In a 4-cell C. elegans embryo the ventral blastomere EMS requires polarity signaling from its posterior sister cell, P2. This signaling event enables EMS to orient its division spindle along the anterior-posterior (A/P) axis and to specify the endoderm fate of its posterior daughter cell, E. Wnt pathway components have been implicated in mediating P2/EMS signaling. However, no single mutants or various mutant combinations of the Wnt pathway components disrupt EMS polarity completely. Here we describe the identification of a pathway that is defined by two tyrosine kinase related proteins, SRC-1 and MES-1, which function in parallel with Wnt signaling to specify endoderm and to orient the division axis of EMS. We show that SRC-1, a C. elegans homolog of c-Src, functions downstream of MES-1 to specifically enhance phosphotyrosine accumulation at the P2/EMS junction in order to control cell fate and mitotic spindle orientation in both the P2 and EMS cells. In the canonical Wnt pathway, GSK-3 is conserved across species and acts as a negative regulator. However, in C. elegans we find that GSK-3 functions in a positive manner and in parallel with other components in the Wnt pathway to specify endoderm during embryogenesis. In addition, we also show that GSK-3 regulates C. elegans germline development, a function of GSK-3 that is not associated with Wnt signaling. It is required for the differentiation of somatic gonadal cells as well as the regulation of meiotic cell cycle in germ cells. Our results indicate that GSK-3 modulates multiple signaling pathways to regulate both embryogenesis and germline development in C. elegans.

Protein folding in the endoplasmic reticulum (ER) is essential for proper cellular function. However, the sensitive environment in the ER can be perturbed by both pathological processes as well as by physiological processes such as a large biosynthetic load placed on the ER. ER stress is a specific type of intracellular stress caused by the accumulation of immature or abnormal misfolded or unfolded proteins in the ER. Simply defined, ER stress is a disequilibrium between ER load and folding capacity. Cells have an adaptive response that counteracts ER stress called the "Unfolded Protein Response” (UPR). The ability to adapt to physiological levels of ER stress is especially important for maintaining ER homeostasis in secretory cells. This also holds true for pancreatic β-cells, which must fold and process large amounts of the hormone insulin. Pancreatic β-cells minimize abnormal levels of glycemia through adaptive changes in the production and regulated secretion of insulin. This process is highly sensitive, so that small degrees of hypo- or hyperglycemia result in altered insulin release. The frequent fluctuation of blood glucose levels in humans requires that β-cells control proinsulin folding in the ER with exquisite sensitivity. Any imbalance between the load of insulin translation into the ER and the actual capacity of the ER to properly fold and process the insulin negatively affects the homeostasis of β-cells and causes ER stress. In this dissertation, we show that Inositol Requiring 1 (IRE1), an ER-resident kinase/endoribonuclease and a central regulator of ER stress signaling, is essential for maintaining ER homeostasis in pancreatic β-cells. Importantly, IRE1 has a crucial function in the body’s normal production of insulin in response to high glucose. Phosphorylation and subsequent activation of IRE1 by transient exposure to high glucose is coupled to insulin biosynthesis, while inactivation of IRE1 by siRNA or inhibition of IRE1 phosphorylation abolishes insulin biosynthesis. IRE1 signaling under these physiological ER stress conditions utilizes a unique subset of downstream components of IRE1 and has a beneficial effect on pancreatic β-cell homeostasis. In contrast, we show that chronic exposure of β-cells to high glucose causes pathological levels of ER stress and hyperactivation of IRE1, leading to the degradation of insulin mRNA. The term “glucose toxicity” refers to impaired insulin secretion by β-cells in response to chronic stimulation by glucose and is characterized by a sharp decline in insulin gene expression. However, the molecular mechanisms of glucose toxicity are not well understood. We show that hyperactivation of IRE1 caused by chronic high glucose treatment or IRE1 overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1 signaling using a dominant negative form of the protein prevents insulin mRNA degradation in β-cells. Additionally, islets from mice heterozygous for IRE1 retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates. This work suggests that the rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism may represent an essential element in the adaptation of β-cells to chronic hyperglycemia. This adaptation is crucial for the maintenance of β-cell homeostasis and may explain in part why the β-cells of diabetic patients with chronic hyperglycemia stop producing insulin without simply undergoing apoptosis. This work implies that prolonged activation of IRE1 signaling is involved in the molecular mechanisms underlying glucose toxicity. This work therefore reveals two distinct activities elicited by IRE1 in pancreatic β-cells. IRE1 signaling activated by transient exposure to high glucose enhances proinsulin biosynthesis, while chronic exposure of β-cells to high glucose causes hyperactivation of IRE1, leading to the degradation of insulin mRNA. Physiological IRE1 activation by transient high glucose levels in pancreatic β cells has a beneficial effect on insulin biosynthesis. However, pathological IRE1 activation by chronic high glucose or experimental drugs negatively affects insulin gene expression. In the future, a system to induce a physiological level of IRE1 activation, and/or reduce the pathological level of IRE1 activation could be used to enhance insulin biosynthesis and secretion in people with diabetes, and may lead to the development of new and more effective clinical approaches to the treatment of this disorder.

