Profiling of signal transduction in human memory T cells

Résumé pour un large public: La vaccination a eu un impact énorme sur la santé mondiale. Mais, quel est le principe d'un vaccin? Il est basé sur la 'mémoire immunologique', qui est une particularité exclusive des systèmes immunitaires des organismes évolués. Suite à une infection par un pathogène, des cellules spécialisées de notre système immunitaire (les lymphocytes) le reconnaissent et initient une réaction immunitaire qui a pour but son élimination. Pendant cette réaction se développent aussi des cellules, appelées cellules lymphocytaires mémoire, qui persistent pour longue durée et qui ont la capacité de stimuler une réaction immunitaire très efficace immédiatement après une seconde exposition à ce même pathogène. Ce sont ces cellules mémoires (lymphocytes B et T) qui sont à la base de la 'mémoire immunologique' et qui sont stimulées lors de la vaccination. Chez l'homme, deux populations distinctes des lymphocytes T mémoires ont été identifiées: les cellules centrales (CM) et effectrices (EM) mémoires. Ces populations sont fonctionnellement hétérogènes et exercent des rôles distincts et essentiels dans l'immunité protectrice. Typiquement, les cellules effectrices mémoires sont capables de tuer immédiatement le pathogène tandis que les cellules centrales mémoires sont responsables d'initier une réponse immunitaire complète. Pourtant, les mécanismes biochimiques qui contrôlent les fonctions de ces cellules ont été jusqu'à présent peu étudiés à cause de la faible fréquence de ces cellules et de la quantité limitée de tissus humains disponibles pour les analyses. La compréhension de ces mécanismes est cruciale pour la réalisation de vaccins efficaces et pour le développement de nouveaux médicaments capables de moduler la réponse immunitaire lymphocytaire. Dans cette thèse, nous avons d'abord développé et amélioré une technologie appelée 'protéine array en phase inverse' qui possède un niveau de sensibilité beaucoup plus élevé par rapport aux technologies classiquement utilisées dans l'étude des protéines. Grâce à cette technique, nous avons pu comparer la composition protéique du système de transmission des signaux d'activation des cellules CM et EM humaines. L'analyse de 8 à 13 sujets sains a montré que ces populations des cellules mémoires possèdent un système de signalisation protéique différent. En effet, les cellules EM possèdent, par rapport aux cellules CM, des niveaux réduits d'une protéine régulatrice (appelée c-Cbl) que nous avons démontré comme étant responsable des fonctions spécifiques de ces cellules. En effet, en augmentant artificiellement l'expression de cette protéine régulatrice dans les cellules EM jusqu'au niveau de celui des cellules CM, nous avons induit dans les cellules EM des capacités fonctionnelles caractéristiques des cellules CM. En conclusion, notre étude a identifié, pour la première fois chez l'homme, un mécanisme biochimique qui contrôle les fonctions des populations des cellules mémoires. Résumé en Français: Les cellules mémoires persistent inertes dans l'organisme et produisent des réactions immunitaires rapides et robustes contre les pathogènes précédemment rencontrés. Deux populations distinctes des cellules mémoires ont été identifiées chez l'homme: les cellules centrales (CM) et effectrices (EM) mémoires. Ces populations sont fonctionnellement hétérogènes et exercent des rôles distincts et critiques dans l'immunité protectrice. Les mécanismes biochimiques qui contrôlent leurs fonctions ont été jusqu'à présent peu étudiés, bien que leur compréhension soit cruciale pour le développement des vaccins et des nouveaux traitements/médicaments. Les limites majeures à ces études sont la faible fréquence de ces populations et la quantité limitée de tissus humains disponibles. Dans cette thèse nous avons d'abord développé et amélioré la technologie de 'protéine array en phase inverse' afin d'analyser les molécules de signalisation des cellules mémoires CD4 et CD8 humaines isolées ex vivo. L'excellente sensibilité, la reproductibilité et la linéarité de la détection, ont permis de quantifier des variations d'expression protéiques supérieures à 20% dans un lysat équivalent à 20 cellules. Ensuite, grâce à l'analyse de 8 à 13 sujets sains, nous avons prouvé que les cellules mémoires CD8 ont une composition homogène de leur système de signalisation tandis que les cellules CD4 EM expriment significativement de plus grandes quantités de SLP-76 et des niveaux réduits de c-Cbl, Syk, Fyn et LAT par rapport aux cellules CM. En outre, l'expression réduite du régulateur négatif c-Cbl est corrélée avec l'expression des SLP-76, PI3K et LAT uniquement dans les cellules EM. L'évaluation des propriétés fonctionnelles des cellules mémoires a permis de démontrer que l'expression réduite du c-Cbl dans les cellules EM est associé à une diminution de leur seuil d'activation. En effet, grâce a la technique de transduction cytosolique, nous avons augmenté la quantité de c-Cbl des cellules EM à un niveau comparable à celui des cellules CM et constaté une réduction de la capacité des cellules EM à proliférer et sécréter des cytokines. Ce mécanisme de régulation dépend principalement de l'activité d'ubiquitine ligase de c-Cbl comme démontré par l'impact réduit du mutant enzymatiquement déficient de c-Cbl sur les fonctions de cellules EM. En conclusion, cette thèse identifie c-Cbl comme un régulateur critique des réponses fonctionnelles des populations de cellules T mémoires et fournit, pour la première fois chez l'homme, un mécanisme contrôlant l'hétérogénéité fonctionnelle des ces cellules. De plus, elle valide l'utilisation combinée des 'RPP arrays' et de la transduction cytosolique comme outil puissant d'analyse quantitative et fonctionnel des protéines de signalisation. Summary : Memory cells persist in a quiescent state in the body and mediate rapid and vigorous immune responses toward pathogens previously encountered. Two subsets of memory cells, namely central (CM) and effector (EM) memory cells, have been identified in humans. These subsets display high functional heterogeneity and assert critical and distinct roles in the control of protective immunity. The biochemical mechanisms controlling their functional properties remain so far poorly investigated, although their clarification is crucial for design of effective T-cell vaccine and drug development. Major limitations to these studies lie in the low frequency of memory T cell subsets and the limited amount of human specimen available. In this thesis we first implemented the innovative reverse phase protein array approach to profile 15 signalling components in human CD8 and CD4 memory T cells isolated ex vivo. The high degree of sensitivity, reproducibility and linearity achieved, allowed an excellent quantification of variations in protein expression higher than 20% in as few as 20-cell equivalent per spot. Based on the analysis of 8 to 13 healthy subjects, we showed that CD8 memory cells have a homogeneous composition of their signaling machinery while CD4 EM cells express statistically significant increased amounts of SLP-76 and reduced levels of c- Cbl, Syk, Fyn and LAT as compared to CM cells. Moreover, in EM but not CM cells, reduced expression of negative regulator c-Cbl correlated with the expression of SLP-76, PI3K and LAT. Subsequently, we demonstrated that the higher functional properties and the lower functional threshold of EM cells is associated with reduced expression of c-Cbl. Indeed, by increasing c-Cbl content of EM cells to the same level of CM cells using cytosolic transduction, we impaired their proliferation and cytokine production. This regulatory mechanism was primarily dependent on c-Cbl E3 ubiquitin ligase activity as evidenced by the weaker impact of enzymatically deficient c-Cbl C381A mutant on EM cell functions. Together, these results identify c-Cbl as a critical regulator of the functional responses of memory T cell subsets and provides, for the first time in humans, a mechanism controlling the functional heterogeneity of memory CD4 cells. Moreover it validates the combined use of RPP arrays and cytosolic transduction approaches as a powerful tool to quantitatively analyze signalling proteins and functionally assess their roles.

