Hypoxie-ischémie cérébrale néonatale : rôle de la voie de JNK et implication de l'autophagie dans la mort neuronale

Résumé : Malgré les immenses progrès réalisés depuis plusieurs années en médecine obstétricale ainsi qu'en réanimation néonatale et en recherche expérimentale, l'asphyxie périnatale, une situation de manque d'oxygène autour du moment de la naissance, reste une cause majeure de mortalité et de morbidité neurologique à long terme chez l'enfant (retard mental, paralysie cérébrale, épilepsie, problèmes d'apprentissages) sans toutefois de traitement pharmacologique réel. La nécessité de développer de nouvelles stratégies thérapeutiques pour les complications de l'asphyxie périnatale est donc aujourd'hui encore essentielle. Le but général de ce travail est l'identification de nouvelles cibles thérapeutiques impliquées dans des mécanismes moléculaires pathologiques induits par l'hypoxie-ischémie (HI) dans le cerveau immature. Pour cela, le modèle d'asphyxie périnatale (proche du terme) le plus reconnu chez le rongeur a été développé (modèle de Rice et Vannucci). Il consiste en la ligature permanente d'une artère carotide commune (ischémie) chez le raton de 7 jours combinée à une période d'hypoxie à 8% d'oxygène. Il permet ainsi d'étudier les lésions de type hypoxique-ischémique dans différentes régions cérébrales dont le cortex, l'hippocampe, le striatum et le thalamus. La première partie de ce travail a abordé le rôle de deux voies de MAPK, JNK et p38, après HI néonatale chez le raton à l'aide de peptides inhibiteurs. Tout d'abord, nous avons démontré que D-JNKI1, un peptide inhibiteur de la voie de JNK présentant de fortes propriétés neuroprotectrices dans des modèles d'ischémie cérébrale adulte ainsi que chez le jeune raton, peut intervenir sur différentes voies de mort dont l'activation des calpaïnes (marqueur de la nécrose précoce), l'activation de la caspase-3 (marqueur de l'apoptose) et l'expression de LC3-II (marqueur de macroautophagie). Malgré ces effets positifs le traitement au D-JNKI1 ne modifie pas l'étendue de la lésion cérébrale. L'action limitée de D-JNKI1 peut s'expliquer par une implication modérée des JNKs (faiblement activées et principalement l'isotype JNK3) après HI néonatale sévère. Au contraire, l'inhibition de la voie de nNOS/p38 par le peptide DTAT-GESV permet une augmentation de 20% du volume du tissu sain à court et long terme. Le second projet a étudié les effets de l'HI néonatale sur l'autophagie neuronale. En effet, l'autophagie est un processus catabolique essentiel au bien-être de la cellule. Le type principal d'autophagie (« macroautophagie » , que nous appellerons par la suite « autophagie ») consiste en la séquestration d'éléments à dégrader (protéines ou organelles déficients) dans un compartiment spécialisé, l'autophagosome, qui fusionne avec un lysosome pour former un autolysosome où le contenu est dégradé par les hydrolases lysosomales. Depuis peu, l'excès ou la dérégulation de l'autoptiagie a pu être impliqué dans la mort cellulaire en certaines conditions de stress. Ce travail démontre que l'HI néonatale chez le raton active fortement le flux autophagique, c'est-à-dire augmente la formation des autophagosomes et des autolysosomes, dans les neurones en souffrance. De plus, la relation entre l'autophagie et l'apoptose varie selon la région cérébrale. En effet, alors que dans le cortex les neurones en voie de mort présentent des caractéristiques mixtes apoptotiques et autophagiques, ceux du CA3 sont essentiellement autophagiques et ceux du CA1 sont principalement apoptotiques. L'induction de l'autophagie après HI néonatale semble donc participer à la mort neuronale soit par l'enclenchement de l'apoptose soit comme mécanisme de mort en soi. Afin de comprendre la relation pouvant exister entre autophagie et apoptase un troisième projet a été réalisé sur des cultures primaires de neurones corticaux exposés à un stimulus apoptotique classique, la staurosporine (STS). Nous avons démontré que l'apoptose induite par la STS était précédée et accompagnée par une forte activation du flux autophagique neuronal. L'inhibition de l'autophagie de manière pharmacologique (3-MA) ou plus spécifiquement par ARNs d'interférence dirigés contre deux protéines autophagiques importantes (Atg7 et Atg5) a permis de mettre en évidence des rôles multiples de l'autophagie dans la mort neuronale. En effet, l'autophagie prend non seulement part à une voie de mort parallèle à l'apoptose pouvant être impliquée dans l'activation des calpaïnes, mais est également partiellement responsable de l'induction des voies apoptotiques (activation de la