Evaluation of peripheral arterial bypass grafts with multi-detector row CT angiography: comparison with duplex US and digital subtraction angiography.

PURPOSE: To assess the technical feasibility of multi-detector row computed tomographic (CT) angiography in the assessment of peripheral arterial bypass grafts and to evaluate its accuracy and reliability in the detection of graft-related complications, including graft stenosis, aneurysmal changes, and arteriovenous fistulas. MATERIALS AND METHODS: Four-channel multi-detector row CT angiography was performed in 65 consecutive patients with 85 peripheral arterial bypass grafts. Each bypass graft was divided into three segments (proximal anastomosis, course of the graft body, and distal anastomosis), resulting in 255 segments. Two readers evaluated all CT angiograms with regard to image quality and the presence of bypass graft-related abnormalities, including graft stenosis, aneurysmal changes, and arteriovenous fistulas. The results were compared with McNemar test with Bonferroni correction. CT attenuation values were recorded at five different locations from the inflow artery to the outflow artery of the bypass graft. These findings were compared with the findings at duplex ultrasonography (US) in 65 patients and the findings at conventional digital subtraction angiography (DSA) in 27. RESULTS: Image quality was rated as good or excellent in 250 (98%) and in 252 (99%) of 255 bypass segments, respectively. There was excellent agreement both between readers and between CT angiography and duplex US in the detection of graft stenosis, aneurysmal changes, and arteriovenous fistulas (kappa = 0.86-0.99). CT angiography and duplex US were compared with conventional DSA, and there was no statistically significant difference (P >.25) in sensitivity or specificity between CT angiography and duplex US for both readers for detection of hemodynamically significant bypass stenosis or occlusion, aneurysmal changes, or arteriovenous fistulas. Mean CT attenuation values ranged from 232 HU in the inflow artery to 281 HU in the outflow artery of the bypass graft. CONCLUSION: Multi-detector row CT angiography may be an accurate and reliable technique after duplex US in the assessment of peripheral arterial bypass grafts and detection of graft-related complications, including stenosis, aneurysmal changes, and arteriovenous fistulas.

ACID-SENSING ION CHANNELS: functional and molecular characterization in putative nociceptors, and modulation by nerve injury and serine protease