The primary goal of my thesis research was to characterize the basis for the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in primary culture diabetic cardiomyocytes. Toward this goal, I have investigated several key regulatory sites in this signaling pathway which could promote the hypersensitive activation of phosphorylase. Specifically, I investigated (1) which adrenergic receptors are involved in mediating the hypersensitive response of glycogen phosphorylase to epinephrine stimulation; (2) whether the presence of fatty acid metabolites affects phosphorylase activation; (3) whether the hypersensitive response of phosphorylase results from altered signal transduction through the β-adrenergic receptor system or from a post-receptor defect; and (4) the potential role for phosphorylase kinase in mediating the hypersensitive response of phosphorylase to catecholamine stimulation. The basis for adrenergic receptor mediation of the catecholamine-induced activation of glycogen phosphorylase was investigated in adult rat cardiomyocytes isolated from normal and alloxan-diabetic animals. Cells derived from diabetic animals exhibited a hypersensitive response to epinephrine stimulation which was apparent 3 hours after cell isolation and was further enhanced upon maintenance of the myocytes in culture for 24 hours. Normal cells initially lacked the hypersensitive response to epinephrine stimulation although upon maintenance of these cells in culture for 24 hours, the hypersensitive response was acquired in vitro. To assess alpha- and beta- adrenergic mediation of the response, normal and diabetic cardiomyocytes were incubated with propranolol, a β-receptor antagonist, prior to direct α1receptor stimulation with phenylephrine. Both normal and diabetic myocytes failed to undergo activation of phosphorylase in 3 or 24 hour cell cultures. In addition, the effects of epinephrine on phosphorylase activation were completely inhibited by propranolol whereas prazosin, an α-receptor antagonist, was unsuccessful. This data suggests that the hypersensitive response of glycogen phosphorylase in normal and diabetic cardiomyocytes is solely mediated through β-adrenergic receptor activation. Since the accumulation of various fatty acid metabolites can affect certain enzymes and signal transduction pathways within the cell, the potential effect of various fatty acid metabolites on phosphorylase activation was investigated. To determine the potential effects of fatty acid metabolites on phosphorylase activation in cultured cardiomyocytes, normal and alloxan-diabetic cells were incubated with either carnitine or palmitoylcarnitine prior to stimulation with epinephrine. Pretreatment of cardiomyocytes with or without carnitine or palmitoylcarnitine for 3 or 24 hours before epinephrine stimulation failed to alter phosphorylase activation. The addition of exogenous carnitine in the absence and presence of insulin was also unsuccessful in attenuating the hypersensitive phosphorylase activation response in 3 and 24 hour, normal and alloxan-diabetic derived cardiomyocytes. To determine if carnitine palmitoyltransferase 1 (CPT-1) activity was responsible for the hypersensitive response of phosphorylase in the diabetic myocytes, both normal and diabetic myocytes were maintained for 3 and 24 hours in the absence and presence of etomoxir, a CPT-1 inhibitor. Subsequent activation of phosphorylase by epinephrine in normal and diabetic myocytes was unaltered in the presence of etomoxir. Collectively, these data fail to support a critical role for fatty acid metabolite involvement in the hypersensitive activation of glycogen phosphorylase in acute, alloxan-diabetic cardiomyocytes. To assess potential G-protein involvement in the response, normal and diabetic-derived myocytes were incubated with either cholera or pertussis toxin prior to hormonal stimulation. Pretreatment of cardiomyocytes with cholera toxin resulted in a potentiated response to epinephrine stimulation whereas pertussis toxin did not affect the activation of this signaling pathway. To determine if the enhanced response of phosphorylase activation resulted from an alteration in adenylyl cyclase activation, the cells were challenged with forskolin. After 3 hours in primary culture, diabetic cardiomyocytes exhibited a hypersensitive response to forskolin stimulation relative to normal cells. However, after 24 hours in culture, both normal and diabetic myocytes responded identically to forskolin challenge. The present data suggest that a cholera toxin sensitive G-protein mediates the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in diabetic cardiomyocytes. This response, which is present in alloxan-diabetic cells, and is induced in vitroin normal cardiomyocytes, is primarily due to a defect at a post-receptor site. To assess the role of phosphorylase kinase in the hypersensitive activation of glycogen phosphorylase in the diabetic heart, phosphorylase kinase activity was measured initially in perfused hearts (to optimize the assay parameters) and subsequently in primary culture cardiomyocytes. Results from these experiments demonstrate that the present method for measuring phosphorylase kinase activity is a reliable indicator of the enzyme's activity in the heart, although the assay conditions must be further optimized before this system can be applied to the measurement of phosphorylase kinase activity in primary cultured cardiomyocytes.

