METABOLIC EFFECTS OF DIETARY PROTEINS

SUMMARY :Non-alcoholic fatty liver disease (NAFLD) is characterized by an elevated intra- hepatocellular lipid (IHCL) concentration (> 5%). The incidence of NAFLD is frequently increased in obese patients, and is considered to be the hepatic component of the metabolic syndrome. The metabolic syndrome, also characterized by visceral obesity, altered glucose homeostasis, insulin resistance, dyslipidemia, and high blood pressure, represents actually a major public health burden. Both dietary factors and low physical activity are involved in the development of the metabolic syndrome. ln animals and healthy humans, high-fat or high-fructose diets lead to the development of several features of the metabolic syndrome including increased intrahepatic lipids and insulin resistance. ln contrast the effects of dietary protein are less well known, but an increase in protein intake has been suggested to exert beneficial effects by promoting weight loss and improving glucose homeostasis in insulin-resistant patients. Increased postprandial thermogenesis and enhanced satiety after protein ingestion may be both involved. The effects of dietary protein on hepatic lipids have been poorly investigated in humans, but preliminary studies in rodents have shown a reduction of hepatic lipids in carbohydrate fed rats and in obese rats. ln this context this work aimed at investigating the metabolic effects of dietary protein intake on hepatic lipid metabolism and glucose homeostasis in humans. The modulation by dietary proteins of exogenous lipid oxidation, net lipid oxidation, hepatic beta-oxidation, triglycerides concentrations, whole-body energy expenditure and glucose tolerance was assessed in the fasting state and in postprandial states. Measurements of IHCL were performed to quantify the amount of triglycerides in the liver. ln an attempt to cover all these metabolic aspects under different point of views, these questions were addressed by three protocols involving various feeding conditions. Study I addressed the effects of a 4-day hypercaloric high-fat high-protein diet on the accumulation of fat in the liver (IHCL) and on insulin sensitivity. Our findings indicated that a high protein intake significantly prevents intrahepatic fat deposition induced by a short- term hypercaloric high-fat diet, adverse effects of which are presumably modulated at the liver level.These encouraging results led us to conduct the second study (Study ll), as we were also interested in a more clinical approach to protein administration and especially if increased protein intakes might be of benefit for obese patients. Therefore the effects of one-month whey protein supplementation on IHCL, insulin sensitivity, lipid metabolism, glucose tolerance and renal function were assessed in obese women. Results showed that whey protein supplementation reduces hepatic steatosis and improves the plasma lipid profile in obese patients, without adverse effects on glucose tolerance or creatinine clearance. However since patients were fed ud-libitum, it remains possible that spontaneous carbohydrate and fat intakes were reduced due to the satiating effects of protein. The third study (Study lll) was designed in an attempt to deepen our comprehension about the mechanisms involved in the modulation of IHCL. We hypothesized that protein improved lipid metabolism and, therefore, we evaluated the effects of a high protein meal on postprandial lipid metabolism and glucose homeostasis after 4-day on a control or a protein diet. Our results did not sustain the hypothesis of an increased postprandial net lipid oxidation, hepatic beta oxidation and exogenous lipid oxidation. Four days on a high-protein diet rather decreased exogenous fat oxidation and enhanced postprandial triglyceride concentrations, by impairing probably chylomicron-TG clearance. Altogether the results of these three studies suggest a beneficial effect of protein intake on the reduction in lHCL, and clearly show that supplementation of proteins do not reduce IHCL by stimulating lipid metabolism, e.g. whole body fat oxidation, hepatic beta oxidation, or exogenous fat oxidation. The question of the effects of high-protein intakes on hepatic lipid metabolism is still open and will need further investigation to be elucidated. The effects of protein on increased postprandial lipemia and lipoproteins kinetics have been little investigated so far and might therefore be an interesting research question, considering the tight relationship between an elevation of plasmatic TG concentrations and the increased incidence of cardiovascular diseases.Résumé :La stéatose hépatique non alcoolique se caractérise par un taux de lipides intra-hépatiques élevé, supérieur à 5%. L'incidence de la stéatose hépatique est fortement augmentée chez les personnes obèses, ce qui mène à la définir comme étant la composante hépatique du syndrome métabolique. Ce syndrome se définit aussi par d'autres critères tels qu'obésité viscérale, altération de l'homéostasie du glucose, résistance à l'insuline, dyslipidémie et pression artérielle élevée. Le syndrome métabolique est actuellement un problème de santé publique majeur.Tant une alimentation trop riche et déséquilibrée, qu'une faible activité physique, semblent être des causes pouvant expliquer le développement de ce syndrome. Chez l'animal et le volontaire sain, des alimentations enrichies en graisses ou en sucres (fructose) favorisent le développement de facteurs associés au syndrome métabolique, notamment en augmentant le taux de lipides intra-hépatiques et en induisant le développement d'une résistance à l'insuline. Par ailleurs, les effets des protéines alimentaires sont nettement moins bien connus, mais il semblerait qu'une augmentation de l'apport en protéines soit bénéfique, favorisant la perte de poids et l'homéostasie du glucose chez des patients insulino-résistants. Une augmentation de la thermogenese postprandiale ainsi que du sentiment de satiété pourraient en être à l'origine.Les effets des protéines sur les lipides intra-hépatiques chez l'homme demeurent inconnus à ce jour, cependant des études préliminaires chez les rongeurs tendent à démontrer une diminution des lipides intra hépatiques chez des rats nourris avec une alimentation riche en sucres ou chez des rats obèses.Dans un tel contexte de recherche, ce travail s'est intéressé à l'étude des effets métaboliques des protéines alimentaires sur le métabolisme lipidique du foie et sur l'homéostasie du glucose. Ce travail propose d'évaluer l'effet des protéines alimentaires sur différentes voies métaboliques impliquant graisses et sucres, en ciblant d'une part les voies de l'oxydation des graisses exogènes, de la beta-oxydation hépatique et de l'oxydation nette des lipides, et d'autre part la dépense énergétique globale et l'évolution des concentrations sanguines des triglycérides, à jeun et en régime postprandial. Des mesures des lipides intra-hépatiques ont aussi été effectuées pour permettre la quantification des graisses déposées dans le foie.Dans le but de couvrir l'ensemble de ces aspects métaboliques sous différents angles de recherche, trois protocoles, impliquant des conditions alimentaires différentes, ont été entrepris pour tenter de répondre à ces questions. La première étude (Etude I) s'est intéressée aux effets d'u.ne suralimentation de 4 jours enrichie en graisses et protéines sur la sensibilité à l'insuline et sur l'accumulation de graisses intra-hépatiques. Les résultats ont démontré que l'apport en protéines prévient l'accumulation de graisses intra-hépatiques induite par une suralimentation riche en graisses de courte durée ainsi que ses effets délétères probablement par le biais de mécanismes agissant au niveau du foie. Ces résultats encourageants nous ont conduits à entreprendre une seconde étude (Etude ll) qui s'intéressait à l'implication clinique et aux bénéfices que pouvait avoir une supplémentation en protéines sur les graisses hépatiques de patients obèses. Ainsi nous avons évalué pendant un mois de supplémentation l'effet de protéines de lactosérum sur le taux de graisses intrahépatiques, la sensibilité à l'insuline, la tolérance au glucose, le métabolisme des graisses et la fonction rénale chez des femmes obèses. Les résultats ont été encourageants; la supplémentation en lactosérum améliore la stéatose hépatique, le profil lipidique des patientes obèses sans pour autant altérer la tolérance au glucose ou la clairance de la créatinine. L'effet satiétogene des protéines pourrait aussi avoir contribué à renforcer ces effets. La troisième étude s'est intéressée aux mécanismes qui sous-tendent les effets bénéfiques des protéines observés dans les 2 études précédentes. Nous avons supposé que les protéines devaient favoriser le métabolisme des graisses. Par conséquent, nous avons cherché a évaluer les effets d'un repas riche en protéines sur la lipémie postprandiale et l'homéostasie glucidique après 4 jours d'alimentation contrôlée soit isocalorique et équilibrée, soit hypercalorique enrichie en protéines. Les résultats obtenus n'ont pas vérifié l'hypothèse initiale ; ni une augmentation de l'oxydation nette des lipides, ni celle d'une augmentation de la béta-oxydation hépatique ou de l'oxydation d'un apport exogène de graisses n'a pu étre observée. A contrario, il semblerait même plutôt que 4 jours d'a]irnentation hyperprotéinée inhibent le métabolisme des graisses et augmente les concentrations sanguines de triglycérides, probablement par le biais d'une clairance de chylornicrons altérée. Globalement, les résultats de ces trois études nous permettent d'attester que les protéines exercent un effet bénéfique en prévenant le dépot de graisses intra-hépatiques et montrent que cet effet ne peut être attribué à une stimulation du métabolisme des lipides via l'augmentation des oxydations des graisses soit totales, hépatiques, ou exogènes. La question demeure en suspens à ce jour et nécessite de diriger la recherche vers d'autres voies d'exploration. Les effets des protéines sur la lipémie postprandiale et sur le cinétique des lipoprotéines n'a que peu été traitée à ce jour. Cette question me paraît néanmoins importante, sachant que des concentrations sanguines élevées de triglycérides sont étroitement corrélées à une incidence augmentée de facteurs de risque cardiovasculaire.

