Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

Ratos foram tratados com salina, óleos de peixe (OP) ou soja (OS) por via intragástrica durante 20 dias antes da indução do IAM. A área de infarto e atividades da creatina quinase no plasma e da caspase 3 no ventrículo esquerdo (VE) foram menores no grupo OP comparado a salina ou OS. Os conteúdos de IL-1β, TNF-α, CINC 2α/β, IL-6 e VEGF-α no VE e de IL-1β, TNF-α, MIP-3, IL-6 e VEGF-α no fígado foram elevados pelo OS. O OP aumentou os conteúdos de ATP e lactato e diminuiu o de glicogênio no VE. A redução do fluxo coronariano no VE dos animais infartados foi abolida pelo OP. A expressão gênica de iNOS, eNOS, HIF-1α, GLUT-1, VEGF-α, p53 e Bax2 no VE aumentou pelo OP. A fração de ejeção, fração de encurtamento e velocidade de encurtamento das fibras cardíacas foram mais elevadas pelo OP. Portanto, o tratamento com OP induziu um estado de pré-condicionamento que conferiu proteção do miocárdio à injúria isquêmica. / Rats were treated with saline, fish (FO) or soybean (SO) oils by gavage for 20 days before myocardial infarction (MI). Infarct size, activities of plasma CK and caspase 3 in the left ventricle (LV) were decreased by FO as compared with saline or SO. The contents of IL-1β, TNF-α, CINC 2α/β, IL-6, VEGF-α in the LV and of IL-1β, TNF-α, MIP-3, IL-6, VEGF-α in the liver were increased by SO. Contents of ATP and lactate in the LV were increased and of glycogen decreased by FO. FO prevented the decrease in the coronary blood flow in the LV of infarcted rats. The mRNA contents of iNOS, eNOS, HIF-1α, GLUT 1, VEGF-α, p53 and Bax2 in the VE were increased by FO. Ejection fraction, fractional shortening and velocity of circumferential fiber-shortening were also increased by FO. So, treatment with FO leads to a preconditioning state that protected the heart from MI injury.

A doença aterosclerótica macrovascular se inicia em fases precoces das alterações do metabolismo glicídico. Este estudo teve por objetivos: 1) avaliar a prevalência de aterosclerose subclínica diagnosticada por métodos não-invasivos em indivíduos com indicação de teste oral de tolerância a glicose; 2) avaliar a distribuição de biomarcadores e de marcadores genéticos nessa população; e 3) determinar os fatores de risco para aterosclerose subclínica em pacientes disglicêmicos. Indivíduos em prevenção primária foram inicialmente submetidos a teste oral de tolerância a glicose e classificados em grupos controle, glicemia de jejum alterada, intolerância à glicose e diabete melito; posteriormente, foram submetidos a pesquisa de aterosclerose subclínica e de biomarcadores, e a avaliação de polimorfismos genéticos e expressão gênica. Foram incluídos 103 pacientes no grupo controle, 80 no grupo glicemia de jejum alterada, 98 no grupo tolerância diminuída à glicose e 59 no grupo diabete melito, com média de idade de 59 + 7,4 anos, sendo 62,4% mulheres. Não foram encontradas diferenças quanto às características clínicas e laboratoriais entre os grupos. Foi observada alta prevalência de aterosclerose subclínica na população (77,1%) e, apesar de não haver diferença entre os grupos, houve tendência a prevalência crescente de acordo com a piora do perfil glicídico. Dentre os biomarcadores, foi encontrada diferença entre os grupos na análise de microalbuminúria, resistina, fator de necrose tumoral alfa e fosfolipase A2 associada a lipoproteína. Não houve diferença com relação aos polimorfismos, mas o grupo glicemia de jejum alterada apresentou maior expressão de mRNA do gene da fosfolipase A2 associada a lipoproteína. Concluímos que indivíduos com indicação de teste oral de tolerância a glicose têm alta prevalência de aterosclerose subclínica, independentemente do perfil glicídico. Após análise multivariada, os fatores que determinaram aterosclerose subclínica foram idade, pressão arterial sistólica, colesterol ligado à lipoproteína de alta densidade, fator de necrose tumoral alfa e uso de estatinas. / Atherosclerotic macrovascular disease begins in early phases of glucose metabolism alterations. The objectives were: 1) To evaluate the prevalence of subclinical atherosclerosis diagnosed by non-invasive methods in patients with an indication for oral glucose tolerance test. 2) To evaluate the distribution of biomarkers and genetic markers in this population. 