Proefschrift Rijksuniversiteit Limburg, Maastricht. / Met lit. opg. - Met een samenvatting in het Nederlands.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Death from acute diarrhea has been reduced since 80Âs. However diarrhoeal diseases account for nearly 1.34 million deaths a year among children under-five years of age, making them the second most common cause of child deaths worldwide. As mortality acute diarrhea was reduced, persistent diarrhea (PD) has become a major enteric diseases infant collaborating to morbidity of affected populations. Small intestinal mucosa injury that becomes prolonged has been named as a central mechanism in the pathophysiology of PD. The protein malnutrition and infection by Escherichia coli enteroaggregative (EAEC) are strongly associated with development PD. The present study investigated the effect of supplementation of Alanyl-Glutamine on monolayer of intestinal cells in the absence of infection, evaluated some parameters of the injury caused by EAEC (strain 239-1) on the intestinal mucosa in vitro and modulation of such injury by Alanyl-Glutamine 10mM. The result show that effects of supplementation of Alanyl-Glutamine 10mM is no different from effects free glutamine 4mM. Compared to E. coli HS, EAEC infection reduced the number of migrating cells, increased the percentage of necrosis, decreased the proliferation and transcription-reverse from small GTPases (RhoA, Rac1 and Cdc42), within 6 hours of contact. The contact EAEC reduced transcription of IL-8 (6h and 0h), NF-κB (6h) and TLR5 (6h and 12h) and increased TNF-α expression. The treatment of infection with Ala-Gln 10mM altered the following parameters: reduced the percentage of necrosis, increase of cell migration, although it has not caused changes in the transcription of Rho GTPases genes, increased transcription IL-8, NF-κB (6h) e TLR5 (6h and 12h). Treatment with dipeptidio significantly reduced the secretion of TNF-α (12h) while increased protein expression of IL-6 (6 e 12h). In conclusion, found that glutamine deprivation decreases cellular response against the infectious stimulus. The EAEC appears to minimize the innate host immune defenses by reducing the transcription of NF-kB and TLR5 and limit the increased transcription of IL-8. The Ala-Gln seems to make the cell more reactive against injurious stimuli, increasing their responsiveness through increased transcriptional IL-8, NF-kB and TLR5. Moreover increased protein expression of IL-6 appears to be one of the mechanisms by which promotes Ala-Gln protective effect on the intestinal epithelial barrier. / A morte por diarreia aguda tem apresentado grande declÃnio desde a dÃcada de 80 em todas as regiÃes do mundo. Entretanto as doenÃas diarreicas ainda sÃo responsÃveis por 1,34 milhÃes de mortes infantis a cada ano e representam a segunda maior causa de mortalidade no grupo etÃrio com idade inferior a 5 anos. Com o declÃnio de mortalidade por casos de diarreia aguda a diarreia persistente (DP) se tornou uma das principais doenÃas entÃricas infantis contribuindo para morbidade das populaÃÃes afetadas. Uma lesÃo no intestino delgado que se torna prolongada tem sido colocada como elemento central da patofisiologia da DP. A desnutriÃÃo proteica e a infecÃÃo por Escherichia coli enteroagregativa (EAEC) estÃo fortemente associados ao desenvolvimento da DP. O presente estudo investigou o efeito da suplementaÃÃo de Alanil-Glutamina sobre monocamada de cÃlulas da mucosa intestinal na ausÃncia de infecÃÃes. Avaliou alguns parÃmetros da lesÃo promovida por EAEC (cepa 239-1) sobre a mucosa intestinal in vitro e a modulaÃÃo de tal dano pela Alanil-Glutamina 10Mm. Os resultados obtidos mostram que os efeitos da suplementaÃÃo de Ala-Gln 10mM nÃo diferem dos efeitos exibidos pela glutamina livre 4mM. Em relaÃÃo a cepa comensal E. coli HS, a infecÃÃo com EAEC reduziu de forma significativa a quantidade de cÃlulas em migraÃÃo, aumentou o percentil de necrose, diminuiu a proliferaÃÃo celular e reduziu significativamente a transcriÃÃo dos genes das pequenas GTPases (RhoA, Cdc42 e Rac1) apÃs 6h de contato com enterÃcitos. O contato com EAEC reduziu a transcriÃÃo de IL-8 (0h e 6h), NF-κB (6h) e TLR5 (6h e 12h) e aumentou a expressÃo proteica de TNF- α (12h). O tratamento da infecÃÃo com Ala-Gln 10mM alterou significativamente os seguintes parÃmetros: aumento da quantidade de cÃlulas em migraÃÃo embora nÃo tenha causado alteraÃÃo na transcriÃÃo dos genes da pequenas GTPases, reduÃÃo do percentual de necrose, aumento da proliferaÃÃo celular, aumento da transcriÃÃo IL-8, NF-κB (6h) e TLR5 (6h e 12h). O tratamento com o dipeptidio reduziu significativamente a secreÃÃo de TNF-α (12h) enquanto aumentou a expressÃo proteica de IL-6 (6 e 12h). Em conclusÃo, verificamos que a privaÃÃo de glutamina diminui a resposta celular frente ao estÃmulo infeccioso. A EAEC, por sua vez, parece minimizar as defesas imunes inatas hospedeiro ao reduzir a transcriÃÃo de NF-kB e TLR5 e limitar o aumento da transcriÃÃo de IL-8. A Ala-Gln parece tornar a cÃlula mais reativa frente aos estÃmulos lesivos, aumentando sua capacidade de resposta atravÃs do aumento transcricional de IL-8, NF-kB e TLR5. Por outro lado o aumento da expressÃo proteica de IL-6 parece ser um dos mecanismos pelas quais Ala-Gln promove efeito protetor sobre a barreira epitelial intestinal.