Over the last decade, the innate immune system has been the subject of extensive research. Often overlooked by the robustness and specificity of the adaptive immune system, the innate immune system is proving to be just as complex. The identification of several families of pattern recognition receptors (PRRs) has revealed an ancient yet multifaceted system of proteins that are responsible for initiating host defense. A wide array of pathogens, from virus to bacteria, is detected using this assortment of receptors. One such family, the Toll-like receptors (TLRs), has been at the forefront of this research. To date, 10 TLRs have been described in the human genome. Activation of TLRs leads to the induction of immune-related genes that ultimately control the response of the host. However, the signaling pathways emanating from activated TLRs and other PRRs are not fully understood. In particular, the pathway leading to the activation of interferon regulatory factor 3 (IRF3), a transcription factor crucial for the induction of type I interferon, remains undefined. IRF3 activation occurs as the consequence of viral infection and through the activation of TLRs 3 and 4 by dsRNA and lipopolysaccharide (LPS), respectively. The focus of this research is to describe components of the IRF3 activation pathway, partly through the analysis of TLR signal transduction. IRF3 normally resides in the cytoplasm of cells. Upon infection with certain viruses and bacteria, IRF3 is activated though phosphorylation at its C-terminus. Phosphorylated IRF3 homodimerizes and associates with co-activators CBP-p300. After translocating to the nucleus, the activate IRF3 complex induces the activation of type 1 interferon and interferon related genes. Little is known about the pathways that lead to the activation of IRF3, especially the kinases involved. In this study we report that the non-canonical IкB kinase homologues, IкB kinase epsilon (IKKε) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-кB activation, are also essential components of the IRF3 signaling pathway. In particular, mouse embryonic fibroblasts from TBK1 deficient mice fail to activate IRF3 in response to both viral infection and stimulation with LPS or poly (IC), a dsRNA analog. Thus, both IKKε and TBK1 play a critical role in innate immunity and host defense. In addition to viral infection, IRF3 activation also occurs via the activation of TLR3 and 4. TLRs signal through a subfamily of Toll-IL-1-Resistance (TIR) domain containing adapter molecules. One such adapter, MyD88, is crucial for all TLRs, with the exception of TLR3. MyD88 participates in a signal transduction pathway culminating in the activation of the transcription factor NF-кB. Studies from MyD88-deficient mice reveal that both TLR3 and 4 still are capable of activating NF-кB, although with slightly delayed kinetics. Another aspect of the MyD88-independent signal transduction pathway is the activation of IRF3. A second TIR domain containing adapter molecule called Mal/Tirap was discovered and originally thought to mediate the MyD88-independent pathway. However, Mal-deficient mice were found to be defective in both TLR2 and 4 mediated NF-кB activation. We hypothesized that other TIR domain containing adapters could mediate this MyD88-independent pathway of TLR3 and 4 leading to the activation of IRF3. Two additional TIR adapters were discovered, TRIF and TRAM. TRIF was shown to mediate TLR3 signal transduction. In this study, we report that both TRIF and TRAM mediate the activation of the MyD88-independent pathway in response to LPS/TLR4 activation. Unlike any of the other known TIR domain containing adapters, TRAM appears to be restricted to the LPS/TLR4 activation pathway while TRIF plays a role in both TLR3 and TLR4 pathways leading to IRF3 target gene expression. Our studies revealed that TRAM could be acting upstream of TRIF in the LPS/TLR4 pathway. To this end, we sought to determine the localization of TRAM within the cell. We found that TRAM localizes to the plasma membrane. TRAM localization is the result of myristoylation since mutation of the predicted myristoylation site (G2A) resulted in the re-distribution of TRAM from the membrane into the cytoplasm. Reconstitution of TRAM-deficient macrophages with TRAM G2A is unable to rescue LPS/TLR4 signal transduction. Thus, myristoylation and membrane association of TRAM are critical for LPS/TLR4 signal transduction. The data generated in this dissertation extends our understanding of the signaling pathways of the innate immune system. Indeed, the molecules and pathways described herein could prove to be beneficial targets for ameliorating symptoms of disease, both autoimmune and pathogen-associated. Finally, the research described here will spur further insight into the complex signaling pathways of a once ignored arm of the immune system.