Combined transcriptomic and proteomic studies reveal non-canonical signaling functions in the jasmonate pathway

Abstract Lipid derived signals mediate many stress and defense responses in multicellular eukaryotes. Among these are the jasmonates, potently active signaling compounds in plants. Jasmonic acid (JA) and 12-oxo-phytodienoic acid (OPDA) are the two best known members of the large jasmonate family. This thesis further investigates their roles as signals using genomic and proteomic approaches. The study is based on a simple genetic model involving two key genes. The first is ALLENE OXIDE SYNTHASE (AOS), encoding the most important enzyme in generating jasmonates. The second is CORONATINE INSENSITIVE 1 (COI1), a gene involved in all currently documented canonical signaling responses. We asked the simple question: do null mutations in AOS and COI1 have analogous effects on the transcriptome ? We found that they do not. If most COI1-dependent genes were also AOS-dependent, the expression of a zinc-finger protein was AOS-dependent but was unaffected by the coi1-1 mutation. We thus supposed that a jasmonate member, most probably OPDA, can alter gene expression partially independently of COI1. Conversely, the expression of at least three genes, one of these is a protein kinase, was shown to be COI1-dependent but did not require a functional AOS protein. We conclude that a non-jasmonate signal might alter gene expression through COIL Proteomic comparison of coi1-1 and aos plants confirmed these observations and highlighted probable protein degradation processes controlled by jasmonates and COI1 in the wounded leaf. This thesis revealed new functions for COI1 and for AOS-generated oxylipins in the jasmonate signaling pathway. Résumé Les signaux dérivés d'acides gras sont des médiateurs de réponses aux stress et de la défense des eucaryotes multicellulaires. Parmi eux, les jasmonates sont de puissants composés de sig¬nalisation chez les plantes. L'acide jasmonique (JA) et l'acide 12-oxo-phytodienoïc (OPDA) sont les deux membres les mieux caractérisés de la grande famille des jasmonates. Cette thèse étudie plus profondément leurs rôles de signalisation en utilisant des approches génomique et protéomique. Cette étude est basée sur un modèle génétique simple n'impliquant que deux gènes. Le premier est PALLENE OXYDE SYNTHASE (AOS) qui encode l'enzyme la plus importante pour la fabrication des jasmonates. Le deuxième est CORONATINE INSENSITIVE 1 (COI1) qui est impliqué dans la totalité des réponses aux jasmonates connues à ce jour. Nous avons posé la question suivante : est-ce que les mutations nulles dans les gènes AOS et COI1 ont des effets analogues sur le transcriptome ? Nous avons trouvé que ce n'était pas le cas. Si la majorité des gènes dépendants de COI1 sont également dépendants d'AOS, l'expression d'un gène codant pour une protéine formée de doigts de zinc n'est pas affectée par la mutation de COI1 tout en étant dépendante d'AOS. Nous avons donc supposé qu'un membre de la famille des jasmonates, probablement OPDA, pouvait modifier l'expression de certains gènes indépendamment de COI1. Inversement, nous avons montré que, tout en étant dépendante de COI1, l'expression d'au moins trois gènes, dont un codant pour une protéine kinase, n'était pas affectée par l'absence d'une protéine AOS fonctionnelle. Nous en avons conclu qu'un signal autre qu'un jasmonate devait modifier l'expression de certains gènes à travers COI1. La comparaison par protéomique de plantes aos et coi1-1 a confirmé ces observations et a mis en évidence un probable processus de dégradation de protéines contrôlé par les jasmonates et COU_ Cette thèse a mis en avant de nouvelles fonctions pour COI1 et pour des oxylipines générées par AOS dans le cadre de la signalisation par les jasmonates.

Identifying quantitative operation principles in metabolic pathways: a systematic method for searching feasible enzyme activity patterns leading to cellular adaptive responses

Background: Optimization methods allow designing changes in a system so that specific goals are attained. These techniques are fundamental for metabolic engineering. However, they are not directly applicable for investigating the evolution of metabolic adaptation to environmental changes. Although biological systems have evolved by natural selection and result in well-adapted systems, we can hardly expect that actual metabolic processes are at the theoretical optimum that could result from an optimization analysis. More likely, natural systems are to be found in a feasible region compatible with global physiological requirements. Results: We first present a new method for globally optimizing nonlinear models of metabolic pathways that are based on the Generalized Mass Action (GMA) representation. The optimization task is posed as a nonconvex nonlinear programming (NLP) problem that is solved by an outer- approximation algorithm. This method relies on solving iteratively reduced NLP slave subproblems and mixed-integer linear programming (MILP) master problems that provide valid upper and lower bounds, respectively, on the global solution to the original NLP. The capabilities of this method are illustrated through its application to the anaerobic fermentation pathway in Saccharomyces cerevisiae. We next introduce a method to identify the feasibility parametric regions that allow a system to meet a set of physiological constraints that can be represented in mathematical terms through algebraic equations. This technique is based on applying the outer-approximation based algorithm iteratively over a reduced search space in order to identify regions that contain feasible solutions to the problem and discard others in which no feasible solution exists. As an example, we characterize the feasible enzyme activity changes that are compatible with an appropriate adaptive response of yeast Saccharomyces cerevisiae to heat shock Conclusion: Our results show the utility of the suggested approach for investigating the evolution of adaptive responses to environmental changes. The proposed method can be used in other important applications such as the evaluation of parameter changes that are compatible with health and disease states.