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Thesis advisor: Thomas N. Seyfried / It has long been posited that all cancer cells are dependent on glucose for energy, termed the "Warburg Effect". As a result of an irreversible injury to the mitochondria, cancer cells are less efficient in aerobic respiration. Therefore, calorie restriction was thought to be a natural way to attenuate tumor growth. Calorie restriction lowers blood glucose, while increasing the circulation of ketone bodies. Ketone bodies are metabolized via oxidative phosphorylation in the mitochondria. Only cells that are metabolically capable of aerobic respiration will thus be able to acquire energy from ketone bodies. To date, calorie restriction has been shown to greatly reduce tumor growth and angiogenesis in the murine CT2A, EPEN, and human U87 brain tumor models. Using the novel VM-M3 model for invasive brain cancer and systemic metastatic cancer, I found that though calorie restriction had some efficacy in reducing brain tumor invasion and primary tumor size, metastatic spread was unaffected. Using a bioluminescent-based ATP assay, I determined the viability of metastatic mouse VM-M3 tumor cells grown in vitro in serum free medium in the presence of glucose alone (25 mM), glutamine alone (4 mM), or in glucose + glutamine. The VM-M3 cells could not survive on glucose alone, but could survive in glutamine alone indicating an absolute requirement for glutamine in these metastatic tumor cells. Glutamine could also maintain viability in the absence of glucose and in the presence of the F1 ATPase inhibitor oligomycin. Glutamine could not maintain viability in the presence of the Krebs (TCA) cycle enzyme inhibitor, 3-nitropropionic acid. The data indicate that glutamine can provide ATP for viability in the metastatic VM-M3 cells through Krebs cycle substrate level phosphorylation in the absence of energy from either glycolysis or oxidative phosphorylation. I therefore developed a metabolic therapy that targeted both glucose and glutamine metabolism using calorie restriction and 6-diazo-5-oxo-L-norleucine (DON), a glutamine analog. Primary tumor growth was about 20-fold less in DON treated mice than in untreated control mice. I also found that DON treatment administered alone or in combination with CR inhibited metastasis to liver, lung, and kidney as detected by bioluminescence imaging and histology. Although DON treatment alone did not reduce the incidence of tumor metastasis to spleen compared to the controls, DON administered together with CR significantly reduced the incidence of metastasis to the spleen, indicating a diet/drug synergy. In addition, the phagocytic capabilities of the VM-M3 tumor cells were enhanced during times of energy stress. This allowed for the digestion of engulfed material to be used in energy production. My data provide proof of concept that metabolic therapies targeting both glucose and glutamine metabolism can manage systemic metastatic cancer. Additionally, due to the phagocytic properties of the VM-M3 cell line also seen in a number of human metastatic cancers, I suggest that a unique therapy targeting metabolism and phagocytosis will be required for effective management of metastatic cancer. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Welkin Johnson / Thesis advisor: Thomas Chiles / B cell activation is an energetically demanding process during which B lymphocytes undergo reprogramming and shift from a resting state to a highly proliferative, metabolically active state. Little is known about the metabolic reprogramming process or the role extracellular nutrients play in the activation response. Here we demonstrate that there are distinct requirements for the nutrients L-glutamine and glucose during activation. We show that cells activated in glucose-depleted conditions are still able to undergo growth and signaling events. In contrast, we show that extracellular L-glutamine is essential for all but the earliest activation events, and cells cultured in L-glutamine-deprived conditions are unable to enter the cell cycle. Consistently, we show that extracellular supplementation of the cell-permeable derivative of α-ketoglutarate (α-KG), a glutaminolytic product, is able to rescue cell activation in the absence of glutamine. We also show the induction of the high affinity amino acid transporter ASCT2 is required for glutamine uptake following B cell receptor (BCR) crosslinking. Specifically, we found that halting glutamine uptake or processing by inhibiting ASCT2 or the glutaminolytic enzyme glutaminase causes activation defects that parallel those observed in glutamine deprived conditions, indicating