A desnutrição é um dos principais problemas de saúde pública do mundo, que contribui significativamente para o aumento da morbidade e mortalidade. Estima-se um total de 815 milhões de pessoas subnutridas no mundo, e apesar da melhoria dos recursos alimentares o número de pessoas desnutridas ainda é alarmante. Estudos de nosso laboratório tem demonstrado, em modelo murino de desnutrição proteica, hipoplasia medular com evidências histológicas de alterações na matriz extracelular (MEC) e permanência da célula-tronco hemopoética (CTH) na fase G0/G1 do ciclo celular em camundongos desnutridos. Dados deste trabalho evidenciaram alterações nas proteínas Akt /mTOR, que podem contribuir para o aumento da expressão autofágica nas CTHs e CTPHs (célula-tronco progenitora). A literatura demonstra que desequilíbrios nutricionais e metabólicos podem induzir ativação autofágica. Autofagia é um processo catabólico que participa da manutenção da homeostase celular, da MEC e na regulação das CTHs, dados deste trabalho demonstram diminuição da quantidade de CTH e CTPH em camundongos desnutridos sem a presença do gene Atg7, proteína participativa no processo autofágico. Já camundongos com deleção da transglutaminase 2 (TG2) e submetidos a privação de nutrientes por 24 horas , apresentou diminuição da quantidade de CTH e aumento da diferenciação da CTPH. A TG2 tem participação na impulsão e formação do fagóforo (processo inicial autofágico). Considerando que a desnutrição proteica leva a comprometimento da hemopoese, alterações no ciclo celular das CTHs e hipoplasia medular com pancitopenia periférica e que privação e ou jejum prolongado de nutrientes pode aumentar a atividade autofágica, concluímos nesse projeto que autofagia é importante para regulação da CTH e diferenciação da CTPH, entretanto a desnutrição proteica e privação de nutrientes estimula de maneira diversa o mecanismo de diferenciação da CTH. / Malnutrition is one of the world\'s major public health problems, which contributes significantly to increased morbidity and mortality. An estimated 815 million people are undernourished in the world, and despite the improvement in food resources the number of undernourished people is still alarming. Studies of our laboratory have demonstrated in murine model of protein malnutrition, medullary hypoplasia with histological evidence of extracellular matrix (ECM) changes and hemopoietic stem cell (HSC) stay in the G0/ G1 phase of the cell cycle in malnourished mice. Data from this work showed alterations in Akt / mTOR proteins, which may contribute to the increase of autophagic expression in HSC and HPC (progenitor stem cell). The literature demonstrates that nutritional and metabolic imbalances can induce autophagic activation. Autophagy is a catabolic process that participates in the maintenance of cellular homeostasis, ECM and in the regulation of HSC, data from this work demonstrate a decrease in the amount of HSC and HPC in malnourished mice without the presence of the Atg7 gene, a participatory protein in the autophagic process. Mice with transglutaminase 2 deletion (TG2) and submitted to nutrient deprivation for 24 hours showed a decrease in the amount of HSC and an increase in the differentiation of HPC. TG2 plays a role in the uptake and formation of phagophore (autophagic initial process). Considering that protein malnutrition leads to hemopoiesis, alterations in the cell cycle of HSC and spinal cord hypoplasia with peripheral pancytopenia, and that prolonged nutrient starvation or fasting may increase the autophagic activity, we conclude in this project that autophagy is important for regulation of HSC and differentiation of HPC, however, protein malnutrition and nutrient deprivation stimulate in a different way the mechanism of differentiation of HSC.

A desnutrição é um dos principais problemas de saúde pública do mundo, que contribui significativamente para o aumento da morbidade e mortalidade. Estima-se um total de 815 milhões de pessoas subnutridas no mundo, e apesar da melhoria dos recursos alimentares o número de pessoas desnutridas ainda é alarmante. Estudos de nosso laboratório tem demonstrado, em modelo murino de desnutrição proteica, hipoplasia medular com evidências histológicas de alterações na matriz extracelular (MEC) e permanência da célula-tronco hemopoética (CTH) na fase G0/G1 do ciclo celular em camundongos desnutridos. Dados deste trabalho evidenciaram alterações nas proteínas Akt /mTOR, que podem contribuir para o aumento da expressão autofágica nas CTHs e CTPHs (célula-tronco progenitora). A literatura demonstra que desequilíbrios nutricionais e metabólicos podem induzir ativação autofágica. Autofagia é um processo catabólico que participa da manutenção da homeostase celular, da MEC e na regulação das CTHs, dados deste trabalho demonstram diminuição da quantidade de CTH e CTPH em camundongos desnutridos sem a presença do gene Atg7, proteína participativa no processo autofágico. Já camundongos com deleção da transglutaminase 2 (TG2) e submetidos a privação de nutrientes por 24 horas , apresentou diminuição da quantidade de CTH e aumento da diferenciação da CTPH. A TG2 tem participação na impulsão e formação do fagóforo (processo inicial autofágico). Considerando que a desnutrição proteica leva a comprometimento da hemopoese, alterações no ciclo celular das CTHs e hipoplasia medular com pancitopenia periférica e que privação e ou jejum prolongado de nutrientes pode aumentar a atividade autofágica, concluímos nesse projeto que autofagia é importante para regulação da CTH e diferenciação da CTPH, entretanto a desnutrição proteica e privação de nutrientes estimula de maneira diversa o mecanismo de diferenciação da CTH. / Malnutrition is one of the world\'s major public health problems, which contributes significantly to increased morbidity and mortality. An estimated 815 million people are undernourished in the world, and despite the improvement in food resources the number of undernourished people is still alarming. Studies of our laboratory have demonstrated in murine model of protein malnutrition, medullary hypoplasia with histological evidence of extracellular matrix (ECM) changes and hemopoietic stem cell (HSC) stay in the G0/ G1 phase of the cell cycle in malnourished mice. Data from this work showed alterations in Akt / mTOR proteins, which may contribute to the increase of autophagic expression in HSC and HPC (progenitor stem cell). The literature demonstrates that nutritional and metabolic imbalances can induce autophagic activation. Autophagy is a catabolic process that participates in the maintenance of cellular homeostasis, ECM and in the regulation of HSC, data from this work demonstrate a decrease in the amount of HSC and HPC in malnourished mice without the presence of the Atg7 gene, a participatory protein in the autophagic process. Mice with transglutaminase 2 deletion (TG2) and submitted to nutrient deprivation for 24 hours showed a decrease in the amount of HSC and an increase in the differentiation of HPC. TG2 plays a role in the uptake and formation of phagophore (autophagic initial process). Considering that protein malnutrition leads to hemopoiesis, alterations in the cell cycle of HSC and spinal cord hypoplasia with peripheral pancytopenia, and that prolonged nutrient starvation or fasting may increase the autophagic activity, we conclude in this project that autophagy is important for regulation of HSC and differentiation of HPC, however, protein malnutrition and nutrient deprivation stimulate in a different way the mechanism of differentiation of HSC.