Minimization of biosynthetic costs in adaptive gene expression responses of yeast to environmental changes

Yeast successfully adapts to an environmental stress by altering physiology and fine-tuning metabolism. This fine-tuning is achieved through regulation of both gene expression and protein activity, and it is shaped by various physiological requirements. Such requirements impose a sustained evolutionary pressure that ultimately selects a specific gene expression profile, generating a suitable adaptive response to each environmental change. Although some of the requirements are stress specific, it is likely that others are common to various situations. We hypothesize that an evolutionary pressure for minimizing biosynthetic costs might have left signatures in the physicochemical properties of proteins whose gene expression is fine-tuned during adaptive responses. To test this hypothesis we analyze existing yeast transcriptomic data for such responses and investigate how several properties of proteins correlate to changes in gene expression. Our results reveal signatures that are consistent with a selective pressure for economy in protein synthesis during adaptive response of yeast to various types of stress. These signatures differentiate two groups of adaptive responses with respect to how cells manage expenditure in protein biosynthesis. In one group, significant trends towards downregulation of large proteins and upregulation of small ones are observed. In the other group we find no such trends. These results are consistent with resource limitation being important in the evolution of the first group of stress responses.

Differences in phasic development rate amongst wheat cultivars independent of responses to photoperiod and vernalization. A viewpoint of the intrinsic earliness hypothesis

Differences amongst wheat cultivars in the rate of reproductive development are largely dependent on differences in their sensitivity to photoperiod and vernalization. However, when these responses are accounted for, by growing vernalized seedlings under long photoperiods, cultivars can still differ markedly in time to ear emergence. Control of rate of development by this ‘third factor’ has been poorly understood and is variously referred to as intrinsic earliness, earliness in the narrow sense, basic vegetative period, earliness per se, and basic development rate. Certain assumptions are made in the concept of intrinsic earliness. They are that differences in intrinsic earliness (i) are independent of the responses of the cultivars to photoperiod and vernalization, (ii) apply only to the length of the vegetative period up to floral initiation (as suggested by several authors), (iii) are maintained under different temperatures, measured either in days or degree days. As a consequence of this, the ranking of cultivars (from intrinsically early to intrinsically late) must be maintained at different temperatures. This paper, by the re-analysis of published data, examines the extent to which these assumptions can be supported. Although it is shown that intrinsic earliness operates independently of photoperiod and vernalization responses, the other assumptions were not supported. The differences amongst genotypes in time to ear emergence, grown under above-optimum vernalization and photoperiod (that is when the response to these factors is saturated), were not exclusively due to parallel differences in the length of the vegetative phase, and the length of the reproductive phase was independent of that of the vegetative phase. Thus, it would be possible to change the relative allocation of time to vegetative and reproductive periods with no change in the full period to ear emergence. The differences in intrinsic earliness between cultivars were modified by the temperature regime under which they were grown, i.e. the difference between cultivars (both considering the full phase to ear emergence or some sub-phases) was not a constant amount of time or thermal time at different temperatures. In addition, in some instances genotypes changed their ranking for ‘intrinsic earliness’ depending on the temperature regime. This was interpreted to mean that while all genotypes are sensitive to temperature they differ amongst themselves in the extent of that sensitivity. Therefore, ‘intrinsic earliness’ should not be considered as a static genotypic characteristic, but the result of the interaction between the genotype and temperature. Intrinsic earliness is therefore likely to be related to temperature sensitivity. Some implications of these conclusions for plant breeding and crop simulation modelling are discussed.

Metabolic responses of guava trees irrigated withdifferent N and K levels in São Francisco Valley

The guava (Psidium guajava L.) cv. Paluma has been cultivated in São Francisco Valley, Northeastern of Brazil, for in natura consumption and processing purposes. In spite of its importance, there are few scientific knowledge regarding guava physiology, nutrition, irrigation and fertigation. The objective of this work was to evaluate the effect of weather conditions and different concentrations of N and K applied by fertigation in foliar contents of reducing sugars, total soluble sugars, starch, sucrose, amino acids, and proteins. The field experiment was carried out at Bebedouro Experimental Field and the biochemical evaluations at the Laboratory of Seed and Plant Physiology, both located at Embrapa Semi-Árido, Petrolina-PE. The doses of 200 g N and 100 g K2O; 400 g N and 200 g K2O; 600 g N and 300 g K2O; and 800 g N and 400 g K2O per plant were applied in an experiment field. The experimental design was totally randomized blocks, with four treatments and five blocks. The weather conditions influenced the plant photosynthesis, which affects the plants metabolism. Guava presented specific responses to N and K fertigation for each parameter evaluated. The weather conditions during the evaluation period influenced guava responses to N and K fertigation.