Résumé Les canaux ioniques ASICs (acid-sensing ion channels) appartiennent à la famille des canaux ENaC/Degenerin. Pour l'instant, quatre gènes (1 à 4) ont été clonés dont certains présentent des variants d'épissage. Leur activation par une acidification rapide du milieu extracellulaire génère un courant entrant transitoire essentiellement sodique accompagné pour certains types d'ASICs d'une phase soutenue. Les ASICs sont exprimés dans le système nerveux, central (SNC) et périphérique (SNP). On leur attribue un rôle dans l'apprentissage, la mémoire et l'ischémie cérébrale au niveau central ainsi que dans la nociception (douleur aiguë et inflammatoire) et la méchanotransduction au niveau périphérique. Toutefois, les données sont parfois contradictoires. Certaines études suggèrent qu'ils sont des senseurs primordiaux impliqués dans la détection de l'acidification et la douleur. D'autres études suggèrent plutôt qu'ils ont un rôle modulateur inhibiteur dans la douleur. De plus, le fait que leur activation génère majoritairement un courant transitoire alors que les fibres nerveuses impliquées dans la douleur répondent à un stimulus nocif avec une adaptation lente suggère que leurs propriétés doivent être modulés par des molécules endogènes. Dans une première partie de ma thèse, nous avons abordé la question de l'expression fonctionnelle des ASICs dans les neurones sensoriels primaires afférents du rat adulte pour clarifier le rôle des ASICs dans les neurones sensoriels. Nous avons caractérisé leurs propriétés biophysiques et pharmacologiques par la technique du patch-clamp en configuration « whole-cell ». Nous avons pu démontrer que près de 60% des neurones sensoriels de petit diamètre expriment des courants ASICs. Nous avons mis en évidence trois types de courant ASIC dans ces neurones. Les types 1 et 3 ont des propriétés compatibles avec un rôle de senseur du pH alors que le type 2 est majoritairement activé par des pH inférieurs à pH6. Le type 1 est médié par des homomers de la sous-unité ASIC1 a qui sont perméables aux Ca2+. Nous avons étudié leur co-expression avec des marqueurs des nocicepteurs ainsi que la possibilité d'induire une activité neuronale suite à une acidification qui soit dépendante des ASICs. Le but était d'associer un type de courant ASIC avec une fonction potentielle dans les neurones sensoriels. Une majorité des neurones exprimant les courants ASIC co-expriment des marqueurs des nocicepteurs. Toutefois, une plus grande proportion des neurones exprimant le type 1 n'est pas associée à la nociception par rapport aux types 2 et 3. Nous avons montré qu'il est possible d'induire des potentiels d'actions suite à une acidification. La probabilité d'induction est proportionnelle à la densité des courants ASIC et à l'acidité de la stimulation. Puis, nous avons utilisé cette classification comme un outil pour appréhender les potentielles modulations fonctionnelles des ASICs dans un model de neuropathie (spared nerve injury). Cette approche fut complétée par des expériences de «quantitative RT-PCR ». En situation de neuropathie, les courants ASIC sont dramatiquement changés au niveau de leur expression fonctionnelle et transcriptionnelle dans les neurones lésés ainsi que non-lésés. Dans une deuxième partie de ma thèse, suite au test de différentes substances sécrétées lors de l'inflammation et l'ischémie sur les propriétés des ASICs, nous avons caractérisé en détail la modulation des propriétés des courants ASICs notamment ASIC1 par les sérines protéases dans des systèmes d'expression recombinants ainsi que dans des neurones d'hippocampe. Nous avons montré que l'exposition aux sérine-protéases décale la dépendance au pH de l'activation ainsi que la « steady-state inactivation »des ASICs -1a et -1b vers des valeurs plus acidiques. Ainsi, l'exposition aux serine protéases conduit à une diminution du courant quand l'acidification a lieu à partir d'un pH7.4 et conduit à une augmentation du courant quand l'acidification alleu à partir d'un pH7. Nous avons aussi montré que cette régulation a lieu des les neurones d'hippocampe. Nos résultats dans les neurones sensoriels suggèrent que certains courants ASICs sont impliqués dans la transduction de l'acidification et de la douleur ainsi que dans une des phases du processus conduisant à la neuropathie. Une partie des courants de type 1 perméables au Ca 2+ peuvent être impliqués dans la neurosécrétion. La modulation par les sérines protéases pourrait expliquer qu'en situation d'acidose les canaux ASICs soient toujours activables. Résumé grand publique Les neurones sont les principales cellules du système nerveux. Le système nerveux est formé par le système nerveux central - principalement le cerveau, le cervelet et la moelle épinière - et le système nerveux périphérique -principalement les nerfs et les neurones sensoriels. Grâce à leur nombreux "bras" (les neurites), les neurones sont connectés entre eux, formant un véritable réseau de communication qui s'étend dans tout le corps. L'information se propage sous forme d'un phénomène électrique, l'influx nerveux (ou potentiels d'actions). A la base des phénomènes électriques dans les neurones il y a ce que l'on appelle les canaux ioniques. Un canal ionique est une sorte de tunnel qui traverse l'enveloppe qui entoure les cellules (la membrane) et par lequel passent les ions. La plupart de ces canaux sont normalement fermés et nécessitent d'être activés