A Monte Carlo-based procedure for independent monitor unit calculation in IMRT treatment plans.

Intensity-modulated radiotherapy (IMRT) treatment plan verification by comparison with measured data requires having access to the linear accelerator and is time consuming. In this paper, we propose a method for monitor unit (MU) calculation and plan comparison for step and shoot IMRT based on the Monte Carlo code EGSnrc/BEAMnrc. The beamlets of an IMRT treatment plan are individually simulated using Monte Carlo and converted into absorbed dose to water per MU. The dose of the whole treatment can be expressed through a linear matrix equation of the MU and dose per MU of every beamlet. Due to the positivity of the absorbed dose and MU values, this equation is solved for the MU values using a non-negative least-squares fit optimization algorithm (NNLS). The Monte Carlo plan is formed by multiplying the Monte Carlo absorbed dose to water per MU with the Monte Carlo/NNLS MU. Several treatment plan localizations calculated with a commercial treatment planning system (TPS) are compared with the proposed method for validation. The Monte Carlo/NNLS MUs are close to the ones calculated by the TPS and lead to a treatment dose distribution which is clinically equivalent to the one calculated by the TPS. This procedure can be used as an IMRT QA and further development could allow this technique to be used for other radiotherapy techniques like tomotherapy or volumetric modulated arc therapy.

