The biochemistry of drug metabolism : an introduction : part 6, Inter-individual factors affecting drug metabolism.

This review is part of a series of review articles on the metabolism of drugs and other xenobiotics published in Chemistry & Biodiversity. After a thorough discussion of metabolic reactions and their enzymes, this article focuses on genetically determined differences in drug and xenobiotic metabolism. After a short introduction on the causes for genetic differences, the first focus is on species differences in drug and xenobiotic metabolism. A major chapter is then dedicated to clinically relevant genetic polymorphisms in human drug metabolism and resultant ethnic differences. The last two chapters deal with sex-dependent differences in drug metabolism and personalized pharmacotherapy related to inter-individual differences in drug metabolism.

Soluble MHC-peptide complexes containing long rigid linkers abolish CTL-mediated cytotoxicity.

Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and co-engage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (> or =80 A) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long D(b)-gp33 complexes strongly and specifically inhibited the D(b)-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.

Quality of assessement of CMR images in the setting of the European cardiovascular magnetic resonance registry : description and validation of standardized criteria

l'imagerie par résonance magnétique (IRMC) est une technologie utilisée depuis les aimées quatre¬-vingts dans le monde de la cardiologie. Cette technique d'imagerie non-invasive permet d'acquérir Ses images du coeur en trois dimensions, dans n'importe quel, plan, sans application de radiation, et en haute résolution. Actuellement, cette technique est devenue un référence dans l'évaluation et 'l'investigation de différentes pathologies cardiaques. La morphologie cardiaque, la fonction des ventricules ainsi que leur contraction, la perfusion tissulaire ainsi que la viabilité tissulaire peuvent être caractérisés en utilisant différentes séquences d'imagerie. Cependant, cette technologie repose sur des principes physiques complexes et la mise en pratique de cette technique se heurte à la difficulté d'évaluer un organe en mouvement permanent. L'IRM cardiaque est donc sujette à différents artefacts qui perturbent l'interprétation des examens et peuvent diminuer la précision diagnostique de cette technique. A notre connaissance, la plupart des images d'IRMC sont analysées et interprétées sans évaluation rigoureuse de la qualité intrinsèque de l'examen. Jusqu'à présent, et à notre connaissance, aucun critère d'évaluation de la qualité des examens d'IRMC n'a été clairement déterminé. L'équipe d'IRMC du CHUV, dirigée par le Prof J. Schwitter, a recensé une liste de 35 critères qualitatifs et 12 critères quantitatifs évaluant la qualité d'un examen d'IRMC et les a introduit dans une grille d'évaluation. L'objet de cette étude est de décrire et de valider la reproductibilité des critères figurant dans cette grille d'évaluation, par l'interprétation simultanée d'examens IRMC par différents observateurs (cardiologues spécialisés en IRM, étudiant en médecine, infirmière spécialisée). Notre étude a permis de démontrer que les critères définis pour l'évaluation des examens d'IRMC sont robustes, et permettent une bonne reproductibilité intra- et inter-observateurs. Cette étude valide ainsi l'utilisation de ces critères de qualité dans le cadre de l'imagerie par résonance magnétique cardiaque. D'autres études sont encore nécessaires afin de déterminer l'impact de la qualité de l'image sur la précision diagnostique de cette technique. Les critères standardisés que nous avons validés seront utilisés pour évaluer la qualité des images dans le cadre d'une étude à échelle européenne relative à l'IRMC : "l'EuroCMR registry". Parmi les autres utilités visées par ces critères de qualité, citons notamment la possibilité d'avoir une référence d'évaluation de la qualité d'examen pour toutes les futures études cliniques utilisant la technologie d'IRMC, de permettre aux centres d'IRMC de quantifier leur niveau de qualité, voire de créer un certificat de standard de qualité pour ces centres, d'évaluer la reproductibilité de l'évaluation des images par différents observateurs d'un même centre, ou encore d'évaluer précisément la qualité des séquences développées à l'avenir dans le monde de l'IRMC.