pour s'ouvrire et générer un influx nerveux. Les canaux ASICs sont activés par l'acidification et sont exprimés dans tout le système nerveux. Cette acidification a lieu notamment lors d'une attaque cérébrale (ischémie cérébrale) ou lors de l'inflammation. Les expériences sur les animaux ont montré que les canaux ASICs avaient entre autre un rôle dans la mort des neurones lors d'une attaque cérébrale et dans la douleur inflammatoire. Lors de ma thèse je me suis intéressé au rôle des ASICs dans la douleur et à l'influence des substances produites pendant l'inflammation sur leur activation par l'acidification. J'ai ainsi pu montrer chez le rat que la majorité des neurones sensoriels impliqués dans la douleur ont des canaux ASICs et que l'activation de ces canaux induit des potentiels d'action. Nous avons opéré des rats pour qu'ils présentent les symptômes d'une maladie chronique appelée neuropathie. La neuropathie se caractérise par une plus grande sensibilité à la douleur. Les rats neuropathiques présentent des changements de leurs canaux ASICs suggérant que ces canaux ont une peut-être un rôle dans la genèse ou les symptômes de cette maladie. J'ai aussi montré in vitro qu'un type d'enryme produit lors de l'inflammation et l'ischémie change les propriétés des ASICs. Ces résultats confirment un rôle des ASICs dans la douleur suggérant notamment un rôle jusque là encore non étudié dans la douleur neuropathique. De plus, ces résultats mettent en évidence une régulation des ASICs qui pourrait être importante si elle se confirmait in vivo de part les différents rôles des ASICs. Abstract Acid-sensing ion channels (ASICs) are members of the ENaC/Degenerin superfamily of ion channels. Their activation by a rapid extracellular acidification generates a transient and for some ASIC types also a sustained current mainly mediated by Na+. ASICs are expressed in the central (CNS) and in the peripheral (PNS) nervous system. In the CNS, ASICs have a putative role in learning, memory and in neuronal death after cerebral ischemia. In the PNS, ASICs have a putative role in nociception (acute and inflammatory pain) and in mechanotransduction. However, studies on ASIC function are somewhat controversial. Some studies suggest a crucial role of ASICs in transduction of acidification and in pain whereas other studies suggest rather a modulatory inhibitory role of ASICs in pain. Moreover, the basic property of ASICs, that they are activated only transiently is irreconcilable with the well-known property of nociception that the firing of nociceptive fibers demonstrated very little adaptation. Endogenous molecules may exist that can modulate ASIC properties. In a first part of my thesis, we addressed the question of the functional expression of ASICs in adult rat dorsal root ganglion (DRG) neurons. Our goal was to elucidate ASIC roles in DRG neurons. We characterized biophysical and pharmacological properties of ASIC currents using the patch-clamp technique in the whole-cell configuration. We observed that around 60% of small-diameter sensory neurons express ASICs currents. We described in these neurons three ASIC current types. Types 1 and 3 have properties compatible with a role of pH-sensor whereas type 2 is mainly activated by pH lower than pH6. Type 1 is mediated by ASIC1a homomultimers which are permeable to Ca 2+. We studied ASIC co-expression with nociceptor markers. The goal was to associate an ASIC current type with a potential function in sensory neurons. Most neurons expressing ASIC currents co-expressed nociceptor markers. However, a higher proportion of the neurons expressing type 1 was not associated with nociception compared to type 2 and -3. We completed this approach with current-clamp measurements of acidification-induced action potentials (APs). We showed that activation of ASICs in small-diameter neurons can induce APs. The probability of AP induction is positively correlated with the ASIC current density and the acidity of stimulation. Then, we used this classification as a tool to characterize the potential functional modulation of ASICs in the spared nerve injury model of neuropathy. This approach was completed by quantitative RT-PCR experiments. ASICs current expression was dramatically changed at the functional and transcriptional level in injured and non-injured small-diameter DRG neurons. In a second part of my thesis, following an initial screening of the effect of various substances secreted during inflammation and ischemia on ASIC current properties, we characterized in detail the modulation of ASICs, in particular of ASIC1 by serine proteases in a recombinant expression system as well as in hippocampal neurons. We showed that protease exposure shifts the pH dependence of ASIC1 activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in the current response if ASIC1 is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provided evidence that this bi-directional regulation of ASIC1a function also occurs in hippocampal neurons. Our results in DRG neurons suggest that some ASIC currents are involved in the transduction of peripheral acidification and pain. Furthermore, ASICs may participate to the processes leading to neuropathy. Some Ca 2+-permeable type 1 currents may be involved in neurosecretion. ASIC modulation by serine proteases may be physiologically relevant, allowing ASIC activation under sustained slightly acidic conditions.