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The esophageal epithelium is a stratified squamous tissue. Maintenance of the esophageal epithelial proliferation-differentiation gradient is critical as esophageal epithelium is the first line barrier to prevent penetration of digestive contents, while abnormal epithelial repair contributes to remodeling and disease development. Autophagy has been demonstrated to play roles in esophageal pathologies both benign and malignant, however, the role of autophagy in normal esophageal biology remains elusive. We hypothesize that autophagy may contribute to the maintenance of the proliferation/differentiation gradient under homeostasis in the esophageal epithelium. To investigate the role of autophagy in esophageal epithelium under homeostatic conditions and in response to the carcinogen 4-nitroquinoline 1-oxide (4NQO), we utilize a novel mouse model with tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy-related 7) conditional knockout. We report that genetic autophagy inhibition in squamous epithelium under homeostatic conditions resulted in enhanced proliferation of esophageal basal cells and increased thickness of epithelium, whether challenging these mice with 4NQO-induced dramatic weight loss that further displayed perturbed epithelial tissue architecture evaluated by histological and biochemical analyses. To characterize cells with high and low levels of autophagic vesicle (AV) content functionally and molecularly, we sorted esophageal basal cells based upon fluorescence of the AV-identifying dye Cyto-ID. We then used transmission electron microscopy validate increased AVs in esophageal basal cells with high AV level (Cyto-IDHigh) as compared to their counterparts with low AV level (Cyto-IDLow). Cyto-IDHigh esophageal basal cells displayed limited organoid formation capability upon initial plating but passaged more efficiently as compared to Cyto-IDLow esophageal basal cells. By RNA-Seq we identified increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations, enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in cells located beyond the outermost basal cell layer. Taken together, these studies provide evidence that ATG7 contributes to homeostasis of esophageal epithelium, in which esophageal basal cells with high level of AVs exhibit limited proliferation. When esophageal basal cells with high AV level are cultured in 3D organoid assays, they exhibit increased self-renewal and enhanced proliferative capacity extending beyond the outermost basal cell layer.Maintenance of the esophageal proliferation-differentiation gradient is a key to support proper functioning of the esophagus and its dysregulation can lead to the development of esophageal pathologies. Published studies provide evidence of epithelial-fibroblast crosstalk in the development of subepithelial fibrosis, a typical type of tissue remodeling found in patients with eosinophilic esophagitis (EoE). The current paradigm presents EoE as a progressive fibrostenotic disease of the esophagus in which aged patients develop fibrosis as a function of disease chronicity. We hypothesize that age of esophageal epithelium may affect EoE presentation. To directly test the impact of age upon EoE disease presentation, we treated young and aged mice with MC903/Ovalbumin to induce EoE inflammation for the same time period. We found increased thickness of lamina propria in aged mice with EoE as compared to their young counterparts, suggesting that age-associated alterations in esophageal biology contribute to EoE-associated fibrosis. To evaluate the impact of esophageal epithelial cell age on EoE-associated fibrosis, we generated primary esophageal epithelial cell lines from young and aged mice and determined the effects of these cells on fibroblast contractility in collagen plug contraction assays in vitro. These studies revealed that esophageal epithelial cells from aged mice limited fibroblast contractility less efficiently than those from their young counterparts. To identify potential signaling pathways through which aged esophageal epithelial cells may stimulate fibrotic remodeling, we conducted cytokine array analysis. We found 6 cytokines/soluble factors that have not previously been linked to EoE but may contribute to fibrotic remodeling. Taken together, this dissertation provides (1) foundation for further studies evaluating the role of autophagy and mechanisms of its regulation in the context of normal homeostasis and carcinogen-induced stress as well as (2) identification of age-associated factors that may contribute to fibrotic remodeling that may aid in the design of strategies toward early detection, prevention, and therapy of fibrostenotic EoE. / Biomedical Sciences