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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Whole yellow pea flour (WPF) and fractionated yellow pea flour (FPF) are novel functional food ingredients that vary in nutritional composition. Consequently, the health benefits of WPF and FPF remain undefined. The purpose of this research was to identify the effects of WPF and FPF on risk factors and morbidities associated with cardiovascular disease, diabetes and obesity as well as the gastrointestinal microbiome. Using USDA recommended dosages of WPF and FPF, clinical endpoints and the colonic microbiome were investigated using a human clinical trial engaging a cross-over design and a diet and energy controlled paradigm. Humans were also utilized to investigate post-prandial glycemic responses and sensory characteristics of novel functional foods formulated with WPF. Finally, Golden Syrian hamsters were used to assess the impact of high doses of WPF and FPF on clinical endpoints and caecal microbial abundance. Results reveal that USDA recommended dosages of WPF and FPF in humans decreased (p<0.05) fasting insulin and estimates of insulin resistance compared to white wheat flour (WF). Android-to-gynoid fat ratios in women were lower (p=0.027) in the WPF group compared to the WF group. FPF decreased (p<0.05) post-prandial energy expenditure alongside a tendency (p<0.075) to reduce carbohydrate oxidation. Novel biscotti and banana bread formulated with WPF induced low post-prandial glycemic responses which were similar to boiled whole yellow peas and significantly lower (p<0.05) than white bread. Sensory analysis of novel WPF biscotti and banana bread demonstrated that WPF-based food products are palatable and acceptable for human consumption. Hamsters consuming diets containing 10% WPF and FPF induced similar reductions (p<0.05) in fasting insulin levels compared to controls. However, animals consuming WPF increased (p<0.05) oxygen consumption while FPF decreased (p<0.05) fasting glucose levels. In addition, terminal restriction fragment length polymorphism analysis revealed that WPF and FPF induced distinct shifts in caecal microbial populations within the phyla Firmicutes. Finally, pyrosequencing analysis of human fecal microbiota demonstrated that FPF and WPF induced shifts in bacterial genera, primarily within Bacteroidetes and Firmicutes. In conclusion, whole and fractionated yellow pea flours are functional food ingredients and can be utilized to manage risk factors for lifestyle-related diseases in humans.

Background: Almost all foals develop transient diarrhoea within the first weeks of life. Studies indicated different viral, bacterial, and parasitic causes, such as rotavirus, Clostridium perfringens, Escherichia coli, and Cryptosporidium are discussed. But little is known about the development of intestinal microflora in foals. The present study investigated whether the supplementation with Bacillus cereus var. toyoi would modify the developing intestinal microflora and consequently reduce diarrhoea in foals. From birth, the foals were randomly assigned to three treatment groups: placebo (10 mL isotonic NaCl, n = 8), low dosage (LD; 5 × 108 cfu B. cereus var. toyoi, n = 7) and high dosage (HD; 2 × 109 cfu B. cereus var. toyoi, n = 10). Treatment groups were supplemented orally once a day for 58 days. Faeces scoring and sampling were performed within the first 24 h after birth and on day 9, 16, 23, 30, 44, 58 of the foal’s life and also on the first day of diarrhoea. Culture-plate methods were used to analyse the bacterial microflora. Results: Eighty-eight per cent of the foals developed diarrhoea (placebo 7/8, LD 5/7, HD 10/10) during the first 58 days of life. Bacillus cereus var. toyoi supplementation had no effect on bacterial microflora. Clostridium perfringens and enterobacteria were equally prevalent in foals with diarrhoea and those who were not afflicted. Conclusions: We conclude that the supplementation of B. cereus var. toyoi had no effect on the occurrence of diarrhoea and health status in the foals.