Face Short-Term Memory-Related Electroencephalographic Patterns can Differentiate Multi- versus Single-Domain Amnestic Mild Cognitive Impairment.

Amnestic mild cognitive impairment (aMCI) is characterized by memory deficits alone (single-domain, sd-aMCI) or associated with other cognitive disabilities (multi-domain, md-aMCI). The present study assessed the patterns of electroencephalographic (EEG) activity during the encoding and retrieval phases of short-term memory in these two aMCI subtypes, to identify potential functional differences according to the neuropsychological profile. Continuous EEG was recorded in 43 aMCI patients, whose 16 sd-aMCI and 27 md-aMCI, and 36 age-matched controls (EC) during delayed match-to-sample tasks for face and letter stimuli. At encoding, attended stimuli elicited parietal alpha (8-12 Hz) power decrease (desynchronization), whereas distracting stimuli were associated with alpha power increase (synchronization) over right central sites. No difference was observed in parietal alpha desynchronization among the three groups. For attended faces, the alpha synchronization underlying suppression of distracting letters was reduced in both aMCI subgroups, but more severely in md-aMCI cases that differed significantly from EC. At retrieval, the early N250r recognition effect was significantly reduced for faces in md-aMCI as compared to both sd-aMCI and EC. The results suggest a differential alteration of working memory cerebral processes for faces in the two aMCI subtypes, face covert recognition processes being specifically altered in md-aMCI.

Multi institutional phase II study of concomitant stereotactic reirradiation and cetuximab for recurrent head and neck cancer.

PURPOSE: Recurrent head and neck cancer is associated to a poor survival prognosis. A high toxicity rate is demonstrated when surgery and/or radiotherapy and/or chemotherapy are combined. Furthermore, the duration of treatment is often not ethically compatible with the expected survival (median survival<1year). Normal tissues tolerance limits the use of reirradiation and stereotactic body radiotherapy (SBRT) could offer precise irradiation while sparing healthy tissues. After completion of a feasibility study, results of a multicentric study (Lille, Nancy & Nice) using SBRT with cetuximab are reported. The aim of the study was to deliver non toxic short course SBRT (2weeks) in order to get the same local control as the one demonstrated with longer protocols. METHODS AND MATERIALS: Patients with inoperable recurrent, or new primary tumor in a previously irradiated area, were included (WHO<3). Reirradiation (RT) dose was 36Gy in six fractions of 6Gy to the 85% isodose line covering 95% of the PTV with 5 injections of concomitant cetuximab (CT). All patients had previous radiotherapy, 85% had previous surgery and 48% previous chemotherapy. RESULTS: Between 11/2007 and 08/2010, 60 were included (46 men and 14 women), 56 received CT+RT, 3 were not treated and 1 received only CT. Median age was 60 (42-87)) and all 56 patients had squamous carcinoma and received concomitant cetuximab. Mean time between previous radiotherapy and the start of SBRT was 38months. Cutaneous toxicity was observed for 41 patients. There was one toxic death from hemorrhage and denutrition. Median follow-up was 11.4months. At 3months, response rate was 58.4% (95% CI: 43.2-72.4%) and disease control rate was 91.7% (95% CI: 80.0-97.7%). The one-year OS rate was 47.5% (95% CI: 30.8-62.4). CONCLUSION: These results suggest that short SBRT with cetuximab is an effective salvage treatment with good response rate in this poor prognosis population with previously irradiated HNC. Treatment is feasible and, with appropriate care to limiting critical structure, acute toxicities are acceptable. This combination may be the reference treatment is this population.