caspase-3 et translocation nucléaire d'AIF). En conclusion, ce travail a montré que l'inhibition de JNK par D-JNKI1 n'est pas un outil neuroprotecteur efficace pour diminuer la mort neuronale provoquée par l'asphyxie périnatalé sévère, et met en lumière deux autres voies thérapeutiques beaucoup plus prometteuses, l'inhibition de nNOS/p38 ou de l'autophagie. ABSTRACT : Despite enormous progress over the last«decades in obstetrical and neonatal medicine and experimental research, perinatal asphyxia, a situation of lack of oxygen around the time of the birth, remains a major cause of mortality and long term neurological morbidity in children (mental retardation, cerebral palsy, epilepsy, learning difficulties) without any effective treatment. It is therefore essential to develop new therapeutic strategies for the complications of perinatal asphyxia. The overall aim of this work was to identify new therapeutic targets involved in pathological molecular mechanisms induced by hypoxia-ischemia (HI) in the immature brain. For this purpose, the most relevant model of perinatal asphyxia (near term) in rodents has been developed (model of Rice and Vannucci). It consists in the permanent ligation of one common carotid artery (ischemia) in the 7-day-old rat combined with a period of hypoxia at 8% oxygen. This model allows the study of the hypoxic-ischemic lesion in different brain regions including the cortex, hippocampus, striatum and thalamus. The first part of this work addressed the role of two MAPK pathways (JNK and p38) after rat neonatal HI using inhibitory peptides. First, we demonstrated that D-JNKI1, a JNK peptide inhibitor presenting strong neuroprotective properties in models of cerebral ischemia in adult and young rats, could affect different cell death mechanisms including the activation of calpain (a marker of necrosis) and caspase-3 (a marker of apoptosis), and the expression of LC3-II (a marker of macroautophagy). Despite these positive effects, D-JNKI1 did not modify the extent of brain damage. The limited action of D-JNKI1 can be explained by the fact that JNKs were only moderately involved (weakly activated and principally the JNK3 isotype) after severe neonatal HI. In contrast, inhibition of nNOS/p38 by the peptide D-TAT-GESV increased the surviving tissue volume by around 20% at short and long term. The second project investigated the effects of neonatal HI on neuronal autophagy. Indeed, autophagy is a catabolic process essential to the well-being of the cell. The principal type of autophagy ("macroautophagy", that we shall henceforth call "autophagy") involves the sequestration of elements to be degraded (deficient proteins or organelles) in a specialized compartment, the autophagosome, which fuses with a lysosome to form an autolysosome where the content is degraded by lysosomal hydrolases. Recently, an excess or deregulation of autophagy has been implicated in cell death in some stress conditions. The present study demonstrated that rat neonatal HI highly enhanced autophagic flux, i.e. increased autophagosome and autolysosome formation, in stressed neurons. Moreover, the relationship between autophagy and apoptosis varies according to the brain region. Indeed, whereas dying neurons in the cortex exhibited mixed features of apoptosis and autophagy, those in CA3 were primarily autophagíc and those in CA1 were mainly apoptotic. The induction of autophagy after neonatal HI seems to participate in neuronal death either by triggering apoptosis or as a death mechanism per se. To understand the relationships that may exist between autophagy and apoptosis, a third project has been conducted using primary cortical neuronal cultures exposed to a classical apoptotic stimulus, staurosporine (STS). We demonstrated that STS-induced apoptosis was preceded and accompanied by a strong activation of neuronal autophagic flux. Inhibition of autophagy pharmacologically (3-MA) or more specifically by RNA interference directed against two important autophagic proteins (Atg7 and AtgS) showed multiple roles of autophagy in neuronal death. Indeed, autophagy was not only involved in a death pathway parallel to apoptosis possibly involved in the activation of calpains, but was also partially responsible for the induction of apoptotic pathways (caspase-3 activation and AIF nuclear translocation). In conclusion, this study showed that JNK inhibition by D-JNKI1 is not an effective neuroprotective tool for decreasing neuronal death following severe perinatal asphyxia, but highlighted two more promising therapeutic approaches, inhibition of the nNOSlp38 pathway or of autophagy.