3) Determine the risk factors for subclinical atherosclerosis in dysglycemic patients. Individuals in primary prevention underwent oral glucose tolerance test and were classified as controls, impaired fasting glucose, decreased glucose tolerance and diabetics and submitted to subclinical atherosclerosis search, evaluation of biomarkers, genetic polymorphisms and gene expression. A group of 103 patients were included as controls, 80 as impaired fasting glucose, 98 as decreased glucose tolerance and 59 as diabetes with a mean age of 59 ± 7.4 years, 62.4% women. No differences were found between clinical and laboratory characteristics of the groups. High prevalence of subclinical atherosclerosis (77.1%) was observed, although there was no significant difference between groups, a tendency of higher prevalence according to worsening of glucose increasing profile was verified. Among the biomarkers difference between groups were found in the analysis of microalbuminuria, resistin, tumor necrosis factor alfa and phospholipase A2 associated with lipoprotein. There was no difference regardind the polymorphisms, but the impaired fasting glucose group had higher expression of PLA2G7. After multivariate analysis, the factors that determined subclinical atherosclerosis were age, systolic blood pressure, HDL-cholesterol, tumor necrosis factor alfa and statins. We concluded that individuals with indication of oral glucose tolerance test have a high prevalence of subclinical atherosclerosis regardless of glucose profile. The factors that determine the presence of subclinical atherosclerosis were age, systolic blood pressure, HDL-cholesterol, tumor necrosis factor alfa and statins.

Made available in DSpace on 2014-08-20T14:37:50Z (GMT). No. of bitstreams: 1 tese_elizabeth_schwegler.pdf: 1198143 bytes, checksum: 306f08a6253b951c8751a8eba9c7194c (MD5) Previous issue date: 2012-08-15 / The peripartum in dairy cows (three weeks before and three weeks after calving) is characterized by large changes in physiological demands in the animal where management practices, particularly nutrition, strongly influences the incidence of peripartum disorders and subsequent milk production. Most of the studies in that period in dairy cows are focused on confined systems where milk production is high. Therefore, the aim of this study was to assess metabolic markers associated with the occurrence of clinical and subclinical mastitis and insulin resistance in dairy cows in semi extensive system. Our study was divided into two major experiments with the following hypothesis: 1) medium milk production primiparous cows in semi extensive system have predictive metabolic markers of clinical and subclinical mastitis in the prepartum, 2) medium milk production pluriparous dairy cows with low rate of glucose metabolism during the prepartum, which is an indicative of insulin resistance, have higher minerals excretion in the postpartum period. In experiment 1, blood concentrations of NEFA in the prepartum period were higher, in contrast, phosphorus and glucose were lower (P <0.05) in animals with clinical mastitis postpartum. In experiment 2, pluriparous dairy cows with low rate of glucose metabolism in the prepartum had higher urinary calcium excretion in both prepartum and postpartum periods, and also the highest NEFA concentration in the prepartum period (P <0.05). The blood concentrations of calcium in dairy cows with high rate of glucose metabolism in the postpartum was elevated from the prepartum period and remained in the postpartum (P <0.05). Dairy cows kept in semi extensive system with moderate production level had predictive markers of mastitis in the prepartum, as previously demonstrated by other authors in more intensified systems of higher requirements for the animal. The highlighted marker was NEFA, however, in the second study it was shown in higher concentrations in the pluriparous dairy cows with low rate of glucose metabolism at prepartum, emphasizing the importance of this marker also in the insulin resistance. / O periparto nas vacas leiteiras (três semanas anteriores e as três posteriores ao parto) é caracterizado por grandes mudanças nas demandas fisiológicas do animal, sendo que, as práticas de manejo, principalmente nutricionais, influenciam intensamente a incidência de desordens no periparto e a subsequente produção de leite. A maioria dos estudos neste período na vaca leiteira é em sistemas confinados de alta produção leiteira, por essa razão o objetivo desta tese foi identificar marcadores metabólicos preditivos com a ocorrência de mastite clínica e subclínica e com a excreção de minerais em vacas leiteiras em sistema semi-extensivo. Nosso estudo foi estratificado em dois trabalhos com as seguintes hipóteses: 1) vacas leiteiras primíparas de média produção em sistema semi-extensivo possuem marcadores metabólicos preditivos de mastite clínica e subclínica no pré-parto; 2) vacas leiteiras pluríparas de média produção com menor taxa de metabolização de glicose, que é um indicativo de