Traumatismes précoces et réponses de stress, leur association aves la santé mentale, l'attachement et l'ocytocine

Lorsqu'un individu est confronté à une situation stressante, une des réponses les plus saillantes est l'activation de l'axe HPA, caractérisée par le déclenchement d'un taux élevé de glucocorticoïdes dans le sang. De manière générale, cette réponse hormonale est adaptative et elle a pour but la mobilisation des ressources physiques et cognitives de l'individu pour une action spécifique (Axelrod & Reisine, 1984; Chrousos & Gold, 1992; N. M. Kaplan, 1988; McEwen, 2004). Cependant, lorsque une personne est confrontée très tôt dans son développement, et de manière répétée, à des situations de stress, cette réponse physiologique peut s'altérer, devenir inadaptée (Anand, 1993; Bremner et al., 1995; Meaney et al., 1996; Mirescu, Peters, & Gould, 2004; Plotsky & Meaney, 1993; Sapolsky, 2000) et être associée à des troubles cognitifs (McEwen & Sapolsky, 1995) et émotionnels (McEwen, 2000). A l'âge adulte, le résultat de ces altérations psychoneuroendocriniennes se traduit au cours de l'activation de l'axe HPA et elles sont visibles lors de situations de stress moins intenses (Graham, Heim, Goodman, Miller, & Nemeroff, 1999; Mirescu et al., 2004; Stam, Bruijnzeel, & Wiegant, 2000; A. Taylor, Fisk, & Glover, 2000). La dysregulation de l'axe HPA semble représenter un facteur de vulnérabilité lié à des dysfonctionnements psychiques et physiologiques chez les adultes (Heim, Ehlert, & Hellhammer, 2000; Heim & Nemeroff, 1999; Heim, Newport, Mletzko, Miller, & Hemeroff, 2008). Cependant, des facteurs de protection peuvent influencer à leur tour ces vulnérabilités. La littérature, basée sur des études translationnelles (animaux, humains), converge vers le postulat selon lequel la dimension relationnelle apportée par l'environnement est fondamentale dans le développement des vulnérabilités physiologiques et psychiques du sujet. Dans ce sens, les relations d'attachement ont été particulièrement étudiées. A l'âge adulte, par exemple, la qualité des représentations d'attachement semble influencer directement l'expression de gènes impliqués dans les réponses hormonales de stress (Biagini, Pich, Carani, Marrama, & Agnati, 1998; Caldji, Diorio, & Meaney, 2000; Dallman, 2000; De Kloet, Rosenfeld, Van Eekelen, Sutanto, & Levine, 1988; Rincon-Cortes & Sullivan, 2014; Romeo, Tang, & Sullivan, 2009; van Oers, de Kloet, Whelan, & Levine, 1998), illustrant ainsi une perspective épigénétique. Traumatismes précoces et réponses de stress, leur association avec la santé mentale, l'attachement et l'ocytocine Deux objectifs principaux définissent ce travail de doctorat. Le premier est de comprendre comment un événement à portée traumatique, qui a eu lieu pendant la période périnatale, l'enfance ou l'adolescence, peut s'inscrire au niveau physiologique (axe hypotalamico- hypophysaire-surrénalien - axe HPA), au niveau psychopathologique ou encore au niveau de la régulation émotionnelle au cours de l'âge adulte. A ce propos, nous avons évalué les réponses physiologiques (telles que le Cortisol, l'ACTH et l'ocytocine), la présence de psychopathologies (relatives à l'axe I du DSM-IV) et les réponses émotionnelles (telles que la perception au stress) au cours d'une situation de stress de nature psychosociale, induite en laboratoire. Le deuxième objectif de ce travail est de savoir si les représentations d'attachement peuvent médiatiser ces effets, chez des individus exposés à différents événements à portée traumatique. Dans ce but, trois populations ont été considérées. La première est relative à des jeunes adultes nés grands prématurés ; la deuxième, concerne des femmes adultes ayant vécu un ou plusieurs abus sexuels au cours de leur enfance ou de leur adolescence et enfin la troisième est constituée de personnes adultes qui ont survécu à une maladie grave (cancer) pendant leur enfance ou leur adolescence. Enfin, ces trois populations sont comparées à des groupes contrôle. La prise en considération de différents types de traumatismes a permis de relever : premièrement, qu'un événement à portée traumatique de nature différente, peut influencer de manière semblable les structures neuronales, par exemple