Analysis of CD4+ and CD8+ T cells by defined MHC-peptide complexes

MHC class II-peptide multimers are important tools for the detection, enumeration and isolation of antigen-specific CD4+ Τ cells. However, their erratic and often poor performance impeded their broad application and thus in-depth analysis of key aspects of antigen-specific CD4+ Τ cell responses. In the first part of this thesis we demonstrate that a major cause for poor MHC class II tetramer staining performance is incomplete peptide loading on MHC molecules. We observed that peptide binding affinity for "empty" MHC class II molecules poorly correlates with peptide loading efficacy. Addition of a His-tag or desthiobiotin (DTB) at the peptide N-terminus allowed us to isolate "immunopure" MHC class II-peptide monomers by affinity chromatography; this significantly, often dramatically, improved tetramer staining of antigen-specific CD4+ Τ cells. Insertion of a photosensitive amino acid between the tag and the peptide, permitted removal of the tag from "immunopure" MHC class II-peptide complex by UV irradiation, and hence elimination of its potential interference with TCR and/or MHC binding. Moreover, to improve loading of self and tumor antigen- derived peptides onto "empty" MHC II molecules, we first loaded these with a photocleavable variant of the influenza A hemagglutinin peptide HA306-318 and subsequently exchanged it with a poorly loading peptide (e.g. NY-ESO-1119-143) upon photolysis of the conditional ligand. Finally, we established a novel type of MHC class II multimers built on reversible chelate formation between 2xHis-tagged MHC molecules and a fluorescent nitrilotriacetic acid (NTA)-containing scaffold. Staining of antigen-specific CD4+ Τ cells with "NTAmers" is fully reversible and allows gentle cell sorting. In the second part of the thesis we investigated the role of the CD8α transmembrane domain (TMD) for CD8 coreceptor function. The sequence of the CD8α TMD, but not the CD8β TMD, is highly conserved and homodimerizes efficiently. We replaced the CD8α TMD with the one of the interleukin-2 receptor a chain (CD8αTac) and thus ablated CD8α TMD interactions. We observed that ΤΙ Τ cell hybridomas expressing CD8αTacβ exhibited severely impaired intracellular calcium flux, IL-2 responses and Kd/PbCS(ABA) P255A tetramer binding. By means of fluorescence resonance energy transfer experiments (FRET) we established that CD8αTacβ associated with TCR:CD3 considerably less efficiently than CD8αβ, both in the presence and the absence of Kd/PbCS(ABA) complexes. Moreover, we observed that CD8αTacβ partitioned substantially less in lipid rafts, and related to this, associated less efficiently with p56Lck (Lck), a Src kinase that plays key roles in TCR proximal signaling. Our results support the view that the CD8α TMD promotes the formation of CD8αβP-CD8αβ dimers on cell surfaces. Because these contain two CD8β chains and that CD8β, unlike CD8α, mediates association of CD8 with TCR:CD3 as well as with lipid rafts and hence with Lck, we propose that the CD8αTMD plays an important and hitherto unrecognized role for CD8 coreceptor function, namely by promoting CD8αβ dimer formation. We discuss what implications this might have on TCR oligomerization and TCR signaling. - Les multimères de complexes MHC classe II-peptide sont des outils importants pour la détection, le dénombrement et l'isolation des cellules Τ CD4+ spécifiques pour un antigène d'intérêt. Cependant, leur performance erratique et souvent inadéquate a empêché leur utilisation généralisée, limitant ainsi l'analyse des aspects clés des réponses des lymphocytes