The intestinal tract of salmonids provides a dynamic interface that not only mediates nutrient uptake but also functions as the first line of defence against ingested pathogens. Exposure of the immune system to beneficial microorganisms and different dietary immunostimulants via the intestine has been shown to prime the immune system and help in the development of immune competence. Furthermore, the morphology and function of teleostean intestines are known to respond to feed components and to ingested and resident bacterial communities. Histological appraisal is still generally considered to be the gold standard for sensitive assessment of the effects of such dietary modulation. The aim of the present study was to improve understanding of salmonid intestinal function, structure and dynamics and to use the knowledge gained to develop a model for analysis, which would allow intestinal health to be assessed with respect to different intestinal communities and feed components. Virtual histology, the process of assessing digital images of histological slides, is gaining momentum as an approach to supplement traditional histological evaluation methodologies and at the same time, image analysis of digitised histological sections provides a practical means for quantifiable assessment of structural and functional changes in tissues, being both objective and reproducible. This project focused on the development of a rapid, practical analytical methodology based on advanced image analysis, that was able to measure and characterise a range of features of the intestinal histology of Atlantic salmon in a quantitative manner. In the first research chapter, the development of a novel histological assessment system based upon advanced image analysis was described, this being developed with the help of a soybean feed model known to induce enteropathy in Atlantic salmon. This tool targeted the evaluation of the extent of morphological changes occurring in the distal intestine of Atlantic salmon following dietary modulation. The final analytical methodology arrived at, could be conducted with minimal user-interaction, allowing rapid and objective assessment of 12 continuous variables per histological frame analysed. The processing time required for each histological frame was roughly 20-25 min, which greatly improved the efficiency of conducting such a quantitative assessment with respect to the time taken for a subjective semi-quantitative alternative approach. Significant agreement between the fully automated and the manual morphometric image segmentation was achieved, however, the strength of this quantitative approach was enhanced by the employment of interactive procedures, which enabled the operator / observer to rectify preceding automated segmentation steps, and account for the specimen’s variations. Results indicated that image analysis provided a viable alternative to a pathologist’s manual scoring, being more practical and time-efficient. In the second research chapter, feeding Atlantic salmon a high inclusion level of unrefined SBM (25 %) produced an inflammatory response in the distal intestine as previously described by other authors. The model feed trial successfully generated differentiable states, although these were not, for the most part, systemically differentiable through the majority of standard immunological procedures used, being only detectable morphologically. Quantitation of morphometric parameters associated with histological sections using the newly developed image analysis tool successfully allowed identification of major morphological changes. Image analysis was thus shown to provide a powerful tool for describing the histomorphological structure of Atlantic salmon distal intestine. In turn, the semi-automated image analysis methods were able to distinguish normal intestinal mucosa from those affected by enteritis. While individual parameters were less discriminatory, use of multivariate techniques allowed better discrimination of states and is likely to prove the most productive approach in further studies. Work described in the third research chapter sought to validate the semi-automated image analysis system to establish that it was measuring the parameters it was purported to be measuring, and to provide reassurance that it could reliably measure pre-determined features. This study, using the same sections for semi-quantitative and quantitative analyses, demonstrated that the quantitative indices performed well when compared to analogous semi-quantitative descriptive parameters of assessment for enteritis prognosis. The excellent reproducibility and accuracy performance levels indicated that the image analysis system was a useful and reliable morphometric method for the quantification of SB-induced enteritis in salmon. Other characteristics such as rapidity, simplicity and adaptability favour this method for image analysis, and are particularly useful where less experienced interpreters are performing the analysis. The work described in the fourth research chapter characterised changes in the morphology of the intestinal epithelial cells occurring as a result of dietary modulation and aspects of inflammatory infiltration, using a selected panel of enzyme and IHC markers. To accomplish this, image analysis techniques were used to evaluate and systematically optimise a quantitative immunolabelling assessment protocol. Digital computer-assisted quantification of labelling for cell proliferation and regeneration; programmed cell death or apoptosis; EGCs and t-cell like infiltrates; mobilisation of stress-related protein regenerative processes and facilitation of nutrient uptake and ion transport provided encouraging results. Through the description of the intestinal cellular responses at a molecular level, such IHC expression profiling further characterised the inflammatory reaction generated by the enteropathic diet. In addition, a number of potential diagnostic parameters were described for fish intestinal health e.g. the relative levels of antigenicity and the spatial distribution of antigens in tissues. Work described in the final research chapter focused on detailed characterisation of intestinal MCs / EGCs in order to try to elucidate their functional role in the intestinal immune responses. Through an understanding of their distribution, composition and ultrastructure, the intention was to better characterise these cells and their functional properties. The general morphology, histochemical characteristics and tissue distribution of these cells were explored in detail using histochemical, IHC and immunogold staining / labelling, visualised using light, confocal and TEM microscopy. Despite these extensive investigations, their physiological function and the content of their granules still remain somewhat obscure, although a role as immunodulatory cells reacting to various exogeneous signals through a finely regulated process and comparable to that causing the degranulation of mammalian MCs is suggested. The histochemical staining properties demonstrated for salmonid MCs / EGCs seem to resemble those of mammalian mucosal mast cells, with both acidophilic and basophilic components in their granules, and a granule content containing neuromodulator / neurotransmitter-peptides such as serotonin, met-enkephalin and substance-p. Consequently, distinguishable bio-chromogenic markers have been identified that are of utility in generating a discriminatory profile for image analysis of such cells.

