The use of positional scanning synthetic combinatorial libraries (PS-SCL) to study T Lympohcyte specificity and degeneracy

Les cellules CD8? T cytolytiques (CTL) sont les principaux effecteurs du système immunitaire adaptatif contre les infections et les tumeurs. La récente identification d?antigènes tumoraux humains reconnus par des cellules T cytolytiques est la base pour le, développement des vaccins antigène spécifiques contre le cancer. Le nombre d?antigènes tumoraux reconnus par des CTL que puisse être utilisé comme cible pour la vaccination des patients atteints du cancer est encore limité. Une nouvelle technique, simple et rapide, vient d?être proposée pour l?identification d?antigènes reconnus par des CTL. Elle se base sur l?utilisation de librairies combinatoriales de peptides arrangées en un format de "scanning" ou balayage par position (PS-SCL). La première partie de cette étude a consisté à valider cette nouvelle technique par une analyse détaillée de la reconnaissance des PS-SCL par différents clones de CTL spécifiques pour des antigènes associés à la tumeur (TAA) connus ainsi que par des clones de spécificité inconnue. Les résultats de ces analyses révèlent que pour tous les clones, la plupart des acides aminés qui composent la séquence du peptide antigénique naturel ont été identifiés par l?utilisation des PS-SCL. Les résultats obtenus ont permis d?identifier des peptides analogues ayant une antigènicité augmentée par rapport au peptide naturel, ainsi que des peptides comportant de multiples modifications de séquence, mais présentant la même réactivité que le peptide naturel. La deuxième partie de cette étude a consisté à effectuer des analyses biométriques des résultats complexes générés par la PS-SCL. Cette approche a permis l?identification des séquences correspondant aux épitopes naturels à partir de bases de données de peptides publiques. Parmi des milliers de peptides, les séquences naturelles se trouvent comprises dans les 30 séquences ayant les scores potentiels de stimulation les plus élevés pour chaque TAA étudié. Mais plus important encore, l?utilisation des PS-SCL avec un clone réactif contre des cellules tumorales mais de spécificité inconnue nous a permis d?identifier I?epitope reconnu par ce clone. Les données présentées ici encouragent l?utilisation des PS-SCL pour l?identification et l?optimisation d?épitopes pour des CTL réactifs anti-tumoraux, ainsi que pour l?étude de la reconnaissance dégénérée d?antigènes par les CTL.<br/><br/>CD8+ cytolytic T lymphocytes (CTL) are the main effector cells of the adaptive immune system against infection and tumors. The recent identification of moleculariy defined human tumor Ags recognized by autologous CTL has opened new opportunities for the development of Ag-specific cancer vaccines. Despite extensive work, however, the number of CTL-defined tumor Ags that are suitable targets for the vaccination of cancer patients is still limited, especially because of the laborious and time consuming nature of the procedures currentiy used for their identification. The use of combinatorial peptide libraries in positionai scanning format (Positional Scanning Synthetic Combinatorial Libraries, PS-SCL)' has recently been proposed as an alternative approach for the identification of these epitopes. To validate this approach, we analyzed in detail the recognition of PS-SCL by tumor-reactive CTL clones specific for multiple well-defined tumor-associated Ags (TAA) as well as by tumor-reactive CTL clones of unknown specificity. The results of these analyses revealed that for all the TAA-specific clones studied most of the amino acids composing the native antigenic peptide sequences could be identified through the use of PS-SCL. Based on the data obtained from the screening of PS-SCL, we could design peptide analogs of increased antigenicity as well as cross-reactive analog peptides containing multiple amino acid substitutions. In addition, the resuits of PS-SCL-screening combined with a recently developed biometric data analysis (PS-SCL-based biometric database analysis) allowed the identification of the native peptides in public protein databases among the 30 most active sequences, and this was the case for all the TAA studied. More importantiy, the screening of PS- SCL with a tumor-reactive CTL clone of unknown specificity resulted in the identification of the actual epitope. Overall, these data encourage the use of PS-SCL not oniy for the identification and optimization of tumor-associated CTL epitopes, but also for the analysis of degeneracy in T lymphocyte receptor (TCR) recognition of tumor Ags.<br/><br/>Les cellules T CD8? cytolytiques font partie des globules blancs du sang et sont les principales responsables de la lutte contre les infections et les tumeurs. Les immunologistes cherchent depuis des années à identifier des molécules exprimées et présentées à la surface des tumeurs qui puissent être reconnues par des cellules T CD8? cytolytiques capables ensuite de tuer ces tumeurs de façon spécifique. Ce type de molécules représente la base pour le développement de vaccins contre le cancer puisqu?elles pourraient être injectées aux patients afin d?induire une réponse anti- tumorale. A présent, il y a très peu de molécules capables de stimuler le système immunitaire contre les tumeurs qui sont connues parce que les techniques développées à ce jour pour leur identification sont complexes et longues. Une nouvelle technique vient d?être proposée pour l?identification de ce type de molécules qui se base sur l?utilisation de librairies de peptides. Ces librairies représentent toutes les combinaisons possibles des composants de base des molécules recherchées. La première partie de cette étude a consisté à valider cette nouvelle technique en utilisant des cellules T CD8? cytolytiques capables de tuer des cellules tumorales en reconnaissant une molécule connue présente à leur surface. On a démontré que l?utilisation des librairies permet d?identifier la plupart des composants de base de la molécule reconnue par les cellules T CD8? cytolytiques utilisées. La deuxième partie de cette étude a consisté à effectuer une recherche des molécules potentiellement actives dans des protéines présentes dans des bases des données en utilisant un programme informatique qui permet de classer les molécules sur la base de leur activité biologique. Parmi des milliers de molécules de la base de données, celles reconnues par nos cellules T CD8? cytolytiques ont été trouvées parmi les plus actives. Plus intéressant encore, la combinaison de ces deux techniques nous a permis d?identifier la molécule reconnue par une population de cellules T CD8? cytolytiques ayant une activité anti-tumorale, mais pour laquelle on ne connaissait pas la spécificité. Nos résultats encouragent l?utilisation des librairies pour trouver et optimiser des molécules reconnues spécifiquement par des cellules T CD8? cytolytiques capables de tuer des tumeurs.