Die Besiedlung des kindlichen Darms beginnt unmittelbar mit der Geburt. Hier findet der erste Kontakt mit verschiedensten Bakterien statt. Deshalb war die zentrale Fragestellung unserer Untersuchung, wie sich die Bakterienarten bei den Geburtsmodi unterscheiden und wie sich die bakterielle Zusammensetzung im Stuhl von Neugeborenen im Laufe der Jahre verändert hat. Wir untersuchten die Stuhlproben von 42 Neugeborenen, wovon 22 vaginal und 20 per Sectio entbunden wurden, am ersten, zweiten und dritten Lebenstag auf Bakterienwachstum. Es wurden die aufgetretenen Bakteriengattungen mittels Kultur bestimmt und von den aufgetretenen Staphylokokken und Enterokokken die Arten und deren Resistenzen mittels des Phönix-Vollautomaten untersucht. Wir konnten zeigen, dass im Gegensatz zu früheren Untersuchungen heutzutage vermehrt Staphylokokken den Darm von Neugeborenen besiedeln. Außerdem fiel auf, dass per Sectio entbundene Kinder eine verminderte Heterogenität in ihrer Stuhlflora haben und erst später besiedelt werden als vaginal entbundene Kinder.

Whole yellow pea flour (WPF) and fractionated yellow pea flour (FPF) are novel functional food ingredients that vary in nutritional composition. Consequently, the health benefits of WPF and FPF remain undefined. The purpose of this research was to identify the effects of WPF and FPF on risk factors and morbidities associated with cardiovascular disease, diabetes and obesity as well as the gastrointestinal microbiome. Using USDA recommended dosages of WPF and FPF, clinical endpoints and the colonic microbiome were investigated using a human clinical trial engaging a cross-over design and a diet and energy controlled paradigm. Humans were also utilized to investigate post-prandial glycemic responses and sensory characteristics of novel functional foods formulated with WPF. Finally, Golden Syrian hamsters were used to assess the impact of high doses of WPF and FPF on clinical endpoints and caecal microbial abundance. Results reveal that USDA recommended dosages of WPF and FPF in humans decreased (p<0.05) fasting insulin and estimates of insulin resistance compared to white wheat flour (WF). Android-to-gynoid fat ratios in women were lower (p=0.027) in the WPF group compared to the WF group. FPF decreased (p<0.05) post-prandial energy expenditure alongside a tendency (p<0.075) to reduce carbohydrate oxidation. Novel biscotti and banana bread formulated with WPF induced low post-prandial glycemic responses which were similar to boiled whole yellow peas and significantly lower (p<0.05) than white bread. Sensory analysis of novel WPF biscotti and banana bread demonstrated that WPF-based food products are palatable and acceptable for human consumption. Hamsters consuming diets containing 10% WPF and FPF induced similar reductions (p<0.05) in fasting insulin levels compared to controls. However, animals consuming WPF increased (p<0.05) oxygen consumption while FPF decreased (p<0.05) fasting glucose levels. In addition, terminal restriction fragment length polymorphism analysis revealed that WPF and FPF induced distinct shifts in caecal microbial populations within the phyla Firmicutes. Finally, pyrosequencing analysis of human fecal microbiota demonstrated that FPF and WPF induced shifts in bacterial genera, primarily within Bacteroidetes and Firmicutes. In conclusion, whole and fractionated yellow pea flours are functional food ingredients and can be utilized to manage risk factors for lifestyle-related diseases in humans.

The cytokine environment of early pregnancy is known to be a key determinant of the development of the pre-implantation embryo, and its subsequent implantation and growth. Factors in male seminal fluid have been identified as regulators of the expression of cytokines in the female tract of mice, humans and other mammalian species, with insemination eliciting a cascade of molecular and cellular events, reminiscent of a classic inflammatory response. In humans, perturbations in seminal fluid signalling have been proposed to predispose to pathologies of pregnancy including implantation failure, recurrent miscarriage and pre-eclampsia. Seminal transforming growth factor-beta (TGFβ) is identified as one key molecule present in seminal fluid responsible for inducing the female post-mating cytokine response in mice. Research in humans however, has shown the seminal TGFβ content of fertile versus infertile couples to be similar, while the content of other known seminal constituents such as interferon-gamma (IFNγ), correlate with reproductive success. This project aimed to investigate the nature of active factors present in seminal fluid in mice, and their interactions in regulating the uterine cytokine environment during early pregnancy, utilising a variety of in vitro and in vivo experimental strategies. Further, the effect of perturbation in the peri-conception