In the thesis the aim is to investigate different aspects of what teachers value when grading in Swedish physical education (PE) and to analyses how sociological background factors impact students’ grades. Grades in PE have included aspects other than those prescribed in the grading criteria, for instance motivation and effort. Teachers sometimes find their value-setting difficult to articulate and refer to a “gut feeling”. In order to explore both explicit and implicit forms of value-setting, the Repertory Grid interview technique is employed. The thesis includes four sub-studies, three interview studies with Swedish PE teachers and a fourth study based on registry data from the Swedish National Agency for Education. The data of all students leaving nine-year compulsory school in 2014 (n=95317) is analysed to explore how sociological background factors, such as migration background, parents’ education, school provider and gender, affect PE grades. The results reveal aspects of grading that are not detectable in the official description of the grading assignment and highlight problems that teachers need to address when grading. Four themes are discerned in the teachers’ grading practices: motivation, knowledge, confidence and social skills. The implementation of a new national curriculum with specified knowledge requirements seems to improve the alignment with the national criteria, but there is still a gap between policy and practice. The knowledge requirements for movement are often interpreted as performances in competitive sports, even if the teachers try to find other interpretations. The odds ratio for getting a higher grade in PE is greater for the variables migration background and parents’ education than for the other investigated variables. The concepts formulated by Bernstein are applied to explore the relations between teachers’ grading practices and cultural and political influences and to discuss how the tensions between different interests could affect teachers’ grading. The conclusion is that the gap between policy and practice confirmed in this study is related to tensions between the interests and purposes of different agents, all of whom strive to influence steering documents and practice. Cultural and political influences need to be considered and facilitate discussions about how to understand which knowledge is valued in PE and who has better possibilities to assimilate it. / Forskningslinjen Utbildning

L’épithélium intestinal est une monocouche de cellules qui tapisse la lumière intestinale, constitué d’un compartiment différencié, les villosités dans l’intestin grêle et les plateaux épithéliaux dans le colon, et d’un compartiment prolifératif, les cryptes de Lieberkühn. Ce tissue se renouvelle de façon rapide et continue tout au long de la vie de l’individu, grâce à la présence de cellules souches adultes dans le fond des cryptes. Ces cellules s’autorenouvellent et donnent naissance à des progéniteurs prolifératifs (capables d’engendrer les différents cytotypes épithéliaux) qui se différencient tout en migrant vers le compartiment différencié. Mon travail de these a porté sur l’étude d’une marqueur putatif de ces cellules souches épithéliales intestinales: Musashi1 (Msi1).Dans ce contexte, mon premier axe d’étude s’est focalisé sur l’isolement et la caractérisation des cellules souches épithéliales intestinales chez la souris. Pour cela, nous avons généré des souris transgéniques exprimant la protéine fluorescente GFP sous le contrôle du promoteur de Msi1. Les cellules souches intestinales de ces souris coexpriment donc Msi1 et la GFP. Ce modèle a été validé et nous à permis de isoler les cellules GFP/Msi1 positives dans l’intestin. A l'aide de différentes approches cellulaires et moléculaires, nous avons confirmé leur nature de cellules souches et nous avons apporté des nouvelles données sur la composition de la zone proliférative de l’épithélium intestinal murin.Le second axe de mes travaux de thèse a porté sur l’étude de la fonction de Msi1 dans l'homéostasie de l’épithélium intestinal chez la souris, par son sur-expression tous au long de l’épithélium. Nous avons montré que la sur-expression de cette protéine, qui est un régulateur des voies Wnt et Notch, perturbe l’architecture intestinale, a propriétés pro-prolifératives et un potentiel tumorigènique. / The intestinal epithelium is a monolayer of cells surrounding the intestinal lumen. It consists of a differentiated compartment, the villi in the small intestine and a flat surface in the colon, and a proliferative compartment, the crypts of Lieberkühn. This tissue self-renews rapidly and continuously throughout life, due to the presence of adult stem cells in the bottom of the crypts. These cells are capable of self-renewing and give rise to proliferating progenitors (capable of generating all the different epithelial cytotypes) that differentiate and migrate toward the differentiated compartment. My thesis focused on the study of the intestinal epithelial stem cells marker Musashi1 (Msi1).In this context, the first part of my thesis work focused on the isolation and characterization of the intestinal epithelial stem cells that express Msi1 in the mouse. For this, we generated transgenic mice expressing the fluorescent protein GFP under the control of the promoter of Msi1. The intestinal stem cells of these mice co-express Msi1 and GFP. This model has been validated and allowed us to isolate GFP+/Msi-expressing cells in the intestine. By using different cellular and molecular approches, we confirmed their nature of stem cells and provided new data on the composition of the proliferative zone in the murine intestinal epithelium.The second part of my thesis has focused on the study of the function of Msi1 in the intestinal epithelium homeostasis in the mouse, by its over- and ectopic expression all along the epithelium. We have shown that the over-expression of this protein, which is a regulator of the Wnt and Notch pathways, perturbs the intestinal architecture, has pro-proliferative properties and tumorigenic potential.