" Arabidopsis Thaliana " response to insect feeding : new components controlling defense gene expression and plant resistance

Abstract: Plants cannot run away to escape attacking herbivores, but they defend themselves by producing anti-digestive proteins and toxic compounds (for example glucosinolates). The first goal of this thesis was to study changes in gene expression after insect attack using microarrays. The responses of Arabidopsis thaliana to feeding by the specialist Pieris rapae and the generalist Spodoptera liffora is were compared. We found that the transcript profiles after feeding by the two chewing insects were remarkably similar, although the generalist induced a slightly stronger response. The second goal was to evaluate the implication of the four signals jasmonic acid (JA), salicylic acid (SA), ethylene (ET), and abscisic acid (ABA) in the control of insect-regulated gene expression. Using signaling mutants, we observed that JA was the predominant signal and that ABA modulated defense gene expression. In contrast, SA and ET appeared to control slightly gene expression, but only after feeding by S. litforalis. The third goal was to establish whether plant responses are really effective against insects. In accordance with the transcript profile, both insects were affected by the JA-dependent defenses, as they performed better on the JA-insensitive mutant. S. littoralis also performed better on ABA-deficient mutants, providing evidence for the role of ABA in defense against insects. When testing indole or aliphatic glucosinolate deficient mutants, we found that they were also more susceptible to insect feeding, providing some of the first genetic evidence for the defensive role of glucosinolates in planta. Finally, a glutathione-deficient mutant, pad2-1, was also more susceptible to insect feeding and we could attribute this phenotype to a lowered accumulation of the major indole glucosinolate. In this thesis, we provide a comprehensive list of insect-regulated genes, including many transcription factors that constitute interesting candidate genes for the further study of insect-induced expression changes. Understanding how the plant responses to insects are regulated will provide tools for a better management of insect pest in the field. Résumé: Les plantes ne peuvent s'échapper pour fuir les insectes qui les attaquent, mais elles se défendent en produisant des protéines anti-digestives et des composés toxiques (par exemple des glucosinolates). Le premier but de cette thèse était d'étudier les changements de l'expression génétique lors d'attaque par des insectes en utilisant des puces à ADN. Nous avons comparé la réponse d'Arabidopsis thaliana à deux espèces d'insectes avec des habitudes alimentaires différentes : le spécialiste Pieris rapae et le généraliste Spodoptera littoralis. Nous avons trouvé que les profils de transcription après l'attaque par les deux insectes sont remarquablement similaires, bien que le généraliste induise une réponse légèrement plus forte. Le deuxième but était de déterminer l'implication de quatre signaux dans le contrôle de la réponse :l'acide jasmonique (JA), l'acide salicylique (SA), l'éthylène (ET), et l'acide abscissique (ABA). En utilisant de mutants de signalisation, nous avons montré que l'acide jasmonique était le signal prédominant et que l'acide abscissique modulait l'expression génétique. D'autre part, l'acide salicylique et l'éthylène contrôlent à un degré moindre l'expression génétique, mais seulement après l'attaque par S. littoralís. Le troisième but était d'établir si les réponses des plantes sont efficaces contre les insectes. En accord avec le profil de transcription, les deux espèces d'insectes se sont mieux développées sur un mutant insensible au JA, indiquant que les défenses contrôlées par ce signal sont cruciales pour la plante. De plus, les larves de S. littorales se sont mieux développées sur des mutants déficients en ABA, ce qui fournit une preuve du rôle de l'acide abscissique dans la défense contre les insectes. En testant des mutants déficients en glucosinolates de type indole ou aliphatique, nous avons trouvé qu'ils étaient plus sensibles aux insectes, démontrant ainsi le rôle défensif des glucosinolates in planta. Finalement, le mutant déficient en glutathion pad2-1 était aussi plus sensible à l'attaque des insectes, et nous avons pu attribuer ce phénotype à une plus faible augmentation d'un indole glucosinolate dans ce mutant. Dans cette thèse, nous avons mis en évidence un nombre important de gènes contrôlés par les insectes, comprenant de nombreux facteurs de transcription qui constituent des candidats intéressants pour`étudier plus en détail les changements d'expression génétique induits par les insectes. Une meilleure compréhension de la réponse des plantes contre l'attaque des insectes devrait nous permettre de développer de nouvelles stratégies pour mieux gérer les ravageurs des cultures.

CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes.

Thymocytes and class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes express predominantly heterodimeric alpha/beta CD8. By interacting with non-polymorphic regions of MHC class I molecules CD8 can mediate adhesion or by binding the same MHC molecules that interact with the T-cell antigen receptor (TCR) function as coreceptor in TCR-ligand binding and T-cell activation. Using TCR photoaffinity labelling with a soluble, monomeric photoreactive H-2Kd-peptide derivative complex, we report here that the avidity of TCR-ligand interactions on cloned cytotoxic T cells is very greatly strengthened by CD8. This is primarily explained by coordinate binding of ligand molecules by CD8 and TCR, because substitution of Asp 227 of Kd with Lys severely impaired the TCR-ligand binding on CD8+, but not CD8- cells. Kinetic studies on CD8+ and CD8- cells further showed that CD8 imposes distinct dynamics and a remarkable temperature dependence on TCR-ligand interactions. We propose that the ability of CD8 to act as coreceptor can be modulated by CD8-TCR interactions.

Transscleral Coulomb-controlled iontophoresis of methylprednisolone into the rabbit eye: influence of duration of treatment, current intensity and drug concentration on ocular tissue and fluid levels.