Study of biomacromolecule conformational changes by Scanning Force Microscopy

L?objectif de ce travail de thèse est l?étude des changements conformationels des biomacromolecules à l?échelle d?une molécule unique. Pour cela on a utilisé la Microscopie à Force Atomique (AFM) appliqué à l?étude des protéines et des acides nucléiques déposés sur une surface. Dans ce type de microscopie, une pointe très fine attachée à l?extrémité d?un levier est balayée au dessus d?une surface. L?interaction de la pointe avec la surface de l?échantillon induit la déflection du levier et ce phénomène permet de reconstruire la topographie de l?échantillon. Très importante dans cette technique est la possibilité de travailler en liquide. Cela permet de étudier les biomolécules en conditions quasi-physiologiques sans qu?elles perdent leur activité. On a étudié GroEL, la chaperonin de E.coli, qui est un homo oligomère avec une structure à double anneau qui joue un rôle très important dans le repliement des protéines dénaturées et celles qui viennent d?être synthétisées. En particulier on a focalisé notre attention sur la stabilité mécanique et sur les changements conformationels qui ont lieu pendant l?activité de GroEL. Une analyse détaillée des changements dans la stabilité mécanique et des effets produits par la liaison et l?hydrolyse de l?ATP est présentée dans ce travail. On a montré que le point le plus faible dans la structure de GroEL est l?interface entre les deux anneaux et que l?étape critique dans l?affaiblissement de la structure est l?hydrolyse de l?ATP. En ce qui concerne le changement conformationel, le passage d?une surface hydrophobe à hydrophile, induit par l?hydrolyse de l?ATP, a été montré. Ensuite on a étudié le changement dans la conformation et dans la topologie de l?ADN résultant de l?interaction avec des molécules spécifiques et en réponse à l?exposition des cellules de E.coli à des conditions de stress. Le niveau de surenroulement est un paramètre très sensible, de façon variée, à tous ces facteurs. Les cellules qui ont crus à de températures plus élevées que leur température optimale ont la tendance à diminuer le nombre de surenroulements négatif pour augmenter la stabilité thermique de leur plasmides. L?interaction avec des agents intercalant induit une transition d?un surenroulement négatif à un surenroulement positif d?une façon dépendante de la température. Finalement, l?effet de l?interaction de l?ADN avec des surfaces différentes a été étudié et une application pratique sur les noeuds d?ADN est présentée.<br/><br/>The aim of the present thesis work is to study the conformational changes of biomacromolecules at the single molecule level. To that end, Atomic Force Microcopy (AFM) imaging was performed on proteins and nucleic acids adsorbed onto a surface. In this microcopy technique a very sharp tip attached at the end of a soft cantilever is scanned over a surface, the interaction of the tip with the sample?s surface will induce the deflection of the cantilever and thus it will make possible to reconstruct the topography. A very important feature of AFM is the possibility to operate in liquid, it means with the sample immersed in a buffer solution. This allows one to study biomolecules in quasi-physiological conditions without loosing their activity. We have studied GroEL, the chaperonin of E.coli, which is a double-ring homooligomer which pays a very important role in the refolding of unfolded and newly synthetized polypeptides. In particular we focus our attention on its mechanical stability and on the conformational change that it undergoes during its activity cycle. A detailed analysis of the change in mechanical stability and how it is affected by the binding and hydrolysis of nucleotides is presented. It has been shown that the weak point of the chaperonin