A atividade do complexo 1 da proteína alvo mecanístico da rapamicina (mTORC1), importante regulador da adiposidade e do metabolismo de lipídeos, está aumentada no tecido adiposo de camundongos obesos. A inibição completa de mTORC1 reduz a adiposidade, enquanto que sua inibição parcial potencializa a obesidade induzida por dieta. Assim, hipotetizamos que um nível ótimo de ativação de mTORC1 é necessário para promover aumento da adiposidade, de forma que sua superativação é tão inibitória para a deposição de gordura quanto sua inibição completa. Para testar esta hipótese, investigamos os efeitos da ativação constitutiva de mTORC1, induzida pela deleção de Tsc1, especificamente em adipócitos na adiposidade in vivo. A deleção de Tsc1 reduziu a massa do tecido adiposo visceral, mas não do subcutâneo, que foi associado ao aumento da lipólise e browning. Além disso, aumentou em ambos tecidos adiposos a massa e atividade oxidativa mitocondrial. Esses dados apoiam nossa hipótese de que é necessário um nível ótimo de ativação de mTORC1 para promover aumento da adiposidade. / The activity of mechanistic target of rapamycin complex 1 (mTORC1), an important regulator of adiposity and lipid metabolism, is increased in adipose tissue of obese mice. Complete mTORC1 inhibition reduces adiposity, whereas partial mTORC1 inhibition enhances diet-induced obesity. Therefore, we hypothesized that an optimal level of mTORC1 activity is required to increase adiposity, in such a manner that mTORC1 overactivation is as inhibitory to fat deposition as its complete inhibition. To test this hypothesis, we investigated the effects of constitutive mTORC1 activation, induced by Tsc1 deletion, specifically in adipocytes on adiposity in vivo. Tsc1 deletion reduced visceral, but not subcutaneous, fat mass, which was associated with increased lipolysis and browning. Moreover, it enhanced mitochondrial mass and oxidative activity in both visceral and subcutaneous fat. These data support our hypothesis that an optimal level of mTORC1 activation is necessary to increase adiposity.

A atividade do complexo 1 da proteína alvo mecanístico da rapamicina (mTORC1), importante regulador da adiposidade e do metabolismo de lipídeos, está aumentada no tecido adiposo de camundongos obesos. A inibição completa de mTORC1 reduz a adiposidade, enquanto que sua inibição parcial potencializa a obesidade induzida por dieta. Assim, hipotetizamos que um nível ótimo de ativação de mTORC1 é necessário para promover aumento da adiposidade, de forma que sua superativação é tão inibitória para a deposição de gordura quanto sua inibição completa. Para testar esta hipótese, investigamos os efeitos da ativação constitutiva de mTORC1, induzida pela deleção de Tsc1, especificamente em adipócitos na adiposidade in vivo. A deleção de Tsc1 reduziu a massa do tecido adiposo visceral, mas não do subcutâneo, que foi associado ao aumento da lipólise e browning. Além disso, aumentou em ambos tecidos adiposos a massa e atividade oxidativa mitocondrial. Esses dados apoiam nossa hipótese de que é necessário um nível ótimo de ativação de mTORC1 para promover aumento da adiposidade. / The activity of mechanistic target of rapamycin complex 1 (mTORC1), an important regulator of adiposity and lipid metabolism, is increased in adipose tissue of obese mice. Complete mTORC1 inhibition reduces adiposity, whereas partial mTORC1 inhibition enhances diet-induced obesity. Therefore, we hypothesized that an optimal level of mTORC1 activity is required to increase adiposity, in such a manner that mTORC1 overactivation is as inhibitory to fat deposition as its complete inhibition. To test this hypothesis, we investigated the effects of constitutive mTORC1 activation, induced by Tsc1 deletion, specifically in adipocytes on adiposity in vivo. Tsc1 deletion reduced visceral, but not subcutaneous, fat mass, which was associated with increased lipolysis and browning. Moreover, it enhanced mitochondrial mass and oxidative activity in both visceral and subcutaneous fat. These data support our hypothesis that an optimal level of mTORC1 activation is necessary to increase adiposity.

