Thèses sur le sujet « Controllo metabolico »

This manuscript presents a thorough investigation and description of metabolic control dynamics in vivo and in silico using as a model de novo pyrimidine biosynthesis. Metabolic networks have been studied intensely for decades, helping develop a detailed understanding of the way cells carry out their biosynthetic and catabolic functions. Biochemical reactions have been defined, pathway structures have been proposed, networks of genetic control have been examined, and mechanisms of enzymatic activity and regulation have been elucidated. In parallel with these types of traditional biochemical analysis, there has been increasing interest in engineering cellular metabolism for commercial and medical applications. Several different mathematical approaches have been developed to model biochemical pathways by combining stoichiometric and/or kinetic information with probabilistic analysis, or deciphering the comparative logic of metabolic networks using genomic-derived data. However, most of the research performed to date has relied on theoretical analyses and non-dynamic physiological states. The studies described in this dissertation provide a unique effort toward combining mathematical analysis with dynamic transition experimental data. Most importantly these studies emphasize the significance of providing a quantitative framework for understanding metabolic control. The pathway of de novo biosynthesis of pyrimidines in Escherichia coli provides an ideal model for the study of metabolic control, as there is extensive documentation available on each gene and enzyme involved as well as on their corresponding mechanisms of regulation. Biochemical flux through the pathway was analyzed under dynamic conditions using middle-exponential growth and steady state cultures. The fluctuations of the biochemical pathway intermediates and end products transitions were quantified in response to physiological perturbation. Different growth rates allowed the comparison of rapid versus long-term equilibrium shifts in metabolic adaptation. Finally, monitoring enzymatic activity levels during metabolic transitions provided insight into the interaction of genetic and biochemical mechanisms of regulation. Thus, it was possible to construct a robust mathematical model that faithfully represented, with a remarkable predictability, the nature of the metabolic response to specific environmental perturbations. These studies constitute a significant contribution to the fields of quantitative biochemistry and metabolic control, which can be extended to other cellular processes as well as different organisms.

This project aimed to investigate the role of the γ-lactone of 3,4-dihydroxybutanoic acid (3,4DB) which was claimed by Japanese investigators to be one of several endogenous γ-lactones involved in the control of food intake. Tissue, plasma and urine organic acid profiles were screened for the γ-lactone of 3,4DB using both GC and GCMS. Careful mass spectral analysis and in vitro acid-γ-lactone exchange analyses with structural validation studies showed that the γ-lactone of 3,4DB did not occur in vivo but that the free acid of 3,4DB did. This rejects previous claims to the contrary. Fasting increased rat plasma 3,4DB with urinary output significantly elevated for the first 24h of fasting. A metabolic route from glutamate to acetate is proposed with 3,4DB as an intermediate. Peripheral administration of glutamate, γ-aminobutyric acid (GABA) and 4-hydroxybutyric acid (4HB) to rats reduced food intake with increasing effects at each stage of the pathway. The γ-lactone 2-buten-4-olide is toxic and other γ-lactones had more potent effects than their free acids on rats. The intragastric administration of GABA stimulated 4HB production in vivo and GABA or 4HB increased plasma 3,4DB levels, thus implying that 3,4DB occurs at an intermediate in our proposed pathway. A likely endogenous source of 3,4DB is as a minor GABA metabolite. The response of the free acid of 3,4DB to fasting followed a similar trend as that reported for its γ-lactone and related γ-lactones are unlikely to occur in vivo. Given the similarities of 4HB (a precursor of 3,4DB) and 3,4DB in structure to the ketone body 3-hydroxybutyrate (3HB) and the similarity of 3,4DB in fasting response to 3HB, both 4HB and 3,4DB may initiate anorexia in fasting.

A depressão metabólica em aves e mamíferos, dada a alta demanda energética destes animais, se apresenta, geralmente, como resposta às condições de escassez de alimentos e baixas temperaturas. Desta forma, este projeto busca explorar, no campo teórico, como o sistema de termorregulação poderia atuar no sentido de maximizar as reservas energéticas minimizando os gastos metabólicos (depressão metabólica). Para tanto, fazemos uso de teorias da engenharia de controle que propiciam ferramental teórico para analisar como se dariam essas minimizações, ou seja, como o sistema nervoso atuaria estabelecendo um controle (set-point hipotalâmico) que minimizasse estes gastos à medida que se desse o processo de termorregulação. Neste contexto, propomos um modelo básico de termorregulação que leva em conta temperatura corpórea, taxa metabólica e temperatura ambiente, no qual o set-point atua como um controle. Mostramos como este modelo de regulação térmica propicia, devido à sua configuração, significativa redução dos distúrbios causados por variações da temperatura ambiente. Através da teoria de controle ótimo, mostramos como o set-point hipotalâmico pode surgir como resultado da minimização de um funcional relacionado ao custo com a termorregulação. Além disso, fez-se uma análise de como a temperatura ambiente pode definir diferentes situações em termos de vantagens da depressão metabólica como mecanismo de minimização de gasto energético. Para este tipo de análise, propomos um índice de razão entre o custo metabólico constante e o obtido sob atuação do controlador durante o período em que se dá o processo. Após um período em depressão metabólica, os indivíduos devem voltar a sua condição de eutermia, e, em situações de baixa temperatura, o custo deste retorno pode suplantar as vantagens para um dado indivíduo. Assim, são analisadas as influencias da massa corpórea, onde se observa aumento do custo em decorrência da entrada em depressão metabólica por parte dos indivíduos de maior massa. Tal aumento de custo é acentuado nas situações de menor temperatura ambiente. Finalmente, uma análise relativa ao tempo para retorno à condição de eutermia é apresentada, sendo que os resultados vão ao encontro das evidencias atuais sobre a flexibilidade estratégica de muitos hibernantes.
Metabolic depression of mammals and birds, animals of high metabolic demands, normally emerges as a response to food shortage and low ambient temperature. The main goal of this research is to explore, in a theoretical perspective, how the thermoregulatory system could extend the energy reserves of these endotherms decreasing metabolic costs under those environmental conditions. To approach the problem, we propose the use of control engineering theories to analyze the way the this minimization could occur, in other words, how the nervous system would act establishing a control (hypothalamic set-point) to minimize those costs during the thermoregulatory process. In this context, we propose a basic thermoregulation model that takes into account body temperature, metabolic rate and environmental temperature, and in which the set-point acts as a control. We show how this model can significantly reduce disturbances generated by ambient temperature. Using optimal control theory, we show how the hypothalamic set-point can emerge as a result of a minimization process of a functional related to thermoregulation costs. Also, how ambient temperature can define different metabolic profiles is explored, in terms of metabolic depression and the necessary return to euthermic conditions. To quantify this analysis we propose an index, based on the ratio between a constant metabolic cost and the metabolic cost defined by the controller. After a period in metabolic depression individuals should return to their euthermic condition, and, in situations of low environmental temperature, it is shown that the cost to return can be larger than the advantages. In this way, analyzing body mass influences we observed increased metabolic depression cost in larger individuals. This cost is even higher under lower environmental temperature. Finally, the cost related to the time elapsed, until the euthermic state is reached again, is considered. These last results are in accordance with current conception about the flexibility in hibernation process.

Magíster en Ciencias de la Ingeniería, Mención Química
Ingeniero Civil Químico
Resultados experimentales señalan que las células animales pueden alcanzar múltiples estados metabólicos con distintas razones de tasa de producción de lactato a tasa de consumo de glucosa (DL=DG), lográndose razones muy por debajo de la razón estequiométrica igual a 2 [mol=mol]. En el presente trabajo de tesis se planteó y ajustó un modelo metabólico que describe el metabolismo de un cultivo de control y se corroboró su falta de capacidad de alcanzar más de un estado estacionario a través de la comparación con datos experimentales y un análisis de estabilidad posterior. La simplificación de dicho modelo inicial, la obtención de un único punto atractor como estado estacionario y el análisis de variaciones de niveles de expresión génica de ciertas enzimas glicolíticas permitió el planteamiento de un modelo de regulación que varía la concentración de la enzima lactato deshidrogenasa (LDH) con el cual se simuló un cultivo hasta alcanzar estado metabólico alterado (DL=DG <0,1 [mol=mol]). El modelo metabólico regulado se utilizó para la simulación de un cultivo continuo alterado para el diseño y ajuste de controladores proporcional (P), basado en modelo lineal y basado en modelo no lineal para la regulación de la concentración de glucosa de entrada frente a perturbaciones en el crecimiento celular. La simulación de la respuesta de lazo cerrado del sistema mostró una fuerte interacción de lazos con el lazo de control de crecimiento celular y los análisis de robustez y de sensibilidad permitieron concluir que el controlador P posee una mayor robustez que el controlador basado en modelo lineal, pero que este último posee una mejor respuesta frente a limitantes que podrían existir a nivel industrial. Por otra parte, el pobre desempeño del controlador basado en modelo no lineal demuestra un desafío de ajuste del mismo producto de las múltiples posibles fuentes del mal desempeño. Como línea de trabajo futuro se puede mejorar la respuesta simulada de los controladores basados en modelo, analizando la eliminación de offset para el caso lineal y los problemas de rendimiento del basado en modelo no lineal.

Para avaliar o efeito de diferentes linhagens de leveduras, Saccharomyces cerevisiae, vivas, na redução de aflatoxicoses e, contribuir para o entendimento do modo de ação destas leveduras sobre aflatoxina B1 marcada com trítio (AFB13H), foram conduzidos três experimentos. No primeiro experimento, foram utilizados ratos Wistar para investigar o efeito de duas linhagens de S. cerevisiae (Y1026 e Y904), e da suplementação com aminoácidos na redução de aflatoxicoses. O bioensaio, em delineamento inteiramente casualizado, foi conduzido por 28 dias para avaliar sete tratamentos (dietas), sendo um livre de aflatoxinas e seis contendo 400g kg-1 de aflatoxinas; destes, cinco com leveduras, a saber: Y1026 (níveis de 0,5; 1,0 e 5,0%) e Y904 (níveis de 1% e 1%+1000ppm de metionina + 1000 ppm de cisteína). Não foram observadas diferenças estatistiticamente significativas no consumo de alimentos, ganho de peso, conversão alimentar e função hepática entre os animais submetidos aos diferentes tratamentos. No entanto, o exame histopatológico revelou que os animais que receberam aflatoxinas sem leveduras sofreram danos mais severos em relação aos que receberam aflatoxinas juntamente com leveduras, os quais apresentaram poucos danos celulares. Conclui-se que, as duas linhagens de leveduras em todas as situações estudadas apresentaram capacidade de reduzir as aflatoxicoses. O segundo ensaio foi conduzido com o objetivo de investigar o efeito de diferentes doses da levedura Y1026 no controle de aflatoxicoses em ratos. Neste bioensaio, os animais foram distribuídos em gaiolas individuais ao acaso, e foram submetidos a 7 tratamentos (dietas) por 60 dias, sendo uma sem aflatoxinas e seis contaminadas com 550 g kg-1 de aflatoxinas, das quais cinco contendo a levedura Y1026 (doses: 0,2; 0,5; 1,0; 2,0 e 5,0%). Foram realizadas análises sobre o aproveitamento do alimento, funções hepáticas e tecido hepático. O consumo de alimentos pelos animais alimentados com dieta livre de aflatoxinas foi maior do que pelos animais que receberam dietas contaminadas com a toxina, os demais parâmetros para julgamento do aproveitamento dos alimentos não diferiram estatisticamente entre os animais que foram submetidos a ambos os tratamentos. A atividade de enzimas hepáticas foi maior nos animais que não receberam a toxina do que nos demais. O exame histopatológico revelou que os animais que receberam aflatoxinas e aflatoxinas mais 0,2 ou 0,5% da levedura apresentaram sinais claros de hepatotoxidez e que os demais animais que ingeriram os níveis mais altos da levedura sofreram poucos danos celulares. Conclui-se que, a dose de levedura Y1026 aplicada foi determinante na redução das aflatoxicoses em ratos Wistar. No último bioensaio, trinta ratos foram alimentados por 28 dias seguindo-se quatro tratamentos (dietas): uma dieta livre de aflatoxinas e as outras três contaminadas com 500 g kg 1 de aflatoxina, sendo uma o controle com aflatoxinas e as outras duas na presença de 1% de levedura Y1026 ou Y904. No 18º dia, seis animais de cada um dos tratamentos que recebiam aflatoxinas foram transferidos para gaiolas metabólicas, onde permaneceram por cinco dias em adaptação e, no 23º dia, estes receberam, por via oral, uma dose simples de 2Ci/animal de AFB13H. Os animais restantes (três ratos em cada um dos tratamentos) foram utilizados nos exames histopatológicos. A radioatividade foi determinada 12, 24, 48, 72, 96 e 120 horas após a introdução do material radioativo nos animais. Os animais que receberam dietas contendo as leveduras apresentaram a absorção, a distribuição e a excreção da radioatividade mais lenta do que aqueles que não receberam os probióticos. O exame histopatológico revelou que os animais que ingeriram leveduras sofreram poucos danos celulares e que aqueles que não receberam levedura foram muito afetados. As leveduras apresentaram habilidade de reduzir aflatoxicoses e modificaram a absorção, a distribuição e a excreção da radioatividade de AFB13H em ratos Wistar.
The capacity of Saccharomyces cerevisiae, from two Strain, to reduce aflatoxicosis and the effect of yeast cells on tritium-labeled B1 aflatoxin (AFB13H) were investigated in three distinct studies. The effects of S. cerevisiae Y1026 and Y904 strains and diets amended with amino acids on the reduction of aflatoxicosis in Wistar rats were evaluated in the first study. A completely randomized block-designed bioassay with Wistar rats was conducted to evaluate seven formulations (Treatments), which consisted of an aflatoxin-free formulation and six formulations with 400 g kg-1 of aflatoxins. Of these, three formulations had the yeast strain Y1026 (at 0.5, 1.0 and 5.0%) and two had the strain Y904 (at 1% and 1%+1000ppm of methionine + 1000 ppm of cysteine). No statistical differences were observed for the food consumption, weights of the body organs, feed conversion and liver function between the animals fed the different treatments. Histopathological analysis revealed that animals fed aflatoxins diet without yeast cells had liver damage caused by the toxins and those that were fed aflatoxin-diet amended with yeast cells had less liver tissue damage. Therefore, the results obtained suggested that the presence of either yeast strain in the formulations caused a reduction in aflatoxicosis. The second study was conducted to investigate the effect of different dosages of the yeast strain Y1026 on the control of aflatoxicosis in rats. The bioassay was conducted with rats randomly placed in individual cages and fed seven different diets (7 treatments) for 60 days. These were an aflatoxin-free formulation and six others containing aflatoxins at 550 g kg-1, of which five had the yeast strain Y1026 (concentrations at 0.2; 0.5; 1.0; 2.0 and 5%). Feed conversion, liver functions indexes and liver tissue parameters were evaluated. The activity of the liver enzymes was greater in animals that fed the toxin-free diet when compared to other animals. Histopathological analysis showed that animals fed aflatoxin containing diets with and without 0.2 or 0.5% yeast cells showed clear signs of hepatotoxicity, while animals that were fed diets with higher concentrations of yeast cells had less liver tissue damage. The concentration of the yeast cells (Y1026) used in the formulations was correlated with the reduction of aflatoxicosis in Wistar rats. The third study fed Wistar rats an aflatoxin-free diet and diets with aflatoxins (at 500 g kg 1) and aflatoxin amended with a 1% concentration of the yeast strains Y1026 or Y904. In this study, six animals from each group fed the aflatoxin-diets were transferred to metabolic cages and received a single oral dose of AFB13H at 2Ci/animal. Three animals of each treatment were kept at the initial conditions and their liver tissues were used for histopathological analysis. Radiation levels in the animals were monitored at 12, 24, 48, 72, 96 and 120 h after receiving the labeled aflatoxin. Animals fed diets with active yeast cells had absorption, distribution and excretion levels of the labeled toxin different than those that did not receive the probiotic. Histopathological analysis showed that animals fed diets with yeast cells had less liver tissue damage while those fed the aflatoxin-diet had significantly higher liver damage. Therefore, these results indicate that active yeast cells have the ability to reduce aflatoxicosis and modify the absorption, distribution and excretion of radioactivity from AFB13H in Wistar rats.