complex is the interface between the two rings and that the critical step to weaken the structure is the hydrolysis of ATP. Concerning the conformational change we have directly measured, with a nanometer scale resolution, the switching from a hydrophobic surface to a hydrophilic one taking place inside its cavity induced by the ATP hydrolysis. We have further studied the change in the DNA conformation and topology as a consequence of the interaction with specific DNA-binding molecules and the exposition of the E.coli cells to stress conditions. The level of supercoiling has been shown to be a very sensitive parameter, even if at different extents, to all these factors. Cells grown at temperatures higher than their optimum one tend to decrease the number of the negative superhelical turns in their plasmids in order to increase their thermal stability. The interaction with intercalating molecules induced a transition from positive to negative supercoiling in a temperature dependent way. The effect of the interaction of the DNA with different surfaces has been investigated and a practical application to DNA complex knots is reported.<br/><br/>Observer les objets biologiques en le touchant Schématiquement le Microscope a Force Atomique (AFM) consiste en une pointe très fine fixée a l?extrémité d?un levier Lors de l?imagerie, la pointe de l?AFM gratte la surface de l?échantillon, la topographie de celui-ci induit des déflections du levier qui sont enregistrées au moyen d?un rayon laser réfléchi par le levier. Ces donnés sont ensuit utilisés par un ordinateur pour reconstituer en 3D la surface de l?échantillon. La résolution de l?instrument est fonction entre autre de la dureté, de la rugosité de l?échantillon et de la forme de la pointe. Selon l?échantillon et la pointe utilisée la résolution de l?AFM peut aller de 0.1 A (sur des cristaux) a quelque dizaine de nanomètres (sur des cellules). Cet instrument est particulierment intéressant en biologie en raison de sa capacité à imager des échantillons immergés dans un liquide, c?est à dire dans des conditions quasiphysiologiques. Dans le cadre de ce travail nous avons étudié les changements conformationels de molécules biologiques soumises à des stimulations externes. Nous avons essentielment concentré notre attention sur des complexes protéiques nommé Chaperons Moléculaires et sur des molécules d?ADN circulaire (plasmides). Les Chaperons sont impliqués entre autre dans la résistance des organismes vivants aux stress thermiques et osmotiques. Leur activité consiste essentielment à aider les autres protéines à être bien pliés dans leur conformation finale et, en conséquence, à eviter que ils soient dénaturées et que ils puissent s?agréger. L?ADN, quant à lui est la molécule qui conserve, dans sa séquence, l?information génétique de tous les organismes vivants. Ce travail a spécifiquement concerné l?étude des changements conformationels des chaperonins suit a leur activation par l?ATP. Ces travaux ont montrés a l?échelle de molécule unique la capacité de ces protéines de changer leur surface de hydrophobique a hydrophilique. Nous avons également utilisé l?AFM pour étudier le changement du nombre des surenroulements des molécules d?ADN circulaire lors d?une exposition à un changement de température et de force ionique. Ces travaux ont permis de montrer comment la cellule regle le nombre de surenroulements dans ces molécules pour répondre et contrôler l?expression génétique même dans de conditions extrêmes. Pour les deux molécules en général, c?était très important d?avoir la possibilité de observer leur transitions d?une conformation a l?autre directement a l?échelle d?une seul molécule et, surtout, avec une résolution largement au dessous des la longueur d?onde de la lumière visible que représente le limite pour l?imagerie optique.