l'hypocortisolémie ; deuxièmement, qu'un dysfonctionnement de l'axe HPA n'aboutit pas nécessairement à la présence de signes de souffrance mentale ; enfin, des effets protecteurs ont été mis en évidence. Ces facteurs sont sous-tendus, d'un point de vue psychologique, par les représentations d'attachement et, d'un point de vue physiologique, par la sécrétion d'ocytocjne périphérique. Traumatismes précoces et réponses de stress, leur association avec la santé mentale, l'attachement et l'ocytocine -- When an individual is faced by a stressful situation, one of the most notable responses is the activation of the HPA axis, which is characterized by a heightened level of glucocortisoids in the blood. In general, this is an adaptive hormonal response which prepares the individual both physically and cognitively for a specific action (Axelrod & Reisine, 1984; Chrousos & Gold, 1992; N. M. Kaplan, 1988; McEwen, 2004). However, should a person be confronted to stressful situations very early and repeatedly in their development, this physiologic response may be altered and become maladapted (Anand, 1993; Bremner et al., 1995; Meaney et al., 1996; Mirescu et al., 2004; Plotsky & Meaney, 1993; Sapolsky, 2000) which can be associated to emotional (McEwen, 2000) and cognitive disorders(McEwen & Sapolsky, 1995). Throughout adulthood, the result of these psychoneuroendocrine alterations affects the activation of the HPA axis and are noticeable during less intense stressful situations (Graham et al., 1999; Mirescu et al., 2004; Stam et al., 2000; A. Taylor et al., 2000). HPA axis dysregulation appears to represent a factor of vulnerability linked to psychological and physical disorders in adults (Heim, Ehlert, et al., 2000; Heim & Nemeroff, 1999; Heim, Newport, et al., 2008). Nonetheless, these vulnerabilities may be influenced by further protection factors. The literature, based on translational studies (animals and humans), suggests that relationships formed in the context of the individual's environment are fundamental in the development of their physiological and psychological vulnerabilities. Thus, attachment relationships have been particularly studied. In adulthood, for example, the quality of attachment representations appear to influence directly the expression of genes involved in the hormonal responses to stress (Biagini et al., 1998; Caldji et al., 2000; Dallman, 2000; De Kloet et al., 1988; Rincon-Cortes & Sullivan, 2014; Romeo et al., 2009; van Oers et al., 1998). With the goal to study these dimensions, two principal objectives define these doctoral study. The first is to understand how an event considered to be traumatic, which took place during early infancy, infancy, or adolescence, could influence physiology (HPA axis), psychopathology or emotional regulation during adulthood. Therefore we have evaluated the presence of psychopathologies (relative to axis I of the DSM), physiological responses (such as Cortisol, ACTH and oxytocin) and emotional responses (such as perception of stress) throughout a psychosocial stress situation, conducted in a laboratory setting. The second objective of this study is to understand if attachment representations can mediate these effects, in individuals exposed to three different types of traumatic events. Therefore, three populations have been considered. The first is young adults who were born prematurely; the second concerns adult women who have suffered sexual abuse, on one or more occasions, during their childhood or adolescence; finally the third group is constituted of people who have survived a grave childhood illness. These populations were all compared to control groups. The consideration of different types of traumatic events has demonstrated, firstly, that different events which are considered to be traumatic can similarly influence neuronal structures, for example hypocortisolism. Secondly, that an HPA axis disorder does not necessarily lead to the presence of mental signs of distress, as is the case for those born very prematurely. Finally, protective effects were demonstrated, distinctively from a psychological point of view, by attachment representations and furthermore by peripheral oxytocin secretion from a physiological perspective.