The brain controls behaviour and has to manage the body’s resources (including energy) at the same time. How the brain coordinates and combines computations for controlling behaviour in response to metabolic state is little understood. I examined internal metabolic state and its role in motor coordination. I found that internal metabolic state modulates human motor coordination, with a lower energy expenditure associated with performing a velocity-controlled centre-out reaching task when in a low metabolic state. One approach to understanding human motor coordination is to consider motor cost functions. Many cost functions have been proposed yet the form and implementation of the cost function in the human motor system remains largely unknown. I have shown how an approximately quadratic metabolic energy cost function can be derived from the physiological properties of muscles and muscle fibres, producing a biophysically plausible cost of motor control. I then used this cost function to predict the manner in which coordination would change during an isometric force production task. I showed my predictions were correct, with motor effort shifting from muscles with higher metabolic energy costs towards muscles with lower metabolic energy costs. I examined the effect of internal metabolic state on muscle fibre recruitment regimes. I found no significant effect here, suggesting that fibre recruitment is computed in an independent manner to muscle coordination and supporting hierarchical control of human motor coordination. To directly uncover the composite cost function of reaching movements, I used model based inverse optimal control to show how differences in hand reaching trajectories between metabolic states can be described by a single parameter representing a trade-off between motor variability and energy expenditure.

Le conoscenze relative al controllo ormonale del metabolismo epatico dei pesci sono ancora piuttosto limitate e per molti anni sono state controverse. Per lungo tempo si è ritenuto che le catecolamine, adrenalina e noradrenalina, agissero nel fegato dei pesci soltanto attraverso i recettori adrenergici di tipo β. Quindi l’assetto recettoriale dei mammiferi, che comprende recettori α e β, era considerato frutto di un processo evolutivo che non aveva ancora avuto luogo nei pesci. Successivamente, nel fegato di vari teleostei è stata dimostrata la presenza di recettori sia α che β. Tuttavia il ruolo fisiologico dei due tipi di recettori non è ancora chiaro. Per esempio, in acciughe e sgombri non è stato fatto alcuno studio sulla risposta alle catecolamine ottenuta attraverso i recettori α e β, nel fegato di trota i recettori α non sono accoppiati alla cascata fisiologica che porta al rilascio di glucosio, e in anguilla e pesce gatto l’azione delle catecolamine attraverso recettori β è predominante rispetto a quella attraverso recettori α. L’utilizzo di ligandi farmacologici non ha portato a chiarimenti significativi, perché la loro specificità per i recettori di mammifero non trova sempre riscontro nei pesci. In questo studio, quindi, abbiamo studiato l’espressione dei geni codificanti per i recettori α e β adrenergici attraverso la tecnica della PCR real time, ottenendo i primi dati in letteratura per quanto riguarda la loro quantificazione assoluta. L’organismo modello utilizzato è stata l’anguilla, teleosteo caratterizzato da un ciclo biologico molto particolare in cui si distinguono nettamente una fase gialla ed una argentina. Le anguille argentine non sono mai state studiate a tale proposito, e date le estreme differenze nella disponibilità e nell’uso delle risorse energetiche in questi due stadi di crescita, il presente studio ha mirato a valutare la differente sensibilità alle catecolamine da parte degli epatociti isolati da anguille gialle ed argentine. I nostri dati hanno confermato quanto solo ipotizzato nei vari studi pubblicati negli ultimi due decenni, ma mai avvalorato da risultati sperimentali, cioè che i recettori α e β sono contemporaneamente espressi negli epatociti dell’anguilla, sia gialla che argentina, e la proporzione tra loro giustifica il ruolo significativamente maggiore giocato dai recettori β. Nelle anguille argentine infatti, come nelle gialle, l’effetto dell’adrenalina sul rilascio di glucosio ottenuto attraverso recettori β è chiaramente predominante. Inoltre, i nostri dati indicano che in due diverse fasi del ciclo vitale dell’anguilla, così come si osserva nell’ontogenesi dei mammiferi, i recettori adrenergici sono espressi in quantità differente.

Skeletal muscle is the most abundant tissue in the whole organism representing more than 40% of the total body mass. This organ is responsible for the 30% of metabolic rate in basal condition, suggesting its great relevance not only for locomotor activity, but also for the control of whole body metabolism. Indeed skeletal muscle is a highly dynamic tissue that modulates its metabolism and mass as a consequence of different physiopathological conditions. One stimulus that triggers major adaptations is exercise, which is also well known to activate autophagy (Grumati, Coletto, Schiavinato, et al., 2011). Physical exercise elicits several beneficial effects acting on mitochondrial content/function, fatty acid oxidation and glucose uptake; however it is considered a disruptive trigger for myofiber homeostasis that needs to be counterbalanced through the activation of transcriptionally regulated pathways ready to contrast mechanical and metabolic stresses produced during contraction. The role of FoxOs transcription factors and TFEB in regulating protein breakdown and autophagy is known (Milan et al., 2015; Settembre et al., 2011). However the role of TFEB in skeletal muscle and its possible effects in controlling exercise-dependent adaptations in this tissue were not proved. TFEB has been proposed as the key factor that coordinates autophagy to lysosomal biogenesis in cell culture, with different evidences showing the regulation of its activity. In particular it is known that an mTORC1 phosphorylation is able to prevent TFEB function by retaining it in the cytoplasm. However, there were no evidences concerning the possible phosphatases involved in TFEB activation. Using a cellular high content screening able to monitor TFEB nuclear translocation during starvation, we identified PPP3CB, the catalytic subunit of calcineurin, as one of the highest hit for TFEB nuclear relocalization. We demonstrated that calcineurin activity is necessary and sufficient to push TFEB in the nucleus, where it can complete its function. Nevertheless, calcineurin is known to be active in skeletal muscle during contraction as a consequence of calcium oscillations. For this reason we wondered whether calcineurin activity could affect TFEB translocation also in adult skeletal muscle during exercise. Using muscles transfected with a TFEB-GFP reporter, we demonstrated that calcineurin activity is necessary and sufficient to promote TFEB nuclear translocation even in adult skeletal muscle during coxntraction. However, the physiological meaning of this nuclear translocation in skeletal muscle remained to be addressed. To answer this question we used gain and loss of function approaches, by mean of viral infection of TFEB overesxpressing vectors, muscle specific TFEB knockout animals and tamoxifen inducible muscle specific TFEB transgenic animals. From microarray analysis of muscles overexpressing and lacking TFEB, we realized that the major pathways affected by genetic manipulation are related to mitochondrial biogenesis and function, lipid utilization and glucose homeostasis. Thus we started to dissect the function of TFEB in skeletal muscle proving that its activation is required for mitochondrial biogenesis that is indeed increased in transgenic muscle. We also found an augmented mitochondrial number and size in transgenic muscle, with only a small percentage of dysfunctional mitochondria in KO animals. These changes were paralleled by a TFEB signature in gene expression of genes involved in mitochondrial biogenesis and functionality. Moreover, these morphometric and gene expression evidences correlate with increased mitochondrial respiration and higher activity of respiratory chain complexes. For this reason transgenic muscles produce more ATP than normal mice, while KO muscles have a lower ATP synthesis because mitochondria present a leak in mitochondrial membrane that dissipate membrane potential. Nevertheless, in order to understand if TFEB is able to promote this mitochondrial program independently from PGC1α, we checked the expression of NRF1/2, TFAM and other genes involved in mitochondrial biogenesis in a model of PGC1α ablation during TFEB overexpression. These data, and complexes activity measurements, demonstrate that TFEB is able per se to activate the transcriptional program directly binding to NRF1 and NRF2 genes promoters without the need of the transcriptional co-activator. At this point, we challenged mice with exercise finding that transgenic mice are more resistant to exhaustive contraction than control; conversely muscle specific TFEB-KO animals display pronounced exercise intolerance due to their lack in ATP production. In order to better explain this latter finding, thanks to metabolic measurements we realized that KO muscles rely more on glucose oxidation both in basal condition and during the first phases of exercise thus explaining the observed exercise intolerance triggered by glycogen storage depletion. Furthermore lactate quantification in serum before and after exercise suggests that KO animal depend more on anaerobic glycolysis with respect to control and transgenic counterpart. To deeply investigate the role of glucose oxidation that seems the cause of exercise intolerance, we monitored glycogen levels in muscle of KO animals in resting condition, revealing a reduction of glycogen storage. For this reason after the early stages of exercise TFEB-KO animals need to rapidly shift their metabolism to fatty acid oxidation that however cannot support energy demand because of the presence of dysfunctional mitochondria. Altogether these findings indicate that TFEB is impinging more on metabolism rather than autophagy, that indeed is not affected by TFEB genetic modulation; more in detail TFEB seems to significantly modulate muscular glucose homeostasis that is altered in KO animals. Reduced glucose uptake and glycogen synthesis during EU clamps explains why glycogen storages are depleted in KO animals, while the transgenic counterpart present more glycogen accumulation. This phenotypic effect is paralleled by a change in glucose related genes expression, with higher levels of glucose transporters and glycogen synthesis regulator in transgenic muscles, even in the absence of PGC1α. Nevertheless TFEB overexpression is also able to drive factors such as nNOS and AMPK activity, thus modulating not only the expression but also the signalling pathways related to glucose homeostasis. In conclusion all these findings strongly support a new vision of TFEB as master regulator of metabolic flexibility during physical exercise in a PGC1α-independent fashion.
Il muscolo scheletrico è il tessuto più abbondante dell’organismo e rappresenta più del 40% della massa corporea. Questo organo è responsabile del 30% della spesa energetica a riposo, suggerendo la sua importanza non solo a livello di locomozione ma anche nel controllo del metabolismo a livello sistemico. Infatti il muscolo scheletrico è un tessuto estremamente dinamico, capace di modulare il suo metabolismo in seguito a stimoli di diversa natura. Uno stimolo che attiva maggiori adattamenti metabolici è l’esercizio, che è noto attivare anche l’autofagia. L’esercizio fisico stimola molti effetti benefici sul contenuto e funzionalità mitocondriale, ossidazione degli acidi grassi e assorbimento del glucosio; tuttavia, è considerato uno stimolo che danneggia la normale omeostasi delle fibre muscolari per cui necessita di essere controbilanciato dall’attivazione di meccanismi trascrizionalmente controllati che contrastano gli stress meccanici e metabolici prodotti durante la contrazione. Il ruolo dei fattori di trascrizione FoxO e TFEB nel regolare la degradazione proteica e l’autofagia è largamente conosciuto. Tuttavia, il ruolo di TFEB nel muscolo scheletrico e i suoi possibili effetti nel regolare gli adattamenti derivanti dall’esercizio in questo tessuto non sono ancora chiari. TFEB è stato proposto come fattore chiave nel coordinare autofagia e biogenesi lisosomiale in cellule in coltura, con diverse evidenze che dimostrano la regolazione della sua attività. In particolare è noto come la fosforilazione operata da mTORC1 sia in grado di prevenire l’attivazione di TFEB sequestrandolo nel citoplasma. Tuttavia, non esistono dati riguardanti le possibili fosfatasi coinvolte nell’attivazione di TFEB. Mediante l’utilizzo di uno High Content Screening in grado di monitorare la traslocazione di TFEB nel nucleo durante la starvation, abbiamo identificato il gene PPP3CB, codificante la subunità catalitica della calcineurina, come uno dei migliori geni coinvolti nella rilocalizzazione di TFEB. Abbiamo dimostrato che l’attività della calcineurina è necessaria e sufficiente per spingere TFEB nel nucleo, dove può espletare la sua funzione. Tuttavia, la calcineurina è noto essere attiva nel muscolo scheletrico durante la contrazione come conseguenza dei transienti di calcio. Per questo motivo ci siamo chiesti se l’attività della calcineurina possa influenzare la traslocazione di TFEB nel nucleo anche nel muscolo scheletrico durante l’esercizio fisico. Utilizzando un reporter TFEB-GFP abbiamo dimostrato che l’attività della calcineurina è necessaria e sufficiente a promuovere la traslocazione nucleare di TFEB anche nel muscolo scheletrico durante la contrazione. Tuttavia il significato fisiologico di questo avvenimento rimane da essere spiegato. Per rispondere a questa domanda abbiamo usato degli approcci di gain e loss of function utilizzando infezioni virali con vettori per l’overespressione di TFEB, una linea di topi con delezione muscolo specifica di TFEB e un’altra linea in cui l’overespressione di TFEB può essere attivata in muscolo grazie al tamoxifen. Da uno studio di espressione genica in muscoli overesprimenti TFEB e TFEB deficienti, abbiamo trovato che le vie di segnale principalmente coinvolte dalle manipolazioni genetiche erano quelle correlate alla biogenesi mitocondriale, utilizzo dei lipidi e omeostasi del glucosio. Abbiamo perciò cominciato a dissezionare il ruolo di TFEB nel muscolo scheletrico provando che la sua attivazione è richiesta per la biogenesi mitocondriale, che è per l'appunto aumentata nei muscoli transgenici. Infatti, in questi abbiamo trovato un aumento nel numero e nella dimensione dei mitocondri, mentre abbiamo riportato solo una piccola percentuale di mitocondri disfunzionali nei muscoli knockout. Questi cambiamenti sono accompagnati da un’attivazione dei geni TFEB-dipendenti responsabili per la biogenesi e funzionalità mitocondriale. Inoltre, questi cambiamenti morfometrici e di espressione genica correlano con un aumento nella respirazione mitocondriale e nell’attività dei complessi della catena respiratoria. Per questo motivo i muscoli transgenici producono più ATP dei wildtype, mentre i muscoli KO presentano una ridotta sintesi di ATP a causa di una disfunzionalità della membrana mitocondriale che dissipa il gradiente protonico. Tuttavia, per capire se questi cambiamenti dipendono direttamente da TFEB indipendentemente da PGC1α, abbiamo monitorato l’espressione di NRF1/2, TFAM e altri geni coinvolti nella biogenesi mitocondriale in un modello in cui PGC1α è deleto e TFEB overespresso. Questi dati di espressione uniti alle misure delle attività dei complessi dimostrano che TFEB è in grado di indurre autonomamente la biogenesi mitocondriale legandosi direttamente ai promotori dei geni NRF1 e NRF2. A questo punto abbiamo sottoposto a esercizio i topi riscontrando che gli animali transgenici resistono maggiormente all’attività fisica; al contrario i topi KO presentano una marcata intolleranza all’esercizio a causa della scarsa produzione di ATP. Per spiegare meglio questo fenomeno, grazie a misurazioni di parametri metabolici abbiamo riscontrato che i topi KO fanno affidamento maggiormente nell’ossidazione del glucosio sia a riposo che durante le fasi iniziali dell’esercizio fisico, spiegando l’intolleranza con la fine delle riserve di glicogeno. Inoltre, le quantificazioni del lattato nel siero prima e dopo l’esercizio suggeriscono che i muscoli KO dipendono maggiormente dalla glicolisi anaerobia a differenza delle controparti wildtype e transgenica. A questo punto, per investigare più in dettaglio il ruolo dell’ossidazione del glucosio che sembra essere alla base dell’intolleranza all’esercizio, abbiamo misurato i livelli di glucosio intramuscolare negli animali KO, notando che a riposo questi presentano una riduzione considerevole delle riserve. Per questo motivo gli animali KO, dopo i primi momenti di esercizio, sono costretti a cambiare il loro metabolismo verso una maggiore ossidazione degli acidi grassi che comunque non riesce a supportare la domanda energetica a causa dei mitocondri disfunzionali. Tutte queste evidenze indicano che TFEB controlla più il metabolismo rispetto all’autofagia la quale non è influenzata dalla modulazione genetica di TFEB; più in dettaglio TFEB sembra controllare direttamente il metabolismo del glucosio che è alterato negli animali TFEB-deficienti. Un ridotto assorbimento del glucosio e una ridotta sintesi del glicogeno durante gli EU-clamps spiegano perché le riserve di glicogeno sono ridotte negli animali KO mentre la controparte transgenica ne accumula in più. Questi effetti fenotipici sono accompagnati da un cambiamento nell’espressione di geni connessi all’omeostasi del glucosio, con maggiore presenza di trascritti per i trasportatori di glucosio and regolatori della sintesi del glicogeno nei muscoli transgenici, anche in assenza di PGC1α. Inoltre, l’overespressione di TFEB è in grado di modulare anche l’attività di nNOS e AMPK, influenzando l’omeostasi del glucosio non solo dal punto di visto trascrizionale, ma impattando anche sulle vie di segnale ad esso correlate. In conclusione tutte queste scoperte sostengono fortemente una nuova visione di TFEB come un fattore chiave nella regolazione della flessibilità metabolica durante l’esercizio fisico in modo indipendente da PGC1α.