Profound lymphopenia has been observed during many acute viral infections, and our laboratory has previously documented a type 1 IFN-dependent loss of most memory (CD44hi) and some naïve (CD44lo) CD8 T cells immediately preceding the development of the antiviral T cell response at days 2-4 following lymphocytic choriomeningitis virus (LCMV) infection. In this thesis, I will examine additional mechanisms involved in the early attrition of CD8 T cells and evaluate whether antigen-specific and non-specific CD8 T cells are equally susceptible. Lastly, I will examine whether the early attrition of CD8 T cells contributes to the generation of an effective immune response. Poly(I:C), a potent inducer of type 1 IFN, was previously shown to cause the attrition and apoptosis of CD8α+CD44hi cells in normal mice, but not in type 1 IFN receptor–deficient mice (IFN1-R KO). I questioned whether additional molecule(s) might contribute to the type 1 IFN-induced apoptosis of CD8α+CD44hi cells. I used a PCR array to determine the expression of 84 apoptosis-related genes at 6 hours post-poly(I:C) treatment, relative to an untreated control. There was an 11-fold increase in CD40 RNA expression in CD8α+CD44hi cells isolated from poly(I:C)-treated mice. CD40 protein expression was also increased on CD8α+CD44hi cells, peaking between 9 and 12 hours following poly(I:C) treatment, before declining thereafter. This increase in CD40 protein expression directly correlated with an increase in Annexin V reactivity, an indicator of early apoptosis. Nevertheless, CD40 was not required for the loss of CD8α+CD44hi cells, as both wildtype and CD40-deficient mice were equally susceptible to the poly(I:C)-induced attrition. Upon further characterization, I found this population of CD40+CD8α+CD44hi cells to be CD11c+B220-Thy1.2- MHCIIhi, which is consistent with a “lymphoid” CD8α+ DC phenotype. Kinetic analysis revealed a type 1 IFN-dependent increase in this CD8α+ DC population at 12 hours post-poly(I:C) treatment. This increase was only observed in the spleen, as no increase in percentage was observed in the peritoneal cavity (PEC), lungs, inguinal lymph nodes (iLN), or peripheral blood. Collectively, these results suggest that the type 1 IFN-dependent increase in splenic CD8α+DCs accounts for the observed increase in Annexin V reactive cells following poly(I:C) treatment. These findings required a re-evaluation of the type 1 IFN-induced attrition of CD8+CD44hi T cells with an anti-CD8β antibody, which is a more exclusive marker for T cells than the anti-CD8α antibody. Kinetic analysis revealed a significant decrease in splenic CD8β+CD44hi T cells at 12 hours post-poly(I:C) treatment. This reduction in splenic CD8β+CD44hi T cells was not due to trafficking to other organs, as the PECs, lungs, iLN, lungs, and peripheral blood all exhibited significant, although varying, decreases in the percentage of CD8β+CD44hi T cells at 12 hour following poly(I:C) treatment. These data support the notion that the type 1 IFN-induced attrition of CD8β+CD44hiT cells was a “global” phenomenon and could not be completely due to migration out of the spleen. The attrition of CD8β+CD44hi T cells was also dependent upon type 1 IFN at 3 days post-LCMV infection, as there was no significant reduction of this population in IFN1-R KO mice. The loss of wildtype CD8β+CD44hi T cells correlated with an increased activation of caspases 3 and 8, which are enzymes that play essential roles in apoptosis and inflammation. A significant loss of CD4+CD44hi T cells, which also correlated with an increased activation of caspases 3 and 8, was observed at 3 days post-LCMV infection. Collectively, these results suggest that attrition of both CD4+CD44hi and CD8β+CD44hiT cell populations is type 1 IFN-dependent and associated with the activation of caspases following LCMV infection. At 3 days post-LCMV infection, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had a higher frequency of cells with fragmented DNA, a hallmark characteristic of the late stages of apoptosis, as revealed by terminal transferase dUTP nick end labeling (TUNEL), relative to uninfected controls. This suggests that the loss of both populations was due to apoptosis. Therefore, I questioned whether the LCMV-induced apoptosis of both CD4+CD44hi and CD8β+CD44hi T cell populations occurred through a mitochondrial-induced pathway involving the pro-apoptotic molecule Bim. The attrition of both CD4+CD44hi and CD8β+CD44hi T cells was significantly higher in wildtype mice compared to Bim KO mice at 3 days post-LCMV infection. Moreover, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had higher frequency of TUNEL+ cells, relative to Bim KO populations. These results suggest that the apoptosis of CD8β+CD44hi and CD4+CD44hiT cells, following LCMV infection, might occur through a mitochondrial-induced pathway involving Bim. Studies have shown “lymphoid” CD8α+ DCs to be involved in the phagocytosis of apoptotic lymphocytes. Therefore, I evaluated whether host CD8α+ DCs are capable of phagocytosing apoptotic lymphocytes by adoptively transferring CFSE-labeled wildtype donor splenocytes (Ly5.1) into congenic wildtype hosts (Ly5.2), followed by inoculation with poly(I:C). There was an increased frequency of donor cells (Ly5.1, CFSE+) within the host CD8α+CD11c+ gate at 9 and 12 hours post-poly(I:C) treatment. The results suggest that type 1 IFN-activated CD8α+DCs might aid in the rapid clearance of apoptotic cells during the type 1 IFN-induced attrition associated with viral infections. I next questioned whether TCR engagement by antigen would render CD8 T cells resistant to attrition. I tested whether a high concentration of antigen (GP33 peptide) would protect LCMV-specific naïve TCR transgenic P14 cells specific for the GP33 epitope of LCMV and GP33-specific LCMV-immune cells from depletion. Both naïve P14 and memory GP33-specific donor CD8 T cells decreased substantially 16 hours after inoculation poly(I:C), regardless of whether a high concentration of GP33 peptide was administered to host mice beforehand. The increased activation status of naïve antigen-specific cells via peptide inoculation did not confer resistance to type 1 IFN-induced depletion. Donor naïve P14 and LCMV-specific memory cells were also depleted from day 2 LCMV-infected (Clone 13) hosts by 16 hours post-transfer. These results indicate that antigen engagement does not protect CD8 T cells from the type 1 IFN-induced attrition associated with viral infections. Computer models indicated that early depletion of memory T cells may allow for the generation for a more diverse T cell response to infection by reducing the immunodomination caused by cross-reactive T cells. To test this in a biological system, I questioned whether the reduced apoptosis of the crossreactive memory CD8 population (NP205), in aged LCMV-immune mice (18-22 months), following heterologous virus challenge (PV), would allow it to dominate the immune response. At day 8 post-PV infection, the cross-reactive memory CD8 T cell response (NP205) was more immunodominating in aged LCMV-immune mice relative to younger LCMV-immune mice. This was indicated by the increased ratio of the cross-reactive NP205 response to the newly arising noncross-reactive, PV-specific NP38 response in older LCMV-mice relative to younger LCMV immune-mice, at day 8 post-PV infection. These data suggest that the early attrition of T cells allows for the generation of a more diverse T cell response to infection by reducing the immunodomination caused by crossreactive T cells. Collectively, these findings offer further insight into the early attrition of T cells associated with viral infections.