Τ CD4+. Dans la première partie de cette thèse, nous montrons que la cause principale de la faible efficacité des multimères de complexes MHC classe II-peptide est le chargement incomplet des molécules MHC par des peptides. Nous montrons également que l'affinité du peptide pour la molécule MHC classe II "vide" n'est pas nécessairement liée au degré du chargement. Grâce à l'introduction d'une étiquette d'histidines (His-tag) ou d'une molécule de desthiobiotine à l'extrémité N-terminale du peptide, des monomères MHC classe II- peptide dits "immunopures" ont pu être isolés par chromatographic d'affinité. Ceci a permis d'améliorer significativement et souvent de façon spectaculaire, le marquage des cellules Τ CD4+ spécifiques pour un antigène d'intérêt. L'insertion d'un acide aminé photosensible entre l'étiquette et le peptide a permis la suppression de l'étiquette du complexe MHC classe- Il peptide "immunopure" par irradiation aux UV, éliminant ainsi de potentielles interférences de liaison au TCR et/ou au MHC. De plus, afin d'améliorer le chargement des molécules MHC classe II "vides" avec des peptides dérivés d'auto-antigènes ou d'antigènes tumoraux, nous avons tout d'abord chargé les molécules MHC "vides" avec un analogue peptidique photoclivable issu du peptide HA306-318 de l'hémagglutinine de la grippe de type A, puis, sous condition de photolyse, nous l'avons échangé avec de peptides à chargement faible (p.ex. NY-ESO-1119-143). Finalement, nous avons construit un nouveau type de multimère réversible, appelé "NTAmère", basé sur la formation chélatante reversible entre les molécules MHC-peptide étiquettés par 2xHis et un support fluorescent contenant des acides nitrilotriacetiques (NTA). Le marquage des cellules Τ CD4+ spécifiques pour un antigène d'intérêt avec les "NTAmères" est pleinement réversible et permet également un tri cellulaire plus doux. Dans la deuxième partie de cette thèse nous avons étudié le rôle du domaine transmembranaire (TMD) du CD8α pour la fonction coréceptrice du CD8. La séquence du TMD du CD8α, mais pas celle du TMD du CD8β, est hautement conservée et permet une homodimérisation efficace. Nous avons remplacé le TMD du CD8α avec celui de la chaîne α du récepteur à l'IL-2 (CD8αTac), éliminant ainsi les interactions du TMD du CD8α. Nous avons montré que les cellules des hybridomes Τ T1 exprimant le CD8αTacβ présentaient une atteinte sévère du flux du calcium intracellulaire, des réponses d'IL-2 et de la liaison des tétramères Kd/PbCS(ABA) P255A. Grâce aux expériences de transfert d'énergie entre molécules fluorescentes (FRET), nous avons montré que l'association du CD8αTacβ avec le TCR:CD3 est considérablement moins efficace qu'avec le CD8αβ, et ceci aussi bien en présence qu'en absence de complexes Kd/PbCS(ABA). De plus, nous avons observé que le CD8αTacβ se distribuait beaucoup moins bien dans les radeaux lipidiques, engendrant ainsi, une association moins efficace avec p56Lck (Lck), une kinase de la famille Src qui joue un rôle clé dans la signalisation proximale du TCR. Nos résultats soutiennent l'hypothèse que le TMD du CD8αβ favorise la formation des dimères de CD8αβ à la surface des cellules. Parce que ces derniers contiennent deux chaînes CD8β et que CD8β, contrairement à CD8α, favorise l'association du CD8 au TCR:CD3 aussi bien qu'aux radeaux lipidiques et par conséquent à Lck, nous proposons que le TMD du CD8α joue un rôle important, jusqu'alors inconnu, pour la fonction coreceptrice du CD8, en encourageant la formation des dimères CD8αβ. Nous discutons des implications possibles sur l'oligomerisation du TCR et la signalisation du TCR.