Bilateral deep brain stimulation: the placement of the second electrode is not necessarily less accurate than that of the first one.

BACKGROUND: Deep brain stimulation (DBS) is recognized as an effective treatment for movement disorders. We recently changed our technique, limiting the number of brain penetrations to three per side. OBJECTIVES: The first aim was to evaluate the electrode precision on both sides of surgery since we implemented this surgical technique. The second aim was to analyse whether or not the electrode placement was improved with microrecording and macrostimulation. METHODS: We retrospectively reviewed operation protocols and MRIs of 30 patients who underwent bilateral DBS. For microrecording and macrostimulation, we used three parallel channels of the 'Ben Gun' centred on the MRI-planned target. Pre- and post-operative MRIs were merged. The distance between the planned target and the centre of the implanted electrode artefact was measured. RESULTS: There was no significant difference in targeting precision on both sides of surgery. There was more intra-operative adjustment of the second electrode positioning based on microrecording and macrostimulation, which allowed to significantly approach the MRI-planned target on the medial-lateral axis. CONCLUSION: There was more electrode adjustment needed on the second side, possibly in relation with brain shift. We thus suggest performing a single central track with electrophysiological and clinical assessment, with multidirectional exploration on demand for suboptimal clinical responses.

Bilateral deep brain stimulation : the placement of the second electrode is not necessarily less accurate than that of the first one

Introduction La stimulation cérébrale profonde est reconnue comme étant un traitement efficace des pathologies du mouvement. Nous avons récemment modifié notre technique chirurgicale, en limitant le nombre de pénétrations intracérébrales à trois par hémisphère. Objectif Le premier objectif de cette étude est d'évaluer la précision de l'électrode implantée des deux côtés de la chirurgie, depuis l'implémentation de cette technique chirurgicale. Le deuxième objectif est d'étudier si l'emplacement de l'électrode implantée était amélioré grâce à l'électrophysiologie. Matériel et méthode Il s'agit d'une étude rétrospective reprenant les protocoles opératoires et imageries à résonnance magnétique (IRM) cérébrales de 30 patients ayant subi une stimulation cérébrale profonde bilatérale. Pour l'électrophysiologie, nous avons utilisé trois canules parallèles du « Ben Gun », centrées sur la cible planifiée grâce à l'IRM. Les IRM pré- et post-opératoires ont été fusionnées. La distance entre la cible planifiée et le centre de l'artéfact de l'électrode implantée a été mesurée. Résultats Il n'y a pas eu de différence significative concernant la précision du ciblage des deux côtés (hémisphères) de la chirurgie. Il y a eu plus d'ajustements peropératoires du deuxième côté de la chirurgie, basé sur l'électrophysiologie, ce qui a permis d'approcher de manière significative la cible planifiée grâce à l'IRM, sur l'axe médio- latéral. Conclusion Il y a plus d'ajustements nécessaires de la position de la deuxième électrode, possiblement en lien avec le « brain shift ». Nous suggérons de ce fait d'utiliser une trajectoire d'électrode centrale accompagnée par de l'électrophysiologie, associé à une évaluation clinique. En cas de résultat clinique sub-optimal, nous proposons d'effectuer une exploration multidirectionnelle.