A soja é uma das culturas mais antigas e praticadas no mundo, tendo sua importância pelo elevado teor de proteínas, sendo utilizada na alimentação humana e animal, além de ser o principal produto de exportação brasileira. Apesar da alta produção nacional, a cultura possui perdas de produtividade pelos ataques de insetos-pragas, dentre eles, a mosca-branca Bemisia tabaci Biótipo B (Hemiptera: Aleyrodidae). Seu controle tradicional é realizado com inseticidas, mas atualmente métodos alternativos estão sendo avaliados, considerando a não contaminação do ambiente e a saúde humana. O presente trabalho teve como objetivo avaliar o potencial de indutores de resistência no processo de defesa vegetal contra a mosca-branca em plantas de soja. Foram realizados dois experimentos na Universidade Tecnológica Federal do Paraná, Campus Dois Vizinhos-PR, no ano de 2017. Sementes de soja da cultivar BRS 284 foram semeadas em vasos de polietileno com capacidade para 10 litros, contendo solo proveniente de lavoura. O cultivo ocorreu em casa de vegetação, sendo que os vasos ficaram dispostos nas bancadas até a fase fenológica V6, onde foram aplicados os indutores conforme os tratamentos. Os tratamentos foram aplicados por microasperção sendo: ASM (0,005%), AS (2Mm), fertilizante foliar composto por fosfito de potássio (0,004%); quitosana (1%) , silício (0,25%) e testemunha (água destilada). O primeiro experimento buscou avaliar o potencial dos indutores quanto a capacidade de ativar mecanismos de defesa vegetal, considerando a presença e a ausência da mosca branca. Para tanto, aplicou- se os indutores, sendo que para a condição de ausência de insetos, os vasos permaneceram em gaiola individual com tela anti-afídica, para evitar o contato com o inseto praga. Então, realizou-se a coleta do material vegetal em intervalos de 0, 24, 48, 96 e 168 horas após a aplicação dos indutores de resistência. Avaliaram-se proteínas totais, açúcares totais e redutores, compostos fenólicos, taninos e a atividade das enzimas peroxidases, fenilalanina amônia-liase (FAL) e quitinase. O segundo experimento buscou avaliar a preferência de oviposição em função da aplicação dos indutores. Após 24 horas da aplicação dos indutores, 500 adultos coletados não sexados de mosca-branca foram liberados no centro dos vasos sobre a bancada, tendo chance de escolha entre os tratamentos. Após 48 horas da infestação inicial, coletaram-se dois folíolos do terço mediano das plantas, de cada tratamento. Os folíolos foram avaliados em microscópio estereoscópio para a quantificação do número de ovos. A área foliar total dos folíolos também foi calculada, utilizando-se o software Image J. Os indutores de resistência possuem capacidade de ativar o metabolismo primário através da síntese de proteínas totais, bem como demonstram potencial na ativação de mecanismos de defesa entre eles, a rota dos fenilpropanóides com a ativação da enzima FAL e a formação de compostos fenólicos. Ainda demonstram ativar enzimas relacionadas a patogenicidade como as peroxidases e quitinase, tais ativações possuem especificidade quanto ao indutor e o tempo de ativação. O uso dos indutores quando desafiados com insetos demonstraram maior efetividade de ativação da enzima FAL, peroxidade e quitinase, enzimas estas relacionadas ao processo de defesa vegetal a insetos. Os indutores ASM, silício e quitosana possuem potencial de redução da oviposição da mosca-branca, o que pode estar relacionado a ativação de mecanismos de defesa vegetal.
Soybean is one of the oldest and most practiced crops in the world,and its importance is due to its high protein content,its use in food for humans and animals, as well as being the main Brazilian export product. Despite the high national production, the crop has productivity losses due to attacks of insect pests, among them, the whitefly Bemisia tabaci Botype B (Hemiptera: Aleyrodidae). Its traditional control is carried out with insecticides, but currently alternative methods are being evaluated considering the noncontamination of the environment and human health. The present work had as objective to evaluate the potential of resistance elicitors in the process of vegetal defense against the whitefly in soybean plants. Two experiments were carried out at the Federal University of Technology - Paraná, Campus Dois Vizinhos-PR, in 2017. Soybean seeds of cultivar BRS 284 were sown in polyethylene pots with capacity of 10 liters, containing soil gathered from crops at UTFPR. Cultivation occurred in a greenhouse, and the vessels were placed on benches until the V6 phenological phase, when the elicitors were applied by microaspersion according to the treatments: ASM (0.005%), SA (2 Mm), foliar fertilizer composed of potassium phosphite (0.004%); chitosan (1%), silicon (0.25%) and control (distilled water). The first experiment aimed to evaluate the potential of the elicitors to activate plant defense mechanisms, considering the presence and absence of the whitefly. For this, the elicitors were applied, and for the condition of absence of insects, the vases remained in individual cages with anti-aphid screen, to avoid contact with the insect pest. Then, the plant material was collected at intervals of 0, 24, 48, 96 and 168 hours after the application of resistance elicitors. Total proteins, total and reducing sugars, phenolic compounds, tannins and the activity of the enzymes peroxidases, phenylalanine ammonia-lyase (FAL) and chitinase were evaluated. The second experiment sought to evaluate the oviposition preference due to the application of the elicitors. After 24 hours of application, 500 not sexed whitefly adults were released in the center of the vases on the bench, having a choice among treatments. After 48 hours of the initial infestation, two leaflets of the median third of the plants were collected from each treatment. The leaflets were evaluated under stereomicroscope for the quantification of the number of eggs. The total leaf area of the leaflets was also calculated using Image J. The resistance elicitors have the capacity to activate the primary metabolism through the synthesis of total proteins, as well as demonstrate the potential in the activation of defense mechanisms among them, the route of the phenylpropanoids with the activation of the enzyme FAL and the formation of phenolic compounds. They also activated pathogenic enzymes such as peroxidases and chitinase, such activations have specificity for the elicitor and the activation time. The use of elicitors when challenged with insects demonstrated greater effectiveness of activation of the enzyme FAL, peroxidase and chitinase, these enzymes related to the process of plant defense against insects. ASM, silicon and chitosan elicitors have the potential to reduce oviposition of the whitefly, which may be related to the activation of plant defense mechanisms.

The liver has a major role in the metabolism of cholesterol, being the main site of lipoprotein assembly and degradation and the only tissue where the metabolism of cholesterol to bile acids occurs. This provides the major pathway for the removal of cholesterol from the body. The results described in this thesis concern the use of specific enzyme inhibitors (58-035, Azacholesterol, Mevinolin) to determine the intracellular use of different sources of cholesterol in monolayers of rat hepatocytes. In particular, the fates of newly synthesized cholesterol from mevalonic acid and cholesterol derived from HDL2 were investigated. Incubation of hepatocyte monolayers with 58-035 resulted in the inhibition of esterification. In the presence of mevalonic acid as a cholesterol source, 58-035 stimulated bile acid synthesis. Azacholesterol inhibited bile acid synthesis, had no effect on cholesterol synthesis, and in the presence of mevalonic acid, stimulated secretion of cholesterol by the hepatocytes; it had no effect on cholesterol esterification. Mevinolin inhibited cholesterol synthesis and as a result inhibited esterification. HDL2, in the presence of mevinolin, was used as a cholesterol source. It stimulated bile acid synthesis and cholesterol esterification. Addition of 58-035 to the system resulted in the inhibition of both esterification and bile acid synthesis. Overall, the results indicated that different intracllular pools of free cholesterol exist and that the inter-relationships of these pools give a complex pattern of flux of intracellular cholesterol between various pathways in the rat hepatocyte.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
A palatoplastia à o procedimento cirÃrgico que visa o fechamento da comunicaÃÃo das cavidades nasal e oral, resultante do nÃo fechamento dos processos palatinos embrionÃrios, na Ãpoca de formaÃÃo da face. O objetivo deste estudo foi avaliar o efeito da L-alanil-glutamina sobre o estresse oxidativo, o controle glicÃmico e a resposta inflamatÃria em pacientes portadores de fissuras lÃbio-palatais, submetidos à palatoplastia. O estudo foi prospectivo, simples cego, randomizado e controlado por placebo, sendo constituÃdo por 30 (trinta) crianÃas do sexo masculino, na faixa etÃria entre 02 e 10 anos de idade, distribuÃdos em 02 grupos: Grupo A - Controle, n = 15, em que foi administrado a cada crianÃa 100ml de soro fisiolÃgico a 0,9% e Grupo B â L-alanil-glutamina, n = 15, em que foi administrada a cada crianÃa uma soluÃÃo de 100ml com L-alanil-glutamina à 20%, 0,5g/kg/dose (DipeptivenÂ) e Soro FisiolÃgico a 0,9%. O Projeto de Pesquisa foi submetido ao Comità de Ãtica em Pesquisa do Hospital Infantil Albert Sabin sendo aprovado sob o registro N 51/06 de 29 de maio de 2006. Foi colhido sangue venoso perifÃrico, em 05 tempos diferentes: T1- 03 horas antes do procedimento cirÃrgico; T2- apÃs a administraÃÃo da soluÃÃo (antes do procedimento cirÃrgico); T3- apÃs o procedimento cirÃrgico; T4- 06 horas de pÃs-operatÃrio e T5- 12 horas de pÃs-operatÃrio. Foram determinadas as concentraÃÃes de: glutationa, SubstÃncias Reativas do Ãcido TiobarbitÃrico(TBARS), glicose, insulina, proteÃna C reativa (PCR), interleucina-6 (IL-6) e interleucina-10 (IL-10). ComparaÃÃes entre os grupos Controle e L-alanil-glutamina foram feitas mediante o uso do teste t para variÃveis nÃo emparelhadas (dados paramÃtricos) ou do teste U de Mann-Whitney (dados nÃo paramÃtricos), sendo considerado como estatisticamente significante um valor P < 0,05. Para os parÃmetros avaliados de peso e idade, nÃo foram constatadas diferenÃas estatisticamente significantes entre os grupos. Em todos os tempos estudados, entre os grupos, nÃo foram verificadas diferenÃas estatisticamente significantes para glicose, insulina, TBARS, glutationa, IL-6 e IL-10. Houve um aumento, no T4 [(Grupo A - 20,472 [11,637-27,780] versus T1 0,0 [0,0-2,226] p<0,05; T2 0,0 [0,0-2,140] p<0.01 e T3 0,625 [0,0-2,153] p<0.01) (Grupo B â 19,317 [11,670-24,048] versus T1 1,888 [0,559-5,295] p<0,05; T2 0,0 [0,0-1,316] p<0,001 e T3 0,0 [0,0-2,757] p<0,05)] e no T5 [(Grupo A â 22,129 [9,721-33,225] versus T1 0,0 [0,0-2,226]p<0,01; T2 0,0 [0,0-2,140] p<0,001; T3 0,625 [0,0-2,153]p<0,001) (Grupo B â 22,177 [11,157-33,407] versus T1 1,888 [0,559-5,295]p<0,01; T2 0,0 [0,0-1,316]p<0,001; T3 0,0 [0,0-2,757]p<0,01)], da IL-6 em relaÃÃo aos tempos T1, T2 e T3, em ambos os Grupos. A infusÃo de L-alanil-glutamina, no grupo B, induziu queda significante nas concentraÃÃes de PCR quando comparadas Ãs do grupo Controle, no T5 (3,6 [3,160-5,05] versus 8,4 [4,1-11,9]) (p=0,0037). Como conclusÃo, reduÃÃo significante na dosagem da PCR, 12 horas apÃs o procedimento cirÃrgico, em crianÃas recipientes de L-alanil-glutamina, reflete menor resposta inflamatÃria.
The palatoplasty is the surgical procedure aimed at closing the communication of the nasal and oral cavities, not resulting from the processes palatins embryonic closing at the time of formation of the face. The objective of this study was to evaluate the effect of L-alanyl-glutamine on the oxidative stress, the glycemic control and inflammatory response in cleft palate and lip patients, submitted to palatoplasty. The study was prospective, single blind, randomized, placebo-controlled, and is comprised of 30 (thirty) male children in the age group between 02 and 10 years of age, divided into 02 groups: Group A-Control, n = 15 , which were administered to each child 100ml of saline solution to 0.9% and Group B- L-alanyl-glutamine, n = 15, which were administered to each child a solution of 100ml with L-alanyl-glutamine to 20 %, 0.5 g / kg / dose (Dipeptiven Â) and saline solution to 0.9%. The Research Project was submitted to the Committee of Ethics in Research of Hospital Infantil Albert Sabin being approved under the registry No 51/06 of May 29, 2006. It was picked peripheral venous blood, at 05 times different: T1- 03 hours prior to the surgical procedure; T2- after administration of the solution (before the surgical procedure), T3- after the surgical procedure, T4- 06 hours post-operative and T5- 12 hours post-operative. The concentrations were determined of: glutathione, Tiobarbituric Acid of Reactivates substances (TBARS), glucose, insulin, C-reactive protein (CRP), interleukin-6 (IL-6) and interleukin-10 (IL-10) . Comparisons between groups Control and L-alanyl-glutamine were made using the t test for not paired variables ( parametric data) or the U test of Mann-Whitney ( not parametric data), and is regarded as statistically significant P value < 0.05. For the parameters evaluated in weight and age, no statistically significant differences were found between groups. In all the times studied, there were no statistically significant differences in glucose, insulin, TBARS, glutathione, IL-6 and IL-10, there was an increase in T4 [(Grupo A - 20,472 [11,637-27,780] versus T1 0,0 [0,0-2,226] p<0,05; T2 0,0 [0,0-2,140] p<0.01 e T3 0,625 [0,0-2,153] p<0.01) (Grupo B â 19,317 [11,670-24,048] versus T1 1,888 [0,559-5,295] p<0,05; T2 0,0 [0,0-1,316] p<0,001 e T3 0,0 [0,0-2,757] p<0,05)] and T5[(Grupo A â 22,129 [9,721-33,225] versus T1 0,0 [0,0-2,226]p<0,01; T2 0,0 [0,0-2,140] p<0,001; T3 0,625 [0,0-2,153]p<0,001) (Grupo B â 22,177 [11,157-33,407] versus T1 1,888 [0,559-5,295]p<0,01; T2 0,0 [0,0-1,316]p<0,001; T3 0,0 [0,0-2,757]p<0,01), of the IL-6, regarding times T1, T2 and T3, for the two groups. In assessing the CRP, children of the L-alanyi-glutamine group, compared to patients in the group Control showed a significant reduction, in T5 (3,6 [3,160-5,05] versus 8,4 [4,1-11,9]) (p=0.0037). In conclusion, significant reduction in the dosage of CRP, 12 hours after the surgical procedure in children who received L-alanyl - glutamine, reflects lower inflammatory response.