A existência de uma proteína mitocondrial desacopladora em plantas, PUMP, foi demonstrada em 1995. A PUMP, como a proteína desacopladora de mitocôndrias de tecido adiposo marrom, UCP1, e outras proteínas homólogas descobertas posteriormente, aumenta a condutividade de membrana a H+. Nucleotídeos de purina, PN, inibem a atividade das proteínas desacopladoras e o mecanismo de condução de H+ depende da presença de ácidos graxos livres, FFA. A atividade e a expressão da PUMP são estimuladas pela exposição ao frio e mudam durante o amadurecimento de frutos. A expressão do gene da PUMP de Arabidopsis thaliana em E. coli permite a obtenção de AtPUMP. Neste trabalho, analisando a funcionalidade da AtPUMP incorporada em proteolipossomos, demonstramos que esta proteína é funcional. A incorporação de proteínas desacopladoras em proteolipossomos fornece um sistema modelo que permite analisar as suas propriedades funcionais e mecanísticas. A condutância a H+ em proteolipossomos contendo PUMP isolada de batata foi claramente ativada por FFA. Contudo, a inibição por PN não se mostrou reprodutível. A AtPUMP, reconstituída em proteolipossomos, foi ativada por FFA com Km\'s aparentes de: 42 µM (ácido linoleico, LA), 55 µM (ácido láurico) e 70 µM (ácido palmítico), e inibida por PN com Ki\'s aparentes de: 0.8 mM (GDP), 0.85 mM (ATP), 0.98 mM (GTP) e 1.4 mM (ADP). O efluxo de H+ ativado por LA mediado por AtPUMP aumentou exponencialmente em função do potencial transmembrânico (Δψ). O coeficiente de partição (KP) entre a fase aquosa e proteolipossomos contendo AtPUMP para o LA de 64170, foi ~1,6 vezes superior que ao KP obtido para o LA em lipossomos. Em ensaios de ligação obtidos usando microcalorimetria de titulação isotérmica (ITC), determinou-se que a AtPUMP tem, provavelmente, dois sítios de ligação para o LA e que essa interação é exotérmica. Em ensaios de microcalorimetria com suspensão de mitocôndrias de batatas, determinou-se que há uma correlação linear entre o calor produzido e o oxigênio consumido (65,6 kcal/mol O2) quando a PUMP foi ativada por LA. Através dos resultados obtidos até então, concluo que a PUMP (AtPUMP) é um desacoplador mitocondrial presente em plantas. Os estudos apresentados aqui, de reconstituição em lipossomos, foram essenciais para a compreensão da regulação da atividade dessa proteína. Além disso, foram obtidas as primeiras medidas diretas de liberação de calor pela PUMP por microcalorimetria. / In 1995, a plant mitochondrial uncoupling protein, PUMP, was first described. PUMP, like the known uncoupling protein from brown adipose tissue, UCP1, increases the inner mitochondrial membrane permeability to H+. H+ transport is dependent on the presence of free fatty acids, FFA, and it is inhibited by purine nucleotides, PN. PUMP expression and activity are stimulated by cold exposure, which may vary during fruit ripening. By expressing a full length cDNA encoding the Arabidopsis UCP in E. coli, the recombinant AtPUMP was obtained. In this study, AtPUMP was incorporated in proteoliposomes and the functionality of the protein was demonstrated. The incorporation of uncoupling proteins in proteoliposomes constitutes a model that allows the functional and the mechanistic analysis of these proteins. The H+ conductance mediated by reconstituted potato PUMP was, undoubtedly, activated by FFA. However, the inhibition by PN was not reproducible. Reconstituted AtPUMP was activated by FFA and the apparent Km\'s were determined: 42 µM (linoleic acid, LA), 55 µM (lauric acid) and 70 µM (palmitic acid). Reconstituted AtPUMP was inhibited by PN, and the apparent Kis were determined: 0.8 mM (GDP), 0.85 mM (ATP), 0.98 mM (GTP) and 1.4 mM (ADP). AtPUMP mediated H+ efflux rate, activated by LA, was exponentially dependent on membrane potential (Δψ). The partition coefficient (KP) between the aqueous phase and the membrane phase was determined for LA. The KP was 1.6 times higher for AtPUMP proteoliposomes than for liposomes. Using Isothermal Titration Microcalorimetry (ITC), we verified that there is a linear correlation between the heat produced and oxygen depletion (65,2 kcal/mol O2) in a suspension of potato mitochondria, when PUMP was activated by LA. Using ITC we also determined that LA might bind to two different sites in AtPUMP. Based on the results obtained, we concluded that PUMP (or AtPUMP) is a plant mitochondrial uncoupler. The reconstitution assays permitted the study of FFA and PN regulation. In addition, our results represent the first direct confirmation that fatty acids regulate heat release in plant mitochondria, a process that may play a role in cold adaptation, fruit ripening and flower blossoming.

