado com rações contendo tais produtos em menor proporção. Por outro lado, em 28 dias as rações contendo MBV ou ABV causaram menor secreção de insulina em ilhotas pancreáticas, o que possibilita poupar as células ß. Os resultados indicam que os produtos estudados têm grande potencial para serem utilizados na elaboração de alimentos destinados à prevenção de determinadas doenças crônicas não-transmissíveis, como diabetes tipo 2. / The study of unavailable carbohydrates has been of great concern due to their proven benefits to health. This work aimed to characterize carbohydrates of unripe bananas, as well as to evaluate physiological effects of their unavailable carbohydrates over glucose tolerance, among other parameters related to it or to individuals\' health in general. The unripe banana mass (MBV) (unripe banana cooked with peel) and the unripe banana starch (ABV) (isolated starch from the fruit in natura) were the main studied products. Short term assays were carried out with humans and rats, while medium term ones were realized with rats. In order to characterize the products and diets used in this work, several parameters were evaluated, such as quantifications of resistant starch (RS), dietary fiber (DF), indigestible fraction (IF) and the study of the in vitro fermentation profile. The physiological effects of the products and diets were evaluated through glycemic curves, glucose tolerance test (GTT), and lipidic and fermentative characteristics. The unripe banana products, as well as the diets elaborated with them, presented high amount of unavailable carbohydrates, in different quantity and quality, but all of them with high fermentative rates, signalized buy an increase in short chain fatty acids (SFCA) production and decrease in pH on colonic fermentation carried out in vivo or in vitro. In the short term assays, the MBV and ABV intakes affected the postprandial glycemic response to a following diet in rats. Similar effect was not noticed in medium term assays after the intake of diets containing unripe banana products in lower proportions for 28 days. On the other hand, in this medium term assays, rats diets containing MBV or ABV lead to lower insulin secretion in isolated pancreatic islets, which may preserve ß cells function. The results of this work point that the unripe banana products have great potential to be applied in special food elaboration aiming the prevention of some non-transmissible chronic diseases.

Orientadores: Carmen Silvia Passos Lima, Maria de Lourdes Setsuko Ayrizono / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T01:40:41Z (GMT). No. of bitstreams: 1 Guimaraes_JoseLuizMiranda_D.pdf: 4302732 bytes, checksum: 2d6604590176d72460b4a606d6ba2dd6 (MD5) Previous issue date: 2012 / Resumo: O desenvolvimento de câncer colorretal (CCR) é resultado de uma complexa interação de variáveis, incluindo elementos externos, como a exposição a agentes ambientais e dietéticos, e fatores internos, de natureza somática ou hereditária. Não está estabelecido se genótipos de polimorfismos de baixa penetrância em genes relacionados com o metabolismo do ácido fólico, como o metilenotetrahidrofolato redutase (MTHFR C677T e MTHFR A1298C), o metionina sintase (MTR A2756G), o metionina sintase redutase (MTRR A66G) e o timidilato sintase (TS 2R3R), estão associados com o risco de ocorrência da doença ou com suas manifestações clínicas. Portanto, o objetivo deste estudo foi verificar se esses polimorfismos gênicos influenciam o risco de ocorrência do adenocarcinoma colorretal esporádico (ACRE) e suas manifestações clínicas e biológicas em pacientes da região sudeste do Brasil. Foram avaliados 113 pacientes com ACRE e 188 controles, considerando os aspectos clínicos como a idade, o sexo, a raça, a localização, o grau de diferenciação do tumor, o estágio e os genótipos de cada gene. Os genótipos dos polimorfismos dos genes MTHFR, MTR, MTRR e TS foram avaliados por meio da reação em cadeia da polimerase (PCR) seguida ou não por digestão enzimática. O significado estatístico das diferenças entre grupos foi calculado por meio do teste da probabilidade exata de Fisher ou qui-quadrado. As determinações dos riscos de ocorrência do ACRE, a que pacientes e controles foram submetidos, foram obtidas por meio das razões das chances (ORs) e calculadas considerando um intervalo de confiança de 95%. Portadores dos genótipos MTRR 66AG+GG, do MTHFR 1298AC+CC+677CT+TT, do MTHFR 677CT+TT+MTR 2756AG+GG, do MTHFR 1298AC+CC + 677CT+TT + MTR 2756AG+GG e MTHFR 1298AC+CC + 677CT+TT + MTRR 66AG+GG apresentaram riscos 1,99, 3,26, 2,22, 10,92 e 14,88 vezes maiores, respectivamente, de desenvolver ACRE do que os outros. Além