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain.

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Navs remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Nav1.7-specific inhibitor, ProTxII, allowed the isolation of Nav1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Nav1.7 and Nav1.8 currents. The redistribution of Nav1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L-/-). SNS-Nedd4L-/- mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Nav1.7 and Nav1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Navs and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Quantitative multi-parameter mapping of R1, PD(*), MT, and R2(*) at 3T: a multi-center validation.

Multi-center studies using magnetic resonance imaging facilitate studying small effect sizes, global population variance and rare diseases. The reliability and sensitivity of these multi-center studies crucially depend on the comparability of the data generated at different sites and time points. The level of inter-site comparability is still controversial for conventional anatomical T1-weighted MRI data. Quantitative multi-parameter mapping (MPM) was designed to provide MR parameter measures that are comparable across sites and time points, i.e., 1 mm high-resolution maps of the longitudinal relaxation rate (R1 = 1/T1), effective proton density (PD(*)), magnetization transfer saturation (MT) and effective transverse relaxation rate (R2(*) = 1/T2(*)). MPM was validated at 3T for use in multi-center studies by scanning five volunteers at three different sites. We determined the inter-site bias, inter-site and intra-site coefficient of variation (CoV) for typical morphometric measures [i.e., gray matter (GM) probability maps used in voxel-based morphometry] and the four quantitative parameters. The inter-site bias and CoV were smaller than 3.1 and 8%, respectively, except for the inter-site CoV of R2(*) (<20%). The GM probability maps based on the MT parameter maps had a 14% higher inter-site reproducibility than maps based on conventional T1-weighted images. The low inter-site bias and variance in the parameters and derived GM probability maps confirm the high comparability of the quantitative maps across sites and time points. The reliability, short acquisition time, high resolution and the detailed insights into the brain microstructure provided by MPM makes it an efficient tool for multi-center imaging studies.

Surgical interventions with fatal outcome: Utility of multi-phase postmortem CT angiography

Cases of fatal outcome after surgical intervention are autopsied to determine the cause of death and to investigate whether medical error caused or contributed to the death. For medico-legal purposes, it is imperative that autopsy findings are documented clearly. Modern imaging techniques such as multi-detector computed tomography (MDCT) and postmortem CT angiography, which is used for vascular system imaging, are useful tools for determining cause of death. The aim of this study was to determine the utility of postmortem CT angiography for the medico-legal death investigation. This study investigated 10 medico-legal cases with a fatal outcome after surgical intervention using multi-phase postmortem whole body CT angiography. A native CT scan was performed as well as three angiographic phases (arterial, venous, and dynamic) using a Virtangio((R)) perfusion device and the oily contrast agent, Angiofil((R)). The results of conventional autopsy were compared to those from the radiological investigations. We also investigated whether the radiological findings affected the final interpretation of cause-of-death. Causes of death were hemorrhagic shock, intracerebral hemorrhage, septic shock, and a combination of hemorrhage and blood aspiration. The diagnoses were made by conventional autopsy as well as by postmortem CT angiography. Hemorrhage played an important role in eight of ten cases. The radiological exam revealed the exact source of bleeding in seven of the eight cases, whereas conventional autopsy localized the source of bleeding only generally in five of the seven cases. In one case, neither conventional autopsy nor CT angiography identified the source of hemorrhage. We conclude that postmortem CT angiography is extremely useful for investigating deaths following surgical interventions. This technique helps document autopsy findings and allows a second examination if it is needed; specifically, it detects and visualizes the sources of hemorrhages in detail, which is often of particular interest in such cases.

Predictors for the emergence of the 2 multi-nucleoside/nucleotide resistance mutations 69 insertion and Q151M and their impact on clinical outcome in the Swiss HIV cohort study.

The 69 insertion and Q151M mutations are multi-nucleoside/nucleotide resistance mutations (MNR). The prevalence among 4078 antiretroviral therapy (ART)-experienced individuals was <1.3%. Combined ART fully prevented MNR in subtype B infections. Case-control studies were performed to identify risk factors. Control subjects were patients with ≥ 3 thymidine-analogue mutations. The 69 insertion study (27 control subjects, 14 case patients) identified didanosine exposure as a risk (odds ratio, 5.0 per year; P = .019), whereas the Q151M study (which included 44 control subjects and 25 case patients) detected no associations. Following detection, individuals with Q151M tended to have lower suppression rates and higher mortality rates, relative to control subjects. Additional studies are needed to verify these findings in non-subtype B infections.