商業信用狀國際統一慣例（以下簡稱ＵＣＰ），自一九三三年制定後，因其內容與實 務操作頗多 合，因此能為全球貿易界廣泛承認與遵循，其後由於實務作法多所演進 ，國際商會為使ＵＣＰ內容能配合潮流需要，乃分別於一九五一、一九六二、一九萋 四及一九八三年因應需要而有所增修， 期使ＵＣＰ能更符合國際貿易發展軌跡， 進而廣泛為全球貿易操作有所遵循。本文即以ＵＣＰ之歷次修訂過程為經，其間國際 貿易發展之探討為緯，作一深入淺出之研究。 全文共約十二萬餘字，計分八章。 第一章緒論。 第二章探討ＵＣＰ之沿革、架構、適用範圍及定義。 第三章探討ＵＣＰ中有關信用狀格式與通知規定之變遷背景。 第四章探討ＵＣＰ中有關銀行義務責任規定變遷之背景。 第五章探討ＵＣＰ中有關單據規定變遷之背景。 第六章探討ＵＣＰ中有關是項規定變遷之背景。 第七章探討ＵＣＰ中有關轉讓規定變遷之背景。 第八章結論。

De par leur importante diversité phénotypique (10000 espèces), les oiseaux ont colonisé la plupart des niches écologiques, aussi " extrêmes " soient-elles. Les zones polaires (Arctique et Antarctiques) et le climat extrêmement froid qui les aractérise, suscitent l'intérêt de nombreuses études. Ce travail de thèse avait pour but d'explorer les différents mécanismes intervenant dans la mise en place de la thermorégulation chez l'oiseau. Nous nous sommes principalement intéressés, par une approche intégrative, de l'animal entier (méthodes de calorimétrie indirecte) à l'expression génique (techniques de RT-PCR), à caractériser les modifications métaboliques et l'implication d'une protéine découplante (avUCP) dans les mécanismes de thermorégulation, et principalement la thermogenèse sans frisson (NST) en réponse à une exposition chronique au froid.Au cours de deux études menées chez le caneton de Barbarie, nous avons démontré l'aspect " adaptatif " de la NST ainsi que l'implication potentielle de l'UCP aviaire dans ce mécanisme en faisant varier tout d'abord la température d'acclimatation puis la durée d'exposition pour caractériser la mise en place de la NST au cours de la croissance. Lors d'une troisième étude, nous nous sommes intéressés à une étape clé de la vie des manchots royaux (passage en mer) caractérisée par un stress thermique important et une activité physique accrue dus aux longs séjours en eau froide. Ce contexte environnemental et physiologique entraine nécessairement des adaptations métaboliques, comme la mise en place d'un métabolisme lipidique efficace soutenant ainsi les dépenses énergétiques accrues lors des voyages en mer.

The prevalence of diabetes is sufficiently high to be classified as an epidemic, and 20-40% of these patients are expected to develop diabetic nephropathy, a leading cause of end-stage renal failure. Studies have identified a correlation between diabetic nephropathy and hypoxia in renal tissue in human studies. Increased oxygen consumption has been associated with the proton transport protein, uncoupling protein-2 (UCP-2), which uncouples the mitochondria. Previous research has reported elevated levels of UCP-2 in diabetic renal tissue. Consequently, it is crucial to determine how different preservation methods affect the detectability of UCP-2 in renal tissue for clinical applications. This study aimed to evaluate the effectiveness of Western blotting for detecting UCP-2 in snap frozen, fresh untreated, formalin-fixed, methyl carnoy-fixed, and RNA later-preserved rat kidneys. Preliminary trials were conducted to identify the optimal antibody combinations, followed by testing on various preserved tissues. The antibodies produced non-reproducible, unspecific, and unselective results. Additionally, technical challenges, such as gels adhering to membranes and low protein concentrations in some samples, rendered the results inconclusive. Further investigations are necessary to explore additional antibodies and variables that may influence the detection of UCP-2 in differently preserved tissues. Overall, this study highlights the complexity and challenges in developing reliable protocols for UCP-2 detection in preserved renal tissue, indicating that significant optimization is still required for consistent results.