No description available.

Submitted by Pedro Silva Filho (pfsilva@ipen.br) on 2017-10-09T19:13:18Z No. of bitstreams: 0 / Made available in DSpace on 2017-10-09T19:13:18Z (GMT). No. of bitstreams: 0 / Estudos têm demonstrado o potencial da terapia fotodinâmica antimicrobiana (aPDT) na inativação de diferentes células microbianas. No geral, são três as fases de crescimento dos microrganismos: fase lag, exponencial e estacionária. Os objetivos deste estudo foram avaliar a susceptibilidade de células de Candida albicans em diferentes fases de crescimento, submetidas à aPDT, associando azul de metileno (50 μM) e luz de emissão vermelha (λ= 660 nm) e investigar alterações morfológicas, mecânicas e bioquímicas, antes e depois da aPDT, por microscopia eletrônica de varredura, de força atômica e por espectroscopia no infravermelho por transformada de Fourier. Os resultados obtidos sugerem que, em parâmetros letais, células em fase estacionária de crescimento (48 h) são menos susceptíveis à aPDT, quando comparadas àquelas em fases lag (6 h) e ex-ponencial (24 h) de crescimento. Entretanto, em parâmetros subletais, células de 6 h e 48 h mostraram a mesma susceptibilidade à aPDT. Em sequência, os experimentos foram realizados em parâmetros considerados subletais para células crescidas por 6 e 48 h. A avaliação morfológica mostrou menor quantidade de matriz extracelular em células de 6 h comparada àquelas de 48 h. A espectroscopia de força atômica mostrou que células em fase lag perderam a rigidez após a aPDT, enquanto que células em fase estacionária mostraram comportamento in-verso. Ainda, células de 48 h diminuíram sua adesividade após a aPDT, enquanto que células de 6 h e 24 h tornaram-se mais adesivas. Os resultados bioquímicos revelaram que as diferenças mais significativas entre as células fúngicas de 6 h e 48 h ocorreram na região de DNA e carboidratos. A aPDT promoveu mais alterações bioquímicas na região de DNA e carboidratos em células de 6 h e em lipídios e ácidos graxos em células de 48 h. Nossos resultados indicam que a fase de crescimento celular desempenha papel importante no sítio de ação da aPDT em células de C. albicans. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-07-29T15:13:45Z (GMT). No. of bitstreams: 1 Tese parte1 Fabricia Rocha Chaves Miotto.pdf: 8322893 bytes, checksum: e402e3e9bcec9054d69df88cb3b48403 (MD5) Previous issue date: 2011-08-18 / This study aimed to evaluate intake, apparent digestibility of nutrients and nitrogen balance (NB) of diets containing babassu mesocarp bran (BMB) in replacement of elephant grass silage (0, 21, 38, 62 and 78%). Twenty sheep were placed in metabolism crates and allotted to a completely randomized design with four replicates. Were collected and recorded food intake and excretion of feces and urine. The intakes of dry matter (DM), organic matter (OM), crude protein (CP), ash, neutral detergent fibrous (NDF), acid detergent fibrous (ADF), hemicellulose and total carbohydrates were not affected by experimental diets. Change was observed in the intakes of non fibrous carbohydrates (NFC) and reduced intake of ether extract (EE) as % of live weight (%LW) and grams per unit of metabolic weight (g/UMW). The total digestible nutrients TDN intake had a quadratic response with maximum points at 37.3 and 37.9% of BMB when expressed in % LW and g/UMW, respectively. The apparent digestibilities of OM, NDF, ADF, CP and NFC reduced to the extent that the BMB has increased in the diets. The digestibility of DM, EE, and NFC had a quadratic response, where the maximum digestibility of DM was estimated at 10% BMB. TDN decreased 0.28% for each percentage point of inclusion in the diets of BMB. The BMB use reduces the fibrous fractions digestibility, but increases the NFC and TDN intake, indicating that this byproduct can be used as an energy source for ruminant diets. Note: Thesis w/seven chapters. Presented summary and abstract of the 2nd chapter / Objetivou-se avaliar consumo, digestibilidade aparente de nutrientes e balanço de nitrogênio (BN) de dietas contendo farelo de mesocarpo de babaçu (FMB) em substituição à silagem de capim elefante (0; 21; 38; 62 e 78%). Utilizaram-se 20 ovinos alojados em gaiolas metabólicas individuais e distribuídos em delineamento inteiramente casualisado com quatro repetições. Foram coletados e registrados o consumo de alimentos e excreções de fezes e urina. Os consumos de matéria seca (MS), matéria orgânica (MO), proteína brutua (PB), cinza, fibra em detergente neutro (FDN), fibra em detergente ácido (FDA), hemicelulose e carboidratos totais não foram influenciados pelas dietas experimentais. Foi observada alteração nos consumos de carboidratos não fibrosos (CNF) e redução nos consumos de extrato etéreo (EE) em % do PV e em gramas por unidade de tamanho metabólico (g/UTM). O consumo de nutrientes digestíveis totais (NDT) teve comportamento quadrático com pontos de máximo em 37,3 e 37,9% de FMB expressos em %PV e g/UTM, respectivamente. As digestibilidades aparentes da MO, FDN, FDA, PB e CNF reduziram na medida em que o FMB aumentou nas dietas. A digestibilidade da MS, EE e CNF tiveram comportamento quadrático, em que a máxima digestibilidade da MS foi estimada com 10% de FMB. O NDT reduziu 0,28% para cada ponto percentual de inclusão de FMB nas dietas. O uso do FMB reduz a digestibilidade de frações fibrosas, contudo aumenta o consumo de CNF e o consumo de NDT, o que indica que esse subproduto pode ser utilizado como fonte energética para dietas de ruminantes. OBS: Tese c/ sete capítulos. Apresentado resumo e abstract do 2° capítulo.