Landslide detection and monitoring capability of boat-based mobile laser scanning along Dieppe coastal cliffs, Normandy

Integrated in a wide research assessing destabilizing and triggering factors to model cliff dynamic along the Dieppe's shoreline in High Normandy, this study aims at testing boat-based mobile LiDAR capabilities by scanning 3D point clouds of the unstable coastal cliffs. Two acquisition campaigns were performed in September 2012 and September 2013, scanning (1) a 30-km-long shoreline and (2) the same test cliffs in different environmental conditions and device settings. The potentials of collected data for 3D modelling, change detection and landslide monitoring were afterward assessed. By scanning during favourable meteorological and marine conditions and close to the coast, mobile LiDAR devices are able to quickly scan a long shoreline with median point spacing up to 10cm. The acquired data are then sufficiently detailed to map geomorphological features smaller than 0.5m2. Furthermore, our capability to detect rockfalls and erosion deposits (>m3) is confirmed, since using the classical approach of computing differences between sequential acquisitions reveals many cliff collapses between Pourville and Quiberville and only sparse changes between Dieppe and Belleville-sur-Mer. These different change rates result from different rockfall susceptibilities. Finally, we also confirmed the capability of the boat-based mobile LiDAR technique to monitor single large changes, characterizing the Dieppe landslide geometry with two main active scarps, retrogression up to 40m and about 100,000m3 of eroded materials.

Pose-based SLAM with probabilistic scan matching algorithm using a mechanical scanned imaging sonar

This paper proposes a pose-based algorithm to solve the full SLAM problem for an autonomous underwater vehicle (AUV), navigating in an unknown and possibly unstructured environment. The technique incorporate probabilistic scan matching with range scans gathered from a mechanical scanning imaging sonar (MSIS) and the robot dead-reckoning displacements estimated from a Doppler velocity log (DVL) and a motion reference unit (MRU). The proposed method utilizes two extended Kalman filters (EKF). The first, estimates the local path travelled by the robot while grabbing the scan as well as its uncertainty and provides position estimates for correcting the distortions that the vehicle motion produces in the acoustic images. The second is an augment state EKF that estimates and keeps the registered scans poses. The raw data from the sensors are processed and fused in-line. No priory structural information or initial pose are considered. The algorithm has been tested on an AUV guided along a 600 m path within a marina environment, showing the viability of the proposed approach

Molecular Characteristics of Neuroblastoma with Special Reference to Novel Prognostic Factors and Diagnostic Applications