The major problems associated with the use of corticosteroids for the treatment of ocular diseases are their poor intraocular penetration to the posterior segment when administered locally and their secondary side effects when given systemically. To circumvent these problems more efficient methods and techniques of local delivery are being developed. The purposes of this study were: (1) to investigate the pharmacokinetics of intraocular penetration of hemisuccinate methyl prednisolone (HMP) after its delivery using the transscleral Coulomb controlled iontophoresis (CCI) system applied to the eye or after intravenous (i.v.) injection in the rabbit, (2) to test the safety of the CCI system for the treated eyes and (3) to compare the pharmacokinetic profiles of HMP intraocular distribution after CCI delivery to i.v. injection. For each parameter evaluated, six rabbit eyes were used. For the CCI system, two concentrations of HMP (62.5 and 150mg ml(-1)), various intensities of current and duration of treatment were analyzed. In rabbits serving as controls the HMP was infused in the CCI device but without applied electric current. For the i.v. delivery, HMP at 10mg kg(-1)as a 62.5mg ml(-1)solution was used. The rabbits were observed clinically for evidence of ocular toxicity. At various time points after the administration of drug, rabbits were killed and intraocular fluids and tissues were sampled for methylprednisolone (MP) concentrations by high pressure liquid chromatography (HPLC). Histology examinations were performed on six eyes of each group. Among groups that received CCI, the concentrations of MP increased in all ocular tissues and fluids in relation to the intensities of current used (0.4, 1.0 and 2.0mA/0.5cm(2)) and its duration (4 and 10min). Sustained and highest levels of MP were achieved in the choroid and the retina of rabbit eyes treated with the highest current and 10min duration of CCI. No clinical toxicity or histological lesions were observed following CCI. Negligible amounts of MP were found in ocular tissues in the CCI control group without application of current. Compared to i.v. administration, CCI achieved higher and more sustained tissue concentrations with negligible systemic absorption. These data demonstrate that high levels of MP can be safely achieved in intraocular tissues and fluids of the rabbit eye, using CCI. With this system, intraocular tissues levels of MP are higher than those achieved after i.v. injection. Furthermore, if needed, the drug levels achieved with CCI can be modulated as a function of current intensity and duration of treatment. CCI could therefore be used as an alternative method for the delivery of high levels of MP to the intraocular tissues of both the anterior and posterior segments.

In vivo assessment of myelination by phase imaging at high magnetic field.

The present study evaluated the potential of using the phase of T2* weighted MR images to characterize myelination during brain development and pathology in rodents at 9.4 T. Phase contrast correlated with myelin content assessed by histology and suggests that most contrast between white and cortical gray matter is modulated by myelin. Ex vivo experiments showed that gray-white matter phase contrast remains unchanged after iron extraction. In dysmyelinated shiverer mice, phase imaging correlated strongly with myelin staining, showing reduced contrast between white and gray matter when compared to healthy controls. We conclude that high-resolution phase images, acquired at high field, allow assessment of myelination and dysmyelination.

MicroRNA-155 Is Required for Effector CD8(+) T Cell Responses to Virus Infection and Cancer.

MicroRNAs (miRNAs) regulate the function of several immune cells, but their role in promoting CD8(+) T cell immunity remains unknown. Here we report that miRNA-155 is required for CD8(+) T cell responses to both virus and cancer. In the absence of miRNA-155, accumulation of effector CD8(+) T cells was severely reduced during acute and chronic viral infections and control of virus replication was impaired. Similarly, Mir155(-/-) CD8(+) T cells were ineffective at controlling tumor growth, whereas miRNA-155 overexpression enhanced the antitumor response. miRNA-155 deficiency resulted in accumulation of suppressor of cytokine signaling-1 (SOCS-1) causing defective cytokine signaling through STAT5. Consistently, enforced expression of SOCS-1 in CD8(+) T cells phenocopied the miRNA-155 deficiency, whereas SOCS-1 silencing augmented tumor destruction. These findings identify miRNA-155 and its target SOCS-1 as key regulators of effector CD8(+) T cells that can be modulated to potentiate immunotherapies for infectious diseases and cancer.

IMRT credentialing for prospective trials using institutional virtual phantoms: results of a joint European Organization for the Research and Treatment of Cancer and Radiological Physics Center project.

BACKGROUND AND PURPOSE: Intensity-modulated radiotherapy (IMRT) credentialing for a EORTC study was performed using an anthropomorphic head phantom from the Radiological Physics Center (RPC; RPCPH). Institutions were retrospectively requested to irradiate their institutional phantom (INSTPH) using the same treatment plan in the framework of a Virtual Phantom Project (VPP) for IMRT credentialing. MATERIALS AND METHODS: CT data set of the institutional phantom and measured 2D dose matrices were requested from centers and sent to a dedicated secure EORTC uploader. Data from the RPCPH and INSTPH were thereafter centrally analyzed and inter-compared by the QA team using commercially available software (RIT; ver.5.2; Colorado Springs, USA). RESULTS: Eighteen institutions participated to the VPP. The measurements of 6 (33%) institutions could not be analyzed centrally. All other centers passed both the VPP and the RPC ±7%/4 mm credentialing criteria. At the 5%/5 mm gamma criteria (90% of pixels passing), 11(92%) as compared to 12 (100%) centers pass the credentialing process with RPCPH and INSTPH (p = 0.29), respectively. The corresponding pass rate for the 3%/3 mm gamma criteria (90% of pixels passing) was 2 (17%) and 9 (75%; p = 0.01), respectively. CONCLUSIONS: IMRT dosimetry gamma evaluations in a single plane for a H&N prospective trial using the INSTPH measurements showed agreement at the gamma index criteria of ±5%/5 mm (90% of pixels passing) for a small number of VPP measurements. Using more stringent, criteria, the RPCPH and INSTPH comparison showed disagreement. More data is warranted and urgently required within the framework of prospective studies.

Effect of orthographic processes on letter identity and letter-position encoding in dyslexic children.