Os frutanos são carboidratos não-disponíveis, classificados como fibra alimentar solúvel e, também, prebióticos. Chegam intactos no intestino grosso (IG) e sofrem fermentação pela microbiota, fornecendo ácidos graxos de cadeia curta (AGCC), gases e biomassa. Os objetivos foram avaliar a fermentação in vivo e in vitro de frutanos da cebola (Allium cepa L.) e seu efeito no intestino grosso de ratos. Durante 38 dias, ratos machos Wistar receberam ração controle (RC) ou suplementada com cebola (10% de frutanos) (RF). Na fermentação in vivo foram avaliados: peso e umidade das fezes, umidade do conteúdo cecal, AGCC, peso total e parede do ceco e pH. Paralelamente, fragmentos do ceco e cólon foram coletados para avaliação morfométrica e proliferação celular. Na fermentação in vitro, as frações indigeríveis das rações (RC e RF) e da cebola foram avaliadas sob diferentes parâmetros, como: pH, AGCC, fermentabilidade e resíduo nãofermentado. A fermentação in vivo causou no grupo frutanos 10%, em relação ao controle, os seguintes efeitos: aumento significante do peso e umidade das fezes, da umidade do conteúdo cecal, do peso e parede do ceco e da concentração de AGCC e diminuição do pH cecal, no grupo frutanos 10% em relação ao controle. Na fermentação in vitro, houve alterações tanto quantitativas como qualitativas de todos os substratos fermentados. De acordo com todas as variáveis analisadas, a RF apresentou maior fermentabilidade quando comparada com a RC. O grupo frutanos 10% apresentou, no ceco, aumento significante no tamanho das criptas, no número de criptas bifurcadas e no índice metafásico em relação ao controle. No cólon não foi evidenciada qualquer mudança microscópica entre os grupos. Os resultados indicam que a ingestão de frutanos causou mudanças significantes nos parâmetros fermentativos, tanto na fermentação in vivo (ratos) quanto na in vitro e na proliferação celular do ceco. A fermentação in vitro mostrou o possível perfil fermentativo dos substratos, a RF teve fermentabilidade maior que a RC. Esse perfil foi confirmado in vivo, onde o grupo frutanos 10% apresentou aumento na produção de AGCC em relação ao controle. O aumento do butirato provavelmente provocou efeito trófico do ceco, evidenciado pelo aumento de peso e de proliferação celular. / Fructans are unavailable carbohydrates, c1assified as soluble dietary fiber and prebiotics. They arrive intact in the large intestine (LI) and are fermented by the microbiota. This fermentation mainly produces short chain fatty acids (SCFA), gases and biomass. The objectives were evaluation of the in vitro and in vivo fermentation of onion fructans (Allium cepa L.) and their effect in the large intestine of rats. Male Wistar rats received, for 38 days, control diet (CD) or onion supplemented diet (10% fructans) (FD). In the in vivo fermentation were evaluated: faeces weight and moisture, moisture of caecal content, SCFA, cecum weight and wall and pH. In parallel, fragments of cecum and colon were collected for morphometric and cellular proliferation evaluation. In the in vitro fermentation, non-digestible fractions of CD and FD were evaluated under different parameters, such as: pH, SCFA, fermentability and non-fermentable residues. In vivo fermentation caused, in the 10% fructans group in relation to the control group, the following effects: a significant increase in faeces weight and moisture, in the moisture of caecal content, in cecum weight and wall, in the concentration of SCFA and a decrease in the caecal pH. The in vitro fermentation showed both quantitative and qualitative changes of all fermented substracts. The FD presented greater fermentability when compared to the CD, according to all variables analyzed. The 10% fructans group presented greater depth and fission of caecal crypts and metaphasic index in relation to the control group. No microscopic changes were noticed in the colon between the groups. The results indicated that the ingestion of fructans caused significant changes on the fermentative parameters, both in vivo (rats) and in vitro fermentation, and on cell proliferation in the cecum. In vitro fermentation indicated a possible fermentative behavior of the substracts and the FD had greater fermentability than the CD. These results were confirmed in vivo, once the 10% fructans group presented an increase in the SCFA production compared to the control group. The increase in butyrate might have caused the trophic effect of the cecum, which was noticed by the increase in weight and cellular proliferation.