disso, os indivíduos com o genótipo MTHFR 677CT+TT e os genótipos MTR 2756AG+GG tiveram um risco de 2,12 e 1,42 vezes maior de desenvolver ACRE com idade menor do que 50 anos. Afro-Brasileiros com o genótipo GG do polimorfismo MTRR A66G tiveram risco 1,98 vezes maior de desenvolver ACRE, e indivíduos com o genótipo MTR 2756AG+GG e os genótipos MTHFR 677CT+TT estiveram sob risco 2,11 e risco 1,62 vezes maiores de ocorrência de tumores indiferenciados e avançados, respectivamente, do que os demais. Portadores dos genótipos MTHFR 1298AC+CC e MTHFR 1298AC+CC + MTRR 66AG+GG estiveram sob riscos 1,42 e 3,07 vezes maiores de tumor no reto, respectivamente, enquanto que portadores dos genótipos MTHFR 677CT+TT e MTHFR 677CT+TT + TS 2R3R+3R3R estiveram sob riscos 1,55 e 5,39 vezes maiores de tumor de cólon, respectivamente, do que portadores dos genótipos selvagens. Estes dados sugerem que polimorfismos dos genes MTHFR, MTR, MTRR e TS, que codificam enzimas que participam do metabolismo do ácido fólico, especialmente em combinação, têm papéis consistentes para o risco de desenvolver ACRE em indivíduos da região sudeste do Brasil / Abstract: The development of colorectal cancer (CRC) is the result of a complex interaction of variables, including external factors such as exposure to environmental agents and dietary factors and internal factors, whether somatic or hereditary. Is not been established genotypes with low penetrance polymorphisms in genes related to metabolism of folic acid such as methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G) and thymidylate synthase (TS 2R3R), are associated with the risk of the disease or its clinical manifestations. Therefore, the aim of this study was to determine whether these genetic polymorphisms influence the risk of sporadic colorectal adenocarcinoma (SCA) and their clinical and biological manifestations in patients from southeast Brazil. For this, we analyzed 113 patients with SCA and 188 controls, considering the clinical aspects such as age, sex, race, location, stage, degree of tumor differentiation and the genotypes of each gene described above. The genotypes of the polymorphisms of the MTHFR, MTR, MTRR and TS were assessed by polymerase chain reaction (PCR) and enzyme digestion. The statistical significance of differences between groups was calculated using the probability test of Fisher's exact or chi-square. Determination of the risks of SCA, the patients and controls were submitted, was obtained through the odds ratios (ORs) and calculated assuming a range of 95%. Carriers of the MTRR 66AG + GG, the MTHFR 1298AC+CC + 677CT+TT, the MTHFR 677CT+TT + MTR 2756AG+GG, the MTHFR 1298AC+CC + 677CT+TT + MTR 2756AG+GG, and the MTHFR 1298AC+CC + 677CT+TT + MTRR 66AG+GG genotypes had a 1.99, a 3.26, a 2.22, a 10.92 and a 14.88-fold increased risks for SCA than others, respectively. In addition, individuals with the MTHFR 677CT+TT and the MTR 2756AG+GG genotypes had a 2.12 and a 1.42-fold increased risks for SCA diagnosed under 50 years. African-Brazilians with the MTRR 66GG genotype had a 1.98-fold increased risk for SCA, and individuals with the MTR 2756AG+GG and the MTHFR 677CT+TT genotypes were under a 2.11 and a 1.62-fold increased risks for undifferentiated and advanced tumors, respectively, than others. Carriers of the MTHFR 1298AC+CC and the MTHFR 1298AC+CC + MTRR 66AG+GG genotypes had a 1.42 and a 3.07-fold increased risks for rectal tumor, respectively, while carriers of the MTHFR 677CT+TT and the MTHFR 677CT+TT + TS 2R3R+3R3R genotypes had a 1.55 and a 5.39-fold increased risks for colon tumor, respectively, than carriers of the wild genotypes. This data suggest that polymorphisms of genes MTHFR, MTR, MTRR and TS, which encode folate-dependent enzymes, particularly in combination, have consistent roles for SCA risk in southeastern Brazil / Doutorado / Fisiopatologia Cirúrgica / Doutor em Ciências

Dietary factors have been implicated in the etiology of colon cancer. The salient components of high-risk diets are thought to be high intakes of meat, especially beef, and fat, especially animal fat, and low intakes of fiber. Low-risk diets are thought to be high in fiber, and low in meat and animal fat. The present study examines the effects of short-term consumption of diets hypothesized to increase or decrease the risk for colon cancer on mutagenic activity of feces. Whether the fecal mutagens responsible for the mutagenic activity observed in the study are directly involved in the etiology of colon cancer is not known. However, most known mutagens are potentially carcinogenic, and fecal mutagenic activity may be an indicator of risk for colon cancer. Six