Molecular Characteristics of Neuroblastoma with Special Reference to Novel Prognostic Factors and Diagnostic Applications Department of Medical Biochemistry and Genetics Annales Universitatis Turkuensis, Medica-Odontologica, 2009, Turku, Finland Painosalama Oy, Turku, Finland 2009 Background: Neuroblastoma, which is the most common and extensively studied childhood solid cancer, shows a great clinical and biological heterogeneity. Most of the neuroblastoma patients older than one year have poor prognosis despite intensive therapies. The hallmark of neuroblastoma, biological heterogeneity, has hindered the discovery of prognostic tumour markers. At present, few molecular markers, such as MYCN oncogene status, have been adopted into clinical practice. Aims: The aim of the study was to improve the current prognostic methodology of neuroblastoma, especially by taking cognizance of the biological heterogeneity of neuroblastoma. Furthermore, unravelling novel molecular characteristics which associate with neuroblastoma tumour progression and cell differentiation was an additional objective. Results: A new strictly defined selection of neuroblastoma tumour spots of highest proliferation activity, hotspots, appeared to be representative and reliable in an analysis of MYCN amplification status using a chromogenic in situ hybridization technique (CISH). Based on the hotspot tumour tissue microarray immunohistochemistry and high-resolution oligo-array-based comparative genomic hybridization, which was integrated with gene expression and in silico analysis of existing transcriptomics, a polysialylated neural cell adhesion molecule (NCAM) and poorly characterized amplicon at 12q24.31 were discovered to associate with outcome. In addition, we found that a previously considered new neuroblastoma treatment target, the mutated c-kit receptor, was not mutated in neuroblastoma samples. Conclusions: Our studies indicate polysialylated NCAM and 12q24.31 amplicon to be new molecular markers with important value in prognostic evaluation of neuroblastoma. Moreover, the presented hotspot tumour tissue microarray method together with the CISH technique of the MYCN oncogene copy number is directly applicable to clinical use. Key words: neuroblastoma, polysialic acid, neural cell adhesion molecule, MYCN, c-kit, chromogenic in situ hybridization, hotspot

Kinetic study of the cobalt electrodeposition onto glassy carbon electrode from ammonium sulfate solutions

We carried out an electrochemical study of the cobalt electrodeposition onto glassy carbon electrode from an aqueous solution containing 10-2 M of CoSO4 + 1 M (NH4)2SO4 at natural pH 4.5. The potentiostatic study indicated a progressive 3D nucleation and growth during the deposition process. The average diffusion coefficient calculated for this system was 2.65 X 10-6 cm² s-1 while the ΔG for the formation of stable nucleus was 6.50 X 10-20 J/nuclei. The scanning electron microscopy images indicated the formation of small and homogeneous nucleus onto GCE of approximately 300 nm.

Optimization of solvent mixtures for extraction from bark of Schinus terebinthifolius by a statistical mixture-design technique and development of a UV-Vis spectrophotometric method for analysis of total polyphenols in the extract

A statistical mixture-design technique was used to study the effects of different solvents and their mixtures on the yield, total polyphenol content, and antioxidant capacity of the crude extracts from the bark of Schinus terebinthifolius Raddi (Anacardiaceae). The experimental results and their response-surface models showed that ternary mixtures with equal portions of all the three solvents (water, ethanol and acetone) were better than the binary mixtures in generating crude extracts with the highest yield (22.04 ± 0.48%), total polyphenol content (29.39 ± 0.39%), and antioxidant capacity (6.38 ± 0.21). An analytical method was developed and validated for the determination of total polyphenols in the extracts. Optimal conditions for the various parameters in this analytical method, namely, the time for the chromophoric reaction to stabilize, wavelength of the absorption maxima to be monitored, the reference standard and the concentration of sodium carbonate were determined to be 5 min, 780 nm, pyrogallol, and 14.06% w v-1, respectively. UV-Vis spectrophotometric monitoring of the reaction under these conditions proved the method to be linear, specific, precise, accurate, reproducible, robust, and easy to perform.