A multi-temporal approach to model endangered species distribution in Europe: the case of the Eurasian otter in Italy

We analysed the relationship between changes in land cover patterns and the Eurasian otter occurrence over the course of about 20 years (1985-2006) using multi-temporal Species Distribution Models (SDMs). The study area includes five river catchments covering most of the otter's Italian range. Land cover and topographic data were used as proxies of the ecological requirements of the otter within a 300-m buffer around river courses. We used species presence, pseudo-absence data, and environmental predictors to build past (1985) and current (2006) SDMs by applying an ensemble procedure through the BIOMOD modelling package. The performance of each model was evaluated by measuring the area under the curve (AUC) of the receiver-operating characteristic (ROC). Multi-temporal analyses of species distribution and land cover maps were performed by comparing the maps produced for 1985 and 2006. The ensemble procedure provided a good overall modelling accuracy, revealing that elevation and slope affected the otter's distribution in the past; in contrast, land cover predictors, such as cultivations and forests, were more important in the present period. During the transition period, 20.5% of the area became suitable, with 76% of the new otter presence data being located in these newly available areas. The multi-temporal analysis suggested that the quality of otter habitat improved in the last 20 years owing to the expansion of forests and to the reduction of cultivated fields in riparian belts. The evidence presented here stresses the great potential of riverine habitat restoration and environmental management for the future expansion of the otter in Italy

The role of PLK-1 in asynchronous cell division of two-cell stage "C. elegans" embryos

Summary Acquisition of lineage-specific cell cycle duration is an important feature of metazoan development. In Caenorhabditis a/egans, differences in cell cycle duration are already apparent in two-cell stage embryos, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. This time difference is under the control of anterior-posterior (A-P) polarity cues set by the PAR proteins. The mechanism by which these cues regulate the cell cycle machinery differentially in AB and P1 are incompletely understood. Previous work established that retardation of P1 cell division is due in part to preferential activation of an ATL1/CHK-1 dependent checkpoint in P1 but how the remaining time difference is controlled was not known at the onset of my work. The principal line of work in this thesis established that differential timing relies also on a mechanism that promotes mitosis onset preferentially in AB. The polo-like kinase PLK-1, a positive regulator of mitotic entry, is distributed in an asymmetric manner in two-cell stage embryos, with more protein present in AB than in P1. We find that PLK-1 asymmetry is regulated by anterior-posterior (A-P) polarity cues through preferential protein retention in the embryo anterior. Importantly, mild inactivation of plk-1 by RNAi delays entry into mitosis in P1 but not in AB, in a manner that is independent of ATL-1/CHK-1. Together, these findings favor a model in which differential timing of mitotic entry in C. elegans embryos relies on two complementary mechanisms: ATL-1/CHK-1 dependent preferential retardation in P1 and PLK-1 dependent preferential promotion in AB, which together couple polarity cues and cell cycle progression during early development. Besides analyzing PLK-1 asymmetry and its role in differential timing of two-cells stage embryos, we also characterized t2190, a mutant that exhibits reduced differential timing between AB and P1. We found this mutant to be a new allele of par-1. Additionally, we analyzed the role of NMY-2 in regulating the asynchrony of two-cell stage embryos, which may be uncoupled from its role in A-P polarity establishment and carried out a preliminary analysis of the mechanism underlying CDC-25 asymmetry between AB and P,. Overall, our works bring new insights into the mechanism controlling cell cycle progression in early C. elegans embryos. As most of the players important in C. elegans are conserved in other organisms, analogous mechanisms may be utilized in polarized cells of other species. Résumé Au cours du développement, les processus de division cellulaire sont régulés dans l'espace et le temps afin d'aboutir à la formation d'un organisme fonctionnel. Chez les Métazoaires, l'un des mécanismes de contrôle s'effectue au niveau de la durée du cycle cellulaire, celle-ci étant specifiée selon la lignée cellulaire. L'embryon du nématode Caenorhabditis elegans apparaît comme un excellent modèle d'étude de la régulation temporelle du cycle cellulaire. En effet, suite à la première division du zygote, l'embryon est alors composé de deux cellules de taille et d'identité différentes, appelées blastomères AB et P1. Ces deux cellules vont ensuite se diviser de manière asynchrone, le grand blastomère antérieur AB se divisant plus rapidement que le petit blastomère postérieur P1. Cette asynchronie de division est sous le contrôle des protéines PAR qui sont impliquées dans l'établissement de l'axe antéro-postérieur de l'embryon. A ce jour, les mécanismes moléculaires gouvernant ce processus d'asynchronie ne sont que partiellement compris. Des études menées précédemment ont établit que le retard de division observé dans le petit blastomère postérieur P1 était dû, en partie, à l'activation préférentielle dans cette cellule de ATL-1/CHK-1, protéines contrôlant la réponse à des erreurs dans le processus de réplication de l'ADN. L'analyse des autres mécanismes responsables de la différence temporelle d'entrée en mitose des deux cellules a été entreprise au cours de cette thèse. Nous avons considéré la possibilité que l'asynchronie de division était du à l'entrée préférentielle en mitose du grand blastomère AB. Nous avons établi que la protéine kinase PLK-1 (polo-like kinase 1), impliquée dans la régulation positive de la mitose, était distribuée de manière asymétrique dans l'embryon deux cellules. PLK-1 est en effet enrichi dans le blastomère AB. Cette localisation asymétrique de PLK-1 est sous le contrôle des protéines PAR et semble établie via une rétention de PLK-1 dans la cellule AB. Par ailleurs, nous avons démontré que l'inactivation partielle de plk-7 par interférence à ARN (RNAi) conduit à un délai de l'entrée en mitose de la cellule P1 spécifiquement, indépendamment des protéines régulatrices ATL-1/CHK-1. En conclusion, nous proposons un modèle de régulation temporelle de l'entrée en mitose dans l'embryon deux cellules de C. elegans basé sur deux mécanismes complémentaires. Le premier implique l'activation préférentielle des protéines ATL-1/CHK-1, et conduit à un retard d'entrée en mitose spécifiquement dans la cellule P1. Le second est basé sur la localisation asymétrique de la protéine kinase PLK-1 dans la cellule AB et induit une entrée précoce en mitose de cette cellule. Par ailleurs, nous avons étudié un mutant appelé t2190 qui réduit la différence temporelle d'entrée en mitose entre les cellules AB et P1. Nous avons démontré que ce mutant correspondait à un nouvel allèle du Bene par-1. De plus, nous avons analysé le rôle de NMY-2, une protéine myosine qui agit comme moteur moléculaire sur les filaments d'active; dans la régulation de l'asynchronie de division des blastomères AB et P1, indépendamment de sa fonction dans l'établissement de l'axe antéro-postérieur. Par ailleurs, nous avons commencé l'étude du mécanisme moléculaire régulant la localisation asymétrique entre les cellules AB et P1 de la protéine phosphatase CDC25, qui est également un important régulateur de l'entrée en mitose. En conclusion, ce travail de thèse a permis une meilleure compréhension des mécanismes gouvernant la progression du cycle cellulaire dans l'embryon précoce de C. elegans. Etant donné que la plupart des protéines impliquées dans ces processus sont conservées chez d'autres organismes multicellulaires, il apparaît probable que les mécanismes moléculaires révélés dans cette étude soit aussi utilisés chez ceux-ci.