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-07-20T19:32:21Z No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-22T15:25:58Z (GMT) No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) / Made available in DSpace on 2016-07-22T15:25:58Z (GMT). No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) Previous issue date: 2011-04-20 / Pitcairnia albiflos Herb. (Bromeliaceae) atualmente se encontra na lista de espécies ameaçadas de extinção. Essa espécie é endêmica dos afloramentos rochosos do município do Rio de Janeiro, RJ, e vem sofrendo com o pisoteio de alpinistas, queimadas, invasão de gramíneas exóticas e extrativismo vegetal. A micropropagação pode ser utilizada como alternativa às condições de risco em que as populações dessa espécie se encontram submetidas, visando à recomposição de populações ameaçadas em ambiente natural, assim como o abastecimento do mercado consumidor. A etapa final da micropropagação é a aclimatização, período em que as plantas ficam mais susceptíveis e sofrem com o estresse oxidativo devido às mudanças nas condições ambientais. No presente trabalho foram avaliadas as atividades enzimáticas antioxidativas da CAT, SOD, POD, PPO e o conteúdo de prolina, além dos teores de pigmentos fotossintéticos em plantas de Pitcairnia albiflos cultivados in vitro, em meios de cultura contendo duas concentrações de sacarose (15 ou 30 g L-1), tampas não vedadas que permitiam trocas gasosas e frascos vedados com tampas e filme plástico de PVC, que impediam a ventilação. Sob essas condições, as plantas foram cultivadas em meios contendo GA3 ou ANA. Após o período de crescimento in vitro, as plantas foram transferidas para condições ex vitro em casa de vegetação. As análises supra-citadas e a determinação dos teores de carboidratos solúveis totais, sacarose, amido, açúcares redutores, conteúdo relativo de água e suculência também foram realizadas nas plantas previamente cultivadas in vitro com 15 ou 30 g L-1 de sacarose e GA3 em tubos com tampas vedadas, após submissão das mesmas a estresse hídrico durante 24, 38 ou 52 dias. Após o período de estresse hídrico, as plantas foram reidratadas durante 34 dias sob irrigação periódica em casa de vegetação. Nos tecidos cultivados in vitro percebeu-se o surgimento de características de hiperidricidade nas plantas cultivadas com 15 g L-1 de sacarose, GA3 e tubos com tampas vedadas, o que foi evidenciado pelo menor acúmulo de prolina, aumento das atividades das enzimas antioxidantes e menor acúmulo de pigmentos fotossintetizantes. Na condição ex vitro, as plantas cultivadas anteriormente em meio de cultura contendo 15 g L-1 de sacarose apresentaram maior atividade das enzimas antioxidantes não havendo, em alguns casos, diferenças significativas em comparação com a concentração mais elevada de sacarose. Nessa condição o acúmulo de prolina foi menor, o que é indicativo de maior estresse oxidativo nessas plantas durante a aclimatização. Durante o estresse hídrico houve queda na atividade de todas as enzimas estudadas, embora essa queda tenha sido mais acentuada para as plantas que inicialmente foram cultivadas com 15 g L-1 de vi sacarose. O acúmulo de prolina aumentou com o prolongamento do estresse hídrico, sendo maior nas plantas que foram cultivadas in vitro com 30 g L-1 de sacarose. Não houve diferenças significativas no conteúdo de pigmentos fotossintetizantes e nas suas relações para nenhuma das concentrações de sacarose, exceto para os carotenóides totais, que apresentaram aumento significativo ao longo do período de estresse hídrico para as plantas previamente cultivadas com a menor concentração de sacarose. Os conteúdos de carboidratos solúveis totais e sacarose aumentaram com o prolongamento do estresse, sendo mais acentuados na concentração de 30 g L-1 de sacarose. Os conteúdos de amido, o conteúdo relativo de água e a suculência apresentaram redução com o aumento do estresse hídrico. Após a reidratação, todas as plantas mostraram capacidade de recuperação, apresentando valores próximos aos dos controles para todas as variáveis analisadas. Ressalta-se, todavia, que as plantas tratadas com 30 g L-1 de sacarose tiveram melhor recuperação quando comparadas com aquelas que foram tratadas com 15 g L-1 de sacarose. Em função dos resultados obtidos, é possível concluir que a concentração de sacarose utilizada in vitro apresenta influência no processo de aclimatização ex vitro e também, posteriormente, no campo, na capacidade de recuperação das plantas à seca quando elas são submetidas a estresse hídrico. As plantas cultivadas in vitro com 15 g L-1 de sacarose se mostraram mais sensíveis à seca e, possivelmente, não sobreviveriam caso fossem