En la acuicultura intensiva existe una práctica creciente encaminada a maximizar tanto el crecimiento como la productividad de los peces mediante altas densidades de producción, dietas altamente energéticas y elevados regímenes de alimentación. Sin embargo, debido al interés en aumentar los mecanismos de control y mejora del bienestar animal, es necesario encontrar nuevos marcadores que evalúen la actividad piscícola. En este sentido, el uso de marcadores mitocondriales de estrés oxidativo y lipotoxicidad constituyen una herramienta indispensable para evaluar los riesgos sobre el bienestar de los peces. Es importante reseñar que cada uno de estos marcadores ofrece una información complementaria e integradora de especial interés cuando se considera una determinada especie, tejido y factor de estrés. Uno de esos mecanismos es la chaperona mitocondrial de la familia de las proteínas de choque térmico 70 (proteína regulada por la glucosa 75, GRP75/mortalina), que protege a los componentes mitocondriales de las especies reactivas de oxígeno (ROS). Un segundo mecanismo es el llevado a cabo por las proteínas desacopladoras (UCP), una familia de transportadores mitocondriales que desacoplan la fosforilación oxidativa mediante la descarga neta del gradiente de protones y la disminución de la producción de ROS en un ambiente rico en ácidos grasos. La caracterización molecular de la GRP75/mortalina y las UCPs en dorada (Sparus aurata) reveló un alto grado de conservación de los rasgos estructurales y parentesco evolutivo de estas familias de proteínas. En el caso de la GRP75/mortalina, esto permitió el uso de anticuerpos heterólogos para el análisis de expresión a nivel de proteína. Sin embargo, para la UCP1 y UCP3 fue necesario la evaluación de la actividad mediante medidas de respiración mitocondrial y la producción de anticuerpos específicos. / Bermejo Nogales, A. (2012). MARCADORES MITOCONDRIALES DE ESTRÉS OXIDATIVO Y LIPOTOXICIDAD EN DORADA (Sparus aurata) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/15189

本文的研究範圍，著重在1983年與1993年修訂統一慣例中對於 ，尤其是開 狀銀行、保兌銀行，與通知銀行部份的有關規定蛂A至於信用狀中的其他 被指定銀行，如讓購銀行、付款銀行□獢B補償銀行、押匯銀行等，僅做 簡要的敘述並未深入討論 蚺摮漸□雯虷傢鷃□瘙q事信用狀交易的功 能、利益、風險與h。然後討論1983修訂統一慣例對於有關銀行地位的規 定，主韘b開狀、保兌、通知銀行，至於其他被指定銀行僅做概略性簸扆u 個案討論各國對於信用狀所引起相關的判決先例檢視統熙W定。然後針 對1993年 4月修訂並自1994年 1月 1日起生效~統一慣例，對於銀行地位 的規定變動的部份做討論。最後就峇@慣例對於未規定的信用狀生效及信 用狀準據法的問題做一 蚺憟D要針對國際商會所制定的信用狀統一 慣1983與1993年兩允鴭韟傢鷃□瘜‘鱆熙W定做討論，並參酌1933、1951 、1962~等各次修訂有關部份的變動情況，但對於信用狀各當事人間鰜Y以 及備受爭議的信用狀的法律性質，不做探討。

Les exigences et les risques du commerce international ont fait apparaître, au début du XXème siècle, une technique juridique adaptée au rapport entre exportateurs et importateurs. Il s'agit tout particulièrement du crédit documentaire qui peut être défini comme l'opération par laquelle la banque de l'acheteur s'engage à régler le prix des marchandises au vendeur, lorsque celui-ci remettra les documents attestant l'expédition des marchandises. Ces derniers peuvent être soit un connaissement, une facture, une lettre de transport, soit même une assurance. Toutefois, l'absence d'une règlementation précise concernant le crédit documentaire a mobilisé les praticiens, ce qui a abouti en 1993 à la naissance des «Règles et Usances Uniformes relatives aux crédits documentaires» (RUU), œuvre de la Chambre de Commerce Internationale. Aujourd'hui faute de législation de source nationale ou internationale régulant le crédit documentaire, elles s'appliquent après adhésion volontaire soit par le système bancaire d'un pays donné, soit par adhésion individuelle de banques,«à tous les crédits documentaires sauf dispositions contraires stipulées expressément par les parties». Ces pratiques sont parallèlement efficaces car elles s'appuient sur des techniques bancaires éprouvées dans la règle de l'inopposabilité des exceptions. / Requirements and risks of international trade have created, in the early twentieth century, a legal technique adapted to the relationship between exporter and importer. This is especially the documentary credit, which can be defined