resistência da insulina, no pré-parto possuem maior excreção de minerais no pós-parto. No experimento 1 as concentrações sanguíneas de NEFA no pré-parto foram maiores e de fósforo e glicose menores (P<0,05), em animais com mastite clínica no pós-parto. No experimento 2 as vacas leiteiras pluríparas com menor taxa de metabolização de glicose no pré-parto apresentaram maior excreção urinária do cálcio no pré e pós-parto, e ainda, o NEFA mais alto no pré-parto (P<0,05). As concentrações sanguíneas de cálcio em vacas leiteiras com maior taxa de metabolização de glicose no pós-parto foi elevada desde o pré parto, mantendo-se no pós (P<0,05). Nas condições estudadas, em vacas leiteiras mantidas em sistema semi-extensivo de mediana produção foi observado que há marcadores preditivos de mastite já no pré-parto, como previamente comprovado por outros autores em sistemas mais intensificados de maiores exigências para o animal. O marcador em destaque foi o NEFA, sendo que o mesmo no segundo estudo se apresentou de forma mais elevada nas vacas leiteiras pluríparas com menor taxa de metabolização de glicose no pré-parto, enfatizando a importância deste marcador também na resistência à insulina.

Les phosphoinositides (PI) sont impliqués dans de nombreux processus biologiques cellulaires, tels que la régulation du cytosquelette d'actine, le trafic vésiculaire membranaire et les voies de signalisation médiées par des récepteurs membranaires. Le métabolisme des phosphoinositides est un processus hautement régulé par un ensemble de PI-kinases et PI-phosphatases. Des mutations dans ces enzymes sont associées à des maladies tels que le syndrome de Lowe, le diabète de type 2, les désordres bipolaires et le cancer.<p>\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Situé dans le noyau suprachiasmatique (SCN) de l’hypothalamus, l'horloge principale contrôle les rythmes des processus comportementaux et métaboliques chez les mammifères. Par exemple, les rythmes quotidiens de veille-sommeil, d'alimentation-jeûne, de glycémie, de tolérance au glucose et de sensibilité à l'insuline sont régulés par l'horloge SCN. La lumière est le synchroniseur principal du SCN, même si de nombreux facteurs autres que la lumière, tels que l'éveil comportemental ou des facteurs métaboliques, peuvent également moduler la phase ou la période des SCN. L'objectif de cette thèse était d'étudier différents aspects des interactions entre l'éveil comportemental, les rythmes circadiens et le métabolisme du glucose. Dans la première partie, nous avons déterminé l'action centrale du Gastrin-Releasing Peptide (GRP), un neuropeptide synthétisé dans le SCN, sur le métabolisme du glucose. Nos résultats indiquent qu’une injection icv de GRP induit une hyperglycémie prolongée. Nous avons également montré qu’une privation de sommeil à court terme conduit à une détérioration de la tolérance au glucose. Dans la deuxième partie, nous avons démontré que l'éveil comportemental induit par la privation de sommeil ou une injection de caféine améliore l’entraînement photique de l'horloge SCN chez un rongeur diurne : le rat roussard du Soudan, Arvicanthis ansorgei. Ces réponses circadiennes chez une espèce diurne qui sont opposées à celles précédemment mises en évidence chez les rongeurs nocturnes pourraient avoir des applications biomédicales. / Located in the hypothalamic suprachiasmatic nucleus (SCN), the master clock generates rhythms of behavioural and metabolic processes in mammals. For example, daily rhythms of sleep-wake, fasting-feeding, plasma glucose concentration, glucose tolerance and insulin sensitivity are regulated by the SCN clock. Light is the primary synchronizer of SCN pacemaker though many light-independent factors such as behavioural arousal and metabolic cues also have phase and period resetting properties. The aim of thesis was to study different aspects of the interactions between behavioural arousal, circadian rhythms and glucose metabolism. In the first part, we extended the study of brain control of glucose metabolism by investigating the central action of gastrin-releasing peptide (GRP), a neuropeptide synthesized in the SCN, on glucose metabolism. Our result indicates that central GRP induces long-lasting hyperglycemia. We also showed that acute sleep deprivation leads to impaired glucose tolerance. In the second part, we demonstrated that behavioural arousal induced by sleep deprivation or caffeine treatment enhances photic-entrainment of the SCN clock in the diurnal Sudanian grass rat, Arvicanthis ansorgei. These circadian responses in a diurnal species are opposite to the earlier findings in nocturnal rodents and may have biomedical applications.