cytokine environment on short and long term pregnancy and postnatal outcomes was investigated. Evaluation of uterine fluids from estrous and mated mice showed a marked upregulation of a number of cytokines following mating, including granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6) and the chemokine KC (rodent IL-8 homologue). Increased production of factors such as GM-CSF and subsequent generation of a receptive uterine environment is thought to be crucial for optimal embryo development and placentation. It has previously been shown that seminal factors such as TGFβ contribute to the uterine post-mating inflammatory response, however other moieties present in seminal fluid, for instance cytokines induced in response to infection such as IFNγ or products from the mucosal microflora, may also play a regulatory role. Using uterine epithelial cells cultured in vitro, it was shown that a variety of immune modulators including the cytokines TGFβ and IFNγ, as well as bacterial products, gram negative lipopolysaccharide (LPS) and gram positive lipoteichoic acid (LTA), can alter basal cytokine production. IFNγ, a pro-inflammatory cytokine secreted by activated natural killer cells and T-cells, is known to interfere with TGFβ signalling in other contexts. Independently TGFβ, LPS and LTA stimulate GM-CSF production while differentially regulating IL-6 and KC production. Conversely IFNγ inhibits GM-CSF production, without effecting IL-6 or KC. Pair wise combinations of TGFβ, LPS and LTA resulted in additive stimulation of GM-CSF, while addition of IFNγ to cultures in conjunction with any of these molecules downregulated GM-CSF and KC stimulation. These in vitro studies indicate factor-specific interactions between seminal fluid constituents and highlight the complex nature of seminal fluid signalling. Consequently we propose that the relative ratio of seminal signalling factors is likely to be more important than the absolute concentration of various regulators, in determining the optimal female reproductive tract response. Using the mouse as an in vivo model, I have in addition demonstrated that LPS and LTA instilled into an estrous uterus can elicit cytokine production comparable to that observed following insemination. Further, these studies have shown that IFNγ instilled into the uterus of a recently mated mouse can reduce the post-copulatory GM-CSF and KC surge. However administration of IFNγ had no effect on near term pregnancy outcomes including fetal or placental weights, fetal crown-rump length, or implantation or resorption rates. The ‘developmental origins of adult disease hypothesis’ proposes the idea that the early uterine environment encountered by the conceptus contributes toward the risk of metabolic disorders in adulthood, hence a long term study of progeny conceived after IFNγ administration was also undertaken. Neo-natal outcomes, such as birth weight, litter size and gestation length were unaltered, as was growth trajectory to 22 weeks of age. Adult metabolic markers, glucose tolerance, organ weight, muscle weight, adiposity and systolic blood pressure were not affected by the perturbation of peri-conceptual cytokine parameters. This work has examined the potential regulatory role of a number of seminal fluid signalling agents in directing the post-mating cytokine response, and has furthermore shown the relatively resilient nature of the early cytokine environment to subtle perturbation. Delineating the identity and roles of seminal fluid factors in early pregnancy brings us closer to an understanding of the key physiological events of early pregnancy and assists in identifying potential risk factors for human pregnancy pathologies. / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Toxoplasma gondii infection in susceptible C57BL/6 mice induces a strong Th1 immune response causing intense inflammation and lesions in ileum in response to microbial antigens and if controlled causes death of animals. The Th1 immune response is similar to that in patients with inflammatory bowel disease (IBD) such as Crohn\'s disease (CD) and ulcerative colitis (UC). Therefore find alternative treatments that have adjuvant effect of conventional treatments are of great interest. The use of probiotics is increasing due to several benefits found in inflammatory diseases. To verify the effects of probiotics Lactobacillus casei Shirota, Lactobacillus acidophilus and Lactococcus lactis NCDO 2118 in C57BL/6 mice, we treat one day before and 7 days after oral infection with 30 cysts of the ME-49 strain. We found that L. casei- and L. acidophilus-treatment mice decreased intestinal parasitism reducing the pathology in small intestine of C57BL/6 mice and prevent death. In addition, the microbiological culture of organs shows L. casei- and L. acidophilus-treatment mice prevent bacterial translocation from the