Multi-scale systems analysis of plant development : beyond cellular signaling and tissue morphodynamics

Les plantes sont essentielles pour les sociétés humaines. Notre alimentation quotidienne, les matériaux de constructions et les sources énergétiques dérivent de la biomasse végétale. En revanche, la compréhension des multiples aspects développementaux des plantes est encore peu exploitée et représente un sujet de recherche majeur pour la science. L'émergence des technologies à haut débit pour le séquençage de génome à grande échelle ou l'imagerie de haute résolution permet à présent de produire des quantités énormes d'information. L'analyse informatique est une façon d'intégrer ces données et de réduire la complexité apparente vers une échelle d'abstraction appropriée, dont la finalité est de fournir des perspectives de recherches ciblées. Ceci représente la raison première de cette thèse. En d'autres termes, nous appliquons des méthodes descriptives et prédictives combinées à des simulations numériques afin d'apporter des solutions originales à des problèmes relatifs à la morphogénèse à l'échelle de la cellule et de l'organe. Nous nous sommes fixés parmi les objectifs principaux de cette thèse d'élucider de quelle manière l'interaction croisée des phytohormones auxine et brassinosteroïdes (BRs) détermine la croissance de la cellule dans la racine du méristème apical d'Arabidopsis thaliana, l'organisme modèle de référence pour les études moléculaires en plantes. Pour reconstruire le réseau de signalement cellulaire, nous avons extrait de la littérature les informations pertinentes concernant les relations entre les protéines impliquées dans la transduction des signaux hormonaux. Le réseau a ensuite été modélisé en utilisant un formalisme logique et qualitatif pour pallier l'absence de données quantitatives. Tout d'abord, Les résultats ont permis de confirmer que l'auxine et les BRs agissent en synergie pour contrôler la croissance de la cellule, puis, d'expliquer des observations phénotypiques paradoxales et au final, de mettre à jour une interaction clef entre deux protéines dans la maintenance du méristème de la racine. Une étude ultérieure chez la plante modèle Brachypodium dystachion (Brachypo- dium) a révélé l'ajustement du réseau d'interaction croisée entre auxine et éthylène par rapport à Arabidopsis. Chez ce dernier, interférer avec la biosynthèse de l'auxine mène à la formation d'une racine courte. Néanmoins, nous avons isolé chez Brachypodium un mutant hypomorphique dans la biosynthèse de l'auxine qui affiche une racine plus longue. Nous avons alors conduit une analyse morphométrique qui a confirmé que des cellules plus anisotropique (plus fines et longues) sont à l'origine de ce phénotype racinaire. Des analyses plus approfondies ont démontré que la différence phénotypique entre Brachypodium et Arabidopsis s'explique par une inversion de la fonction régulatrice dans la relation entre le réseau de signalisation par l'éthylène et la biosynthèse de l'auxine. L'analyse morphométrique utilisée dans l'étude précédente exploite le pipeline de traitement d'image de notre méthode d'histologie quantitative. Pendant la croissance secondaire, la symétrie bilatérale de l'hypocotyle est remplacée par une symétrie radiale et une organisation concentrique des tissus constitutifs. Ces tissus sont initialement composés d'une douzaine de cellules mais peuvent aisément atteindre des dizaines de milliers dans les derniers stades du développement. Cette échelle dépasse largement le seuil d'investigation par les moyens dits 'traditionnels' comme l'imagerie directe de tissus en profondeur. L'étude de ce système pendant cette phase de développement ne peut se faire qu'en réalisant des coupes fines de l'organe, ce qui empêche une compréhension des phénomènes cellulaires dynamiques sous-jacents. Nous y avons remédié en proposant une stratégie originale nommée, histologie quantitative. De fait, nous avons extrait l'information contenue dans des images de très haute résolution de sections transverses d'hypocotyles en utilisant un pipeline d'analyse et de segmentation d'image à grande échelle. Nous l'avons ensuite combiné avec un algorithme de reconnaissance automatique des cellules. Cet outil nous a permis de réaliser une description quantitative de la progression de la croissance secondaire révélant des schémas développementales non-apparents avec une inspection visuelle classique. La formation de pôle de phloèmes en structure répétée et espacée entre eux d'une longueur constante illustre les bénéfices de notre approche. Par ailleurs, l'exploitation approfondie de ces résultats a montré un changement de croissance anisotropique des cellules du cambium et du phloème qui semble en phase avec l'expansion du xylème. Combinant des outils génétiques et de la modélisation biomécanique, nous avons démontré que seule la croissance plus rapide des tissus internes peut produire une réorientation de l'axe de croissance anisotropique des tissus périphériques. Cette prédiction a été confirmée par le calcul du ratio des taux de croissance du xylème et du phloème au cours de développement secondaire ; des ratios élevés sont effectivement observés et concomitant à l'établissement progressif et tangentiel du cambium. Ces résultats suggèrent un mécanisme d'auto-organisation établi par un gradient de division méristématique qui génèrent une distribution de contraintes mécaniques. Ceci réoriente la croissance anisotropique des tissus périphériques pour supporter la croissance secondaire. - Plants are essential for human society, because our daily food, construction materials and sustainable energy are derived from plant biomass. Yet, despite this importance, the multiple developmental aspects of plants are still poorly understood and represent a major challenge for science. With the emergence of high throughput devices for genome sequencing and high-resolution imaging, data has never been so easy to collect, generating huge amounts of information. Computational analysis is one way to integrate those data and to decrease the apparent complexity towards an appropriate scale of abstraction with the aim to eventually provide new answers and direct further research perspectives. This is the motivation behind this thesis work, i.e. the application of descriptive and predictive analytics combined with computational modeling to answer problems that revolve around morphogenesis at the subcellular and organ scale. One of the goals of this thesis is to elucidate how the auxin-brassinosteroid phytohormone interaction determines the cell growth in the root apical meristem of Arabidopsis thaliana (Arabidopsis), the plant model of reference for molecular studies. The pertinent information about signaling protein relationships was obtained through the literature to reconstruct the entire hormonal crosstalk. Due to a lack of quantitative information, we employed a qualitative modeling formalism. This work permitted to confirm the synergistic effect of the hormonal crosstalk on cell elongation, to explain some of our paradoxical mutant phenotypes and to predict a novel interaction between the BREVIS RADIX (BRX) protein and the transcription factor MONOPTEROS (MP),which turned out to be critical for the maintenance of the root meristem. On the same subcellular scale, another study in the monocot model Brachypodium dystachion (Brachypodium) revealed an alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis. In the latter, increasing interference with auxin biosynthesis results in progressively shorter roots. By contrast, a hypomorphic Brachypodium mutant isolated in this study in an enzyme of the auxin biosynthesis pathway displayed a dramatically longer seminal root. Our morphometric analysis confirmed that more anisotropic cells (thinner and longer) are principally responsible for the mutant root phenotype. Further characterization pointed towards an inverted regulatory logic in the relation between ethylene signaling and auxin biosynthesis in Brachypodium as compared to Arabidopsis, which explains the phenotypic discrepancy. Finally, the morphometric analysis of hypocotyl secondary growth that we applied in this study was performed with the image-processing pipeline of our quantitative histology method. During its secondary growth, the hypocotyl reorganizes its primary bilateral symmetry to a radial symmetry of highly specialized tissues comprising several thousand cells, starting with a few dozens. However, such a scale only permits observations in thin cross-sections, severely hampering a comprehensive analysis of the morphodynamics involved. Our quantitative histology strategy overcomes this limitation. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with an automated cell type recognition algorithm, it allows precise quantitative characterization of vascular development and reveals developmental patterns that were not evident from visual inspection, for example the steady interspace distance of the phloem poles. Further analyses indicated a change in growth anisotropy of cambial and phloem cells, which appeared in phase with the expansion of xylem. Combining genetic tools and computational modeling, we showed that the reorientation of growth anisotropy axis of peripheral tissue layers only occurs when the growth rate of central tissue is higher than the peripheral one. This was confirmed by the calculation of the ratio of the growth rate xylem to phloem throughout secondary growth. High ratios are indeed observed and concomitant with the homogenization of cambium anisotropy. These results suggest a self-organization mechanism, promoted by a gradient of division in the cambium that generates a pattern of mechanical constraints. This, in turn, reorients the growth anisotropy of peripheral tissues to sustain the secondary growth.