Genome-wide transcriptional responses to shade: Linking shade avoidance and auxin

Plants have the ability to use the composition of incident light as a cue to adapt development and growth to their environment. Arabidopsis thaliana as well as many crops are best adapted to sunny habitats. When subjected to shade, these plants exhibit a variety of physiological responses collectively called shade avoidance syndrome (SAS). It includes increased growth of hypocotyl and petioles, decreased growth rate of cotyledons and reduced branching and crop yield. These responses are mainly mediated by phytochrome photoreceptors, which exist either in an active, far-red light (FR) absorbing or an inactive, red light (R) absorbing isoform. In direct sunlight, the R to FR light (R/FR) ratio is high and converts the phytochromes into their physiologically active state. The phytochromes interact with downstream transcription factors such as PHYTOCHROME INTERACTING FACTOR (PIF), which are subsequently degraded. Light filtered through a canopy is strongly depleted in R, which result in a low R/FR ratio and renders the phytochromes inactive. Protein levels of downstream transcription factors are stabilized, which initiates the expression of shade-induced genes such as HFR1, PIL1 or ATHB-2. In my thesis, I investigated transcriptional responses mediated by the SAS in whole Arabidopsis seedlings. Using microarray and chromatin immunoprecipitation data, we identified genome-wide PIF4 and PIF5 dependent shade regulated gene as well as putative direct target genes of PIF5. This revealed evidence for a direct regulatory link between phytochrome signaling and the growth promoting phytohormone auxin (IAA) at the level of biosynthesis, transport and signaling. Subsequently, it was shown, that free-IAA levels are upregulated in response to shade. It is assumed that shade-induced auxin production takes predominantly place in cotyledons of seedlings. This implies, that IAA is subsequently transported basipetally to the hypocotyl and enhances elongation growth. The importance of auxin transport for growth responses has been established by chemical and genetic approaches. To gain a better understanding of spatio-temporal transcriptional regulation of shade-induce auxin, I generated in a second project, an organ specific high throughput data focusing on cotyledon and hypocotyl of young Arabidopsis seedlings. Interestingly, both organs show an opposite growth regulation by shade. I first investigated the spatio-transcriptional regulation of auxin re- sponsive gene, in order to determine how broad gene expression pattern can be explained by the hypothesized movement of auxin from cotyledons to hypocotyls in shade. The analysis suggests, that several genes are indeed regulated according to our prediction and others are regulated in a more complex manner. In addition, analysis of gene families of auxin biosynthetic and transport components, lead to the identification of essential family members for shade-induced growth re- sponses, which were subsequently experimentally confirmed. Finally, the analysis of expression pattern identified several candidate genes, which possibly explain aspects of the opposite growth response of the different organs.