L'avènement des méthodes de séquençage moléculaire à large échelle a permis l'identification d'altérations du microbiote digestif spécifiquement associés à l'obésité notamment un ratio Bacteroidetes/Firmicutes diminué chez les obèses. Depuis, de nombreux travaux ont décrit de nouvelles altérations associées à l'obésité, notamment une augmentation des représentants du genre Lactobacillus mais l'ensemble de ces résultats sont souvent l'objet de controverses. Afin de clarifier si le genre Lactobacillus était associé à l'obésité, nous avons réalisé deux études cas témoins (la deuxième étant le prolongement de la première avec un effectif de 263 individus) qui nous ont permis d'identifier que les altérations du microbiote digestif sont plus reproductibles au niveau de l'espèce. A ce titre nous avons retrouvé une plus grande concentration de Lactobacillus reuteri dans le microbiote digestif de sujets obèses alors que les concentrations de Bifidobacterium animalis, Methanobrevibacter smithii et Escherichia coli étaient diminuées. Nous avons pu établir une relation dose-dépendante entre la concentration de Lactobacillus reuteri et l'indice de masse corporelle. Par ailleurs, nous avons réalisé une méta-analyse sur les résultats des études publiées et avons retrouvé une association entre les genres Bifidobacterium (6 études, 348 individus) et Methanobrevibacter (3 études, 195 individus) avec l'absence d'obésité (…) / The revolution of large scale molecular sequencing methods allowed the identification of specific alterations in the gut microbiota associated with obesity such as a decreased Bacteroidetes / Firmicutes ratio in obese individuals. Since then, many studies have described different alterations associated with obesity, including an increase in members of the Lactobacillus genus, but results are often controversial. To clarify whether the genus Lactobacillus was associated with obesity, we conducted two case-control studies (the second being the follow-up of the first study with a total of 263 individuals) allowing us to understand that gut microbiota alterations are more reproducible at the species level. We found a greater concentration of Lactobacillus reuteri in the gut microbiota of obese while concentrations of Bifidobacterium animalis, Methanobrevibacter smithii and Escherichia coli were reduced. We were able to establish a dose-dependent relationship between the concentration of Lactobacillus reuteri and body mass index. In addition, we performed a meta-analysis on the results of published studies and we found an association between the Bifidobacterium (6 studies, 348 individuals) and Methanobrevibacter (3 studies, 195 individuals) with absence of obesity. (…)

Les lipides polaires (LP) laitiers (~2% des lipides du lait) présentent un potentiel bioactif élevé, notamment lié à leur richesse en sphingomyéline (SM, ~25% des LP). Nos hypothèses sont que les LP laitiers peuvent exercer certains de leurs effets bénéfiques par l'intermédiaire de la SM, notamment sur l'intégrité de la barrière intestinale et le microbiote, ce qui pourrait contribuer à réduire l'inflammation métabolique. Nous avons testé à long terme in vivo l'impact de régimes hyperlipidiques (HF) supplémentés en LP laitiers. In vitro, nous avons étudié l'effet des LP laitiers et de la SM (laitière ou d'oeuf) sur l'expression génique des protéines de jonctions serrées. Nos travaux in vitro ont également permis de tester que l'interleurkine-8 (IL-8), impliquée dans la maturation de l'épithélium intestinal, serait un acteur des modifications intestinales en réponse aux LP laitiers et/ou à la SM. L'impact à court terme d'un gavage chez la souris avec des LP laitiers ou de la SM laitière a également été étudié. Après 8 semaines de régime HF supplémenté en LP laitiers (1,6%) les souris présentent un moindre gain de poids en comparaison au régime HF. Nous observons une augmentation de Bifidobacterium animalis pour le groupe contenant 1,1% de LP laitiers. Le groupe nourri avec une supplémentation de 1,6% de LP laitiers présente une diminution de Lactobacillus reuteri et des cryptes coliques plus profondes. Nous retrouvons également une plus forte teneur en acide gras spécifiques des LP laitiers (C23:0, C24:0 et C24:1, présents dans la SM laitière) dans les lipides fécaux. Ces acides gras sont corrélés à la teneur en Lactobacillus spp. Parmi les protéines de jonctions serrées impliquées dans la perméabilité paracellulaire, seule l'expression de ZO-1 tend à être augmentée dans le duodénum. In vitro, lorsque les cellules Caco-2/TC7 sont incubées avec des micelles mixtes supplémentées en SM pure, une augmentation de l'expression génique des protéines de jonctions serrées, ainsi qu'une augmentation de la concentration d'IL-8 en apicale et en basolatérale, sont observées. Ces effets sont également retrouvés avec la SM d'oeuf, contrairement aux LP