IκB-ζ controls the constitutive NF-κB target gene network and survival of ABC DLBCL.

Constitutive activation of the nuclear factor-κ B (NF-κB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-κB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IκB protein IκB-ζ to be upregulated in ABC compared with germinal center B-cell-like (GCB) DLBCL primary patient samples. Knockdown of IκB-ζ by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IκB-ζ knockdown demonstrated a significant downregulation of a large number of known NF-κB target genes, indicating an essential role of IκB-ζ in regulating a specific set of NF-κB target genes. To further investigate how IκB-ζ mediates NF-κB activity, we performed immunoprecipitations and detected a physical interaction of IκB-ζ with both p50 and p52 NF-κB subunits, indicating that IκB-ζ interacts with components of both the canonical and the noncanonical NF-κB pathway in ABC DLBCL. Collectively, our data demonstrate that IκB-ζ is essential for nuclear NF-κB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies.

Tomosyn-1 is involved in a post-docking event required for pancreatic beta-cell exocytosis.

Although the assembly of a ternary complex between the SNARE proteins syntaxin-1, SNAP25 and VAMP2 is known to be crucial for insulin exocytosis, the mechanisms controlling this key event are poorly understood. We found that pancreatic beta-cells express different isoforms of tomosyn-1, a syntaxin-1-binding protein possessing a SNARE-like motif. Using atomic force microscopy we show that the SNARE-like domain of tomosyn-1 can form a complex with syntaxin-1 and SNAP25 but displays binding forces that are weaker than those observed for VAMP2 (237+/-13 versus 279+/-3 pN). In pancreatic beta-cells tomosyn-1 was found to be concentrated in cellular compartments enriched in insulin-containing secretory granules. Silencing of tomosyn-1 in the rat beta-cell line INS-1E by RNA interference did not affect the number of secretory granules docked at the plasma membrane but led to a reduction in stimulus-induced exocytosis. Replacement of endogenous tomosyn-1 with mouse tomosyn-1, which differs in the nucleotide sequence from its rat homologue and escapes silencing, restored a normal secretory rate. Taken together, our data suggest that tomosyn-1 is involved in a post-docking event that prepares secretory granules for fusion and is necessary to sustain exocytosis of pancreatic beta-cells in response to insulin secretagogues.

Analysis of the enantiomers of citalopram and its demethylated metabolites using chiral liquid chromatography.

A procedure using a chirobiotic V column is presented which allows separation of the enantiomers of citalopram and its two N-demethylated metabolites, and of the internal standard, alprenolol, in human plasma. Citalopram, demethylcitalopram and didemethylcitalopram, as well as the internal standard, were recovered from plasma by liquid-liquid extraction. The limits of quantification were found to be 5 ng/ml for each enantiomer of citalopram and demethylcitalopram, and 7.5 ng/ml for each enantiomer of didemethylcitalopram. Inter- and intra-day coefficients of variation varied from 2.4% to 8.6% for S- and R-citalopram, from 2.9% to 7.4% for S- and R-demethylcitalopram, and from 5.6% to 12.4% for S- and R- didemethylcitalopram. No interference was observed from endogenous compounds following the extraction of plasma samples from 10 different patients treated with citalopram. This method allows accurate quantification for each enantiomer and is, therefore, well suited for pharmacokinetic and drug interaction investigations. The presented method replaces a previously described highly sensitive and selective high-performance liquid chromatography procedure using an acetylated 3-cyclobond column which, because of manufactural problems, is no longer usable for the separation of the enantiomers of citalopram and its demethylated metabolites.

Neural control of vascular reactions: impact of emotion and attention.

This study investigated the neural regions involved in blood pressure reactions to negative stimuli and their possible modulation by attention. Twenty-four healthy human subjects (11 females; age = 24.75 ± 2.49 years) participated in an affective perceptual load task that manipulated attention to negative/neutral distractor pictures. fMRI data were collected simultaneously with continuous recording of peripheral arterial blood pressure. A parametric modulation analysis examined the impact of attention and emotion on the relation between neural activation and blood pressure reactivity during the task. When attention was available for processing the distractor pictures, negative pictures resulted in behavioral interference, neural activation in brain regions previously related to emotion, a transient decrease of blood pressure, and a positive correlation between blood pressure response and activation in a network including prefrontal and parietal regions, the amygdala, caudate, and mid-brain. These effects were modulated by attention; behavioral and neural responses to highly negative distractor pictures (compared with neutral pictures) were smaller or diminished, as was the negative blood pressure response when the central task involved high perceptual load. Furthermore, comparing high and low load revealed enhanced activation in frontoparietal regions implicated in attention control. Our results fit theories emphasizing the role of attention in the control of behavioral and neural reactions to irrelevant emotional distracting information. Our findings furthermore extend the function of attention to the control of autonomous reactions associated with negative emotions by showing altered blood pressure reactions to emotional stimuli, the latter being of potential clinical relevance.

The p63 target HBP1 is required for skin differentiation and stratification.