healthy adult subjects consumed the following diets in sequence a baseline diet for one week, a low-risk lacto-ovo vegetarian, high fiber diet for two weeks, and a high-risk, high meat, low fiber diet for two weeks. Quantitative daily food intake records were kept, and daily bowel habits were recorded. Fecal samples were collected at the end of each diet period. Analyses were performed of the diets for food and nutrient intake, and of feces for percent dry weight and pH. Mutagenic activity of the fecal samples was assayed using the fluctuation test for mutagens. The subjects' habitual diets, although omnivorous, were found to closely resemble a low-risk diet pattern. Analysis of the vegetarian and high meat diets confirmed that the subjects had consumed foods which respectively represented the components of high-risk and low-risk diets. The overall fecal mutagenic activity obtained with samples on the high meat diet was higher than with the vegetarian or baseline diets using Salmonella typhimurium TA 98 and TA 100. The trend towards higher mutagenicity on the high meat diet over the vegetarian diet was consistent for all six subjects using TA 100, and for five of the six using TA 98. The vegetarian and baseline diets resulted in similar overall mutagenic activity. Analysis of the fecal sample parameters using the Kruskal-Wallis one-way analysis of variance showed no significant differences among fecal samples from the three diet periods with respect to wet weight, dry weight, percent dry weight, pH or number of daily bowel movements. However, a sign-test analysis showed a significant trend (p<0.05) towards fewer bowel movements on the high meat diet than on the vegetarian diet. There were significant differences among subjects for all of the fecal sample parameters (p<0.01 or p<0.001). Spearman rank correlations were significantly positive between mutagenic activities using bacterial strains Salmonella typhimurium TA 98 and TA 100 for the baseline diet (p<0.01) and the vegetarian diet (p<0.05). There were also significant positive correlations (p<0.001) between pH and fecal mutagenicity on the high meat' diet using tester strain TA 100, and between wet weight and dry weight. The results of this study indicate that the overall mutagenic activity of human feces can be increased over a period of two weeks by the consumption of a diet high in meat and low in fiber, which is considered to be a high-risk diet for colon cancer. / Land and Food Systems, Faculty of / Graduate

Mutagens in feces have been suggested to be an indicator for risk of colon cancer. Groups consuming vegetarian diets are known to have lower mortality from colon cancer. The purpose of this study was to assess mutagenic activity in feces of persons habitually consuming vegetarian or non-vegetarian diets and to try to identify dietary factors or other health habits which contributed to fecal mutagenicity. Eleven strict vegetarians, six ovo-lacto vegetarians and twelve non-vegetarians, all from the Greater Vancouver area, participated in this study. Data on certain demographic variables and health habits, as well as dietary intake (food frequency and food records), were taken. One fecal sample was collected from each subject for the study. Aqueous extracts of the feces were prepared and analyzed for mutagens using the fluctuation test with Salmonella typhimurium TA100 and TA98. Levels of mutagenicity on each organism were then statistically correlated with frequency of consumption of food groups, nutrient intake, demographic data and health habits. Ovo-lacto vegetarians and strict vegetarians, as groups, had significantly lower levels of fecal mutagens than non-vegetarians in the TA100 assay. With TA98, only the strict vegetarians had lower levels of mutagens compared to the non-vegetarians. The presence of several different mutagenic compounds was indicated. Significant negative correlations were found with mutagenicity on TA98 for all subjects with the following dietary variables: fruits and juices, fiber and iron. Similar negative correlations were found for total carbohydrate and Southgate fiber intakes and mutagenicity on TA100. Within the group of non-vegetarians, there were negative correlations with mutagenicity on TA98 and total protein and with mutagenicity on TA100 and calcium. With the demographic variables and health habits, no clear pattern emerged to indicate factors which would predict lowered mutagenicity for all subjects. It is concluded that vegetarians have lower levels of fecal mutagenicity and that several dietary factors are likely to contribute to this phenomenon. / Land and Food Systems, Faculty of / Graduate