Decoding auditory EEG responses in healthy and clinical populations: A comparative study.

BACKGROUND: Analyses of brain responses to external stimuli are typically based on the means computed across conditions. However in many cognitive and clinical applications, taking into account their variability across trials has turned out to be statistically more sensitive than comparing their means. NEW METHOD: In this study we present a novel implementation of a single-trial topographic analysis (STTA) for discriminating auditory evoked potentials at predefined time-windows. This analysis has been previously introduced for extracting spatio-temporal features at the level of the whole neural response. Adapting the STTA on specific time windows is an essential step for comparing its performance to other time-window based algorithms. RESULTS: We analyzed responses to standard vs. deviant sounds and showed that the new implementation of the STTA gives above-chance decoding results in all subjects (in comparison to 7 out of 11 with the original method). In comatose patients, the improvement of the decoding performance was even more pronounced than in healthy controls and doubled the number of significant results. COMPARISON WITH EXISTING METHOD(S): We compared the results obtained with the new STTA to those based on a logistic regression in healthy controls and patients. We showed that the first of these two comparisons provided a better performance of the logistic regression; however only the new STTA provided significant results in comatose patients at group level. CONCLUSIONS: Our results provide quantitative evidence that a systematic investigation of the accuracy of established methods in normal and clinical population is an essential step for optimizing decoding performance.

Factors modifying drug and placebo responses in randomized trials for bipolar mania

Factors modifying drug and placebo responses in randomized trials for bipolar mania. Yildiz A, Vieta E, Tohen M, Baldessarini RJ. Source Department of Psychiatry, Dokuz Eylül University, Izmir, Turkey. agul_yildiz@hotmail.com Abstract Randomized placebo-controlled trials (RCTs) are standard for assessing efficacy and safety of treatments. We pursued preliminary indications that some factors are associated differentially with responses to placebo or drugs in RCTs for bipolar mania. We meta-analysed data from RCTs to assess influences of study-site count, subjects' age, sex distribution, diagnostic subgroups, clinical features, trial-completion rates, and publication year on mean difference (MD) in mania ratings between intake and final assessments. In 38 RCTs involving 3812 placebo-treated and 6988 drug-treated patients, symptomatic improvement was similar in placebo arms of trials of effective (6.77, 95% CI 5.77-7.76) and ineffective (7.61, 95% CI 5.47-8.75) drugs. Lesser placebo responses (MD) and greater drug-placebo differences (Hedges' g) were associated with fewer study sites, younger patients' age, and male sex. More patients with initial psychotic features and more trial completion in drug arms were associated with greater drug-associated improvement (MD) and drug-placebo contrast (Hedges' g), whereas more mixed-state diagnoses decreased both measures. Identifying modifying factors can support more efficient and cost-effective designs of therapeutic trials. In trials for mania, fewer sites may limit placebo response and enhance drug-placebo contrasts.

Vanishing chromosomal inversion clines in Drosophila subobscura from Chile: Is behavioral thermoregulation to blame?