Genetic experiments established that p63 is crucial for the development and maintenance of pluristratified epithelia. In the RNA interference (RNAi) screening for targets of p63 in keratinocytes, we identified the transcription factor, High Mobility Group (HMG) box protein 1 (HBP1). HBP1 is an HMG-containing repressor transiently induced during differentiation of several cell lineages. We investigated the relationship between the two factors: using RNAi, overexpression, chromatin immunoprecipitations and transient transfections with reporter constructs, we established that HBP1 is directly repressed by p63. This was further confirmed in vivo by evaluating expression in p63 knockout mice and in transgenics expressing p63 in basal keratinocytes. Consistent with these findings, expression of HBP1 increases upon differentiation of primary keratinocytes and HaCaT cells in culture, and it is higher in the upper layers of human skin. Inactivation of HBP1 by RNAi prevents differentiation of keratinocytes and stratification of organotypic skin cultures. Finally, we analyzed the keratinocyte transcriptomes after HBP1 RNAi; in addition to repression of growth-promoting genes, unexpected activation of differentiation genes was uncovered, coexisting with repression of other genes involved in epithelial cornification. Our data indicate that suppression of HBP1 is part of the growth-promoting strategy of p63 in the lower layers of epidermis and that HBP1 temporally coordinates expression of genes involved in stratification, leading to the formation of the skin barrier.

Feasibility and electromagnetic compatibility study of the ClearPEM front-end electronics for simultaneous PET-MR imaging

In this work we present a first feasibility study of the ClearPEM technology for simultaneous PET-MR imaging. The mutual electromagnetic interference (EMI) effects between both systems were evaluated on a 7 T magnet by characterizing the response behavior of the ClearPEM detectors and front-end electronics to pulsed RF power and switched magnetic field gradients; and by analyzing the MR system performance degradation from noise pickup into the RF receiver chain, and from magnetic susceptibility artifacts caused by PET front-end materials.

Tectonics of the Simplon massif and Lepontine gneiss dome: deformation structures due to collision between the underthrusting European plate and the Adriatic indenter

this study presents a review of published geological data, combined with original observations on the tectonics of the simplon massif and the Lepontine gneiss dome in the Western Alps. New observations concern the geometry of the Oligocene Vanzone back fold, formed under amphibolite facies conditions, and of its root between Domodossola and Locarno, which is cut at an acute angle by the Miocene, epi- to anchizonal, dextral centovalli strike-slip fault. the structures of the simplon massif result from collision over 50 Ma between two plate boundaries with a different geometry: the underthrusted European plate and the Adriatic indenter. Detailed mapping and analysis of a complex structural interference pattern, combined with observations on the metamorphic grade of the superimposed structures and radiometric data, allow a kinematic model to be developed for this zone of oblique continental collision. the following main Alpine tectonic phases and structures may be distinguished: 1. NW-directed nappe emplacement, starting in the Early Eocene (similar to 50 Ma); 2. W, SW and S- verging transverse folds; 3. transpressional movements on the dextral simplon ductile shear zone since similar to 32 Ma; 4. formation of the Bergell - Vanzone backfolds and of the southern steep belt during the Oligocene, emplacement of the mantle derived 31 - 29 Ma Bergell and Biella granodiorites and porphyritic andesites as well as intrusions of 29-25 Ma crustal aplites and pegmatites; 5. formation of the dextral discrete Rhone-Simplon line and the centovalli line during the Miocene, accompanied by the pull-apart development of the Lepontine gneiss dome - Dent blanche (Valpelline) depression. It is suggested that movements of shortening in fan shaped NW, W and sW directions accompanied the more regular NW- to WNW-directed displacement of the Adriatic indenter during continental collision.

Improved HIV-1 RNA quantitation by COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 using a novel dual-target approach.

BACKGROUND: HIV-1 RNA viral load is a key parameter for reliable treatment monitoring of HIV-1 infection. Accurate HIV-1 RNA quantitation can be impaired by primer and probe sequence polymorphisms as a result of tremendous genetic diversity and ongoing evolution of HIV-1. A novel dual HIV-1 target amplification approach was realized in the quantitative COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (HIV-1 TaqMan test v2.0) to cope with the high genetic diversity of the virus. OBJECTIVES AND STUDY DESIGN: The performance of the new assay was evaluated for sensitivity, dynamic range, precision, subtype inclusivity, diagnostic and analytical specificity, interfering substances, and correlation with the COBAS AmpliPrep/COBAS TaqMan HIV-1 (HIV-1 TaqMan test v1.0) predecessor test in patients specimens. RESULTS: The new assay demonstrated a sensitivity of 20 copies/mL, a linear measuring range of 20-10,000,000 copies/mL, with a lower limit of quantitation of 20 copies/mL. HIV-1 Group M subtypes and HIV-1 Group O were quantified within +/-0.3 log(10) of the assigned titers. Specificity was 100% in 660 tested specimens, no cross reactivity was found for 15 pathogens nor any interference for endogenous substances or 29 drugs. Good comparability with the predecessor assay was demonstrated in 82 positive patient samples. In selected clinical samples 35/66 specimens were found underquantitated in the predecessor assay; all were quantitated correctly in the new assay. CONCLUSIONS: The dual-target approach for the HIV-1 TaqMan test v2.0 enables superior HIV-1 Group M subtype coverage including HIV-1 Group O detection. Correct quantitation of specimens underquantitated in the HIV-1 TaqMan test v1.0 test was demonstrated.