intestinal lumen to organs such as liver, spleen, lung and blood, moreover the microbiological analysis of feces showed that there were fewer gram negative bacteria species in feces of probiotic treated mice. The NAG, MPO and EPO assay that are characteristic of macrophages, and eosinophils neutrophils respectively showed that there was a decrease in the activity of enzymes MPO and NAG and a small increase in activity and EPO. Finally, the qPCR analysis revealed that treatment with L. acidophilus decreased IFN-&#947; and TNF-&#945; mRNA induced by infection with T. gondii and L.casei treatment increased the expression of Foxp3 and IL-10. Our results demonstrate the ability of probiotics to control the inflammation response and reduce mortality caused by ileitis. However, further studies are needed to find out what the precise mechanisms that probiotics L. casei and L. acidophilus modulate the host immune response. / A infecção por Toxoplasma gondii em camundongos susceptíveis C57BL/6 com 30 cistos da cepa ME-49 induz uma forte resposta do sistema imune do tipo Th1 causando uma intensa inflamação com lesão no íleo em resposta a antígenos da microbiota e se não for controlada causa a morte dos animais. Essa resposta é semelhante à encontrada em pacientes com doenças inflamatórias intestinais (IBD) como a doença de Crohn (CD) e colite ulcerativa (UC). Portanto encontrar tratamentos alternativos que tenham efeito adjuvante aos tratamentos convencionais é de grande interesse. O uso de probióticos em produtos fermentados vem aumentando devido aos vários benefícios encontrados em pacientes com doenças inflamatórias que utilizam esses produtos. Com o objetivo de investigar o efeito da administração dos probióticos Lactobacillus casei Shirota, Lactobacillus acidophilus e Lactococcus lactis NCDO 2118 no intestino delgado, tratamos camundongos C57BL/6 um dia antes e 7 dias depois da infecção oral com 30 cistos da cepa ME-49 de T. gondii. O tratamento com L. casei ou L. acidophilus diminuiu o parasitismo intestinal, reduziu a patologia associada ao intestino delgado e evitou a morte dos animais. Além disso, a cultura microbiológica dos órgãos revelou que L. casei e L. acidophilus foi capaz de impedir a translocação de bactérias do lúmen intestinal para órgãos como fígado, baço, pulmão e também para o sangue. O cultivo microbiológico das fezes mostrou que houve menos espécies de bactérias gram negativas encontradas nas fezes dos animais tratados com probióticos. A análise das enzimas NAG, MPO e EPO que são características de macrófagos, neutr´filos e eosinófilos, respectivamente mostrou que houve diminuição na atividade das enzimas MPO e NAG e um pequeno aumento na atividade e EPO. Por último, a análise em qPCR revelou que o tratamento com L. acidophilus diminuiu a expressão de IFN-&#947; e TNF-&#945; induzido pela infecção por T. gondii e o tratamento com L.casei aumentou a expressão de Foxp3 e IL-10. Esses resultados demonstram a capacidade dos probióticos em controlar a resposta inflamação e reduzir a mortalidade causada pela ileíte. Contudo, serão necessários mais estudos para descobrir quais os mecanismos precisos que os probióticos L. casei e L. acidophilus utilizam para modular a resposta imune em favor do hospedeiro. / Mestre em Imunologia e Parasitologia Aplicadas

Allergic diseases, for example asthma and eczema, are nowadays considered belonging to the most common chronic diseases amongst children in the West, but the cause for this increase in allergy prevalence is unknown. Since studies have indicated a connection between children's exposure of microorganisms during infancy and risk of developing allergic disease, it is suggested that this exposure is a crucial factor in question of allergy development or not. Other studies have established differences in microflora composition between healthy children and children with allergic disease, and several studies have shown that probiotic therapy can give positive results in both prevention and treatment of allergic diseases. The aim of this master's thesis was to develop a method, using PCR and denaturing gradient gel electrophoresis, to study the diversity of Lactobacillus spp. in fecal samples retrieved from a study of the probiotic strain L. reuteri ATCC 55730. The developed method was successful in detecting lactobacilli in fecal samples, but three other bacterial genera commonly found in humans were also amplified. Comparison of average numbers of detected bacterial strains and lactobacilli strains between samples belonging to the probiotics and placebo groups, respectively, showed higher numbers for the probiotics group. Also, the only fecal samples that contained L. reuteri belonged to the probiotics group. Although the results are far from statistically significant, they support the theories that probiotics may influence the intestinal microbiota.