ELETRODOS DE FTO MODIFICADOS POR ELETRODEPOSIÇÃO DIRETA DE OURO: PRODUÇÃO, CARACTERIZAÇÃO E APLICAÇÃO COMO SENSOR ELETROQUÍMICO

Chemically modified electrodes have been studied to obtain new and better electrochemical sensors. Transparent conductive oxides, such as fluorine-doped tin-oxide (FTO), shows electrical conductivity comparable to metals and are potential candidates for new sensors. In this work, FTO was modified by gold electrodeposition from chlorine-auric acid solution using cyclic voltammetry (CV) technique. A set of different materials were produced, varying the scan number. Scanning electron microscopy and electrochemical impedance spectroscopy were performed for the characterization of electrodes surfaces. From this analysis was possible to observe the resistive, capacitive and difusional aspects from all kind of modified electrodes produced, establishing a relationship between this parameters and the scan number. The electrode with 100 scans of CV presented better characteristics for an electrochemical sensor; it has the lowest global impedance and rising of capacitive behavior (related to electrical double layer formation) at lower frequencies. This electrode was tested for paracetamol and caffeine detection. The results showed a high specificity, decreased oxidation potential (0.58 V and 0.97 Vvs. SCE, for paracetamol and caffeine, respectively) and low detection limits (0.82 and 0.052 µmol L-1).

Use of nuclear technique in samples for agricultural purposes

The concern related to environment is growing. Due to this, it is needed to determine chemical elements in a large range of concentration. The neutron activation technique (NAA) determines the elemental composition by the measurement of artificial radioactivity in a sample that was submitted to a neutron flux. NAA is a sensitive and accurate technique with low detection limits. An example of application of NAA was the measurement of concentrations of rare earth elements (REE) in waste samples of phosphogypsum (PG) and cerrado soil samples (clayey and sandy soils). Additionally, a soil reference material of the International Atomic Energy Agency (IAEA) was also analyzed. The REE concentration in PG samples was two times higher than those found in national fertilizers, (total of 4,000 mg kg-1 ), 154 times greater than the values found in the sandy soil (26 mg kg-1 ) and 14 times greater than the in clayey soil (280 mg kg-1 ). The experimental results for the reference material were inside the uncertainty of the certified values pointing out the accuracy of the method (95%). The determination of La, Ce, Pr, Nd, Pm, Sm, Eu, Tb, Dy, Ho, Er, Tm, Yb and Lu in the samples and reference material confirmed the versatility of the technique on REE determination in soil and phosphogypsum samples that are matrices for agricultural interest.

Uncertainties of the 137Cs technique for validation of soil redistribution modelling in a semiarid meso-scale watershed

Scarcity of long-term series of sediment-related variables has led watershed managers to apply mathematical models to simulate sediment fluxes. Due to the high efforts for installation and maintenance of sedimentological gauges, tracers have been pointed out as an alternative to validate soil redistribution modelling. In this study, the 137Cs technique was used to assess the WASA-SED model performance at the Benguê watershed (933 km²), in the Brazilian semiarid. Qualitatively, good agreement was found among the 137Cs technique and the WASA-SED model results. Nonetheless, quantitatively great differences, up to two orders of magnitude, were found between the two methods. Among the uncertainties inherent to the 137Cs technique, definition of the reference inventory seems to be a major source of imprecision. In addition, estimations of water and sediment fluxes with mathematical models usually also present high uncertainty, contributing to the quantitative differences of the soil redistribution estimates with the two methods.

Reference range of uterine artery Doppler parameters between the 11th and 14th pregnancy weeks in a population sample from Northeast Brazil