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The nematode Pratylenchus brachyurus known as nematode lesions, affects the soybean crop caused significant damage, this means that there is a need to develop alternatives that supply the control of pathogens by aggregating in productivity. Then the objective is to verify the influence of homeopathic Mercurius solubilis in different potencies in soybean plants and on control of the nematode. For this, three experiments were carried out in climatized greenhouse, testing the potencies of 6, 12, 24, 50, 100, 200 and 400CH (centesimal Hahnemannian) of Mercurius solubilis, ethanol 30% and healthy plants (untreated and not inoculated) were used as control treatment. The treatments were applied weekly from the V3 growth stage of soybeans. Three days after the first treatment, inoculation of nematodes was done. After 50 and 70 days after inoculation of the first and second experiment respectively, were made assessments of the aerial part height, stem diameter, number of pods per plant, dry weight of aerial part, dry weight of leaf + petiole + stem, dry mass of total pods, dry weight per pod, fresh weight of root, and were count juvenile, adults and eggs in the soil and in roots, and determined the reproduction factor (RF). In the third experiment, were quantified enzymes involved in secondary metabolism of plants, peroxidase (POX), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO). The sample of roots were taken at intervals of 0, 3, 7 and 14 days after treatment (DAT) and in the 3rd DAT, inoculation was made. In laboratory was conducted a experiment to evaluate in vitro motility and mortality, a distilled water solution containing 100 ml-1 juveniles and adults were placed in plastic container and add 7 mL of in vivo treatments tested at a dilution of 0.1%. The experiments were conducted in a randomized block design. The potencies 24CH, 50CH, 200CH and 100CH reduce the number of adults and juveniles in soil, as well as the reproduction factor, furthermore 100CH is able to interfere in the productive aspects, increasing 107.5% in the number of pods when compared to the control ethanol 30%, as well as dynamization 6CH and 12CH. To POX the enzymatic activity was higher for dynamizations 6CH, 100CH and 400CH, 3, 7 and 14 DAT respectively. The PAL activity presented increases of 79.93%, 80.72% and 84.10% in dynamizations 6CH, 12CH and 24CH respectively compared to control treatment, 3 DAT. 14 days after the first treatment, 400CH dynamization showed an increase in the enzymatic activity of 53.41% and 32.21% when compared to the control ethanol 30% and absolute control respectively. The dynamization 24CH when compared to absolute control showed an increase of 41.10% in the enzymatic activity. So Mercurius solubilis may be a potential alternative for the control of the nematode
O nematoide Pratylenchus brachyurus conhecido como nematoide das lesões, afeta a cultura da soja causado danos expressivos, isso faz com que haja a necessidade de desenvolver alternativas que supram o controle dos patógenos, agregando em produtividade. Objetivou-se então, verificar a influência do medicamento homeopático Mercurius solubilis em diferentes dinamizações nas plantas de soja e no controle de P. brachyurus. Para tanto, foram conduzidos três experimentos em casa de vegetação climatizada, testando-se as dinamizações de 6, 12, 24, 50, 100, 200 e 400CH (centesimal hahnemanniana) de Mercurius solubilis, Etanol 30% e plantas sadias (não tratada e não inoculada) foram utilizadas como tratamento testemunha. Os tratamentos foram aplicados semanalmente a partir do estádio fenológico V3 da soja. Três dias após o primeiro tratamento, foi feita a inoculação dos nematoides. Decorridos 50 e 70 dias após a inoculação do primeiro e segundo experimento respectivamente, foram realizadas as avaliações de altura de parte aérea, diâmetro do coleto, número de vagens por planta, massa seca de parte aérea, massa seca de folha+pecíolo+caule, massa seca total de vagens, massa seca por vagem, massa fresca de raiz, e contagem de juvenis, adultos e ovos presentes no solo e na raiz, e determinado o fator de reprodução (FR). No terceiro experimento, foram quantificadas enzimas envolvidas no metabolismo secundário das plantas, peroxidase (POX), fenilalanina amônia-liase (FAL) e a polifenoloxidase (PFO). As coletas das amostras de raízes foram realizadas no intervalo de 0, 3, 7 e 14 dias após o tratamento (DAT) sendo que no 3º DAT foi feita a inoculação. Em laboratório foi realizado experimento in vitro para avaliação de motilidade e mortalidade, uma solução de água destilada contendo 100 juvenis e adultos mL-1 foi depositada em recipiente plástico, e adicionados 7 mL dos tratamentos testados in vivo na diluição de 0,1%. Os experimentos foram conduzidos em delineamento em blocos casualizados. As dinamizações 24CH, 50CH, 100CH e 200CH reduzem o número de juvenis e adultos presentes no solo, assim como o fator de reprodução, além disso, a dinamização 100CH é capaz de interferir em aspectos produtivos pelo aumento de 107,5% no número de vagens quando comparada à testemunha etanol 30%, assim como a dinamização 6CH e 12CH. Para POX a atividade enzimática foi superior para as dinamizações 6CH, 100CH e 400CH, em 3, 7 e 14 DAT respectivamente. A atividade de FAL apresentou incrementos de 79,93%, 80,72% e 84,10% nas dinamizações 6CH, 12CH e 24CH respectivamente em relação à testemunha absoluta, 3 DAT. 14 dias após o primeiro tratamento, a dinamização 400CH mostrou um aumento na atividade enzimática de 53,41% e 32,21% quando comparada à testemunha etanol 30% e testemunha absoluta respectivamente. A dinamização 24CH quando comparada a testemunha absoluta mostrou um acréscimo de 41,10% na atividade enzimática. Assim Mercurius solubilis pode ser uma alternativa potencial para o controle de P. brachyurus

Glioblastomas (GBM) are the most frequent and aggressive tumors of the human central nervous system (CNS). Highly vascularized, infiltrating, and resistant to current therapies, they affect patients at different ages. The median survival of patients is shorter than 18 months. A growing body of evidence indicates that glioblastoma cancer stem-like cells (GSC) play a major role in the development of these tumors and their resistance to current therapies. Development of effective therapies requires understanding the molecular mechanisms that govern GSC properties, including the signals that sustain their interactions with the tumor stroma, and determining the extent to which these mechanisms differ between GSC and neural stem cells (NSC). Our studies of the signals that underlie the interactions between GSC or “non-stem” GBM cells demonstrated that the deposition of an extracellular matrix (ECM) rich in Tenascin-C and poor in Fibronectin, by “non-stem” GBM cells, induced human endothelial cell death and impaired tubulogenesis in vitro. Furthermore, we described for the first time, in vitro and in vivo, an angiogenic function for HDGF (hepatoma derived growth factor) secreted only by GSC. Comparison of the effects of the natural polyphenol Resveratrol (RSV) on GSC and NSC showed that RSV induced in a specific manner GSC cell cycle arrest in a Sirtuin2-dependent manner. My main body of works focused on the metabolic pathways that participate in GSC maintenance. It took advantage of the recent demonstration that the cluster of microRNAs, miR-302-367, irreversibly commits GSC into a non stem-like state, and suppresses their ability to initiate tumours in vivo (Fareh et al. , 2012). Metabolome profiling of GSC and GSC-miR-302-367 (measure of the intracellular and secreted levels of 271 metabolites by mass spectrometry) pointed notably to changes in the GABA metabolic pathway. We showed that changes in GABA metabolism translated to an over-production of the GABA by-product, γ-hydroxybutyrate (GHB), which inhibited GSC clonal and self-renewal properties, and decreased the nuclear expression of the transcription factor Nanog, essential for the maintenance of stem-like properties in GSC. GHB decreased also GSC proliferation, an effect accompanied by the increased expression of CDKN1 (cyclin-dependent kinase inhibitor or p21) that limits the cell cycle progression. GHB effects on GSC were accompanied with impaired activity of TET2 methylcytosine dioxygenase, an enzyme necessary for the initiation of DNA demethylation. GHB exerted similar effects on GSC derived from distinct adult and pediatric high-grade glioma and bearing different mutations. Finally, research into the mechanisms by which miR-302-367 induces GHB overproduction showed a direct targeting of the transcript of ALDH5A1 gene by miR-302-367. The knockdown of ALDH5A1 by siRNA led to increased levels of GHB and reduced GSC proliferation. These results demonstrate an unprecedented role for GABA by-products in the control of GSC maintenance and epigenetic regulation. Alltogether, the results of these studies identify novel molecular mechanisms governing GSC maintenance that belong to cell metabolism and neoangiogenesis processes
Des nombreux travaux indiquent une participation cruciale de cellules souches cancéreuses au développement et à la résistance aux thérapies des glioblastomes (GBM), les tumeurs primaires les plus fréquentes et les plus agressives du SNC. Mes travaux ont eu pour but d’identifier les voies moléculaires qui gouvernent les propriétés des cellules souches de glioblastome (CSG) et leurs interactions avec le micro-environnement, et de déterminer dans quelle mesure ces voies s’apparentent à celles qui gouvernent le comportement des cellules souches neurales (CSN). Les études des signaux qui sous-tendent les interactions entre les GSC ou les cellules plus différenciées des GBM avec les cellules endothéliales ont abouti à l’identification d’un déséquilibre de la matrice extra-cellulaire sécrétée par les cellules de GBM, qui induit la mort cellulaire des cellules endothéliales humaines et une tubulogenèse défectueuse in vitro. De plus, nous avons décrit par la première fois, in vitro et in vivo, une fonction angiogénique pour HDGF (Hepatoma derived growth factor) et sa sécrétion par les CSG. La comparaison des effets du polyphénol naturel Resvératrol (RSV) sur les GSC et les CSN a montré une sensibilité spécifique des CSG aux effets inhibiteurs du RSV sur le cycle cellulaire due à leur expression de la déacétylase SIRT2, qui n’est pas présente dans les CSN. Mes principaux travaux ont porté sur les voies métaboliques contrôlant le maintien des CSG. Ils se sont appuyés sur l’expression du groupe de micro-ARN, miR-302-367, qui inhibe de façon irréversible les propriétés souches et tumorigènes des CSG (Fareh et coll. , 2012). Nous avons postulé qu’un tel changement de phénotype cellulaire devait s’accompagner d’altérations métaboliques participant à la perte des propriétés fonctionnelles des CSG induite par miR-302-367. La comparaison des profils métaboliques des CSG et des CSG-miR-302-367 (spectrométrie de masse, 271 métabolites extra- et intra-cellulaires mesurés) a révélé une altération de la voie de synthèse du GABA dans les CSG-miR-302-367 marquée notamment par une augmentation de la production de γ-hydroxybutyrate (GHB), un catabolite du GABA. De façon remarquable, l’exposition de CSG naïves au GHB a reproduit les effets inhibiteurs du miR-302-367 sur les propriétés clonales et d’auto-renouvellement des CSG, ainsi que la diminution de l’expression nucléaire du facteur de transcription « souche » Nanog, essentiel au maintien des propriétés souche des CSG. Le GHB diminue aussi la prolifération des CSG, un effet accompagné d’une augmentation de l’expression de CDKN1 (cyclin-dependent kinase inhibitor 1A ou p21), qui limite la progression du cycle cellulaire. Les effets du GHB se retrouvent sur des GSC issues de GBM de l’adulte comme de gliomes de haut-grade de l’enfant, porteurs de mutations distinctes. Ils apparaissent liés à son action inhibitrice sur l’activité de la méthylcytosine dioxygénase TET2, nécessaire à l’initiation de la déméthylation de l’ADN. Enfin, la recherche des mécanismes par lesquels l’expression de miR-302-367 aboutit à la surproduction de GHB a montré un ciblage direct du transcrit du gène ALDH5A1 par miR-302-367. Le knockdown d’ALDH5A1 par des siARN conduit à une augmentation des niveaux de GHB et à la réduction de la prolifération des CSG. Ces études révèlent la participation des régulations métaboliques au contrôle des propriétés des CSG, et ouvre de nouvelles voies pour le ciblage thérapeutique des glioblastomes