transferidas dos tubos de ensaio diretamente para o campo. As plantas cultivadas in vitro com 30 g L-1 de sacarose aparentemente eram mais resistentes ao processo de aclimatização ex vitro, apresentando maiores chances de sobrevivência em campo, maior tolerância à seca e maior capacidade de recuperação após períodos prolongados de estresse hídrico. / Pitcairnia albiflos Herb. (Bromeliaceae) is currently in the list of endangered species. This species is endemic of the inselbergs of the city of Rio de Janeiro, RJ, and has been suffering with the mountaineer’s trampling, wildfires, invasion of exotic grasses and plant extraction. The micropropagation can be used as an alternative to the risk conditions under which populations of this species are submitted, aiming at the recomposition of endangered populations in the natural environment, as well as the supply of the consumer market. The final stage of the micropropagation is the acclimatization, period in which the plants become more susceptible and suffer from oxidative stress due to the changes in the environmental conditions. In the present study it were evaluated the antioxidative enzymatic activities of CAT, SOD, POD, PPO and proline content, besides the levels of photosynthetic pigments in plants of Pitcairnia albiflos grown in vitro in culture mediums containing two sucrose concentrations (15 or 30 g L-1). Part was covered with unsealed lids that allowed gas exchanges and part was kept in sealed flasks with lids and PVC plastic film, that didn’t allow the ventilation. Under these conditions, the plants were cultivated in culture mediums containing GA3 or NAA. After the in vitro growth period, the plants were transferred to ex vitro conditions at a greenhouse. The above mentioned analyses and the determination of total soluble carbohydrate levels, sucrose, starch, reducer sugars, relative water content and succulence were also performed on the plants previously grown in vitro with 15 or 30 g L-1 of sucrose and GA3 in tubes with sealed lids, after the submission of these to water stress during 24, 38 or 52 days. After the water stress period, the plants were rehydrated for 34 days under regular irrigation at the greenhouse. In the in vitro cultivated tissues it was noted the emergence of hyperhydricity characteristics in the plants grown with 15 g L-1 of sucrose, GA3 and tubes with sealed lids, which was evidenced by the lowest proline accumulation, the increased in the antioxidative enzymatic activities and the lowest accumulation of photosynthetic pigments. In the ex vitro condition, the plants previously grown in culture mediums containing 15 g L-1 of sucrose presented larger antioxidative enzymatic activities, which did not show, in some cases, significant differences compared with the largest concentration of sucrose. In this condition, the proline accumulation was lower, which is an indicative of larger oxidative stress in these plants during acclimatization. During the water stress, there was a fall in the activity of all studied enzymes, although that fall had been more evident in the plants that were initially cultivated with 15 g L-1 of sucrose. The proline accumulation increased with the extension of the water stress, being larger in the plants grown in vitro with 30 g L-1 of sucrose. There were no viii significant differences in the content of photosynthetic pigments and in their relation with any sucrose concentrations, except for total carotenoids, which significantly increased over the period of water stress for the plants previously grown with the lowest concentration of sucrose. The contents of total soluble carbohydrates and sucrose increased with the extension of the stress, being more accentuated in the 30 g L-1 of sucrose concentration. The contents of starch, the relative content of water and succulence presented a reduction with the increase of water stress. After the rehydration, all plants showed recovery capacity, presenting values close to those from the control groups for all the analyzed variables. It should be noted, however, that the plants treated with 30 g L-1 of sucrose had better recovery compared with those that were treated with 15 g L-1 of sucrose. According to the obtained results, it is possible to conclude that the concentration of sucrose used in vitro presents influence on the process of ex vitro acclimatization and also, later in the field, in the recovery capacity of the plants to drought when they are submitted to water stress. The plants grown in vitro with 15 g L-1 of sucrose were more sensitive to drought and, possibly, would not survive if they were transferred from the test tubes directly to the field. The plants grown in vitro with 30 g L-1 of sucrose were apparently more resistant to the ex vitro acclimatization process, presenting greater survival chances in the field, larger drought tolerance and larger recovery capacity after extended periods of water stress.