The ability to identify letters and encode their position is a crucial step of the word recognition process. However and despite their word identification problem, the ability of dyslexic children to encode letter identity and letter-position within strings was not systematically investigated. This study aimed at filling this gap and further explored how letter identity and letter-position encoding is modulated by letter context in developmental dyslexia. For this purpose, a letter-string comparison task was administered to French dyslexic children and two chronological age (CA) and reading age (RA)-matched control groups. Children had to judge whether two successively and briefly presented four-letter strings were identical or different. Letter-position and letter identity were manipulated through the transposition (e.g., RTGM vs. RMGT) or substitution of two letters (e.g., TSHF vs. TGHD). Non-words, pseudo-words, and words were used as stimuli to investigate sub-lexical and lexical effects on letter encoding. Dyslexic children showed both substitution and transposition detection problems relative to CA-controls. A substitution advantage over transpositions was only found for words in dyslexic children whereas it extended to pseudo-words in RA-controls and to all type of items in CA-controls. Letters were better identified in the dyslexic group when belonging to orthographically familiar strings. Letter-position encoding was very impaired in dyslexic children who did not show any word context effect in contrast to CA-controls. Overall, the current findings point to a strong letter identity and letter-position encoding disorder in developmental dyslexia.

Role of β-TrCP1, variant and homologue in HIV-1 life cycle

Summary The CD4 molecule plays a key role in AIDS pathogenesis, it is required for entry of the virus into permissive cells and its subsequent down-modulation of the cell surface is a hallmark of HN-1 infected cells. The virus encodes no less than three proteins that participate in this process: Nef, Vpu and Env. Vpu protein interacts with CD4 within the endoplasmic reticulum of infected cells, where it targets CD4 for degradation through the interaction with a cellular protein named ß-TrCP1. This F-box protein functions as the substrate recognition subunit of the SCF ß-Trcr E3 ubiquitin ligase, which normally induce the ubiquitination and subsequent degradation of various proteins such as ß-catenin and IxBa. Mammals possess a homologue of ß-TrCP1, HOS, also named ß-TrCP2 which has a cytoplasmic subcellular distribution. Structural analysis of the ligand-binding domain of both homologues shows striking surface similarities. Both F-box proteins have a redundant role in a number of cellular processes; however the potential role of ß-TrCP2 in HIV-1 infected cells has not been evaluated. In the present study, we assessed the existence of génetic variants of BRTC, encoding ß-TrCP1, and evaluated whether these variants would affect CD4 down-modulation. Additionally, we determined whether ß-TrCP2 shares with its homologue structural and functional properties that would allow it to bind Vpu, modulate CD4 expression, and thus participate in HN-1 pathogenesis. We identified a single nucleotide polymorphism present in the human population with an allelic frequency of 0.03 that leads to the substitution of alanine 507 by a serine. However, we showed by transient transfection in HeLa CD4+ cells that this variant behaves as ß-TrCP1 with respect to CD4 down-modulation. We established transient expression systems in HeLa CD4+ cells to test whether ß-TrCP2 is implicated in Vpu-mediated CD4 down-modulation. We show by coimmunoprecipitation experiments that ß-TrCP2 binds Vpu and is able to induce CD4 down-modulation as efficiently as ß-TrCP1. In two different cell lines, HeLa CD4+ and Jurkat, Vpu-mediated CD4 down-modulation could not be completely reversed through the silencing of endogenous ß-TrCP 1 or ß-TrCP2 individually, but required both genes to be silenced simultaneously. We evaluated the role of ß-TrCP1 and ß-TrCP2 in HIV-1 life cycle using silencing prior to actual viral infection. Both ß-TrCP1 and ß-TrCP2 contributed to CD4 down-modulation during aone-cycle viral infection iri Ghost cells. In addition, the combined silencing of both homologues in the absence of env and nef reversed CD4 down-modulation, showing that ß-TrCP 1 and ß-TrCP2 represent the main and additive effectors of HIV-1 encoded Vpu. In addition, we showed that silencing of ß-TrCPI but not ß-TrCP2 induced a decrease of HIV-1 LTR-driven expression. In a transient transfection system with Tat and a LTR luciferase reporter, both homologues modulated LTR-driven expression. The present study revealed that ß-TrCP2 represents a novel protein participating in HIV-1 cycle and complete comprehension of the complex interplay occurring between the two F-Box will improve our understanding of HIV-1 infection. Résumé La molécule CD4 joue un rôle clef dans la pathogenèse du SIDA ; elle est requise pour l'entrée du virus dans les cellules permissives et la diminution de sa concentration au niveau de la surface cellulaire est une importante caractéristique des cellules infectées par le VIH-1. Le virus encode pas moins de trois protéines qui participent à ce processus Nef, Vpu et Env. La protéine Vpu lie CD4 au niveau du réticulum endoplasmique et induit sa dégradation en interagissant avec une protéine cellulaire nommée ß-TrCP 1. Cette protéine de type F-Box est une sous unité du complexe ubiquitine-ligase E3 SCFß-TrCP. Elle permet la reconnaissance du substrat par le complexe qui induit l'ubiquitination et la subséquente dégradation de diverses protéines cellulaires comme la ß-catenin ou IκBα. Les mammifères possèdent un homologue à ß-TrCP1appelé ß-TrCP2 (ou HOS). L'analyse comparative du domaine permettant la reconnaissance des substrats des deux homologues montre de frappantes similarités. Le rôle de ß-TrCP2 dans le cycle viral du VIH-1 n'a pas encore été évalué. Lors de cette étude, nous avons recherché l'existence de variants génétique de BTRC (codant pour ß-TrCP1) et nous avons évalué si ces variants pourraient affecter la dégradation des molécules CD4 induite par le virus. Nous avons ainsi identifié un polymorphisme présent dans la population humaine avec une fréquence allélique de 0.03 qui consiste en une substitution de l'alanine 507 par une sérine. Nous avons cependant montré par transfection dans des cellules HeLa CD4+ que ce variant se comporte comme ß-TrCP 1 en ce qui concerne la modulation de CD4. De plus, nous avons déterminé si ß-TrCP2 partageait avec son homologue des propriétés structurelles et fonctionnelles qui lui permettraient de lier Vpu, moduler la concentration de CD4 et ainsi prendre part à la pathogenèse du SIDA. Pour ce faire, nous avons établi un système d'expression temporaire dans des cellules HeLa CD4+. Par co-immunoprécipitation, nous avons montré que ß-TrCP2 lie Vpu et est capable d'induire la dégradation de CD4 aussi efficacement que ß-TrCP1. Dans deux différentes lignées cellulaires, HeLa CD4+ et Jurkat, la dégradation de CD4 n'a pu être complètement inhibée par le silencing individuel de ß-TrCP 1 ou ß-TrCP2, mais nécessitait le silencing simultané des 2 gènes. Nous avons évalué le rôle des deux homologues dans le cycle viral du VIH-1 en infectant des cellules Ghost avec le virus après avoir effectué un silencing des deux protéines. Nous avons ainsi montré que ß-TrCP 1 et ß-TrCP2 contribuent de manière additive à la dégradation de CD4 induite par une infection du VIH-1. Le silencing combiné des deux homologues inhiba complètement cette dégradation en l'absence de env et nef, prouvant qu'aucune autre voie ne participe à ce processus: En outre, nous avons montré que le silencing de ß-TrCP 1 mais pas celui de ß-TrCP2 induisait une diminution de l'expression virale sous contrôle du LTR. Nous n'avons cependant pas été en mesure de reconstituer cet effet en exprimant Tat et un gène reporteur sous contrôle du LTR dans des cellules HeLa CD4+. Le présent travail révèle que ß-TrCP2 représente une nouvelle protéine participant dans le cycle viral du VIH-1. Une complète compréhension de l'effet de chacun des deux homologues sur le cycle viral permettra d'améliorer notre compréhension de l'infection par le VIH-1.