as a transaction in which the buyer's bank agrees to pay the price of goods to the seller, when he will submit documentation confirming the shipping of the goods. This can be either a bill of lading an invoice, or even an insurance police... However, the absence of specific regulations concerning the documentary credit decided the practitioners to give birth, in 1993, of the «Uniform Customs and Practice on Documentary Credit» (UCP), the creation of the International Chamber of Commerce. Today, because of the lack of legislation regulating national or international documentary credit source, these rules apply, after voluntary decision of the parties or by the banking system of a said country or by individual adoption by the banks, to « all documentary credits unless otherwise as expressly provided by the parties». These practices are also effective because they rely on proven banking techniques in the rule of unenforceable exceptions.

Les sécrétines de l’hormone de croissance (GHRPs) sont de petits peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance à partir de l’hypophyse via leur liaison au récepteur de la ghréline GHS-R1a. Le GHRP hexaréline a été utilisé afin d’étudier la distribution tissulaire de GHS-R1a et son effet GH-indépendant. Ainsi, par cette approche, il a été déterminé que l’hexaréline était capable de se lier à un deuxième récepteur identifié comme étant le récepteur scavenger CD36. Ce récepteur possède une multitude de ligands dont les particules oxLDL et les acides gras à longue chaîne. CD36 est généralement reconnu pour son rôle dans l’athérogénèse et sa contribution à la formation de cellules spumeuses suite à l’internalisation des oxLDL dans les macrophages/monocytes. Auparavant, nous avions démontré que le traitement des macrophages avec l’hexaréline menait à l’activation de PPARƔ via sa liaison à GHS-R1a, mais aussi à CD36. De plus, une cascade d’activation impliquant LXRα et les transporteurs ABC provoquait également une augmentation de l’efflux du cholestérol. Une stimulation de la voie du transport inverse du cholestérol vers les particules HDL entraînait donc une diminution de l’engorgement des macrophages de lipides et la formation de cellules spumeuses. Puisque CD36 est exprimé dans de multiples tissus et qu’il est également responsable du captage des acides gras à longue chaîne, nous avons voulu étudier l’impact de l’hexaréline uniquement à travers sa liaison à CD36. Dans le but d’approfondir nos connaissances sur la régulation du métabolisme des lipides par CD36, nous avons choisi des types cellulaires jouant un rôle important dans l’homéostasie lipidique n’exprimant pas GHS-R1a, soient les adipocytes et les hépatocytes. L’ensemble de mes travaux démontre qu’en réponse à son interaction avec l’hexaréline, CD36 a le potentiel de réduire le contenu lipidique des adipocytes et des hépatocytes. Dans les cellules adipeuses, l'hexaréline augmente l’expression de plusieurs gènes impliqués dans la mobilisation et l’oxydation des acides gras, et induit également l’expression des marqueurs thermogéniques PGC-1α et UCP-1. De même, hexaréline augmente l’expression des gènes impliqués dans la biogenèse mitochondriale, un effet accompagné de changements morphologiques des mitochondries; des caractéristiques observées dans les types cellulaires ayant une grande capacité oxydative. Ces résultats démontrent que les adipocytes blancs traités avec hexaréline ont la capacité de se transformer en un phénotype similaire aux adipocytes bruns ayant l’habileté de brûler les acides gras plutôt que de les emmagasiner. Cet effet est également observé dans les tissus adipeux de souris et est dépendant de la présence de CD36. Dans les hépatocytes, nous avons démontré le potentiel de CD36 à moduler le métabolisme du cholestérol. En réponse au traitement des cellules avec hexaréline, une phosphorylation rapide de LKB1 et de l’AMPK est suivie d’une phosphorylation inhibitrice de l’HMG-CoA réductase (HMGR), l’enzyme clé dans la synthèse du cholestérol. De plus, la