a requirement for glutaminolysis during the very early stages of activation. We found that -KG does not contribute to epigenetic remodeling, but is necessary for mammalian target of rapamycin complex 1 (mTORC1) activation. In turn, mTORC1 activity is required for upregulation of the glucose transporter Glut1 during the initial 24 hours of activation, as well as increased glucose uptake. These findings indicate a distinct metabolic profile that begins with glutamine uptake, and acts through mTORC1 signaling to later promote glucose uptake. Finally, we show that nutrients contribute to functional differentiation events during B cell activation. Glucose is required to support biogenesis of the endoplasmic reticulum as well as differentiation into plasma-like cells, while glutamine is required to support differentiation into IL-10 secreting regulatory B cell subsets. The requirement for glutamine for in vitro B10 cell differentiation is the first reported link between nutrient signaling and regulatory B cell development, and is a novel finding in the field. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Orientador: Caio Antonia Carbonari / Coorientador: Te-ming Paul Tseng / Banca: Edivaldo Domingues Velini / Banca: Leandro Tropaldi / Banca: Marcelo Rocha Corrêa / Banca: Ivana Paula Ferraz Santos de Brito / Resumo: As plantas daninhas causam grandes perdas de produtividade nas culturas devido à competição imposta por estas espécies. Para controlar tais espécies, é necessário adotar práticas efetivas de manejo. O glufosinate, é um herbicida aplicado em pós emergência, com amplo espectro de controle em planta daninhas, podendo ser utilizado como uma alternativa viável por apresentar diferente mecanismo de ação. Estudos de efeito de glufosinate em aplicação diurna e noturna são importantes, para verificar se existe diferença em seu comportamento em função do horário de aplicação. Neste trabalho, as plantas daninhas Digitaria insularis, Lolium multiflorum e Eleusine indica, Ipomoea grandifolia e Euphorbia heterophylla foram submetidas a aplicação diurna e noturna de glufosinate para verificar os seus efeitos no acúmulo de amônia, taxa de transporte de elétrons (ETR) e controle desta espécies. Para atingir estes objetivos a tese foi dividida em 3 capítulos: (1) Efeitos da aplicação diurna e noturna e glufosinate no acúmulo de amônia, taxa de transporte de elétrons e controle das plantas daninhas Digitaria insularis, Lolium multiflorum e Eleusine indica; (2) Efeitos da aplicação diurna e noturna de glufosinate em no acúmulo de amônia, taxa de transporte de elétrons e controle de Euphorbia heterophylla e Ipomoea grandifolia; (3) Efeito de glufosinate no acúmulo de amônia em plantas daninhas aplicadas em diferentes condições de luz e formulações do herbicida, e a velocidade de resposta à ETR. A... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The weeds cause great losses of productivity in the crops due to the competition imposed imposed by these species. To control such species, it is necessary to adopt effective management practices. Glufosinate is a non-seletive herbicide applied post-emergence, with a broad spectrum of weed control, and can be used as a viable alternative for presenting different mode of action. Glufosinate effects studies in day and night application are important, to verify if there is a difference in their behavior depending on the application time. In this study, Digitaria insularis, Lolium multiflorum, Eleusine indica, Ipomoea grandifolia and Euphorbia heterophylla were submitted to day and night application of glufosinate to verify their effects on the ammonia accumulation, electron transport rate (ETR) and weed control. To achieve these goals the thesis was divided into 3 chapters: (1) Effects of day and night glufosiante application on ammonia accumulation, electron transport rates and weed control of Digitaria insularis, Lolium multiflorum and Eleusine indica; (2) Effects of day and night glufosiante application on ammonia accumulation, electron transport rates and weed control of Euphorbia heterophylla and Ipomoea grandifolia; (3) Effect of glufosinate on ammonia accumulation in applied weeds in diferente conditions of light and herbicide formulations, and the reponse of ETR. The day and night application of glufosinate generated ammonia accumulation directly porportional to the dose used. The night application of glufosinate on weeds kept in the dark not increase the ammonia content. The ETR evaluated 12 hours after The ETR evaluated 12 hours after application (HAA) of glufosiante performed at 7:30 a.m, was reduced; while, for the same period of time, after application at 7:30 p.m., there was not reduction. However, 24 HAA the results were similar, regardless of the treatment time. The ETR evaluations performed... / Doutor

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Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 1997. / Includes bibliographical references. Also available on the Internet.