laitiers totaux. L'incubation d'IL-8 recombinante humaine conduit à une augmentation de l'expression génique des protéines de jonctions serrées. Un gavage avec de la SM laitière pure chez la souris induit une augmentation de l'expression des homologues murins de l'IL-8 (KC et Mip-2). Cette étude suggère que les LP laitiers peuvent limiter la prise de poids induite par un régime HF et moduler le microbiote intestinal. La présence de produits d'hydrolyse spécifiques de la SM pourrait expliquer les effets sur le côlon et le microbiote intestinal. Les résultats in vitro, suggèrent un impact spécifique de la SM sur la barrière intestinale. L'IL-8 semble impliquée dans la régulation de l'expression des protéines de jonctions serrées. Ces résultats contribuent à expliquer les effets bénéfiques démontrés des LP laitiers. L'exploration mécanistique des effets directs et/ou indirects de la SM et de l'IL-8 sur la barrière intestinale reste à élucider / Interest is growing for the metabolic impact of milk polar lipids (MPL, ~2% of dairy lipids), which present a high bioactive potential, particularly related to their content in sphingomyelin (SM, ~ 25% of MPL). Our hypotheses are that MPL can exert some of their beneficial effects through SM, including the integrity of the intestinal barrier and the microbiota, which could contribute to reduce metabolic inflammation. We tested the metabolic impact of the addition of MPL in a high-fat (HF) diet in mice on the modulation of the intestinal barrier. In vitro, we studied the effect of SM (milk or egg) on tight junction protein We also tested in vitro, that interleurkin-8 (IL-8), which is involved in the maturation of the intestinal epithelium, is an actor of intestinal changes in response to MPL and/or MSM. The short-term impact in mice of MPL or milk SM was also studied. After 8 weeks of diet, the supplementation with 1.6% of MPL prevented the HF-diet-induced body weight gain. In caecal microbiota, addition of 1.1% of MPL induced a specific increase in Bifidobacterium spp., in particular B. animalis. The group fed with a 1.6% MPL-supplementation showed a specific decrease in Lactobacteria reuteri and colonic crypt depth were greatest. We also found a higher content of fatty acids specific of MPL (C23:0, C24:0 and C24:1, found in milk SM) in fecal lipids of mice. These fatty acids are correlated with Lactobacillus spp. Among the tight junction proteins involved in paracellular permeability, only the expression of ZO-1 tended to be increased in the duodenum. In vitro, when Caco-2/TC7 cells were incubated with mixed micelles supplemented with pure SM, an increase in the gene expression of tight junction proteins (ZO-1, occludin, JAM-1, claudin-1) and an increase in apical and basolateral IL-8 concentration were observed. These effects were also found with egg SM, unlike total MPL. Incubation of recombinant human IL-8 led to an increase in gene expression of tight junction proteins. Gavage with pure milk- SM in mice induced an increase in the expression of murine homologs of IL-8 (KC and Mip-2). Our results show that MPL can limit HF-induced body weight gain and modulate the abundance of beneficial bacteria of the gut microbiota. The presence of SM-specific hydrolysis products may explain the effects on the colon and gut microbiota. In vitro results suggest a specific impact of pure SM on the intestinal barrier. IL-8 appears to be involved in the regulation of tight junction protein expression. This can contribute to explain reported beneficial effects of MPL in mice regarding HF induced metabolic disorders. The mechanistic exploration of direct and / or indirect effects of SM and IL-8 on the intestinal barrier remains to be elucidated

Human milk is recognized as the main component for growth, metabolism, and immune development in infants. Furthermore, during lactation, human milk is an important source of microorganisms for the intestinal colonization of newborns. Mother-related factors have been associated with the human milk microbiota composition. Nevertheless, apparently, there has not been any study in which the maternal diet was evaluated as a modulator of the human milk microbiota. Therefore, the aim of this study was to investigate the impact of the maternal diet on the human milk microbiota composition of healthy women, and subsequently, to evaluate the effect of fructooligosaccharides supplementation on the human milk microbiota. This study consisted of two parts; the first was a cross-sectional study, including 94 lactating women recruited at the University Hospital of the University of São Paulo (HU/USP), to investigate the association between the maternal nutrient intake during pregnancy and lactation over the first month and the human milk microbiota. The second part consisted of a randomized, placebo-controlled clinical trial with 53 lactating, classified as FOS