Resistência inter e intra-agregados em ensaios de cisalhamento direto de um nitossolo vermelho distrófico

Para solos agregados, a envoltória da resistência ao cisalhamento pode ser dividida em dois segmentos, com declividades e interceptos diferentes. Um primeiro segmento mais inclinado e com intercepto menor representaria a envoltória de ruptura definida pelo atrito e coesão interagregados, enquanto o segundo segmento, menos inclinado e com intercepto maior, seria a envoltória definida pelo atrito e coesão intra-agregados. O presente estudo foi realizado com o objetivo de avaliar se a envoltória de resistência ao cisalhamento de agregados do horizonte superficial de um Nitossolo Vermelho pode ser subdividida em segmentos distintos, com coeficientes angulares diferentes, e se isso está relacionado à existência de agregados nele. Inicialmente, amostras coletadas na superfície de um Nitossolo Vermelho distrófico latossólico de textura argilosa foram submetidas ao ensaio de cisalhamento direto com pressões normais de 24,4; 48,9; 98,2; 196,4; 294,6; 392,8 e 491,8 kPa. Posteriormente, o ensaio foi realizado com amostras de agregados de cinco classes de diâmetro: < 0,25; 0,25-0,50; 0,50-1,00; 1,00-2,00 e 2,00-4,00 mm, empregando-se pressões normais de 24,4; 48,9; 73,5; 147,3; 294,6; 441,9; 589,2 e 736,6 kPa. Em todos os ensaios, verificou-se que as envoltórias obtidas podem ser divididas em dois segmentos de reta. Os resultados indicam que a agregação deve ser considerada na definição das cargas normais a serem utilizadas no ensaio de cisalhamento direto, bem como na análise dos resultados, pois ela tem efeitos sobre a envoltória de resistência ao cisalhamento. Para o Nitossolo Vermelho distrófico usado neste estudo, constatou-se que, na umidade em que as amostras foram ensaiadas, os agregados definem a resistência ao cisalhamento do solo para cargas normais de até 294,6 kPa.

NS4B Self-Interaction through Conserved C-Terminal Elements Is Required for the Establishment of Functional Hepatitis C Virus Replication Complexes.

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.

Molecular modeling of peptide-MHC class I complexes : applications to peptide based immunotherapy

Summary The specific CD8+ T cell immune response against tumors relies on the recognition by the T cell receptor (TCR) on cytotoxic T lymphocytes (CTL) of antigenic peptides bound to the class I major histocompatibility complex (MHC) molecule. Such tumor associated antigenic peptides are the focus of tumor immunotherapy with peptide vaccines. The strategy for obtaining an improved immune response often involves the design of modified tumor associated antigenic peptides. Such modifications aim at creating higher affinity and/or degradation resistant peptides and require precise structures of the peptide-MHC class I complex. In addition, the modified peptide must be cross-recognized by CTLs specific for the parental peptide, i.e. preserve the structure of the epitope. Detailed structural information on the modified peptide in complex with MHC is necessary for such predictions. In this thesis, the main focus is the development of theoretical in silico methods for prediction of both structure and cross-reactivity of peptide-MHC class I complexes. Applications of these methods in the context of immunotherapy are also presented. First, a theoretical method for structure prediction of peptide-MHC class I complexes is developed and validated. The approach is based on a molecular dynamics protocol to sample the conformational space of the peptide in its MHC environment. The sampled conformers are evaluated using conformational free energy calculations. The method, which is evaluated for its ability to reproduce 41 X-ray crystallographic structures of different peptide-MHC class I complexes, shows an overall prediction success of 83%. Importantly, in the clinically highly relevant subset of peptide-HLAA*0201 complexes, the prediction success is 100%. Based on these structure predictions, a theoretical approach for prediction of cross-reactivity is developed and validated. This method involves the generation of quantitative structure-activity relationships using three-dimensional molecular descriptors and a genetic neural network. The generated relationships are highly predictive as proved by high cross-validated correlation coefficients (0.78-0.79). Together, the here developed theoretical methods open the door for efficient rational design of improved peptides to be used in immunotherapy. Résumé La réponse immunitaire spécifique contre des tumeurs dépend de la reconnaissance par les récepteurs des cellules T CD8+ de peptides antigéniques présentés par les complexes majeurs d'histocompatibilité (CMH) de classe I. Ces peptides sont utilisés comme cible dans l'immunothérapie par vaccins peptidiques. Afin d'augmenter la réponse immunitaire, les peptides sont modifiés de façon à améliorer l'affinité et/ou la résistance à la dégradation. Ceci nécessite de connaître la structure tridimensionnelle des complexes peptide-CMH. De plus, les peptides modifiés doivent être reconnus par des cellules T spécifiques du peptide natif. La structure de l'épitope doit donc être préservée et des structures détaillées des complexes peptide-CMH sont nécessaires. Dans cette thèse, le thème central est le développement des méthodes computationnelles de prédiction des structures des complexes peptide-CMH classe I et de la reconnaissance croisée. Des applications de ces méthodes de prédiction à l'immunothérapie sont également présentées. Premièrement, une méthode théorique de prédiction des structures des complexes peptide-CMH classe I est développée et validée. Cette méthode est basée sur un échantillonnage de l'espace conformationnel du peptide dans le contexte du récepteur CMH classe I par dynamique moléculaire. Les conformations sont évaluées par leurs énergies libres conformationnelles. La méthode est validée par sa capacité à reproduire 41 structures des complexes peptide-CMH classe I obtenues par cristallographie aux rayons X. Le succès prédictif général est de 83%. Pour le sous-groupe HLA-A*0201 de complexes de grande importance pour l'immunothérapie, ce succès est de 100%. Deuxièmement, à partir de ces structures prédites in silico, une méthode théorique de prédiction de la reconnaissance croisée est développée et validée. Celle-ci consiste à générer des relations structure-activité quantitatives en utilisant des descripteurs moléculaires tridimensionnels et un réseau de neurones couplé à un algorithme génétique. Les relations générées montrent une capacité de prédiction remarquable avec des valeurs de coefficients de corrélation de validation croisée élevées (0.78-0.79). Les méthodes théoriques développées dans le cadre de cette thèse ouvrent la voie du design de vaccins peptidiques améliorés.