The influence of tobacco consumption on the relationship between schizotypy and hemispheric asymmetry.

Tobacco use is positively associated with severity of symptoms along the schizophrenia spectrum. Accordingly it could be argued that neuropsychological performance, formerly thought to be modulated by schizotypy, is actually modulated by drug use or an interaction of drug use and schizotypy. We tested whether habitual cigarette smokers as compared to non-smokers would show a neuropsychological profile similar to that observed along the schizophrenia spectrum and, if so, whether smoking status or nicotine dependence would be more significant modulators of behavior than schizotypy. Because hemispheric dominance has been found to be attenuated along the schizophrenia spectrum, 40 right-handed male students (20 non-smokers) performed lateralized left- (lexical decisions) and right- (facial decision task) hemisphere dominant tasks. All individuals completed self-report measures of schizotypy and nicotine dependence. Schizotypy predicted laterality in addition to smoking status: While positive schizotypy (Unusual Experiences) was unrelated to hemispheric performance, Cognitive Disorganization predicted reduced left hemisphere dominant language functions. These latter findings suggest that Cognitive Disorganization should be regarded separately as a potentially important mediator of thought disorganization and language processing. Additionally, increasing nicotine dependence among smokers predicted a right hemisphere shift of function in both tasks that supports the role of the right hemisphere in compulsive/impulsive behavior.