liaison d'hexaréline à CD36 provoque le recrutement d’insig-2 à HMGR, l’étape d’engagement dans sa dégradation. La dégradation de HMGR par hexaréline semble être dépendante de l’activité de PPARƔ et de l’AMPK. Dans le but d’élucider le mécanisme d’activation par hexaréline, nous avons démontré d’une part que sa liaison à CD36 provoque une déphosphorylation de Erk soulevant ainsi l’inhibition que celui-ci exerce sur PPARƔ et d’autre part, un recrutement de l’AMPK à PGC-1α expliquant ainsi une partie du mécanisme d’activation de PPARƔ par hexaréline. Les résultats générés dans cette thèse ont permis d’élucider de nouveaux mécanismes d’action de CD36 et d'approfondir nos connaissances de son influence dans la régulation du métabolisme des lipides. / Growth hormone releasing peptides (GHRPs) are small synthetic peptides aimed at stimulating GH release from the pituitary through their binding to ghrelin receptor known as growth hormone secretagogue receptor 1a (GHS-R1a). Using the GHRP, hexarelin to study tissue distribution of GHS-R1a and its GH-independent effect, it was observed that hexarelin was capable of binding to a second receptor identified as scavenger receptor CD36. While having multiple ligands, CD36 is mainly known for binding and internalizing oxLDL and long chain fatty acids. CD36 is thought to play a detrimental role in macrophage derived foam cell formation and development of atherosclerosis. Previously, we have shown that in macrophages, expressing both GHS-R1a and CD36, hexarelin promoted an activation of PPARƔ via GHS-R1a but also through its binding to CD36. This activation led to the induction of the LXRα-ABC transporters pathway and an increase in cholesterol efflux, reducing lipid-laden macrophage content. This positive effect on macrophages was reproduced in apolipoprotein E-null mice on a high fat diet treated with hexarelin. A significant reduction in the size of atherosclerotic lesions was observed while similar increases in the expression of PPARƔ, LXRα and ABC transporters occurred in isolated peritoneal macrophages. CD36 also plays a role in fatty acid uptake, and to further investigate the impact of the interaction of hexarelin with CD36, we aimed at evaluating the role of CD36 in regulating lipid metabolism in cells devoid of GHS-R1a such as adipocytes and hepatocytes. In the present thesis, we demonstrated through its interaction with hexarelin, the ability of CD36 to decrease intracellular lipid content in both adipocytes and hepatocytes. In adipocytes, hexarelin was able to increase the expression of several genes involved in fatty acid mobilization, fatty acid oxidation but also to induce the expression of the thermogenic markers, PGC-1α and UCP-1. In addition, hexarelin increased the expression of genes involved in mitochondrial biogenesis which was accompanied by mitochondrial morphological changes in agreement with what is usually seen in highly oxidative cells. In support of these findings, we also observed an increase in the activity of cytochrome c oxidase (a component of the respiratory chain) which could reflect an increase in oxidative phosphorylation. The results generated with cultured white adipocytes suggest the ability of hexarelin to promote changes toward a brown fat-like phenotype which also occurred in vivo and was dependent on the presence of CD36. In hepatocytes, CD36 was capable of regulating cholesterol metabolism by rapidly phosphorylating LKB1 and AMPK which subsequently resulted in the inactivating phosphorylation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Hexarelin via CD36 also induced the recruitment of insig-2 to HMGR, the committed step in HMGR degradation while lifting the exerted inhibitory effect of Erk on nuclear receptor PPARƔ activity, and promoting the recruitment of AMPK to PPARƔ coactivator PGC-1α, suggesting an enhanced transcriptional potential of PPARƔ. The results generated during my graduate studies represent unique and novel mechanisms by which CD36 is capable of regulating lipid metabolism.