Solute carriers (SLCs) are membrane-bound transporter proteins, important for nutrient, ion, drug and metabolite transport across membranes. A quarter of the human genome codes for membrane-bound proteins, and SLCs make up the largest group of transporter proteins. Due to their ability to transport a large repertoire of substances across, not just the plasma membrane, but also the membrane of internal organelles, they hold a key position in maintaining homeostasis affecting metabolic pathways. Unfortunately, some of the more than 400 identified SLCs are still not fully characterized, even though a quarter of these are associated with human disease. In addition, there are about 30 membrane-bound proteins with strong resemblance to SLCs, of which very little is known. The aim of this thesis is to characterize some of these putative SLCs, focusing on their localization and function in the central nervous system. Since many of the known SLCs play a vital part in metabolism and related pathways, the response to different nutritional conditions has been used as a key method. MFSD14A and MFSD14B, characterized in Paper I, are putative SLCs belonging to the Major Facilitator Superfamily (MFS) and found to be neuronal, differentially expressed in the mouse central nervous system and transiently upregulated in mouse embryonic cortex cultures due to amino acid deprivation. They were also altered in areas of the mouse brain after starvation as well as after high fat diet. In Paper II, the effect on gene regulation due to complete amino acid starvation was monitored in a mouse hypothalamic cell line and 47 different genes belonging to SLCs, or putative SLCs, were found to be affected. Of these, 15 genes belonged to already known amino acid transporters, whereas 32 were putative SLCs with no known function or SLCs not known to react to amino acids. The three SV2 proteins, SV2A, SV2B and SV2C, were studied in Paper III using human neuroblastoma cell lines. The high metabolic state of cancers often result in an upregulation and alteration of transporter proteins, and alterations of the SV2 proteins were found following different treatments performed in this study. Paper IV focused on putative SLCs of MFS type and their role in glucose metabolism. Mouse embryonic cortex cultures were subjected to glucose starvation and the gene expression of 19 putative transporters were analyzed. All but four of the putative transporters were affected either at 3h or 12h of glucose deprivation. In conclusion, several SLCs and putative SLCs studied in this thesis are strongly affected by alteration in metabolism, either due to amino acids or glucose or both. This makes the putative SLCs dynamic membrane-bound proteins, possibly transporters, highly affected by nutritional status and most likely regulated to maintain homeostasis.

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

La progression tumorale repose sur l'acquisition progressive d'anomalies génétiques qui vont conduire à la prolifération dérégulée de ces cellules. Il existe cependant des systèmes de protection contre cette progression tumorale que l'on appelle systèmes de sauvegarde. Ainsi, pour se transformer, la cellule tumorale doit franchir ces barrières anti-tumorales. Les résultats de mon travail de thèse, qui avait pour objectif initial d'identifier les altérations moléculaires précoces de l'oncogenèse, m'ont permis de mettre en évidence un nouveau mécanisme de sauvegarde anti-tumoral. Pour cette étude, un modèle d'étude in vitro de l'initiation et de la progression tumorale déclenchée par l'oncogène RET développé par notre équipe a été utilisé. Grâce à l'utilisation de ce système, nous avons pu montrer que le Réticulum Endoplasmique (RE) est un senseur efficace de l'altération du métabolisme glucidique déclenchée par les signalisations oncogéniques, et que le stress qu'il subit alors, conduit à l'apoptose. Ce travail a permis de mettre mis en évidence que les cellules malignes qui franchissent cette barrière peuvent alors bénéficier d'un effet pro-tumorale du SRE. Ainsi, les résultats présentés dans ce manuscrit offrent une meilleure compréhension du rôle complexe que joue le SRE dans la cancérogénèse / Carcinogenesis involves not only inactivation of tumourigenesis barriers, but also alterations in energy metabolism to fulfil the synthetic and bioenergetic requirements for fast and uncontrolled growth. Our study supports a model in which the ER acts as a node between altered glucose metabolism and tumourigenesis barriers. This major site in the cell for protein folding and maturation, can sense glucose limitation that results from oncogenic-mediated increased glucose demand, and consequently trigger unfolded protein response-dependent apoptosis. As such, the ER functions as a surveillance mechanism that suppresses the emergence of tumour cells. Overcoming this early barrier involves a specific attenuation of the pro-apoptotic PERK-CHOP branch of the unfolded protein response, a cellular adaptation that in turn may favour malignant progression. These observations bring new insights into the complex role of the unfolded protein response during tumourigenesis