Nitrile hydratase activity in Rhodococcus rhodochrous DAP 96253 can be induced with multiple inducers that include urea, cobalt (Co), iron (Fe) and nickel (Ni). When induced with Co/urea, cells of R. rhodochrous DAP 96253 expressed the highest level of nitrile hydratase activity (~200 units/min·mg-cdw) when compared with the other inducers tested. Cells induced with Co had the second highest nitrile hydratase activity (~7 units/min·mg-cdw), whereas in the uninduced cells, nitrile hydratase activity was lower than 1 unit/min·mg-cdw. Similarly in R. rhodochrous DAP 96622, when induced with Co/urea, the nitrile hydratase activity of R. rhodochrous DAP 96622 cells was around 50 units/min·mg-cdw which was the highest of all inducers tested. When induced with Co only, the nitrile hydratase activity of R. rhodochrous DAP 96622 was around 20 units/min·mg-cdw, and the nitrile hydratase activity of R. rhodochrous DAP 96622 uninduced was the same as the nitrile hydratase activity of uninduced R. rhodochrous DAP 96253. When Co/urea induced R. rhodochrous DAP 96253 cell lysate was examined on gradient SDS-PAGE and analyzed by Image Quant TL, the nitrile hydratase bands (both α and β subunits) accounted for more than 55% of the total cytosolic proteins. Whereas in Co/urea induced R. rhodochrous DAP 96622, the nitrile hydratase bands accounted for around 25% of the total cytosolic proteins. According to matrix-assisted laser desorption ionization time-of-flight mass spectrometry results, amidase in R. rhodochrous DAP 96253 was approximately 38 kDa from the nitrilase/cyanide hydratase family and amidase in R. rhodochrous DAP 96622 was 55 kDa from the amidase signature family. In addition, the nitrile hydratase regulation system in both R. rhodochrous DAP 96253 and DAP 96622 strains are different. Moreover, the nitrile hydratase regulation system in R. rhodochrous DAP 96253 is different from R. rhodochrous J1. Purified nitrile hydratase from R. rhodochrous DAP 96253 may form a protein complex with glutamine synthetase, resulting in a nitrile hydratase activity of approximately 1500 units/mg-proteins, and nitrile hydratase from R. rhodochrous DAP 96622 is not a protein complex and results in a nitrile hydratase activity of 950 units/mg-proteins.

Primary spinal cord injury (SCI) results from direct mechanical damage to the spinal cord. The resulting pathochemical and pathophysiological events, including oxidative stress and inflammation, lead to secondary injury. The ability to decrease secondary injury may lead to improved recovery. Increasing glutathione production after SCI leads to decreased secondary injury. Glutamine is an important precursor to glutathione following trauma. Skeletal muscle phenotype is strongly influenced by neuromuscular activity. SCI causes myosin heavy chain (MyHC) profiles to shift towards faster isoforms in slow muscles and slower isoforms in fast muscles. The hypothesis was that glutamine, as a precursor of glutathione, administration to SCI rats would lead to better functional recovery and a more preserved MyHC phenotype in locomotory muscles. <p> Rats were assigned to one of four groups; healthy, laminectomy only, untreated SCI, and SCI treated with an intraperitoneal injection of 1mmol/kg glutamine every 12 hours for one week after injury. SCIs were performed at T6 with a modified aneurism clip. Functional recovery was measured weekly using the Basso-Beattie-Bresnahan scale and the angle board method. Six weeks later, all rats were killed, and their extensor digitorum longus and soleus muscles excised and weighed. MyHC composition of the muscles was determined using SDS-PAGE.<p>The hypothesis that glutamine treatment following SCI would lead to better functional recovery and a more preserved MyHC profile was validated. Glutamine treated rats received significantly higher BBB scores (p<0.01) and angle board scores (p<0.001) than untreated SCI rats. Glutamine treatment also reduces muscle atrophy in the soleus muscle, but not the extensor digitorum longus (EDL). In untreated rats the soleus muscle accounted for significantly (p<0.001) less of the percentage of total body weight than the soleus muscle from glutamine treated rats. Finally, SCI rats with preserved functional abilities displayed a significantly better preserved MyHC profile compared to untreated SCI rats. In the soleus healthy rats contain 94% type 1 myosin, treated rats maintained 68% which was significantly (p<0.001) greater than 28% maintained by untreated rats. In the EDL healthy rats contain 55% type 2b myosin, treated rats maintained 32% which was greater than 26% type 2b myosin maintained by untreated rats.