Monitoramento Ambiental da Estrada São Domingos – Assomada, Ilha de Santiago, Cabo Verde, com Ênfase na Compartimentação Geomorfológica

Este trabalho tem por objetivo a aferição dos impactos ambientais ocorridos ao longo da estrada São Domingos-Assomada, na ilha de Santiago na República de Cabo Verde, África Ocidental no período de 2003 a 2009, com ênfase sobre os compartimentos geomorfológicos e seus materiais estruturadores, bem como estabelecer medidas de minimização dos referidos impactos. O estudo ainda procurou mostrar que o monitoramento ambiental das obras da rodovia tem uma série de vantagens porque pode identificar tendências nocivas sobre as variáveis ambientais e sociais antes que seja tarde demais para minimizar ou prevenir seus impactos. Ao longo da pesquisa foram destacadas as principais atividades que originaram impactos ambientais causados pelas obras tanto na fase de reabilitação como na fase de exploração da via. Buscou-se ainda mostrar que a crescente intervenção antrópica ao longo da estrada tem repercutido cada vez mais intensamente sobre a dinâmica do relevo e nos processos de erosão do solo associados às práticas inadequadas de uso da terra. Os impactos ambientais ocorridos durante a exploração da via resultaram, sobretudo, na degradação dos compartimentos geomorfológicos, por estes se constituírem no principal suporte das demais organizações espaciais. Metodologicamente este trabalho abrangeu uma pesquisa aplicada de cunho descritivo e exploratório visando melhor identificar as alterações nos sistemas ambientais, decorrentes da implantação da estrada e das atividades antrópicas realizadas no seu entorno. A fim de estimar a repercussão das intervenções junto à população imediatamente afetada foram aplicadas entrevistas voltadas à avaliação da percepção dos riscos ambientais. A coleta de dados geomorfológicos em campo foi realizada mediante a observação in loco das condições ambientais e sua posterior cartografação em gabinete. Os resultados dessas foram cotejados com o levantamento da literatura especializada a fim de permitir o enquadramento coerente do grau dos agravos ambientais analisados. Os resultados do estudo apontaram que a degradação dos compartimentos geomorfológicos e suas formações superficiais não ocorrem unicamente em função da estrada, mas também pelas práticas tradicionais de uso da terra. Ainda foi possível constatar que no caso da estrada São Domingos – Assomada, as obras de recuperação não levam em conta à dinâmica do relevo, ou seja, não consideram os parâmetros morfogenéticos. Dessa forma, tais obras essenciais de infra-estrutura acabam, muitas vezes, durando pouco tempo, desperdiçando recursos financeiros que são escassos nos países em desenvolvimento.

Modeling complex wells with the multi-scale finite-volume method

In this paper, an extension of the multi-scale finite-volume (MSFV) method is devised, which allows to Simulate flow and transport in reservoirs with complex well configurations. The new framework fits nicely into the data Structure of the original MSFV method,and has the important property that large patches covering the whole well are not required. For each well. an additional degree of freedom is introduced. While the treatment of pressure-constraint wells is trivial (the well-bore reference pressure is explicitly specified), additional equations have to be solved to obtain the unknown well-bore pressure of rate-constraint wells. Numerical Simulations of test cases with multiple complex wells demonstrate the ability of the new algorithm to capture the interference between the various wells and the reservoir accurately. (c) 2008 Elsevier Inc. All rights reserved.