La pertinence en science forensique. Une (en)quête épistémologique et empirique

A criminal investigation requires to search and to interpret vestiges of a criminal act that happened in a past time. The forensic investigator arises in this context as a critical reader of the investigation scene, in search of physical traces that should enable her to tell a story of the offence/crime which allegedly occurred. The challenge of any investigator is to detect and recognise relevant physical traces in order to provide forensic clues for investigation and intelligence purposes. Inspired by this obser- vation, the current research focuses on the following questions : What is a relevant physical trace? And, how does the forensic investigator know she is facing one ? The interest of such questions is to provide a definition of a dimension often used in forensic science but never studied in its implications and operations. This doctoral research investigates scientific paths that are not often explored in forensic science, by using semiotic and sociological tools combined with statistical data analysis. The results are shown following a semiotic path, strongly influenced by Peir- ce's studies, and a second track, called empirical, where investigations data were analysed and forensic investigators interviewed about their work practices in the field. By the semiotic track, a macroscopic view is given of a signification process running from the discove- red physical trace at the scene to what is evaluated as being relevant for the investigator. The physical trace is perceived in the form of several signs, whose meaning is culturally codified. The reasoning should consist of three main steps : 1- What kind of source does the discovered physical trace refer to ? 2- What cause/activity is at the origin of this source in the specific context of the case ? 3- What story can be told from these observations ? The stage 3 requires to reason in creating hypotheses that should explain the presence of the discovered trace coming from an activity ; the specific activity that is related to the investigated case. To validate this assumption, it would depend on their ability to respond to a rule of relevancy. The last step is the symbolisation of the relevancy. The rule would consist of two points : the recognition of the factual/circumstantial relevancy (Is the link between the trace and the case recognised with the formulated hypothesis ? ) and appropriate relevancy (What investment is required to collect and analyse the discovered trace considering the expected outcome at the investigation/intelligence level?). This process of meaning is based on observations and a conjectural reasoning subject to many influences. In this study, relevancy in forensic science is presented as a conventional dimension that is symbolised and conditioned by the context, the forensic investigator's practice and her workplace environment (culture of the place). In short, the current research states relevancy results of the interactions between parameters from situational, structural (or organisational) and individual orders. The detection, collection and analysis of relevant physical traces at scenes depends on the knowledge and culture mastered by the forensic investigator. In the study of the relation relevant trace-forensic investigator, this research introduces the KEE model as a conceptual map that illustrates three major areas of forensic knowledge and culture acquisition, involved in the research and evaluation of the relevant physical trace. Through the analysis of the investigation data and interviews, the relationship between those three parameters and the relevancy was highlighted. K, for knowing, embodies a rela- tionship to the immediate knowledge allowing to give an overview of the reality at a specific moment ; an important point since relevancy is signified in a context. E, for education, is considered through its relationship with relevancy via a culture that tends to become institutionalised ; it represents the theoretical knowledge. As for the parameter E, for experience, it exists in its relation to relevancy in the adjustments of the strategies of intervention (i.e a practical knowledge) of each practitioner having modulated her work in the light of success and setbacks case after case. The two E parameters constitute the library resources for the semiotic recognition process and the K parameter ensures the contextualisation required to set up the reasoning and to formulate explanatory hypotheses for the discovered physical traces, questioned in their relevancy. This research demonstrates that the relevancy is not absolute. It is temporal and contextual; it is a conventional and relative dimension that must be discussed. This is where the whole issue of the meaning of what is relevant to each stakeholder of the investigation process rests. By proposing a step by step approach to the meaning process from the physical trace to the forensic clue, this study aims to provide a more advanced understanding of the reasoning and its operation, in order to streng- then forensic investigators' training. This doctoral research presents a set of tools critical to both pedagogical and practical aspects for crime scene management while identifying key-influences with individual, structural and situational dimensions. - Une enquête criminelle consiste à rechercher et à faire parler les vestiges d'un acte incriminé passé. L'investigateur forensique se pose dans ce cadre comme un lecteur critique des lieux à la recherche de traces devant lui permettre de former son récit, soit l'histoire du délit/crime censé s'être produit. Le challenge de tout investigateur est de pouvoir détecter et reconnaître les traces dites pertinentes pour fournir des indices forensiques à buts d'enquête et de renseignement. Inspirée par un tel constat, la présente recherche pose au coeur de ses réflexions les questions suivantes : Qu'est-ce qu'une trace pertinente ? Et, comment fait le forensicien pour déterminer qu'il y fait face ? L'intérêt de tels questionnements se comprend dans la volonté de définir une dimension souvent utili- sée en science forensique, mais encore jamais étudiée dans ses implications et fonctionnements. Cette recherche se lance dans des voies d'étude encore peu explorées en usant d'outils sémiotiques et des pratiques d'enquêtes sociologiques combinés à des traitements statistiques de données. Les résultats sont représentés en suivant une piste sémiotique fortement influencée par les écrits de Peirce et une seconde piste dite empirique où des données d'interventions ont été analysées et des investigateurs forensiques interviewés sur leurs pratiques de travail sur le terrain. Par la piste sémiotique, une vision macroscopique du processus de signification de la trace en élément pertinent est représentée. La trace est perçue sous la forme de plusieurs signes dont la signification est codifiée culturellement. Le raisonnement se formaliserait en trois principales étapes : 1- Quel type de source évoque la trace détectée? 2- Quelle cause/activité est à l'origine de cette source dans le contexte précis du cas ? 3- Quelle histoire peut être racontée à partir de ces observations ? Cette dernière étape consiste à raisonner en créant des hypothèses devant expliquer la présence de la trace détectée suite à une activité posée comme étant en lien avec le cas investigué. Pour valider ces hypothèses, cela dépendrait de leur capacité à répondre à une règle, celle de la pertinence. Cette dernière étape consiste en la symbolisation de la pertinence. La règle se composerait de deux points : la reconnaissance de la pertinence factuelle (le lien entre la trace et le cas est-il reconnu dans l'hypothèse fournie?) et la pertinence appropriée (quel est l'investissement à fournir dans la collecte et l'exploitation de la trace pour le bénéfice attendu au niveau de l'investigation/renseignement?). Tout ce processus de signification se base sur des observations et un raisonnement conjectural soumis à de nombreuses influences. Dans cette étude, la pertinence en science forensique se formalise sous les traits d'une dimension conventionnelle, symbolisée, conditionnée par le contexte, la pratique de l'investigateur forensique et la culture du milieu ; en somme cette recherche avance que la pertinence est le fruit d'une interaction entre des paramètres d'ordre situationnel, structurel (ou organisationnel) et individuel. Garantir la détection, la collecte et l'exploitation des traces pertinentes sur les lieux dépend de la connaissance et d'une culture maîtrisées par le forensicien. Dans l'étude du rapport trace pertinente-investigateur forensique, la présente recherche pose le modèle SFE comme une carte conceptuelle illustrant trois grands axes d'acquisition de la connaissance et de la culture forensiques intervenant dans la recherche et l'évaluation de la trace pertinente. Par l'analyse des données d'in- terventions et des entretiens, le rapport entre ces trois paramètres et la pertinence a été mis en évidence. S, pour savoir, incarne un rapport à la connaissance immédiate pour se faire une représentation d'une réalité à un instant donné, un point important pour une pertinence qui se comprend dans un contexte. F, pour formation, se conçoit dans son rapport à la pertinence via cette culture qui tend à s'institutionnaliser (soit une connaissance théorique). Quant au paramètre E, pour expérience, il se comprend dans son rapport à la pertinence dans cet ajustement des stratégies d'intervention (soit une connaissance pratique) de chaque praticien ayant modulé leur travail au regard des succès et échecs enregistrés cas après cas. F et E formeraient la bibliothèque de ressources permettant le processus de reconnaissance sémiotique et S assurerait la contextualisation nécessaire pour poser le raisonnement et formuler les hypothèses explicatives pour les traces détectées et questionnées dans leur pertinence. Ce travail démontre que la pertinence n'est pas absolue. Elle est temporelle et contextuelle, c'est une dimension conventionnelle relative et interprétée qui se doit d'être discutée. C'est là que repose toute la problématique de la signification de ce qui est pertinent pour chaque participant du processus d'investigation. En proposant une lecture par étapes du processus de signification depuis la trace à l'indice, l'étude vise à offrir une compréhension plus poussée du raisonnement et de son fonctionnement pour renforcer la formation des intervenants forensiques. Cette recherche présente ainsi un ensemble d'outils critiques à portée tant pédagogiques que pratiques pour la gestion des lieux tout en identifiant des influences-clé jouées par des dimensions individuelles, structurelles et situationnelles.