Inter-relações entre as propriedades físicas e os coeficientes da curva de retenção de água de um latossolo sob diferentes sistemas de uso

O comportamento físico e hídrico do solo fundamenta as práticas de seu uso e manejo. O objetivo deste trabalho foi avaliar as inter-relações entre as propriedades físicas e os coeficientes da curva de retenção de água de um Latossolo Vermelho distrófico sob diferentes sistemas de uso. Em 2004, foram selecionadas três áreas contíguas utilizadas por mais de vinte anos com mata (nativa), pousio (Brachiaria decumbens) e cultivo (culturas anuais). Foram coletadas 25 amostras de solo com estrutura não deformada na camada de 0-0,15 m de profundidade, que foram utilizadas para determinação das curvas de retenção de água do solo. As curvas de retenção de água foram ajustadas pela equação de van Genuchten, obtendo-se os coeficientes qs, qr, a e n. Também foram mensuradas as variáveis densidade do solo, teor de C orgânico e teor de argila do solo (grupo I). Essas variáveis e os coeficientes das curvas de retenção do solo (grupo II) foram submetidos às análises de correlações canônicas. Verificou-se que a intensificação do uso do solo (mata, pousio e cultivo) resultou em maiores valores de densidade do solo e na redução dos teores de C orgânico e argila. O primeiro par das variáveis canônicas indicou dependência de qs em relação a densidade, e o segundo par distinguiu os sistemas de uso do solo verificado pela dependência de qr em relação aos teores de argila e de C orgânico. As modificações dos teores de C orgânico e da densidade do solo sugerem a degradação da qualidade física e hídrica do solo.

Inter-relação biomassa vegetal subterrânea-estabilidade de agregados-erosão hídrica em solo submetido a diferentes formas de manejo