Members of the AKHIRPCH family of peptides were identified in corpora cardiaca of the dragonfly Anax imperator (Anisoptera: Aeshnidae), Orthetrumjuliajalsum (Anisoptera: Libellulidae) and the damselflies Pseudagrion inconspicuum and Ischnura senegalensis (Zygoptera: Coenagrionidae). After isolation ofthe peptides by reversed phase high performance liquid chromatography, the primary structures were established by Edman sequencing and mass spectrometry (Ani-AKH: pGlu-Val-Asn-Phe-Ser-Pro-Ser-TrpNH 2 ), (Lia-AKH: pGlu-Val-Asn-Phe¬ Thr-Pro-Ser- TrpNH 2 ) and (Psi-AKH: pGlu- Val-Asn-Phe- Thr-Pro-Gly- TrpNH 2 ). One corpus cardiac of A. imperator contains about 40 pmol Ani-AKH, O. julia 19-24 pmol Lia¬ AKH and P. inconspicuum about 2.4 pmol Psi-AKH. Injection of Ani-AKH (3.4 pmol) increased the concentration of haemolymph lipids in A. imperator. Lia-AKH (l pmol) similarly had an adipokinetic effect in 0. julia. Psi-AKH (I pmol) had an adipokinetic effect, as well as a small hyperglycaemic effect in P. inconspicuum. The AKH peptides of other Odonata were investigated. In the suborder Anisoptera, Ani-AKH was identified in representatives of the Aeshnidae, Cordulegasteridae, and possibly the Corduliidae. Lia¬ AKH was identified in representatives of the Libellulidae and Gomphidae. In the suborder Zygoptera, Psi-AKH was identified in representatives of the families Chlorolestidae, Lestidae and Chlorocyphidae, and possibly the Calopterygidae and Protoneuridae. Classification of Odonata according to their flight behaviour as "perchers" or "fliers" is supported by parameters of energy metabolism. Lipid metabolism seems to have a greater importance in fliers than perchers. The lipid concentration in the haemolymph is highest in the flier A. imperator, intermediate in the percher 0. julia and lowest in the percher P. inconspicuum. There are indications that mitochondria isolated from flight muscles of A. imperator may have a higher capacity for lipid oxidation than 0. julia. The contribution of carbohydrates to flight metabolism seems to be more important in perchers than in fliers. The concentration of carbohydrates in the haemolymph is highest in P. inconspicuum, intermediate in O. julia and lowest inA. imperator. The maximal activity of phosphofructokinase (a rate-limiting enzyme of glycolysis) is higher in the percher, O. julia, than in the flier, A. imperator. The lipid concentration in the haemolymph is higher than that of the carbohydrates in O. julia, A. imperator and P. inconspicuum. Palmitoyl-carnitine is oxidised at high rates by isolated mitochondria from flight muscles of O. julia andA. imperator, similar to Locusta migratoria. Lipid is the major fuel utilised during flight in O. julia. Carbohydrates (in the haemolymph) and proline (in the haemolymph and flight muscles) are utilised as minor fuels. It is concluded that the processes of lipid metabolism provide the major source of energy during flight in Odonata. The AKH peptides seem to play a role in regulating lipid mobilisation during flight in Odonata.

This thesis addresses the problem of how to examine a metabolic pathway and identify what are the key elements, specifically with respect to rate-limitation. The aim is to be able to analyze a pathway, identify the bottlenecks and implement genetic modifications to remove these bottlenecks. This is done by defining the system of interest and developing a predictive model using kinetic data. The model predictions can then be verified using fermentation data and genetic techniques to make the appropriate changes for improved performance. The test system chosen for this study was the TOL meta-cleavage pathway for the degradation of benzoate. This system was chosen on the basis of the application of pathway engineering principles to other systems. The modelling strategy and software was developed using principles from metabolic control theory and biochemical systems theory. By applying this to the TOL pathway using kinetic data, the control coefficients for the pathway were obtained as well as the system parameters required for the optimization of the pathway. The simulated results obtained from this model must be validated by experiment. Errors can arise both from incorrect assumptions in the model and from the fact that the kinetic data taken from individual in vitro experiments may not be applicable to the in vivo system. The effect of the presence of the TOL pathway on the behaviour of E.coli JM107 during fermentation was investigated and the transient concentration data necessary to identify the bottlenecks in the pathway measured. This data is then used to calculate the flux control coefficients for the TOL pathway. The predictive results were verified by the fermentation data which identified the first two enzymes in the pathway as having significant flux control coefficients. This final chapter also addresses the issue of flux analysis, that is, the calculation of the fluxes in the system to determine where fluxes to unwanted by-products occur and to indicate points of control. A graphical user interface is used to provide a user-friendly and intuitive means of building and customising metabolic pathways which can then be interfaced with instrumentation to provide on-line flux analysis.

Diameters of microvessels undergo continuous structural adaptation in response to hemodynamic and metabolic stimuli. To ensure adequate flow distribution, metabolic responses are needed to increase diameters of vessels feeding poorly perfused regions. Possible modes of metabolic control include release of signaling substances from vessel walls, from the supplied tissue and from red blood cells (RBC). Here, a theoretical model was used to compare the abilities of these metabolic control modes to provide adequate tissue oxygenation, and to generate blood flow velocities in agreement with experimental observations. Structural adaptation of vessel diameters was simulated for an observed mesenteric network structure in the rat with 576 vessel segments. For each mode of metabolic control, resulting distributions of oxygen and deviations between simulated and experimentally observed flow velocities were analyzed. It was found that wall-derived and tissue-derived growth signals released in response to low oxygen levels could ensure adequate oxygen supply, but RBC-derived signals caused inefficient oxygenation. Closest agreement between predicted and observed flow velocities was obtained with wall-derived growth signals proportional to vessel length. Adaptation in response to oxygen-independent release of a metabolic signal substance from vessel walls or the supplied tissue was also shown to be effective for ensuring tissue oxygenation due to a dilution effect if growth signal substances are released into the blood. The present results suggest that metabolic signals responsible for structural adaptation of microvessel diameters are derived from vessel walls or from perivascular tissue.

Sensitivity analysis studies how changes in the parameters affect the system's variables. Its application to metabolic systems (Metabolic Control Analysis, MCA) was traditionally developed under certain assumptions:i) the steady state is stable (the effect on the steady state values only is studied).ii) each reaction is catalyzed by one enzyme, the rates being proportional to the corresponding enzyme concentration.iii) the parameters are changed by a small (strictly speaking infinitesimal) amount. In the present work MCA is extended to deal with the instantaneous values of time-dependent metabolite concentrations and fluxes. Their summation and connectivity relationships are derived. In some cases it is more convenient to characterize the time courses by time-invariant variables (such as period and amplitude in oscillating systems). Summation relationships for time-invariant variables are also derived. Stability analysis shows that a linear chain of four enzyme-catalized reactions, where the third metabolite is a negative effector of the first enzyme constitutes a 'minimal' oscillator. The model is used to gain insight into the control of oscillations. The control exerted by enzyme concentrations and other parameters that are not proportional to the rate is appropriately described by parameter-unspecified coefficients (C_v). A proof of the theorems of steady-state MCA in terms of C_v is given. By a similar procedure an attempt is made to derive the theorems in terms of C_v for time-dependent systems, which is only successful for the particular case of constant π-matrix. The effect that a simultaneous change in all the enzyme concentrations by the same factor α (Coordinate-Control Operation. CCO) has on the variables of time-dependent metabolic systems is investigated. This factor α can have any arbitrary large value. The metabolic variables are classified according to the relationships they fulfil when the CCO is applied. A method is given to test these relationships in experimental systems and quantify deviations from the predicted behaviour.

Active insulin receptors (IR) phosphorylate insulin receptor substrate (IRS), but it is not clear whether IRS is phosphorylated mainly by IR at the plasma membrane or by internalized IR in the cytosol. In this thesis, structural identifiability analysis and parameter sensitivity analysis is performed for models of the first steps in the metabolic insulin signaling pathway. In particular, the identifiability of the kinetic parameters governing IRS phosphorylation are investigated.

Given measurements of the relative increase in phosphorylation degree of IR and IRS, the structural identifiability analysis revealed that the parameters governing IRS phosphorylation are non-identifiable, but their ratio is identifiable. This is sufficient to study whether phosphorylation of IRS proceeds more rapidly by IR at the plasma membrane or by internalized IR in the cytosol. In the examined model structure, internalization of insulin receptors is shown to be necessary to reproduce the experimental data.

Sensitivity analysis of the parameters governing IRS phosphorylation showed that many parameters need to be known in order to obtain ``practical identifiability'' of the interesting parameters.

Is it possible for a minimal model of anaerobic muscle contraction to describe measured data? There have been many models trying to describe separate parts of the human body with various results. In this thesis a model has been created to describe all the essential biochemical reactions of anaerobic muscle metabolism during contraction but with as few states and parameters as possible. A toolbox in Matlab was used for simulation and also for parameter estimation. The best model eventually got validated to see statistically how well it can describe the measured data. During the simulations an unnecessary assumption got revealed which helped us to understand the system better. The vision of a whole-body model may not be so far into the future as many think and the first step is to understand smaller biochemical systems like muscle contraction.

Myocardial and skeletal muscle high energy phosphate metabolism is abnormal in heart failure, but the pathophysiology is not understood. Plasma non-esterified fatty acids (NEFA) increase in heart failure due to increased sympathetic drive, and regulate the transcription of mitochondrial uncoupling protein-3 (UCP3), through peroxisome proliferator-activated receptor-α. The aim of the work in this thesis was to determine whether cardiac PCr/ATP ratios and skeletal muscle PCr kinetics during exercise were related to cardiac and skeletal muscle UCP3 levels respectively, thus providing a mechanism for the apparent mitochondrial dysfunction observed in heart failure. Patients having cardiac surgery underwent pre-operative testing, including cardiac and gastrocnemius 31P magnetic resonance spectroscopy. Intra-operatively, ventricular, atrial and skeletal muscle biopsies were taken for measurement of mitochondrial protein levels by immunoblotting, along with mitochondrial function by tissue respiration rates. Fasting plasma NEFA concentrations increased in patients with ventricular dysfunction and with New York Heart Association (NYHA) class. Ventricular UCP3 levels increased and cardiac PCr/ATP decreased with NYHA class, however, demonstrated no relationship to each other. In skeletal muscle, maximal rates of oxidative ATP synthesis (Qmax) related to functional capacity. Skeletal muscle UCP3 levels increased with NYHA class but were unrelated to skeletal muscle Qmax. Tissue respiration experiments revealed no relationship between ventricular function and indices of mitochondrial coupling, furthermore, indices of mitochondrial coupling were unrelated to tissue UCP3 levels. No evidence was found to support mitochondrial uncoupling, mediated through UCP3, as a cause of the abnormalities in cardiac and skeletal muscle high energy phosphate metabolism.