group (n = 28), which received 4.5 g of fructooligosaccharides + 2 g of maltodextrin or placebo group (n = 25), which received 2 g of maltodextrin, over a period of 20 days. The DNA was isolated and used as template for amplification and sequencing by the Illumina MiSeq® System. Overall, the maternal diet during lactation (\"short-term\" food intake) influenced specific bacterial groups, including positive correlations between polyunsaturated fatty acids/linoleic fatty acids and Bifidobacterium. However, only the maternal diet during pregnancy (\"long-term\" food intake) was statistically significant (p = 0.02) for the clustering analyzes (community structure analyzes), in which higher levels of vitamin C intake during pregnancy was related to cluster 2, driven by the Staphylococcus genus. After the intervention period on the maternal diet, no differences were found for relative abundance of genera between the placebo and the FOS groups. However, the distances of the trajectories covered by the samples from the beginning to the end of the supplementation was higher for the FOS group (p = 0.0007). According to our results, the maternal age affects the response for FOS supplementation (p = 0.02), though no patterns in the differences of relative abundances were found between the groups. Our results suggest that the maternal diet may influence the human milk microbiota, and the diet during pregnancy is a stronger factor over the bacterial community structure. Minor changes were found by the maternal short-term food intake or the maternal intervention with the prebiotic, and the changes seem to be individual-dependent and influenced by the maternal age, particularly in the intervention study. / O leite humano é, reconhecidamente, o principal componente para o crescimento e o desenvolvimento metabólico e imunológico de lactentes. Adicionalmente, durante a lactação, o leite humano consiste em uma importante fonte de micro-organismos para a formação da microbiota intestinal de neonatos. Fatores relacionados à mãe têm sido associados à composição da microbiota do leite humano. Entretanto, poucos estudos avaliaram a dieta materna como componente modulador da microbiota do leite humano. Os objetivos deste estudo foram investigar o impacto da dieta materna sobre a composição da microbiota do leite humano de mães saudáveis e, posteriormente, avaliar a influência da intervenção com fruto-oligossacarídeo na microbiota do leite humano, durante 20 dias de lactação. O estudo foi dividido em duas partes; a primeira parte consistiu de um estudo transversal, com 94 lactantes atendidas no Hospital Universitário da Universidade de São Paulo (HU/USP), a fim de investigar a associação entre o consumo materno de nutrientes durante a gestação e durante o primeiro mês de lactação e a microbiota do leite humano. A segunda parte consistiu em um ensaio clínico, aleatorizado, placebo-controlado, com 53 lactantes, classificadas em grupo FOS, que recebeu 4.5 g de fruto-oligossacarídeo + 2 g de maltodextrina (n = 28) ou grupo placebo, que recebeu 2 g de maltodextrina (n = 25), suplementados por 20 dias. O DNA das amostras de leite foi isolado e utilizado como molde para amplificação e sequenciamento em Illumina MiSeq® System. Em geral, a dieta materna durante a lactação (consumo a curto prazo) apresentou influência pontual sobre diversos grupos de micro-organismos, incluindo correlações positivas entre ácidos graxos poli-insaturados/linoleico e o gênero Bifidobacterium. No entanto, somente a dieta materna durante a gestação (consumo a longo prazo) foi estatisticamente significante (p = 0.02) para as análises de agrupamento das amostras (análises de estrutura de comunidade), sendo o maior teor de vitamina C consumido durante a gestação relacionado ao agrupamento 2, direcionado por maiores populações do gênero Staphylococcus. Após o período de intervenção na dieta materna, não foram encontradas diferenças entre a abundância relativa de gêneros entre os grupos placebo e FOS. No entanto, as distâncias do percurso das amostras do início até o final da suplementação foram maiores para o grupo FOS (p = 0.0007). De acordo com os resultados, a idade materna influencia essa resposta à suplementação por FOS (p = 0.02), embora, não tenham sido encontrados padrões nítidos nas diferenças de abundância relativa entre os grupos. Os resultados obtidos sugerem que a dieta materna consiste em um fator de modulação da microbiota do leite humano, sendo a dieta durante a gestação um fator mais intenso sobre a estrutura da comunidade bacteriana do leite humano. No entanto, o consumo a curto prazo ou a intervenção alimentar com prebiótico sobre a dieta materna apresentou influência pontual sobre a dinâmica da microbiota do leite, ainda que mudanças observadas sejam indivíduo-dependentes e influenciadas pela idade materna, como no caso do estudo de intervenção.