Unification of multi-species vertebrate anatomy ontologies for comparative biology in Uberon.

BACKGROUND: Elucidating disease and developmental dysfunction requires understanding variation in phenotype. Single-species model organism anatomy ontologies (ssAOs) have been established to represent this variation. Multi-species anatomy ontologies (msAOs; vertebrate skeletal, vertebrate homologous, teleost, amphibian AOs) have been developed to represent 'natural' phenotypic variation across species. Our aim has been to integrate ssAOs and msAOs for various purposes, including establishing links between phenotypic variation and candidate genes. RESULTS: Previously, msAOs contained a mixture of unique and overlapping content. This hampered integration and coordination due to the need to maintain cross-references or inter-ontology equivalence axioms to the ssAOs, or to perform large-scale obsolescence and modular import. Here we present the unification of anatomy ontologies into Uberon, a single ontology resource that enables interoperability among disparate data and research groups. As a consequence, independent development of TAO, VSAO, AAO, and vHOG has been discontinued. CONCLUSIONS: The newly broadened Uberon ontology is a unified cross-taxon resource for metazoans (animals) that has been substantially expanded to include a broad diversity of vertebrate anatomical structures, permitting reasoning across anatomical variation in extinct and extant taxa. Uberon is a core resource that supports single- and cross-species queries for candidate genes using annotations for phenotypes from the systematics, biodiversity, medical, and model organism communities, while also providing entities for logical definitions in the Cell and Gene Ontologies. THE ONTOLOGY RELEASE FILES ASSOCIATED WITH THE ONTOLOGY MERGE DESCRIBED IN THIS MANUSCRIPT ARE AVAILABLE AT: http://purl.obolibrary.org/obo/uberon/releases/2013-02-21/ CURRENT ONTOLOGY RELEASE FILES ARE AVAILABLE ALWAYS AVAILABLE AT: http://purl.obolibrary.org/obo/uberon/releases/

Targeted therapy against multi-resistant bacteria in leukemic and hematopoietic stem cell transplant recipients: guidelines of the 4th European Conference on Infections in Leukemia (ECIL-4, 2011).

The detection of multi-resistant bacterial pathogens, particularly those to carbapenemases, in leukemic and stem cell transplant patients forces the use of old or non-conventional agents as the only remaining treatment options. These include colistin/polymyxin B, tigecycline, fosfomycin and various anti-gram-positive agents. Data on the use of these agents in leukemic patients are scanty, with only linezolid subjected to formal trials. The Expert Group of the 4(th) European Conference on Infections in Leukemia has developed guidelines for their use in these patient populations. Targeted therapy should be based on (i) in vitro susceptibility data, (ii) knowledge of the best treatment option against the particular species or phenotype of bacteria, (iii) pharmacokinetic/pharmacodynamic data, and (iv) careful assessment of the risk-benefit balance. For infections due to resistant Gram-negative bacteria, these agents should be preferably used in combination with other agents that remain active in vitro, because of suboptimal efficacy (e.g., tigecycline) and the risk of emergent resistance (e.g., fosfomycin). The paucity of new antibacterial drugs in the near future should lead us to limit the use of these drugs to situations where no alternative exists.

Multi-slice three-dimensional myocardial strain tensor quantification using zHARP.

In this article we propose a novel method for calculating cardiac 3-D strain. The method requires the acquisition of myocardial short-axis (SA) slices only and produces the 3-D strain tensor at every point within every pair of slices. Three-dimensional displacement is calculated from SA slices using zHARP which is then used for calculating the local displacement gradient and thus the local strain tensor. There are three main advantages of this method. First, the 3-D strain tensor is calculated for every pixel without interpolation; this is unprecedented in cardiac MR imaging. Second, this method is fast, in part because there is no need to acquire long-axis (LA) slices. Third, the method is accurate because the 3-D displacement components are acquired simultaneously and therefore reduces motion artifacts without the need for registration. This article presents the theory of computing 3-D strain from two slices using zHARP, the imaging protocol, and both phantom and in-vivo validation.

Multi-analyte detection for biological fluids. Towards continous monitoring of glucose, ionized calcium and pH using a viscometric affinity biosensor.

We present a viscometric affinity biosensor that can potentially allow continuous multi-analyte monitoring in biological fluids like blood or plasma. The sensing principle is based on the detection of viscosity changes of a polymeric solution which has a selective affinity for the analyte of interest. The chemico-mechanical sensor incorporates an actuating piezoelectric diaphragm, a sensing piezoelectric diaphragm and a flow-resisting microchannel for viscosity detection. A free-standing Anodic Alumina Oxide (AAO) porous nano-membrane is used as selective interface. A glucose-sensitive sensor was fabricated and extensively assessed in buffer solution. The sensor reversibility, stability and sensitivity were excellent during at least 65 hours. Results showed also a good degree of stability for a long term measurement (25 days). The sensor behaviour was furthermore tested in fetal bovine serum (FBS). The obtained results for glucose sensing are very promising, indicating that the developed sensor is a candidate for continuous monitoring in biological fluids. Sensitive solutions for ionized calcium and pH are currently under development and should allow multi-analyte sensing in the near future.