Introduzione. Le alterazioni metaboliche sono una caratteristica delle cellule tumorali, le quali devono sostenere un’attiva proliferazione senza incorrere in una carenza energetica. Pertanto, la ricerca di approcci metabolici che interferiscono con la crescita tumorale è attivamente perseguita. La maggior parte delle cellule tumorali è contraddistinta da un aumentato consumo di glucosio, necessario per rifornire il ciclo di Krebs di intermedi estratti a scopo biosintetico (anaplerosi). Tuttavia, a seconda delle mutazioni oncogeniche presenti nel fenotipo tumorale, altri nutrienti oltre al glucosio possono essere utilizzati a scopo anaplerotico. Tra essi spicca la glutamina (Gln), il più abbondante amminoacido nel sangue, precursore di altri aminoacidi, di nucleotidi e del glutatione nonché attivatore di mTOR. Tumori che utilizzano Gln a scopo anaplerotico hanno un aumentato fabbisogno di tale aminoacido e vengono perciò definiti “glutamine addicted”. Tuttavia non sono ancora stati identificati markers di “glutamine addiction” e, in particolare, il ruolo della Glutamina Sintetasi (GS), enzima chiave del metabolismo della Gln, non è ancora stato chiarito. L’espressione di GS è regolata a livello proteico dalla concentrazione intracellulare di Gln, ma, a livello trascrizionale, la sua espressione è dovuta all’oncogene β-catenina. Difatti l’iper-espressione di GS è un marker diagnostico di mutazione della β-catenina nel carcinoma epatocellulare (HCC), un tumore maligno del fegato con prognosi infausta. Pertanto l’espressione di GS causata dall’iperattivazione della β-catenina potrebbe indicare un metabolismo tumorale dipendente da Gln. Scopo della tesi. Lo scopo di questo studio è valutare gli effetti della deplezione di Gln in linee umane di HCC, per verificare l’ipotesi che iperespressione di GS dovuta a mutazioni di β-catenina possa essere un marker di “glutamine addiction”. Inoltre è stato valutato anche il ruolo di GS nell’adattamento alla deprivazione di Gln di altri due modelli cellulari umani, per ottenere una stima generale sul ruolo metabolico dell’enzima in condizioni di stress nutrizionale. Metodi. La deplezione di Gln è stata ottenuta combinando l’enzima batterico L-asparaginasi (ASNase), in uso nella terapia della leucemia linfoblastica acuta, con la metionina-L-sulfoximina (MSO), un inibitore irreversibile di GS. Sono stati valutati gli effetti di ASNase e MSO sulla vitalità cellulare e sulla attività di mTOR in linee tumorali umane di HCC mutate in β-catenina (in vitro e in xenografts in modelli murini), in due linee di oligodendroglioma umano e in cellule mesenchimali staminali (MSC) umane derivate dal midollo osseo di donatori. Risultati. Sia in vitro che in vivo, la deplezione di Gln ottenuta tramite ASNase e MSO, inibisce marcatamente la proliferazione delle linee cellulari mutate in β-catenina e positive per GS. Inoltre, mentre ASNase inibisce significativamente l’attività di mTOR, l’aggiunta di MSO, pur causando un’ulteriore diminuzione del contenuto intracellulare di Gln, determina un’attivazione paradossale della chinasi, suggerendo che l’inibitore di GS interagisca con dei sensori per aminoacidi a monte di mTOR. Nei modelli di oligodendroglioma umano GS-negativi ASNase causa una massiva morte cellulare, mentre l’aggiunta di MSO risulta inefficace. D’altra parte MSO previene l’adattamento delle MSC alla deplezione di Gln, suggerendo che GS possa contribuire alla funzione trofica, recentemente dimostrata, delle MSC nei confronti dei blasti leucemici. Conclusioni. I risultati ottenuti indicano un coinvolgimento delle mutazioni di β-catenina nel favorire un fenotipo neoplastico dipendente da Gln e dimostrano che la deplezione farmacologica di Gln rappresenta un approccio metabolico per la terapia dei tumori al fegato caratterizzati da mutazioni di β-catenina. Inoltre GS sembra necessaria per l’adattamento alla deplezione di Gln in tutti i modelli cellulari testati. Questi risultati suggeriscono che GS possa essere un mezzo diagnostico e/o un target terapeutico in determinati tipi di tumore.
Background. Deranged metabolism is a hallmark of cancer cells which need to sustain uncontrolled cell growth while maintaining a proper energy balance. Means to interfere with the cancer-associated metabolic alterations to control tumor cell proliferation are actively pursued. Most tumors rely on enhanced glucose consumption to supply Krebs cycle intermediates (a process called anaplerosis), which are continuously diverted to the macromolecular biosynthesis needed by proliferation. However, nutrients other than glucose may support anaplerosis in tumors, likely depending on the oncogenic mutations present in the particular type of cancer. Glutamine (Gln), the most abundant amino acid in blood, is a precursor of other amino acids (i.e. asparagine), nucleotides and glutathione, and stimulates mTOR. Tumors that rely on Gln instead of glucose for anaplerosis exhibit an enhanced requirement for the amino acid (“glutamine addiction”); however, reliable markers of “glutamine addiction” are still needed; in particular, the role of Glutamine Synthetase (GS), a key enzyme of Gln metabolism, has not been yet defined in this context. GS expression is known to be regulated at the protein level by intracellular Gln content, but, at transcriptional level, its expression is driven by the oncogene β-catenin. Consistently, its over-expression is a marker of β-catenin mutations in hepatocellular carcinoma (HCC), a liver cancer with poor prognosis. We have hypothesized that β-catenin-dependent GS expression may be a marker of a metabolic drift towards Gln-dependence and, hence, of Gln-addiction. Aim of the thesis. The purpose of this study is to investigate the effects of Gln depletion on human HCC cells so as to verify the hypothesis that β-catenin-dependent GS expression points to a Gln-addicted phenotype. In addition, the role of GS in the adaptation to Gln depletion of two other human cell models has been also assessed so as to achieve a general appraisal of the metabolic role of the enzyme under conditions of nutritional stress. Methods. Gln depletion was achieved with the bacterial enzyme Lasparaginase (ASNase), a drug employed in the treatment of acute lymphoblastic leukemia, and the irreversible GS inhibitor methionine-Lsulfoximine (MSO). We have evaluated ASNase and MSO effects on cell viability and mTOR activity in β-catenin-mutated HCC cell lines, in two GSnegative human oligodendroglioma cells, and in bone marrow mesenchymal stem cells (MSCs). Moreover, ASNase and MSO effects were also assessed on HCC xenograft models. Results. Gln depletion, obtained with ASNase and MSO, markedly hinders the proliferation of β-catenin-mutated, GS-positive, HCC cells in vitro and in vivo. The determination of mTOR activity in Gln-depleted HCC cells indicated that ASNase markedly inhibits the kinase, while MSO caused its paradoxical activation, suggesting that the GS inhibitor may deceive the intracellular amino acid sensors that regulate mTOR. ASNase causes massive cells death in GS-negative human oligodendroglioma cell lines, where MSO is ineffective. On the other hand, MSO prevents the adaptation of MSCs to Gln-depletion, suggesting that GS activity may contribute to the recently described trophic function of MSCs towards leukemic blasts. Conclusions. These results indicate a role of β-catenin mutations in promoting Gln addiction in HCC and suggest that pharmacological Gln depletion represents a metabolic approach for the therapy of β-catenin-mutated liver cancers. Moreover, GS activity seems crucial for adaptation to Gln shortage, not only in HCC cells, but also in MSCs and oligodendroglioma cells. These evidences suggest that GS constitute a useful diagnostic tool and/or therapeutic target in selected types of human tumors.

2015 - 2016
Regulatory CD4+CD25+ T (Treg) cells play a central role in the maintenance of immune self-tolerance and homeostasis. Although Treg cells operate through multiple mechanisms, it appears that the expression of the transcription factor Forkhead-box-P3 (FoxP3) is crucial for their function. Here we describe human peripheral Treg (pTreg) cells that develop from CD4+CD25- T (Tconv) cells following suboptimal stimulation via the T cell antigen receptor (TCR). This population of pTreg cells, which we call inducible Treg (iTreg) cells, is characterized by high FoxP3 expression, strong suppressive capacity and an active proliferative and metabolic state. The development of iTreg cells tightly depends on glycolysis, which controls FoxP3 splicing variants containing exon 2 (FoxP3-E2), through the glycolytic enzyme enolase-1. Remarkably, iTreg cells suppressive activity is impaired in autoimmune diseases such as relapsing remitting multiple sclerosis (RR-MS), and associates with the reduction of FoxP3-E2 expression, secondarily to impaired glycolysis and IL-2/IL-2R/STAT-5 signalling. These results suggest a novel mechanism that links glucose metabolism to the induction of specific FoxP3 splicing variants, via enolase-1, that directly impact on human Treg cell function, in health and in autoimmunity. [edited by author]
Le cellule T regolatorie CD4+CD25+ (Treg) svolgono un ruolo centrale nel mantenimento dell’omeostasi e della tolleranza immunitaria. Sebbene le cellule Treg operino attraverso diversi meccanismi, sembra che l'espressione del fattore di trascrizione Forkhead-box-P3 (FoxP3) è fondamentale per la loro funzione. Qui descriviamo le cellule Treg periferiche (pTreg) umane che si sviluppano dalle cellule T CD4+CD25- (Tconv) dopo stimolazione subottimale del recettore delle cellule T (TCR). Questa popolazione di cellule pTreg, chiamata cellule Treg indotte (iTreg), è caratterizzata da un'elevata espressione di FoxP3, da una forte capacità soppressoria e da uno stato proliferativo e metabolico attivo. Lo sviluppo delle cellule iTreg dipende fortemente dalla glicolisi, che controlla le varianti di splicing di FoxP3 contenenti l'esone 2 (FoxP3-E2), attraverso l'enzima glicolico enolasi-1. In particolare, l’attività soppressoria delle cellule iTreg è compromessa nelle malattie autoimmuni come la sclerosi multipla recidivante-remittente (RR-MS) e si associa alla riduzione dell'espressione di FoxP3-E2, secondariamente alla compromissione della glicolisi e della via di segnalazione IL-2 / IL-2R / STAT-5. Questi risultati suggeriscono un nuovo meccanismo che collega il metabolismo del glucosio all'induzione di specifiche varianti di splicing di FoxP3, attraverso l'enolasi-1, che ha un impatto diretto sulla funzionalità delle cellule Treg, sia in condizioni fisiologiche che in corso di autoimmunità. [a cura dell'autore]
XXIX n.s.

2012 - 2013
Obesity is a chronic disorder characterized by a tonic low-grade activation of the innate immune system that affects steady-state measures of metabolic homeostasis over time. In addition, obesity is often accompanied by elevations in tissue and circulating FFA concentrations. Systemic levels of FFAs can induce inflammatory cascades in adipocytes and macrophages through TLR4-dependent effect. Signaling through TLR4 activates a broad range of intracellular cascades that include stimulation of IKK-β, NF-kB, JNK and AP1. Indeed, in addition to store excess calories in the form of lipid, adipose tissue produces classical cytokines and chemokines such as MCP-1, IL-8 and CCL5. CCL5, as other chemokines, participates in mediating leukocyte infiltration of adipose tissue. Moreover circulating CCL5 concentrations are elevated in obesity, impaired glucose tolerance (IGT) and type 2 diabetes. In this study I have investigated the molecular mechanisms involved in the metabolic control of CCL5 expression in adipocytes. Cytokine/growth factor screening of conditioned media from 3T3-L1 preadipocytes and adipocytes revealed that adipocytes secreted higher amount of CCL5 compared to their undifferentiated precursors. Higher concentrations of glucose and fatty acids (oleate and palmitate) increased CCL5 secretion by 3T3-L1 adipocytes. Moreover, both oleate and palmitate enhanced CCL5 mRNA levels and induced an activation of JNK, NF-kB, MAPK and PI3K/AKT pathways. In cells treated with JSH23, a NF-kB inhibitor, the effect of FFAs on CCL5 mRNA levels was reduced thus indicating a direct involvement of NF-kB. Treatment of the cells with SP600125, a JNK inhibitor, also significantly reduced the stimulatory effect of oleate and palmitate on CCL5 mRNA and interestingly prevented FFA-induced NF-kB binding to CCL5 promoter. I have also obtained evidence that insulin exerted an inhibitory effect on CCL5 mRNA and counteracted fatty acid-induced stimulation. Both PD98059 and LY294002, inhibitors of MAPK and PI3K, respectively, increased CCL5 expression levels reverted anti-inflammatory effect of insulin in presence of fatty acids. Consistently, insulin exposure reduced NF-kB recruitment onto CCL5 promoter, and almost completely prevented fatty acid effect. In conclusion, oleate and palmitate induce CCL5 mRNA, possibly via JNK and NF-kB pathways. Fatty acid effect on CCL5 is largely prevented by insulin and may involve PI3K/AKT and MAPK. [edited by author]
XII n.s.

Nuclear receptors play crucial roles in the transcriptional regulation of many biological processes such as development and cellular differentiation. ERRalpha is known, along with coactivator PGC-1alpha, to playa central role in the control of energy metabolism in cardiac and skeletal muscle. They activate the expression of many genes involved in mitochondrial oxidative metabolism. Here we identified PROX1, a factor that was previously shown to broadly influence metabolism, as a regulator of this pathway. Indeed, PROX1 interacts in vitro and in vivo with both ERRalpha and PGC-1alpha. To provide more insight on the hepatic functions of ERRalpha and PROX1, we performed ChIP-on-chip using mouse liver, identifying a large number of ERRalpha and PROX1 genomic targets and reinforcing their role in energy metabolism. Over 40% of the target genes were found to be common to both factors and we observed that PROX1 could be recruited to ERRalpha binding sites and act as a negative regulator o fthe ERRalpha/POC-1alpha pathway.

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Heart transplantation (HT) represents one of the greatest advances in medicine over the last decades. It is indicated for patients with severe heart disease unresponsive to clinical treatment and conventional surgery, poor short-term prognosis and a 1- year mortality rate over 40%. HT has improved survival worldwide (80% in the first year, 70% in five years and 60% in ten years). However, the procedure has been associated with weight change and increased risk of secondary conditions such as diabetes, hypertension, dyslipidemia and obesity due to immunosuppressive therapy following transplantation. The objective of this study was to determine the impact of weight change on the metabolic stability of HT patients. The study was retrospective with data collected from the records of 82 adult patients (83% male; average age 45.06?12.04 years) submitted to HT between October 1997 and December 2005 at a transplantation service in Cear? (Brazil). The selected outcome variables (biopathological profile, weight and body mass index―BMI) were related to biochemical and metabolic change. The results were expressed in terms of frequency, measures of central tendency, Student s t test and Pearson s correlation coefficients. The analysis showed that following HT the average global BMI increased from 23.77?3.68kg/m2 to 25.48?3.92kg/m2 in the first year and to 28.38?4.97kg/m2 in the fifth. Overweight/obese patients (BMI ≥ 25 kg/m2) had higher average levels of glucose, total cholesterol, low-density lipoprotein and triglycerides than patients with eutrophy/malnutrition (BMI < 25 kg/m2). In conclusion, overweight/obese patients were likely to present higher average levels of glucose, triglycerides, total cholesterol and fractions than patients with eutrophy/malnutrition, indicating a direct and significant relation between nutritional status and weight change in the metabolic profile of HT patients
O Transplante Card?aco (TC) tornou-se um dos grandes avan?os da medicina nas ?ltimas d?cadas. ? um procedimento indicado para pacientes com doen?a card?aca avan?ada, refrat?ria ao tratamento cl?nico e cir?rgico convencional, progn?stico reservado em curto prazo e mortalidade acima de 40% no prazo de um ano na evolu??o natural da doen?a. Em todo o mundo seus resultados t?m evidenciado melhora significante na sobrevida, sendo considerada de 80% no primeiro ano, 70% em cinco anos e 60% em dez anos. No entanto, as altera??es de peso ap?s o procedimento frequentemente ocorrem e aumentam os riscos de doen?as secund?rias como diabetes, hipertens?o, dislipidemia e obesidade, complica??es que est?o associadas ? terapia imunossupressora indispens?vel ap?s o TC. O objetivo deste estudo foi determinar o impacto da variabilidade de peso na estabilidade metab?lica de pacientes transplantados do cora??o. O desenho do estudo foi do tipo retrospectivo documental, realizado com 82 pacientes adultos submetidos ao TC entre outubro de 1997 a dezembro de 2005 em centro transplantador no Cear?, sendo 83% do sexo masculino e 17% do sexo feminino com idade m?dia de 45,06?12,04 anos. As vari?veis estudadas foram o perfil biopatol?gico, o peso e o ?ndice de massa corporal (IMC), relacionadas ?s altera??es bioqu?micas-metab?licas. Os dados foram descritos usando frequ?ncias, medidas de tend?ncia central, teste t de Student e coeficiente de correla??o de Pearson. Ap?s a an?lise dos dados, verificou-se que a m?dia global do IMC aumentou de 23,77?3,68 kg/m2 antes do TC, para 25,48?3,92 kg/m2 no primeiro ano e para 28,38?4,97 kg/m2 no quinto. Os pacientes com sobrepeso/ obesidade (IMC ≥25 kg/m2) apresentaram valores m?dios de glicose, colesterol total, lipoprote?na de baixa XIV densidade (LDL) e triglic?rides maiores que os pacientes com eutrofia/ desnutri??o (IMC < 25 kg/m2). Diante dos resultados encontrados nesse estudo, conclui-se que os pacientes com sobrepeso/ obesidade est?o propensos a apresentar n?veis de glicose, colesterol total, LDL e triglic?rides mais elevados que os pacientes com eutrofia/ desnutri??o, o que demonstra que houve uma rela??o direta e significativa entre o estado nutricional e a variabilidade de peso no perfil metab?lico de pacientes transplantados card?acos