Molecular mechanisms of insulin exocytosis role of small monomeric GTP-ASES

SUMMARYInsulin secretion from pancreatic beta-cells is a fundamental condition for the maintenance of blood glucose levels. During the last decades, important components of the molecular machinery controlling hormone release have been characterized. My PhD thesis was dedicated to the study of new signaling pathways regulating insulin exocytosis and in particular to the role of small monomelic guanine triphosphatase or GTPases controlling the last events of hormone release.The first part of my thesis focused on Ras-like (Ral) RalA and RalB proteins. We investigated the mechanisms leading to activation of Ral proteins in pancreatic beta-cells and analyzed their impact on different steps of the insulin-secretory process. Our results have shown that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-IE cells by RNA interference led to a decrease in secretagogue-induced hormone release. The activation of the GTPase, followed by FRET imaging, is triggered by increases in intracellular Ca and cAMP. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane, detected by TIRF microscopy and with strong impairment in PLD1 activation in response to secretagogues. RalA was found to be activated by the exchange factor RalGDS, which regulates hormone secretion induced by secretagogues and the docking step of insulin-containing granules at the plasma membrane. In the second part of this work we have shown that a member of the Rab family, Rab37, is present on insulin-containing secretory granules of pancreatic beta-cells. In addition, our experiments have suggested that Rab37 is required to obtain an optimal insulin secretory response induced by secretogogues and is important for the docking step of insulin-containing granules at the plasma membrane.

Pre-Mesozoic Alpine basements - Their place in the European Paleozoic framework

Prior to their Alpine overprinting, most of the pre-Mesozoic basement areas in Alpine orogenic structures shared a complex evolution, starting with Neoproterozoic sediments that are thought to have received detrital input from both West and East Gondwanan cratonic sources. A subsequent Neoproterozoic-Cambrian active margin setting at the Gondwana margin was followed by a Cambrian-Ordovician rifting period, including an Ordovician cordillera-like active margin setting. During the Late Ordovician and Silurian periods, the future Alpine domains recorded crustal extension along the Gondwana margin, announcing the future opening of the Paleotethys oceanic domain. Most areas then underwent Variscan orogenic events, including continental subduction and collisions with Avalonian-type basement areas along Laurussia and the juxtaposition and the duplication of terrane assemblages during strike slip, accompanied by contemporaneous crustal shortening and the subduction of Paleotethys under Laurussia. Thereafter, the final Pangea assemblage underwent Triassic and Jurassic extension, followed by Tertiary shortening, and leading to the buildup of the Alpine mountain chain. Recent plate-tectonic reconstructions place the Alpine domains in their supposed initial Cambrian-Ordovician positions in the eastern part of the Gondwana margin, where a stronger interference with the Chinese blocks is proposed, at least from the Ordovician onward. For the Visean time of the Variscan continental collision, the distinction of the former tectonic lower-plate situation is traceable but becomes blurred through the subsequent oblique subduction of Paleotethys under Laurussia accompanied by large-scale strike slip. Since the Pennsylvanian, this global collisional scenario has been replaced by subsequent and ongoing shortening and strike slip under rising geothermal conditions, and all of this occurred before all these puzzle elements underwent the complex Alpine reorganization.

Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity.

Resistance to alkylating agents via direct DNA repair by O(6)-methylguanine methyltransferase (MGMT) remains a significant barrier to the successful treatment of patients with malignant glioma. The relative expression of MGMT in the tumor may determine response to alkylating agents, and epigenetic silencing of the MGMT gene by promoter methylation plays an important role in regulating MGMT expression in gliomas. MGMT promoter methylation is correlated with improved progression-free and overall survival in patients treated with alkylating agents. Strategies to overcome MGMT-mediated chemoresistance are being actively investigated. These include treatment with nontoxic pseudosubstrate inhibitors of MGMT, such as O(6)-benzylguanine, or RNA interference-mediated gene silencing of MGMT. However, systemic application of MGMT inhibitors is limited by an increase in hematologic toxicity. Another strategy is to deplete MGMT activity in tumor tissue using a dose-dense temozolomide schedule. These alternative schedules are well tolerated; however, it remains unclear whether they are more effective than the standard dosing regimen or whether they effectively deplete MGMT activity in tumor tissue. Of note, not all patients with glioblastoma having MGMT promoter methylation respond to alkylating agents, and even those who respond will inevitably experience relapse. Herein we review the data supporting MGMT as a major mechanism of chemotherapy resistance in malignant gliomas and describe ongoing studies that are testing resistance-modulating strategies.