Chromosomal inversion clines paralleling the long-standing ones in native Palearctic populations of Drosophila subobscura evolved swiftly after this species invaded the Americas in the late 1970s and early 1980s. However, the new clines did not consistently continue to converge on the Old World baseline. Our recent survey of Chilean populations of D. subobscura shows that inversion clines have faded or even changed sign with latitude. Here, we investigate the hypothesis that this fading of inversion clines might be due to the Bogert effect, namely, that flies' thermoregulatory behavior has eventually compensated for environmental variation in temperature, thus buffering selection on thermal-related traits. We show that latitudinal divergence in thermal preference (T-p) has evolved in Chile for females, with higher-latitude flies having a lower mean T-p. Plastic responses in T-p also lessen latitudinal thermal variation because flies developed at colder temperatures prefer warmer microclimates. Our results are consistent with the idea that active behavioral thermoregulation might buffer environmental variation and reduce the potential effect of thermal selection on other traits as chromosomal arrangements.

Common Variants of TLR1 Associate with Organ Dysfunction and Sustained Pro- Inflammatory Responses during Sepsis

Background: Toll-like receptors (TLRs) are critical components for host pathogen recognition and variants in genes participating in this response influence susceptibility to infections. Recently, TLR1 gene polymorphisms have been found correlated with whole blood hyper-inflammatory responses to pathogen-associated molecules and associated with sepsis-associated multiorgan dysfunction and acute lung injury (ALI). We examined the association of common variants of TLR1 gene with sepsis-derived complications in an independent study and with serum levels for four inflammatory biomarker among septic patients. Methodology/Principal Findings: Seven tagging single nucleotide polymorphisms of the TLR1 gene were genotyped in samples from a prospective multicenter case-only study of patients with severe sepsis admitted into a network of intensive care units followed for disease severity. Interleukin (IL)-1 b, IL-6, IL-10, and C-reactive protein (CRP) serum levels were measured at study entry, at 48 h and at 7th day. Alleles -7202G and 248Ser, and the 248Ser-602Ile haplotype were associated with circulatory dysfunction among severe septic patients (0.001<=p <= 0.022), and with reduced IL-10 (0.012<= p <=0.047) and elevated CRP (0.011<= p <=0.036) serum levels during the first week of sepsis development. Additionally, the -7202GG genotype was found to be associated with hospital mortality (p =0.017) and ALI (p =0.050) in a combined analysis with European Americans, suggesting common risk effects among studies Conclusions/Significance: These results partially replicate and extend previous findings, supporting that variants of TLR1 gene are determinants of severe complications during sepsis.

Psychometric properties and dimensional structure of the Spanish version of the Coping Responses Inventory - Adult Form

One of the goals of psychological assessment focuses on the adaptation of its instruments to different populations. The objective of this study is to establish the psychometric properties and dimensional structure of the Spanish version of the Coping Responses Inventory- Adult Form (CRI-Adult, Moos, 1993). The following criteria were analyzed: a) descriptive statistics; b) internal consistency reliability (Cronbach"s alpha, and intercorrelations between scales); c) test-retest reliability (4-week interval); d) dimensionality of CRI-Adult (exploratory factor analysis); e) construct validity (confirmatory factor analysis); f) convergent criterion validity (correlations between CRI-Adult and Coping Strategies Indicator, CSI, Amirkhan, 1990), and g) predictive criterion validity (correlations between CRI-Adult, and SCL-90-R, Derogatis, 1983). The results, obtained with 800 adults from Barcelona and surrounding area (334 men and 466 women, aged between 18 to 76 years) indicate that the Spanish version of CRIAdult has satisfactory psychometric properties that allow using this test with guarantee.

Differential monocular vs. binocular pupil responses from melanopsin-based photoreception in patients with anterior ischemic optic neuropathy.

We examined the effect of anterior ischemic optic neuropathy (AION) on the activity of intrinsically photosensitive retinal ganglion cells (ipRGCs) using the pupil as proxy. Eighteen patients with AION (10 unilateral, 8 bilateral) and 29 age-matched control subjects underwent chromatic pupillometry. Red and blue light stimuli increasing in 0.5 log steps were presented to each eye independently under conditions of dark and light adaptation. The recorded pupil contraction was plotted against stimulus intensity to generate scotopic and photopic response curves for assessment of synaptically-mediated ipRGC activity. Bright blue light stimuli presented monocularly and binocularly were used for melanopsin activation. The post-stimulus pupil size (PSPS) at the 6th second following stimulus offset was the marker of intrinsic ipRGC activity. Finally, questionnaires were administered to assess the influence of ipRGCs on sleep. The pupil response and PSPS to all monocularly-presented light stimuli were impaired in AION eyes, indicating ipRGC dysfunction. To binocular light stimulation, the PSPS of AION patients was similar to that of controls. There was no difference in the sleep habits of the two groups. Thus after ischemic injury to one or both optic nerves, the summated intrinsic ipRGC activity is preserved when both eyes receive adequate light exposure.