O aumento da biomassa vegetal subterrânea e da estabilidade dos agregados do solo pelo seu manejo adequado aumentam sua capacidade para infiltrar água da chuva e resistir à erosão hídrica. Baseado nesta premissa, foi realizado um estudo de erosão em campo, sob chuva simulada, na Estação Experimental Agronômica da Universidade Federal do Rio Grande do Sul - EEA/UFRGS, em Eldorado do Sul (RS), no verão de 2003/2004, utilizando um experimento de manejo do solo com 7,5 anos de duração. O objetivo da pesquisa foi investigar relações entre variáveis de manejo e de erosão, relacionadas à história de uso e manejo do solo e suas condições físicas superficiais momentâneas, criadas por tratamentos de seu preparo e de cobertura por resíduo cultural, os quais foram aplicados imediatamente antes da realização de testes de erosão com chuva simulada. Utilizou-se um Argissolo Vermelho distrófico típico, com textura superficial franco-argilo-arenosa e declividade média de 0,115 m m-1. Foram estudadas quatro seqüências culturais, com ou sem preparo (gradagem) e cobertura (resíduo cultural) do solo. Realizaram-se três testes de erosão com chuva simulada, cada um na intensidade de 64 mm h-1 e duração de 1,5 h, usando o aparelho simulador de braços rotativos. Avaliaram-se a massa de raízes mortas das plantas, o diâmetro médio ponderado de agregados do solo, a taxa constante de infiltração de água no solo e a perda total de água e de solo por erosão. A infiltração de água no solo foi maior e, inversamente, a perda de água menor no solo recém-mobilizado (superfície solta e rugosa) do que no solo não-mobilizado (superfície firme e praticamente lisa), mesmo o primeiro estando descoberto e o segundo tanto coberto quanto descoberto, com diferenças entre as seqüências culturais. No solo não-mobilizado e coberto, a perda foi pequena em todas as seqüências culturais, enquanto no solo descoberto, tanto não-mobilizado quanto recém-mobilizado, ela foi pequena somente naquelas seqüências culturais que propiciaram altos valores de massa de raízes mortas das plantas e de diâmetro médio ponderado de agregados do solo. No solo recém-mobilizado e descoberto, a diminuição da perda de solo também se deveu à rugosidade superficial criada pelo preparo. As relações da perda total de água e da perda total de solo, com a massa de raízes mortas das plantas e com o diâmetro médio ponderado de agregados do solo, foram significativas a 5 %.

The mechanisms of agglomeration: Evidence from the effect of inter-industry relations on the location of new firms

The objective of this paper is to explore the relative importance of each of Marshall's agglomeration mechanisms by examining the location of new manufacturing firms in Spain. In particular, we estimate the count of new firms by industry and location as a function of (pre-determined) local employment levels in industries that: 1) use similar workers (labor market pooling); 2) have a customer- supplier relationship (input sharing); and 3) use similar technologies (knowledge spillovers). We examine the variation in the creation of new firms across cities and across municipalities within large cities to shed light on the geographical scope of each of the three agglomeration mechanisms. We find evidence of all three agglomeration mechanisms, although their incidence differs depending on the geographical scale of the analysis.

Characterization of the role of Rho activating signalling complexes in G protein-coupled receptor induced cardiac fibrosis