Stimulus statistics shape oscillations in nonlinear recurrent neural networks.

Rhythmic activity plays a central role in neural computations and brain functions ranging from homeostasis to attention, as well as in neurological and neuropsychiatric disorders. Despite this pervasiveness, little is known about the mechanisms whereby the frequency and power of oscillatory activity are modulated, and how they reflect the inputs received by neurons. Numerous studies have reported input-dependent fluctuations in peak frequency and power (as well as couplings across these features). However, it remains unresolved what mediates these spectral shifts among neural populations. Extending previous findings regarding stochastic nonlinear systems and experimental observations, we provide analytical insights regarding oscillatory responses of neural populations to stimulation from either endogenous or exogenous origins. Using a deceptively simple yet sparse and randomly connected network of neurons, we show how spiking inputs can reliably modulate the peak frequency and power expressed by synchronous neural populations without any changes in circuitry. Our results reveal that a generic, non-nonlinear and input-induced mechanism can robustly mediate these spectral fluctuations, and thus provide a framework in which inputs to the neurons bidirectionally regulate both the frequency and power expressed by synchronous populations. Theoretical and computational analysis of the ensuing spectral fluctuations was found to reflect the underlying dynamics of the input stimuli driving the neurons. Our results provide insights regarding a generic mechanism supporting spectral transitions observed across cortical networks and spanning multiple frequency bands.

Deleterious Actions Of Vegf On The Blood Retinal Barrier And Photoreceptor Survival In The Light-damage Model

Purpose: The retinal balance between pro- and anti-angiogenic factors is critical for angiogenesis control, but is also involved in cell survival. We previously reported upregulation of VEGF and photoreceptor (PR) cell death in the Light-damage (LD) model. Preliminary results showed that anti-VEGF can rescue PR from cell death. Thus, we investigated the role of VEGF on the retina and we herein described the effect of anti-VEGF antibody delivered by lentiviral gene transfer in this model.Methods: To characterize the action of VEGF during the LD, we exposed Balb/c mice subretinally injected with LV-anti-VEGF, or not, to 5'000 lux for 1h. We next evaluated the retinal function, PR survival and protein expression (VEGF, VEGFR1/2, Src, PEDF, p38MAPK, Akt, Peripherin, SWL-opsin) after LD. We analyzed Blood retinal barrier (BRB) integrity on flat-mounted RPE and cryosections stained with β-catenin, ZO-1, N-cadherin and albumin.Results: Results indicate that the VEGF pathway is modulated after LD. LD leads to extravascular albumin leakage and BRB breakdown: β-catenin, ZO-1 and N-cadherin translocate to the cytoplasm of RPE cells showing loss of cell cohesion. This phenomenon is in adequacy with the VEGF time-course expression. Assessment of the retinal function reveals that PR rescue correlates with the level of LV-anti-VEGF expression. Rhodopsin content was higher in the LV-anti-VEGF group than in controls and measures of the ONL thickness indicate that LV-anti-VEGF preserves by 82% the outer nuclear layer from degeneration. Outer segments (OS) appeared well organized with an appropriate length in the LV-anti-VEGF group compared to controls, and the expression of SWL-opsin is maintained in the OS without being mislocalized as in the LV-GFP group. Finally, LV-anti-VEGF treatment prevents BRB breakdown and maintained RPE cell integrity.Conclusions: This study involves VEGF in LD and highlights the prime importance of the BRB integrity for PR survival. Taken together, these results show that anti-VEGF is neuroprotective in this model and maintains functional PR layer in LD-treated mice.

Accuracy of out-of-field dose calculation of tomotherapy and cyberknife treatment planning systems: A dosimetric study.

PURPOSE: Late toxicities such as second cancer induction become more important as treatment outcome improves. Often the dose distribution calculated with a commercial treatment planning system (TPS) is used to estimate radiation carcinogenesis for the radiotherapy patient. However, for locations beyond the treatment field borders, the accuracy is not well known. The aim of this study was to perform detailed out-of-field-measurements for a typical radiotherapy treatment plan administered with a Cyberknife and a Tomotherapy machine and to compare the measurements to the predictions of the TPS. MATERIALS AND METHODS: Individually calibrated thermoluminescent dosimeters were used to measure absorbed dose in an anthropomorphic phantom at 184 locations. The measured dose distributions from 6 MV intensity-modulated treatment beams for CyberKnife and TomoTherapy machines were compared to the dose calculations from the TPS. RESULTS: The TPS are underestimating the dose far away from the target volume. Quantitatively the Cyberknife underestimates the dose at 40cm from the PTV border by a factor of 60, the Tomotherapy TPS by a factor of two. If a 50% dose uncertainty is accepted, the Cyberknife TPS can predict doses down to approximately 10 mGy/treatment Gy, the Tomotherapy-TPS down to 0.75 mGy/treatment Gy. The Cyberknife TPS can then be used up to 10cm from the PTV border the Tomotherapy up to 35cm. CONCLUSIONS: We determined that the Cyberknife and Tomotherapy TPS underestimate substantially the doses far away from the treated volume. It is recommended not to use out-of-field doses from the Cyberknife TPS for applications like modeling of second cancer induction. The Tomotherapy TPS can be used up to 35cm from the PTV border (for a 390 cm(3) large PTV).