A maioria dos estudos da interação parasito-hospedeiro tem focado na interação de um único patógeno. Porém, o hospedeiro em um ambiente natural é comumente exposto a múltiplos patógenos sequencialmente ou mesmo simultaneamente. Diversos estudos têm utilizado o modelo de Ligadura e perfuração do Ceco (CLP) para estudar a sepse, mas nenhum deles apresentou modelo de coinfecção ou estudo avaliando o papel de infecções prévias no desfecho da sepse polimicrobiana experimental. Neste contexto, nossa hipótese é de que a infecção crônica por parasitos poderia alterar o curso da resposta durante a sepse polimicrobiana. Para testar essa hipótese, animais C57BL/6 ou BALB/c foram infectados com 5 ou 20 cistos da cepa ME 49 de Toxoplasma gondii e 40 dias após a infecção os animais foram induzidos à sepse polimicrobiana. Em nosso estudo, 100% dos animais cronicamente infectados por T. gondii morreram num período de 24 horas após CLP. O mesmo não foi observado quando animais foram infectados cronicamente com os parasitos Leishmania major e Trypanosoma cruzi ou com o fungo Paracoccidioides brasiliensis. Um dado interessante em nosso estudo foi que, nos animais previamente infectados com T. gondii, constatamos melhora na eliminação de bactérias liberadas pela CLP e aumento do recrutamento celular para o sítio da infecção. Apesar de esses animais apresentarem melhora na resposta contra as bactérias, verificamos a presença de lesão intestinal e maior infiltrado inflamatório neste órgão, associado a um aumento da produção de citocinas pró-inflamatórias (IFN-, TNF-, IL-6 e IL-1) e consequente aumento de óxido nítrico (NO), num período de 24 horas depois da CLP. Verificamos que as células TCD4+ e TCD8+ são responsáveis pela produção de IFN- e TNF- nesse modelo de coinfecção, e em modelo in vitro, que macrófagos podem ser responsáveis pela produção de IL-1 dependente de ativação do inflamassoma NLRP3/ASC/Caspase 1. Neste estudo, observamos que a rápida resposta contra a CLP acontece em função da presença de células de memória de padrão Th1, induzidas na infecção por T. gondii. Dessa forma, esse trabalho mostra que a infecção crônica por T. gondii agrava a sepse polimicrobiana subletal, por aumentar a produção de citocinas pró-inflamatórias IL-6, TNF- e IL-1, com a indução de hipotensão, predispondo ao choque séptico. / Most studies of parasite-host interaction have focused on the interaction of a single pathogen with cells or organism of the host. However, in a natural enviroment, the host is commonly exposed to multiple pathogens sequentially or even simultaneously. Several studies have used the model of cecal ligation and puncture (CLP) to study sepsis, but none of them evaluated the effect of the presence of previous infections to the outcome of polymicrobial sepsis. In this context, we hypothesized that chronic infection with Toxoplasma gondii could alter the course of host response against polymicrobial sepsis. To test this hypothesis, C57BL/6 or BALB/c mice were orally infected with 5 or 20 cysts of ME-49 strain of T. gondii and 40 days post infection, they were subjected to CLP. When mice were chronically infected with T. gondii, 100% of the animals died within 24 hours after CLP. The same phenomenons were not observed in animals previously infected with other parasites, such as Leishmania major and Trypanosoma cruzi or the fungus Paracoccidioides brasiliensis. Interestingly, when we evaluated the response against the CLP in animals that were infected with T. gondii, we found an improvement in the killing of bacteria released by CLP and an increase in recruitment of inflammatory cells to the site of infection. However, despite the fact that these animals have improved response against the bacterial infection, they presented intestinal damage and increased inflammatory infiltrate in this organ. The animals also had increased pro-inflammatory cytokines (IFN-, TNF-, IL-6 and IL-1), and nitric oxide (NO) detected within 24 hours after CLP. We also found that the TCD4+ and TCD8+ cells were responsible to produce IFN- and TNF-, and, using an in vitro model, we verified that macrophages are primarily responsible for the production of IL-1 in a pathway dependent on the activation of NLRP3/ASC/Caspase 1 inflamassoma. In this study, we found that early response against CLP happens due to the presence of mainly Th1 memory cells, induced by T. gondii infection. Finally, we found that chronic infection with T. gondii aggravates sublethal polymicrobial sepsis by increasing the cytokines IL-6, TNF- and IL-1, with induction of hypotension that predispose to septic shock.