Patients with insulin-dependent diabetes mellitus (IDDM) must adhere to a complex treatment regimen. Adherence is often poor during adolescence, when the regimen behaviours may interact negatively with many of the developmental tasks. In a randomized controlled outcome study, 15 adolescents and their families participated in a family-based intervention for improving adherence to the diabetic regimen. Fourteen comparison group adolescents with IDDM spent a comparable amount of time learning stress management techniques. Adolescents in both groups attended a diabetes management review session with the clinic nurse. Inclusion criteria included mean glycosylated hemoglobin (HbAlc) levels $>$9.0% over the previous nine months. At a 6-month follow-up, adolescents in the family intervention group were testing blood glucose levels more regularly (p $$1% (0% vs. 29%, $\chi\sp2$ = 6.32, p =.05). Methodological issues were discussed.

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Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil
A leishmaniose é uma antropozoonose causada por protozoários do gênero Leishmania e é considerada uma das principais doenças negligenciadas. Modelos experimentais são amplamente utilizados para uma melhor compreensão da doença e dos mecanismos relacionados à resistência e susceptibilidade à infecção. Macrófagos de camundongos CBA controlam a infecção por Leishmania major ao passo que são permissivos a Leishmania amazonensis. Além disso, estudos baseados em abordagem proteômica demonstraram padrões distintos de expressão proteica em macrófagos derivados de medula óssea (BMMΦ) infectados por essas espécies de Leishmania. Dentre as proteínas diferentemente expressas, foram identificadas proteínas envolvidas no metabolismo de ferro moduladas positivamente em macrófagos infectados por L. amazonensis. Adicionalmente, embora ainda existam controvérsias, diversos estudos têm abordado a participação do elemento ferro na interação parasito-hospedeiro e no estabelecimento das infecções por tripanossomatídeos, incluindo Leishmania. Assim, para melhor compreender os mecanismos envolvidos nessa doença, o presente estudo buscou explorar o modelo comparativo de resistência e suscetibilidade do camundongo CBA para determinar o papel do ferro na infecção por Leishmania. Nossa hipótese é que a expressão de proteínas envolvidas no metabolismo de ferro é modulada diferentemente em macrófagos de camundongos CBA infectados por L. amazonensis, em comparação à L. major, favorecendo a sobrevivência intracelular do parasito. Nosso objetivo foi avaliar a expressão de proteínas que participam do metabolismo de ferro, como receptor de transferrina (Tf), CD71, e heme oxigenasse-1, HO-1, e determinar o efeito da modulação da disponibilidade de ferro na infecção por Leishmania. Observamos maior expressão de HO-1 em BMMΦ infectados por L. amazonensis (18,34 ± SD ng/mL), quando comparados a BMMΦ infectados por L. major (7,07 ± SD ng/mL), utilizando ELISA. Maior expressão de CD71 também foi observada na infecção por L. amazonensis (MFI 2.103) em comparação à infecção por L. major (MFI 472), utilizando FACS, além de uma maior ligação e captação de HoloTf (Tf carregada com ferro). Embora tenha sido observado que essas proteínas encontram-se diferentemente expressas em BMMΦ infectados por essas duas espécies de Leishmania, não foram observadas diferenças significativas na concentração intracelular do ferro. Em seguida, ensaios funcionais a partir da modulação da disponibilidade intracelular de ferro foram realizados com o objetivo de avaliar seu papel no desfecho da infecção por Leishmania. Os resultados mostraram que a depleção de ferro reduz em 90% o percentual de BMMΦ infectados por L. amazonensis e 70% dos infectados por L. major. Adicionalmente, a suplementação de ferro aumenta o percentual de BMMΦ infectados por L. amazonensis, de 69,64 para 82,79%, e a carga parasitária, de 2,996 para 4,001 parasitos/célula, assim como a viabilidade intracelular de L. amazonensis e L. major. Em conjunto, os dados obtidos nesse estudo indicam que, apesar de L. amazonensis modular positivamente a expressão de proteínas envolvidas no metabolismo de ferro, esse metal apresenta um papel importante na infecção pelas duas espécies de Leishmania, favorecendo a sobrevivência intracelular desse parasito.
Leishmaniasis is an anthropozoonosis caused by the protozoan parasite Leishmania and is considered one of the main neglected diseases. Animal models are widely used to better understand the disease and the mechanisms involved in resistance and susceptibility to infection. CBA mouse macrophages control the infection by L. major, while are permissive to L. amazonensis. Proteomic studies showed different protein profiles in bone marrow macrophages (BMMΦ) infected these species of Leishmania. We also observed that proteins involved in iron metabolism were positively modulated in L. amazonensis-infected macrophages. In addition, although literature review showed controverse data, several studies have addressed the role iron plays in host-parasite interaction and the establishment of trypanosomatids infections, including Leishmania. To better understand the mechanisms of the disease, this study sought to evaluate in a comparative model of resistance and susceptibility, using CBA macrophages, the role iron plays in Leishmania infection. Our hypothesis is that the expression of proteins involved in iron metabolism is differently modulated in CBA mice macrophages infected with L. amazonensis in comparison to L. major, favoring the intracellular survival of the parasite. Our goal was to evaluate the expression of proteins involved in iron metabolism of CBA mice macrophages, such as transferrin receptor (Tf), CD71, and heme oxygenase 1 (HO-1) and determine the effect of the modulation of intracellular iron in Leishmania infection. Using ELISA, we confirmed a higher expression of HO-1 in L. amazonensis- (18.34 ng/mL) compared to L. major-infected CBA macrophages (7.07 ng/mL). Using FACS analysis, CD71 showed to be higher expressed in L. amazonensis- (MFI 2.103) than in L. major-infected macrophages (MFI 472), in addition to higher binding and take up of HoloTf in these cells. Although it has been observed that proteins involved in iron metabolism were differently expressed in BMMΦ infected with these Leishmania species, no significant differences were observed in intracellular iron concentration. To further evaluate the role iron plays in the outcome of Leishmania infection, we modulated iron availability to Leishmania-infected cells using iron chelates or iron supplements. The results show that iron depletion reduces in 90% L. amazonensis infection and in 70% L. major infection. In addition, iron supplementation increased the percentage of L. amazonensis-infected cells from 69.64 to 82.79% and parasite load from 2,996 to 4,001 Leishmania/cell, as well as in the intracellular viability of both Leishmania species. In sum, these data indicate that although there is a positive modulation of proteins involved in iron metabolism in L. amazonensis infection, this metal seems to favor the survival of both parasite species in CBA macrophages.

El principal objectiu d'aquest treball ha estat estudiar la producció de metabòlits amb activitat antibiòtica per soques de l'espècie Pseudomonas fluorescens de la col·lecció EPS, i també avaluar la seva potencialitat com a agents de biocontrol. Es va disposar també de diverses soques de P. fluorescens, cedides per altres investigadors, que van utilitzar-se com a referència perquè algunes són actives en control biològic i produeixen metabòlits secundaris d'interès en el biocontrol de malalties de plantes.
La present memòria s'estructura en cinc capítols, que són, introducció al control biològic, descripció de l'etapa de selecció de soques i cerca dels metabòlits produïts, estudi de la producció d'HCN per la soca EPS288, estudi de la producció de l'antibiòtic 2,4-diacetilfloroglucinol (DAPG), i finalment, el darrer capítol, on s'ha estudiat la producció de DAPG sobre material vegetal i la capacitat de colonitzar arrels per diverses soques d'interès.
En l'etapa de prospecció, va demostrar-se que un 37% del total de les soques de la col·lecció EPS produïen HCN, totes de l'espècie P. fluorescens, i un 90% d'aquestes provenien de les arrels de plantes. Es va confirmar la producció dels metabòlits secundaris 2,4-diacetilfloroglucinol, àcid fenazina-1-carboxílic, i pirrolnitrina, per diverses soques de la col·lecció EPS seleccionades mitjançant tècniques moleculars. Així, de la col·lecció EPS, les soques EPS317 i EPS808 produeixen DAPG, la EPS263 àcid fenazina-1-carboxílic i pirrolnitrina i, EPS894, EPS895, EPS945 produeixen àcid fenazina-1-carboxílic.
La producció d'HCN es va estudiar més exhaustivament en la soca EPS288, seleccionada per la seva activitat antifúngica i candidata a agent de biocontrol contra Stemphylium vesicarium, causant de la estemfiliosi de la perera, i contra Penicillium expansum, causant de la podridura blava en conservació de fruita a postcollita. Per aquest estudi, es va dissenyar i validar un sistema per recollir l'HCN a partir de cultius en medi líquid. S'ha demostrat que la temperatura d'incubació, la concentració cel·lular de sembra i la composició del medi de cultiu afectaven a la producció d'HCN. Els medis complexos i la glicina n'afavorien la síntesi i la font de carboni no afectava. La soca EPS288 va produir més HCN que P. fluorescens CHA0, soca de referència productora d'HCN i descrita com a activa en processos de biocontrol de fongs fitopatògens.
En l'estudi de la producció de DAPG, les soques de la col·lecció EPS i de referència, es van comparar en diversos medis de cultiu estudiant l'efecte de la complexitat i consistència del medi, i l'addició de ferro o de glucosa. Va demostrar-se que la producció de DAPG depèn principalment de la soca i de les característiques del medi de cultiu. La glucosa estimula la producció, mentre que el ferro pràcticament no afecta, i en general, el medi sòlid i complex estimula la producció de DAPG. Tanmateix, aquests efectes varien en alguna de les soques assajades donant lloc a comportaments singulars.
En el seguiment del creixement amb un sistema automàtic es va comprovar que la velocitat específica de creixement i la concentració cel·lular assolida al final del cultiu, estaven condicionades per la composició del medi de cultiu.
En les proves d'antagonisme vers fitopatògens que foren seleccionats com a indicadors, va observar-se que tant l'antagonisme in vitro com la inhibició d'infeccions sobre material vegetal estaven parcialment relacionades amb la producció dels metabòlits secundaris estudiats. La promoció del creixement de portaempelts per aquestes soques depenia de la soca i de l'hoste, però no es pogué establir una relació causa-efecte amb el metabòlits produïts. També es va comprovar que algunes de les soques podien sobreviure en ferides de pomes i de peres, on produïren DAPG.
Mutants resistents a rifampicina de diverses soques de la col·lecció EPS i de referència es van inocular en llavors de pomera i de tomatera que es van sembrar i incubar en condicions controlades. Es va fer el seguiment de la població bacteriana total i resistent a rifampicina present a les arrels durant 72 dies. Totes les soques van colonitzar les arrels de les plantes, mantenint una elevada població durant 37 dies, cap d'elles va estimular el creixement ni mostrar efectes fitotòxics, no afectant tampoc signicativament a la població bacteriana espontània de les arrels.
La soca EPS808, una de les seleccionades pel treball, va aconseguir uns nivells de producció de DAPG, una velocitat de creixement i una supervivència relativa a les arrels similar a altres soques de referència descrites com a bons agents de biocontrol. En conseqüència, se la considera una candidata a agent de biocontrol que hauria de ser objecte de futurs estudis d'eficàcia.
El principal objetivo de este trabajo ha sido estudiar la producción de metabolitos con actividad antibiótica por cepas de la especie Pseudomonas fluorescens de la colección EPS, y también evaluar su potencialidad como agentes de biocontrol. Se dispuso de varias cepas de P. fluorescens, cedidas por otros investigadores, que se utilizaron como referencia ya que algunas de estas son activas en el control biológico, y producen metabolitos secundarios de interés para el biocontrol de enfermedades de plantas.
La presente memoria se estructura en cinco capítulos, revisión bibliográfica del control biológico, descripción de la etapa de selección de cepas y de los metabolitos producidos, estudio de la producción de HCN por la cepa EPS288, estudio de la producción de 2,4-diacetilfloroglucinol(DAPG), y finalmente, el último capítulo, donde se ha estudiado la producción de DAPG sobre material vegetal y la capacidad de colonizar las raíces de diversas plantas.
En la etapa de prospección se demostró que el 37% del total de las cepas de la colección EPS producían HCN, todas ellas de la especie P. fluorescens, de las cuales el 90% había sido aislada de las raíces de plantas. Se confirmó la producción de los metabolitos secundarios 2,4-diacetilfloroglucinol, ácido fenazina-1-carboxílico y pirrolnitrina, por diversas cepas de la colección EPS seleccionadas mediante técnicas moleculares. De la colección EPS las cepas EPS317 y EPS808 producen DAPG, la EPS263 ácido fenazina-1-carboxílico y pirrolnitrina y las cepas EPS894, EPS895 y EPS945 producen ácido fenazina-1-carboxílico.
La producción de HCN se estudió en detalle por la cepa EPS288, esta había sido seleccionada por su actividad antifúngica y es candidata a agente de biocontrol contra Stemphylium vesicarium, causante de la estemfiliosis del peral, y contra Penicillium expansum, causante de la enfermedad del moho azul en la conservación de la fruta en poscosecha. Para este estudio se diseñó y se validó un sistema para la recogida del HCN que se desprende de cultivos en medio líquido. La temperatura de incubación, la concentración en la siembra y la composición del medio de cultivo afectaban a la producción de HCN. Los medios de cultivo complejos y la glicina estimulaban su síntesis, mientras que la fuente de carbono no afectaba. La cepa EPS288 produjo más HCN que la P. fluorescens CHA0, cepa de referencia productora de HCN y descrita como activa en procesos de control biológico de hongos fitopatógenos.
En el estudio de la producción de DAPG, las cepas de la colección EPS y de referencia, se compararon en diferentes medios de cultivo estudiando el efecto de la complejidad y la consistencia del medio de cultivo, y de la adición de hierro o de glucosa. Se demostró que la producción de DAPG depende principalmente de la cepa y de la composición del medio de cultivo. La glucosa estimula la producción, mientras que el hierro casi no tiene efecto. Sin embargo, estos efectos varían en alguna de las cepas ensayadas dando lugar a comportamientos singulares.
El seguimiento del crecimiento mediante un sistema automático permitió verificar que la velocidad específica de crecimiento y la concentración celular en la fase estacionaria estaban condicionadas por la composición del medio de cultivo.
En las pruebas de antagonismo frente a hongos fitopatógenos seleccionados como indicadores, se observó que el antagonismo in vitro y la inhibición de infecciones sobre material vegetal correlacionaban parcialmente con la producción de los metabolitos secundarios estudiados. La promoción del crecimiento de portainjertos con esas cepas era función del huésped y de la cepa. Se comprobó que algunas de las cepas sobrevivían en heridas de manzanas y de peras dónde sintetizaban DAPG.
Mutantes resistentes a rifampicina de diversas cepas de la colección EPS y de referencia se inocularon en semillas de manzano y de tomate las cuales se sembraron e incubaron en ambiente controlado. La población bacteriana en las raíces de las plantas se monitorizó durante un periodo de 72 días. Todas las cepas colonizaron las raíces de las plantas manteniendo una elevada población durante, al menos, 37 días. Ninguna de la cepas estimuló el crecimiento ni mostró fitotoxicidad, y no afectaron significativamente a la población bacteriana espontánea de las raíces.
La cepa EPS808, una de las seleccionadas para el presente trabajo, demostró una producción de DAPG, velocidad específica de crecimiento y una supervivencia relativa en las raíces, similares a otras cepas de referencia descritas como buenos agentes de biocontrol. Por lo tanto, se considera a esta cepa como una candidata a agente de biocontrol, que tendría que ser objeto de futuros estudios de eficacia.
The main objective of this work is to study the production of biologically active secondary metabolites produced by Pseudomonas fluorescens strains from EPS collection. Moreover, their potential as biocontrol agents was also evaluated. We used P. fluorescens strains, provided by foreign scientists, that were utilised as a reference because these strains are active as biocontrol agents and produce secondary metabolites involved in biological control.
The present work is divided into five chapters which deal with an introduction to biological control, the selection of strains, the detection and identification of metabolites produced by these bacteria, the study of HCN production by EPS288 strain, the study of 2,4-diacetylphloroglucinol (DAPG) production, and finally, the DAPG production based on fruits and plants, and root colonisation by selected strains.
It was shown that 37% of isolates in EPS collection produced cyanide, all were P. fluorescens, and 90% of them were isolated at roots of different plants. The secondary metabolites 2,4-diacetilphloroglucinol (DAPG), phenazine-1-carboxilic acid (PCA) and pyrrolnitrin were identified and its production by some EPS collection strains, selected with molecular techniques, was demonstrated. The strains EPS317 and EPS808 produce DAPG, EPS263 produces phenazine-1-carboxilic acid and pyrrolnitrin, and the strains EPS894, EPS895, EPS945 produce phenazine-1-carboxilic acid.
The production of cyanide acid by P. fluorescens EPS288 was studied more exhaustively. This strain was selected as a biocontrol agent due to its antifungal activity against Stemphylium vesicarium, that causes brown spot on pear, and Penicillium expansum, that causes the blue mould of fruits during postharvest. A device to collect cyanide produced by bacteria from broth cultures was set up and validated. The incubation temperature, the initial cell density and the composition of growth media clearly affected HCN production by EPS288 strain. Complex media and glycine stimulated HCN production, however, it was not affected by carbon source in defined growth media. The EPS288 strain produced more cyanide in Castric growth media than P. fluorescens CHA0, a reference strain employed in biological control of fungal pathogens.
The reference strains and those from EPS collection which produced DAPG were compared considering the factors complexity and consistence of growth media. The effect of iron and glucose addition were also evaluated. The strain and growth media characteristics were the predominant factors in the DAPG production. Glucose increased DAPG production, while iron addition had no considerable effect, and as general rule, solid and complex media stimulated DAPG production. However these effects are variable depending on the strain.
The monitoring of bacterial growth and DAPG production with an automated absorbance reader showed that the growth rate and cellular yield were affected by growth media.
In antagonism tests against selected fungal pathogens, the in vitro inhibition and inhibition of infections over organic material were, in part, correlated with secondary metabolite production. The rootstock growth promotion caused by some of these strains was dependent on the strain and the host, but a cause-effect relationship with antibiotics produced could not be clearly established. Some strains were able to colonise and produce DAPG in wounds on apples and pears.
Rifampicine resistant spontaneous mutants of the reference and of EPS collection strains were isolated and inoculated on apple and tomato seeds, which were sown and grown under a controlled conditions. The evolution of inoculated strains and the whole bacterial population at the roots were monitored during 72 days. All selected strains efficiently colonised plant roots at high levels for at least 37 days, and nor phytotoxic either plant growth promotion was observed by any strain. Moreover, they did not significantly affected the spontaneous bacterial population at roots.
The EPS808 strain exhibited a 2,4-diacetilphloroglucinol production level, a specific growth rate and a relative root population as high as the reference strains that are referenced to be good biocontrol agents. Thus we conclude that this strain can be considered as a candidate for biological control agent, but further efficacy studies are required.