La réduction des excursions glycémiques postprandiales a été proposée comme un moyen pour limiter le risque de développement du diabète de type 2. L’intérêt s’est donc porté sur les outils nutritionnels susceptibles de moduler la biodisponibilité des glucides et ainsi leur impact sur la glycémie postprandiale. Les travaux réalisés au cours de cette thèse avaient pour but d’étudier les effets de différents ingrédients modifiant la biodisponibilité du glucose, non seulement sur la glycémie postprandiale à court terme (2 heures) mais aussi sur les cinétiques du débit d’apparition et de disparition de glucose (total, exogène et endogène - isotopes stables) et les autres paramètres métaboliques de la phase postprandiale au cours de la journée. Dans la première étude (β-glucanes), nous avons montré que l’addition de fibres β-glucanes à un repas glucidique chez des sujets sains en surpoids ralentit l’absorption du glucose dans le plasma. Ceci a prolongé la réponse insulinique et par conséquent l’inhibition de la lipolyse et de la production endogène de glucose. Dans la deuxième étude (Eurostarch), nous avons montré que la diminution de la biodisponibilité du glucose au petit-déjeuner (amidon lentement digestible, index glycémique bas) diminue l’apparition du glucose exogène dans le plasma et pourrait avoir un effet second-repas chez des sujets sains en surpoids. Mais nous n’avons pas mis en évidence d’amélioration de ces effets métaboliques à plus long terme (5 semaines). Dans la troisième étude présentée (Nutriose), nous avons montré que l’addition de dextrine résistante NUTRIOSE®10 (fermentescible) au petit-déjeuner chez des sujets sains, diminue les réponses glycémiques, insuliniques postprandiales et le profil de ghréline au cours de la journée (en comparaison à une maltodextrine). En parallèle, la prolongation observée de la fermentation et l’oxydation du NUTRIOSE®10 pourraient fournir de l’énergie en phase postprandiale tardive. En conclusion, l’analyse des paramètres métaboliques au-delà de 2 heures après le repas, a permis de mettre en évidence les effets métaboliques à plus long terme de la modulation de l’apparition du glucose dans le plasma (ralentissement, prolongation, réduction) sur les cinétiques du glucose, la réponse insulinique, la lipolyse et l’oxydation des substrats / The reduction of the postprandial glycemic excursions has been proposed as to limit risk of type 2 diabetes. There has been growing interest in the development of dietary ingredients that could potentially modulate carbohydrates bioavailability and thus their impact on postprandial glycemia. The aim of this thesis was to investigate the effects of the the modulation of glucose bioavailability by different ingredients on 2-hour glycemic response but also on glucose kinetics (total, exogenous and endogenous – stable isotopes) and on other daylong metabolic parameters. In the first study (β-glucanes), we showed that the addition of β-glucan fiber to a carbohydrate meal in healthy overweight subjects reduced the appearance of glucose in plasma. As a consequence, insulin response was also prolonged and induced a prolonged inhibition on lipolysis and endogenous glucose production. In the second study (eurostarch), the reduction in glucose availability (slowly available glucose, low GI) at breakfast decreased plasma exogenous glucose appearance and tended to improve glucose control at the subsequent lunch. But we did not observe the improvement of such metabolic effects in the long-term (5 weeks). In the last study, we showed that the addition of a resistant dextrin, NUTRIOSE®10, decreased postprandial glycemic and insulinemic response as well as daylong satiety-related ghrelin profile, compared to maltodextrin. In parallel, the prolonged fermentation and oxidation pattern of NUTRIOSE®10 up to 10 hours after ingestion at breakfast could induced an extended energy release with NUTRIOSE®10 in the late postprandial phase. In conclusion, the follow-up of metabolic parameters beyond 2 hours after the meal have highlighted the longer-term metabolic effects of the modulation of glucose appearance in plasma (delay, extension, reduction) on glucose kinetics, insulin response, lipolysis and nutrients oxidation