O diabetes tipo 1 (DM1) é uma doença autoimune caracterizada pela destruição das células ? presentes nas ilhotas pancreáticas por linfócitos T autorreativos, especialmente Th1 e Th17, levando o indivíduo a um estado de hiperglicemia. Embora existam diversos estudos que abordam a resposta imune adaptativa no contexto do DM1, poucos trabalhos tentaram elucidar o papel da resposta imune inata no desenvolvimento da doença. Neste contexto, observamos que camundongos WT pré-diabéticos possuem um aumento significativo na expressão gênica e proteica do receptor AIM2 e de moléculas relacionadas à sua via de ativação e sinalização (Caspase-1, IL-1? e IL-18) nos linfonodos pancreáticos (LNPs) e no íleo. Posteriormente, foi verificado que camundongos deficientes do receptor AIM2 tornaram-se mais suscetíveis ao DM1, comprovado por elevados níveis de glicose sanguínea e menor produção de insulina em relação aos animais selvagens (WT) após a administração com estreptozotocina (STZ). Tal suscetibilidade está relacionada a um processo de disbiose e aumento da translocação de bactérias da microbiota intestinal para os LNPs de camundongos AIM2-/-. De maneira interessante, o inflamassoma AIM2 foi ativado apenas na presença de DNA fecal de animais diabéticos, que possui uma microbiota em disbiose, uma vez que resultou na produção significativa da citocina IL-1?. Também foi constatado que a ativação do receptor AIM2 na mucosa intestinal regulou a expressão gênica e proteica de proteínas de junção celular, peptídeos antimicrobianos e mucinas, como forma de minimizar a translocação de bactérias da microbiota para os LNPs. Adicionalmente, foi visto que a ativação do receptor AIM2 contribui para a indução de células Th17 intestinais, para a migração de neutrófilos no intestino, assim como para a expressão das citocinas IL-23, IL-17 e IL-22 no íleo. Por fim, mostramos que o receptor AIM2 modulou negativamente a ativação de células dendríticas expressando TLR4 e TLR9, que correlacionou com o aumento de células Tc1 patogênicas nos LNPs. De forma geral, nossos resultados demonstram que a ativação do receptor AIM2 na mucosa intestinal desempenha um importante papel em controlar a homeostase da microbiota intestinal, manter a integridade da barreira intestinal, e consequentemente. / Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of ? cells present in the pancreatic islets by autoreactive T lymphocytes, especially Th1 and Th17, leading to a state of hyperglycemia. There are many studies that address the role of adaptive immune response, so only some studies have attempted to elucidate the role of the innate immune response in the context of T1D. In this regard, we observed that pre-diabetic WT mice have a significant increase in the gene and protein expression of the AIM2 receptor and in molecules related to its activation and signaling pathways (Caspase-1, IL- 1? and IL-18) in the pancreatic lymph nodes (PLNs) and in the ileum. Subsequently, it was verified that AIM2 receptor deficient mice became more susceptible to T1D, as proved by blood glucose levels and lower insulin production compared to wild-type mice (WT) after administration of streptozotocin (STZ). This susceptibility was related to a process of dysbiosis and increased translocation of bacteria from gut microbiota to PLNs in AIM2-/- mice. Interestingly, the AIM2 inflammasome was activated in the presence of fecal DNA from diabetic mice, which has a gut microbiota in dysbiosis, since resulted in significant production of IL-1?. It was found that activation of the AIM2 receptor in the intestinal mucosa regulated the gene and protein expression of tightjunction proteins, antimicrobial peptides and mucins in order to minimizing a bacterial translocation of the microbiota to the PLNs. In addition, it was seen that activation of the AIM2 receptor contributes to induction of intestinal Th17 cells, to neutrophil migration in the intestine, as well as for expression of IL-23, IL-17 and IL-22 cytokines in the ileum. Finally, we show that the AIM2 receptor negatively modulated the activation of dendritic cells expressing TLR4 and TLR9, which correlated with the increase of pathogenic Tc1 cells in the PLNs. In general, the results demonstrate that activation of the AIM2 receptor in the intestinal mucosa plays an important role in controlling the composition of gut microbiota homeostasis, maintaining the intestinal barrier function, and consequently reducing the bacterial translocation to the PLNs, conferring a protective effect to the immunopathogeny against to DM1.