The main aim of this thesis was to increase our understanding of the metabolic responses associated with short-term high-fat overfeeding. To this end, four separate studies are described in this thesis; each of which involved the provision of a high-fat, high-energy diet to young, healthy, lean individuals. The first of these experimental chapters (Chapter 2) determined the effects of a 7-day, high-fat (65%), high-energy (+50%) diet on postprandial metabolic and endocrine responses to a mixed meal challenge. This chapter demonstrates that 7-days of overfeeding impaired glycaemic control in our subject cohort but did not influence the response of selected gut hormones (acylated ghrelin, GLP-1 and GIP). In a mechanistic follow up study utilising stable isotope tracer methodology we then demonstrate that overfeeding-induced impairments in glycaemic control are attributable to subtle alterations in plasma glucose flux, rather than the overt tissue-specific adaptations (e.g. increased EGP, or reduced glucose disposal) that have previously been reported (Chapter 3). In an attempt to delineate the time-course of diet-induced impairments in glycaemic control, we then investigated the effects of 1-day of overfeeding (+80% energy with 73% of total energy coming as fat) (Chapter 4). Results demonstrate that a single day of overfeeding elicits responses which are comparable to 7-days of high-fat overfeeding; highlighting the rapidity with which excessive high-fat food intake can negatively influence glucose metabolism. In chapter 5 we utilised stable isotope tracer and muscle biopsy techniques to demonstrate that 7-days of high-fat overfeeding impairs glycaemic control but does not influence the fed-state mixed muscle protein fractional synthesis rate (FSR). In conclusion, the findings of this thesis demonstrate that while short-term high-fat overfeeding negatively influences whole-body glucose metabolism, skeletal muscle protein metabolism appears to be relatively unaffected in young, lean, healthy humans.

The detection of endogenous anabolic androgenic steroids (EAAS) is one of the most difficult analytical challenges in the doping control field. The main problem for their detection is to distinguish between normally endogenous concentrations and those observed after the exogenous administration of an EAAS. The screening methods for EAAS are currently based on the determination of the steroid profile and the application of the athlete biological passport. The inclusion of new steroid metabolites can improve the screening capabilities of the steroid profile. Thus, the objective of the thesis is to elucidate and characterize new testosterone metabolites that can be implemented to the current steroid profile and to evaluate their usefulness for doping control analysis. Four unreported testosterone metabolites were detected and characterized by using liquid chromatography coupled to tandem mass spectrometry approaches. These compounds were demonstrated to come from degradation of cysteine conjugates. The formation of these conjugates implies an addition of a double bond as a phase I metabolism followed by conjugation with glutathione and the subsequent transformation to cysteine conjugates in urine. In order to determinate the usefulness of the cysteinyl compounds for doping control purposes, a quantitative method for the indirect determination of these compounds was developed and validated. Using this method, reference population limits were established by the analysis of 174 urine samples. Additionally, different factors that can potentially influence the excretion of these compounds were evaluated. Finally, the usefulness of these cysteinyl metabolites for the detection of EAAS misuse was evaluated by the analysis of samples collected after different EAAS administration. The use of these metabolites seems to improve in some cases the detection capabilities of the current marker used in routine analysis.
La detecció d’esteroides androgènics anabolitzants endògens (EAAE) és un dels reptes analítics més difícils en la lluita contra el dopatge. El problema més important per a la seva detecció és distingir entre concentracions endògenes i aquelles que s’observen després de l’administració exògena d’un EAAE. Els mètodes de cribatge per a la detecció d’EAAE estan basats en la determinació del perfil esteroïdal i la introducció d’aquest en el passaport biològic de l’atleta. La inclusió de nous metabòlits d’esteroides pot ajudar a millorar les capacitats de cribatge del perfil esteroïdal. Per tant, l’objectiu d’aquesta tesis és detectar i caracteritzar nous metabòlits d’EAAE que puguin implementar-se en l’actual perfil esteroïdal i l’avaluació de la seva utilitat en la lluita contra el dopatge. Quatre metabòlits desconeguts de la testosterona van ser detectats i caracteritzats mitjançant la utilització de la cromatografia líquida acoblada a l’espectrometria de masses en tàndem. L’origen d’aquests compostos es va demostrar que provenia de la degradació de conjugats amb cisteïna. La formació d’aquests conjugats implica l’addició d’un doble enllaç com a reacció metabòlica de fase I acompanyat per la conjugació amb glutationa i la subseqüent degradació d’aquesta a cisteïna en orina. Per tal de poder veure la seva aplicació en el camp del dopatge, es va desenvolupar i validar un mètode per la quantificació indirecta d’aquests compostos en orina. Utilitzant aquest mètode es van establir límits de referència basats en l’anàlisi de 174 mostres de orina. Addicionalment, diferents factors descrits que poden afectar l’excreció en orina d’aquests compostos també van ser estudiats en detall. Finalment, es va avaluar la utilitat d’aquests metabòlits conjugats amb cisteïna per a la detecció de l’abús d’EAAE mitjançant l’ anàlisis de mostres després de l’administració de diferents EAAE. L’ús d’aquests metabòlits va millorar (en alguns casos) els temps de detecció comparant-los amb els actuals marcadors utilitzats en rutina.

Thesis (Ph. D.)--University of California, San Diego, 2010.
Title from first page of PDF file (viewed May 28, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (leaves 142-151).

Problems associated with fuel preference, metabolic capacity and regulation were addressed using isolated mitochondria and enzyme analyses of cardiac and skeletal muscles of spiny dogfish (Squalus acanthias), common carp (Cyprinus carpio), skipjack tuna (Katsuwonus pelamis) and rainbow (Oncorhynchus my kiss). Chondrichthian muscle is unusual because it possesses no detectible activities of carnitine palmitoyl transferase (CPT), which is involved in transport of fatty acids into mitochondria, yet demonstrates activity of B-hydroxyacyl CoA dehydrogenase, a fatty acid B-oxidation enzyme. This paradox was addressed by searching for alternate pathways for mitochondrial fatty acid utilization possibly involving peroxisomal fatty acid processing. Dogfish red muscle and ventricle mitochondria did not utilize long, medium or short chain free fatty acids or fatty acyl carnitines under a variety of conditions. These substrates include the putative products of peroxisomal B-oxidation, which was not detectible in muscles of 2 chondrichthians. It was concluded that dogfish muscles do not utilize fatty acids by direct or indirect muscle pathways, but may rely on ketone bodies as "lipid" fuel. Carp and tuna red and white skeletal muscle, compact and spongy myocardium and atrium were compared to examine the importance of mitochondrial differences in determining tissue aerobic capacity. The spectrum of tissues examined possess a 26-fold difference in mitochondrial content, as indicated by citrate synthase activity. Higher aerobic capacities (tuna vs. carp) are attributed to a combination of three factors: i. greater recruitable muscle mass/kg body mass, ii. greater mitochondrial volume density/g tissue iii. increased mitochondrial specific activity. The relative importance of each factor varied between tissues. In general, the more aerobic tissues (ventricle, red muscle) differ primarily in recruitable mass/kg body mass. There was less than a 2-fold difference in mitochondrial content/g or mitochondrial specific activity between tuna and carp tissues. In less aerobic tissues (white muscle, atrium), a several fold greater mitochondrial content in tuna contributed to greater aerobic capacity. Coupled with the greater aerobic capacities of tuna muscles, was a shift in fuel preference toward fatty acid utilization, as indicated by CPT/mg mitochondrial protein and CPT/hexokinase ratios. Tuna ventricle mitochondria may operate close to their maximal in vitro rates (State 3) in situ at cardiac VO₂MAX. In contrast, trout white muscle mitochondria in vivo operate at a small fraction of their in vitro maximum. The maximal aerobic demands of white muscle probably occur during recovery from burst exercise, when a high lactate load is converted to glycogen in situ: This added cost of recovery represents an ATP demand that is only 3.5% of the maximal mitochondrial capacity. This capacity is suppressed in vivo by highly inhibitory ATP/ADP ratios and limiting phosphate. The primary signal for increased ATP synthesis associated with recovery is not ATP/ADP but probably phosphate, elevated due to phosphocreatine hydrolysis and adenylate catabolism in the purine nucleotide cycle. At low ADP availability and sub-optimal phosphate (<5mM), acidosis enhances respiration. At high respiratory rates mitochondrial pyruvate oxidation is sensitive to pyruvate concentration over the physiological range (apparent Km= 35-40 μM). The sensitivity is lost at the low rates that approximate in vivo respiration. Changes in lactate do not affect the kinetics of pyruvate oxidation. Fatty acid oxidation may spare pyruvate and lactate for use in glyconeogenesis, primarily through allosteric inhibition of pyruvate dehydrogenase, rather than covalent modification.
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