O experimento foi realizado na unidade de creche do Laboratório de Pesquisa em Suínos (LPS) da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo (FMVZ-USP), campus de Pirassununga (SP), sendo utilizados setenta e dois leitões recém desmamados (21 dias), com peso médio de 6,5 kg. O delineamento experimental foi em blocos casualizados, num arranjo fatorial 2 x 2, sendo os fatores: probiótico e desafio, os quais definiram os tratamentos: T 1 - emprego do probiótico, com desafio (CPCD); T 2 - emprego do probiótico, sem o desafio (CPSD); T 3 - ausência do probiótico, com desafio (SPCD); T 4 - ausência do probiótico e sem o desafio (SPSD). As faixas de ganho médio de peso diário consideradas foram: dos 21 aos 35 dias (GMPD 35); dos 35 aos 49 dias (GMPD 49); dos 49 aos 63 dias (GMPD 63) e dos 21 aos 63 dias (GMPD Total). Os leitões foram submetidos ao desafio em sala separada, 14 dias após a distribuição nos tratamentos. O desafio foi realizado com uma amostra de E. coli positiva para as toxinas LT e ST b e para as fímbrias K 88 e F 18. A colheita das fezes foi feita após o desafio, em três períodos (35, 42 e 49 dias de idade dos leitões), para contagem de coliformes fecais, sendo analisadas pelo Laboratório de Sanidade Suína da FMVZ-USP, em São Paulo. A ração oferecida aos leitões foi semelhante à empregada comercialmente nas criações, havendo somente suplementação ou não do probiótico. O experimento avaliou o desempenho dos leitões através do ganho de peso diário (GMPD) e peso (P), acrescido da verificação da consistência das fezes e da contagem de coliformes fecais. Os resultados mostraram interação significativa entre probiótico e desafio quanto ao GMPD 49, indicando nesse período, o ponto de inflexão que caracterizou o início da mudança no comportamento desta variável. Com relação à consistência das fezes, o probiótico agiu reduzindo o percentual de fezes cremosas, confirmando sua ação protetora da mucosa intestinal, reduzindo a perda de líquido e, conseqüentemente, a diarréia. Além de atuar melhorando os parâmetros relativos ao desempenho e à consistência das fezes, a utilização do probiótico promoveu uma alteração na contagem de E. coli, reduzindo-a para um valor inferior, 14 dias após o desafio, reforçando assim a sua ação benéfica junto á manutenção da integridade intestinal / The experiment was conducted in the nursery unit at the Swine Research Laboratory from the FMVZ, University of São Paulo, Pirassununga campus, where we used 72 piglets recently weaned (about 21 days) with the average weight of 6,5 kg. The experiment was outlined in random blocks system, 2 x 2 factorial arrangements, being the factors: probiotic and challenge. The treatment was: T1 ? use of the probiotic, with challenge (CPCD); T2 ? use of the probiotic, without the challenge (CPSD); T3 ? no probiotic, with challenge (SPCD); T4 ? no probiotic and no challenge (SPSD). The intervals of weight daily gain considered were 21 to 35 days (GMPD 35); 35 to 49 days (GMPD 49); 49 to 63 days (GMPD 63) and 21 to 63 days (GMPD Total). The piglets were kept in two different rooms. One of the groups was challenged 14 days after the distribution in the treatments, in a separated room and for the challenge we used a sample of E.coli positive for the toxins LT and ST b and also for the pilli K88 and F18. The samples of feces were collected after the challenge in three periods (35, 42 and 49 days of age of the piglets) and sent to the Swine Health Laboratory at the FMVZ-USP, São Paulo campus, where the count of some species of intestinal bacteria was performed. The feed offered to the piglets was the same used in commercial swine herds. The only difference was the use or not of the probiotic. The experiment evaluated the development of the piglets through the amount of weight (AW) they gained and the average daily gain (ADG) of weight, plus the verification of the consistency of the feces and the count of some intestinal bacteria. The results showed a significant interaction between the probiotic and the challenge, as related to GMPD 49, indicating in this period the point of inflexion that characterized the beginning of the change in behavior of this variable. Analyzing the consistency of the feces we have notice that the probiotic has reduced the percentage of creamy feces, confirming its protective action in the intestinal mucus, reducing the loss of liquid and consequently the diarrhea. Together with the positive effect on the performance, the probiotic reduced the abnormal consistency of the feces, specially the creamy ones, as well as decreased the counting of E.coli 14 days after the challenge, keeping the intestinal integrity