PURPOSE: To establish reference values for the first trimester uterine artery resistance index (UtA-RI) and pulsatility index (UtA-PI) in healthy singleton pregnant women from Northeast Brazil. METHODS: A prospective observational cohort study including 409 consecutive singleton pregnancies undergoing routine early ultrasound screening at 11 - 14 weeks of gestation was performed. The patients responded to a questionnaire to assess maternal epidemiological characteristics. The left and right UtA-PI and UtA-RI were examined by color and pulsed Doppler by transabdominal technique and the mean UtA-PI, mean UtA-RI and the presence of bilateral protodiastolic notching were recorded. Quartile regression was used to estimate reference values. RESULTS: The mean±standard deviation UtA-RI and UtA-PI were 0.7±0.1 and 1.5±0.5, respectively. When segregated for gestation age, mean UtA-PI was 1.6±0.5 at 11 weeks, 1.5±0.6 at 12 weeks, 1.4±0.4 at 13 weeks and 1.3±0.4 at 14 weeks' gestation and mean UtA-RI was 0.7±0.1 at 11 weeks, 0.7±0.1 at 12 weeks, 0.6±0.1 at 13 weeks and 0.6±0.1 at 14 weeks' gestation. Uterine artery bilateral notch was present in 261 (63.8%) patients. We observed that the 5th and 95th percentiles of the UtA-PI and UtA-RI uterine arteries were 0.7 and 2.3 and, 0.5 and 0.8, respectively. CONCLUSION: Normal reference range of uterine artery Doppler in healthy singleton pregnancies from Northeast Brazil was established. The 95th percentile of UtA-PI and UtA-RI values may serve as a cut-off for future prediction of pregnancy complications studies (i.e., pre-eclampsia) in Northeast Brazil.

Reference values for lung function tests: III. Carbon monoxide diffusing capacity (transfer factor)

Carbon monoxide diffusing capacity (DLCO) or transfer factor (TLCO) is a particularly useful test of the appropriateness of gas exchange across the lung alveolocapillary membrane. With the purpose of establishing predictive equations for DLCO using a non-smoking sample of the adult Brazilian population, we prospectively evaluated 100 subjects (50 males and 50 females aged 20 to 80 years), randomly selected from more than 8,000 individuals. Gender-specific linear prediction equations were developed by multiple regression analysis with single breath (SB) absolute and volume-corrected (VA) DLCO values as dependent variables. In the prediction equations, age (years) and height (cm) had opposite effects on DLCOSB (ml min-1 mmHg-1), independent of gender (-0.13 (age) + 0.32 (height) - 13.07 in males and -0.075 (age) + 0.18 (height) + 0.20 in females). On the other hand, height had a positive effect on DLCOSB but a negative one on DLCOSB/VA (P<0.01). We found that the predictive values from the most cited studies using predominantly Caucasian samples were significantly different from the actually measured values (P<0.05). Furthermore, oxygen uptake at maximal exercise (VO2max) correlated highly to DLCOSB (R = 0.71, P<0.001); this variable, however, did not maintain an independent role to explain the VO2max variability in the multiple regression analysis (P>0.05). Our results therefore provide an original frame of reference for either DLCOSB or DLCOSB/VA in Brazilian males and females aged 20 to 80 years, obtained from the standardized single-breath technique.

Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression

The objective of the present study was to develop a quantitative method to evaluate laser-induced choroidal neovascularization (CNV) in a rat model using Heidelberg Retina Angiograph 2 (HRA2) imaging. The expression of two heparan sulfate proteoglycans (HSPG) related to inflammation and angiogenesis was also investigated. CNV lesions were induced with argon laser in 21 heterozygous Zucker rats and after three weeks a fluorescein angiogram and autofluorescence exams were performed using HRA2. The area and greatest linear dimension were measured by two observers not aware of the protocol. Bland-Altman plots showed agreement between the observers, suggesting that the technique was reproducible. After fluorescein angiogram, HSPG (perlecan and syndecan-4) were analyzed by real-time RT-PCR and immunohistochemistry. There was a significant increase in the expression of perlecan and syndecan-4 (P < 0.0001) in retinas bearing CNV lesions compared to control retinas. The expression of these two HSPG increased with increasing CNV area. Immunohistochemistry demonstrated that the rat retina damaged with laser shots presented increased expression of perlecan and syndecan-4. Moreover, we observed that the overexpression occurred in the outer layer of the retina, which is related to choroidal damage. It was possible to develop a standardized quantitative method to evaluate CNV in a rat model using HRA2. In addition, we presented data indicating that the expression of HSPG parallels the area of CNV lesion. The understanding of these events offers opportunities for studies of new therapeutic interventions targeting these HSPG.