Dans certaines conditions pathologiques, telles que l'hypertension artérielle ou l'infarctus du myocarde, le coeur répond à une augmentation de la post-charge par des processus de remodelage aboutissant à une hypertrophie du ventricule gauche. L'hypertrophie cardiaque est caractérisée par une croissance hypertrophique des cardiomyocytes, ainsi que par une différenciation des fibroblastes en un phenotype présentant une capacité accrue de synthèse protéiques, nommés myofibroblastes. Ceci résulte en une accumulation excessive des constituants de la matrice extracellulaire, ou autrement dit fibrose. En raison de son effet délétère sur la contractilité du coeur, menant sur le long terme à une insuffisance cardiaque, de nombreux efforts ont été déployés, afin de définir les mécanismes moléculaires impliqués dans la réponse profibrotique. A ce jour, de nombreuses études indiquent que la petite GTPase RhoA pourrait être un médiateur important de la réponse profibrotique du myocarde. Cependant, les facteurs d'échanges impliqués dans la transduction de signaux profibrotiques, via la régulation de son activité au niveau des fibroblastes cardiaques, n'ont pas encore été identifiés. De précédentes études menées dans le laboratoire, ont identifiées une nouvelle protein d'ancrage de la PKA, exprimée majoritairement dans le coeur, nommée AKAP-Lbc. Il a été montré que cette protéine, en plus de sa fonction de protein d'ancrage, possédait une activité de facteur d'échange de nucléotide guanine (GEF) pour la petite GTPase RhoA. Au niveau des cardiomyocytes, il a été montré que l'AKAP-Lbc participe à une voie de signalisation pro-hypertrophique, incluant la sous-unité alpha de la protéine G hétérotrimerique G12 et RhoA. Chose intéressante, des observations antérieures à cette étude, indiquent que dans le coeur, l'AKAP-Lbc est également exprimée dans les fibroblastes. Cependant aucunes études n'a encore reporté de fonction pour ce facteur d'échange dans les fibroblastes cardiaques. Dans ce travail, les résultats obtenus indiquent que dans les fibroblastes cardiaques, I'activation de RhoA par l'AKAP-Lbc est impliquée dans la transmission de signaux profibrotiques, en aval des récépteurs à l'angiotensine II. En particulier, nous avons observé que la suppression de l'expression de l'AKAP-Lbc dans les fibroblastes ventriculaires de rat adultes, réduisait fortement Γ activation de Rho induite par l'angiotensine II, la déposition de collagène, la capacité migratoire des fibroblastes ainsi que leur différenciation en myofibroblastes. A notre connaissance, l'AKAP-Lbc est le premier RhoGEF identifié comme médiateur de la réponse profibrotique dans les fibroblastes cardiaques. - In pathological conditions such as chronic hypertension or myocardial infarction, the myocardium is subjected to various biomechanical and biochemical stresses, and undergoes an adverse ventricular remodelling process associated with cardiomyocytes hypertrophy and excess deposition of extracellular matrix proteins resulting in fibrosis. During the fibrotic response, cardiac fibroblasts differentiate into a more mobile and contractile phenotype termed myofibroblasts. These cells, possess a greater synthetic ability to produce ECM proteins and have been implicated in diseases with increased ECM deposition including cardiac fibrosis. Because fibrosis impairs myocardial contractility and is associated with the progression to heart failure, a major cause of lethality worldwide, many efforts have been made to define the molecular players involved in this process. During these last years, increasing evidence suggests a role for the small GTPase RhoA in mediating the fibrotic response in CFbs. However the identity of the exchange factors that modulate its activity and transduce fibrotic signals in CFbs is still unknown. Earlier work in our laboratory identified a novel PKA anchoring protein expressed in the heart termed AKAP-Lbc that has been shown to function as anchoring protein as well as a guanine nucleotide exchange factor (GEF) for the small GTPase RhoA. In response to several hypertrophic stimuli we have shown that RhoGEF activity of AKAP-Lbc mediated by Gan promotes the activation of a signaling pathway including RhoA, leading to cardiomyocytes hypertrophy. Within the heart, previous observations made in the laboratory indicated that AKAP-Lbc was also expressed in fibroblasts. However its role in cardiac fibroblasts remained to be determined. In the present study, we show that AKAP-Lbc is critical for activating RhoA and transducing profibrotic signals downstream of angiotensin II receptors in cardiac fibroblasts. In particular, our results indicate that suppression of AKAP-Lbc expression by infecting adult rat ventricular fibroblasts with lentiviruses encoding AKAP-Lbc specific short hairpin RNAs strongly reduces angiotensin II-induced RhoA activation, collagen deposition as well as cell migration and differentiation. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor involved in a profibrotic signalling pathway at the level of cardiac fibroblasts.

Impact of precipitation on the water table level of different ombrotrophic raised bog complexes, central Estonia

Sadannan vaikutus vedenpinnan tasoon kohosuolla

The mechanisms of agglomeration: Evidence from the effect of inter-industry relations on the location of new firms

The objective of this paper is to explore the relative importance of each of Marshall's agglomeration mechanisms by examining the location of new manufacturing firms in Spain. In particular, we estimate the count of new firms by industry and location as a function of (pre-determined) local employment levels in industries that: 1) use similar workers (labor market pooling); 2) have a customer- supplier relationship (input sharing); and 3) use similar technologies (knowledge spillovers). We examine the variation in the creation of new firms across cities and across municipalities within large cities to shed light on the geographical scope of each of the three agglomeration mechanisms. We find evidence of all three agglomeration mechanisms, although their incidence differs depending on the geographical scale of the analysis.

Inter-firm labor mobility and knowledge diffusion: a theoretical approach

[cat] Analitzem una economia amb dues característiques principals: la mobilitat dels treballadors implica transferència de coneixement i la productivitat de l’empresa augmenta amb l’intercanvi de coneixement. Cada empresa desenvolupa un tipus de coneixement que serà trasmès a la resta de la indústria mitjançant la mobilitat de treballadors. Estudiem dues estructures de mercat laboral i utilitzant un anàlisi comparatiu derivem les implicacions del model. Els resultats revelen com la mobilitat de treballadors depèn en la varietat i nivell del coneixement, la presència de costos de mobilitat, les institucions, la capacitat d’absorvir coneixement per part de les empreses i la mida de la indústria. Els resultats no depenen de l’estructura del mercat laboral.

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.