O diabetes tipo 1 (DM1) é uma doença autoimune caracterizada pela destruição das células ? presentes nas ilhotas pancreáticas por linfócitos T autorreativos, especialmente Th1 e Th17, levando o indivíduo a um estado de hiperglicemia. Embora existam diversos estudos que abordam a resposta imune adaptativa no contexto do DM1, poucos trabalhos tentaram elucidar o papel da resposta imune inata no desenvolvimento da doença. Neste contexto, observamos que camundongos WT pré-diabéticos possuem um aumento significativo na expressão gênica e proteica do receptor AIM2 e de moléculas relacionadas à sua via de ativação e sinalização (Caspase-1, IL-1? e IL-18) nos linfonodos pancreáticos (LNPs) e no íleo. Posteriormente, foi verificado que camundongos deficientes do receptor AIM2 tornaram-se mais suscetíveis ao DM1, comprovado por elevados níveis de glicose sanguínea e menor produção de insulina em relação aos animais selvagens (WT) após a administração com estreptozotocina (STZ). Tal suscetibilidade está relacionada a um processo de disbiose e aumento da translocação de bactérias da microbiota intestinal para os LNPs de camundongos AIM2-/-. De maneira interessante, o inflamassoma AIM2 foi ativado apenas na presença de DNA fecal de animais diabéticos, que possui uma microbiota em disbiose, uma vez que resultou na produção significativa da citocina IL-1?. Também foi constatado que a ativação do receptor AIM2 na mucosa intestinal regulou a expressão gênica e proteica de proteínas de junção celular, peptídeos antimicrobianos e mucinas, como forma de minimizar a translocação de bactérias da microbiota para os LNPs. Adicionalmente, foi visto que a ativação do receptor AIM2 contribui para a indução de células Th17 intestinais, para a migração de neutrófilos no intestino, assim como para a expressão das citocinas IL-23, IL-17 e IL-22 no íleo. Por fim, mostramos que o receptor AIM2 modulou negativamente a ativação de células dendríticas expressando TLR4 e TLR9, que correlacionou com o aumento de células Tc1 patogênicas nos LNPs. De forma geral, nossos resultados demonstram que a ativação do receptor AIM2 na mucosa intestinal desempenha um importante papel em controlar a homeostase da microbiota intestinal, manter a integridade da barreira intestinal, e consequentemente. / Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of ? cells present in the pancreatic islets by autoreactive T lymphocytes, especially Th1 and Th17, leading to a state of hyperglycemia. There are many studies that address the role of adaptive immune response, so only some studies have attempted to elucidate the role of the innate immune response in the context of T1D. In this regard, we observed that pre-diabetic WT mice have a significant increase in the gene and protein expression of the AIM2 receptor and in molecules related to its activation and signaling pathways (Caspase-1, IL- 1? and IL-18) in the pancreatic lymph nodes (PLNs) and in the ileum. Subsequently, it was verified that AIM2 receptor deficient mice became more susceptible to T1D, as proved by blood glucose levels and lower insulin production compared to wild-type mice (WT) after administration of streptozotocin (STZ). This susceptibility was related to a process of dysbiosis and increased translocation of bacteria from gut microbiota to PLNs in AIM2-/- mice. Interestingly, the AIM2 inflammasome was activated in the presence of fecal DNA from diabetic mice, which has a gut microbiota in dysbiosis, since resulted in significant production of IL-1?. It was found that activation of the AIM2 receptor in the intestinal mucosa regulated the gene and protein expression of tightjunction proteins, antimicrobial peptides and mucins in order to minimizing a bacterial translocation of the microbiota to the PLNs. In addition, it was seen that activation of the AIM2 receptor contributes to induction of intestinal Th17 cells, to neutrophil migration in the intestine, as well as for expression of IL-23, IL-17 and IL-22 cytokines in the ileum. Finally, we show that the AIM2 receptor negatively modulated the activation of dendritic cells expressing TLR4 and TLR9, which correlated with the increase of pathogenic Tc1 cells in the PLNs. In general, the results demonstrate that activation of the AIM2 receptor in the intestinal mucosa plays an important role in controlling the composition of gut microbiota homeostasis, maintaining the intestinal barrier function, and consequently reducing the bacterial translocation to the PLNs, conferring a protective effect to the immunopathogeny against to DM1.

Colorectal cancer (CRC) is the second most common cause of cancer-related death in the UK and Western world. More than 90% of sporadic CRCs harbour mutations in the multi-functional tumour suppressor gene Adenomatous polyposis coli (<i>Apc</i>). The most commonly studied function of APC is its role as a scaffold for the β-catenin destruction complex involved in Wnt signalling. However, APC binds many other proteins. For example, it directly binds to and stabilises microtubules and actin. These non-Wnt related functions of APC are poorly understood. My PhD examines non-Wnt functions of APC. To this end, I created degron-tagged APC in DT40 cells that allowed for the rapid, conditional degradation of endogenous APC. The aim was to identify the immediate effects on cellular processes. Then, to identify the contribution of different APC domains by measuring the ability to rescue any defects when reintroducing fragments of APC. However, creation of these degron-tagged <i>Apc </i>knock-in cell lines resulted in hypomorphic phenotypes and auxin-associated off-target effects. Nonetheless, I compared the response of APC^high, APC^low, and APC^minimal cells to DNA damaging agents and Taxol® but found no significant differences. Subsequently, I focused on the relationship between APC and apoptosis. Previous observations suggested that deficiency in <i>Apc </i>rendered cells less sensitive to low doses of Taxol®. However, <i>Apc </i>deficient cells were more readily killed when Taxol® was combined with the Bcl-2 inhibitor, ABT-737. One possible explanation is the increase in Bcl-2 protein upon <i>Apc </i>depletion. However, I found that ABT-737, Taxol® and <i>Apc </i>depletion each cause activation of the unfolded protein response. This suggests that these treatments elicit a stress response that can stimulate apoptosis. Moreover, the same treatments also cause changes in mitochondria. Importantly, all of these effects do not require an increase in the β-catenin protein. Together, my data reveal novel links between APC and apoptosis that could be exploited clinically.