Characterization of polycystin-1 in ADPKD pathogenetic mechanism : biogenesis and functional implications by genetic approaches in mouse

La polykystose rénale autosomique dominante (ADPKD) est une des maladies génétiques les plus communes. ADPKD se manifeste le plus souvent au stade adulte par la présence de kystes rénaux, et bien souvent de kystes hépatiques, avec une progression très variable. ADPKD mène à une insuffisance rénale: les seuls recours sont la dialyse puis la transplantation rénale. Les mutations dispersées sur les gènes PKD1 (majoritairement; la protéine polycystine-1, PC1) et PKD2 (la protéine polycystine-2, PC2) sont responsables de l’ADPKD. Le mécanisme pathogénétique de perte de fonction (LOF) et donc d’un effet récessif cellulaire est évoqué comme causatif de l’ADPKD. LOF est en effet supporté par les modèles murins d’inactivation de gènes PKD1/PKD2, qui développent de kystes, quoique in utéro et avec une rapidité impressionnante dans les reins mais pas dans le foie. Malgré de nombreuses études in vitro, le rôle de PC1/PC2 membranaire/ciliaire reste plutôt hypothétique et contexte-dépendant. Ces études ont associé PC1/PC2 à une panoplie de voies de signalisation et ont souligné une complexité structurelle et fonctionnelle exceptionnelle, dont l’implication a été testée notamment chez les modèles de LOF. Toutefois, les observations patho-cellulaires chez l’humain dont une expression soutenue, voire augmentée, de PKD1/PC1 et l’absence de phénotypes extrarénaux particuliers remet en question l’exclusivité du mécanisme de LOF. Il était donc primordial 1) d’éclaircir le mécanisme pathogénétique, 2) de générer des outils in vivo authentiques d’ADPKD en terme d’initiation et de progression de la maladie et 3) de mieux connaitre les fonctions des PC1/PC2 indispensables pour une translation clinique adéquate. Cette thèse aborde tous ces points. Tout d’abord, nous avons démontré qu’une augmentation de PKD1 endogène sauvage, tout comme chez l’humain, est pathogénétique en générant et caractérisant en détail un modèle murin transgénique de Pkd1 (Pkd1TAG). Ce modèle reproduit non seulement les caractéristiques humaines rénales, associées aux défauts du cil primaire, mais aussi extrarénales comme les kystes hépatiques. La sévérité du phénotype corrèle avec le niveau d’expression de Pkd1 ce qui supporte fortement un modèle de dosage. Dans un deuxième temps, nous avons démontré par les études de complémentations génétiques que ces deux organes reposent sur une balance du clivage GPS de Pc1, une modification post-traductionelle typique des aGPCR, et dont l’activité et l’abondance semblent strictement contrôlées. De plus, nous avons caractérisé extensivement la biogénèse de Pc1 et de ses dérivés in vivo générés suite au clivage GPS. Nous avons identifié une toute nouvelle forme et prédominante à la membrane, la forme Pc1deN, en plus de confirmer deux fragments N- et C-terminal de Pc1 (NTF et CTF, respectivement) qui eux s’associent de manière non-covalente. Nous avons démontré de façon importante que le trafic de Pc1deN i.e., une forme NTF détachée du CTF, est toutefois dépendant de l’intégrité du fragment CTF in vivo. Par la suite, nous avons généré un premier modèle humanisant une mutation PKD1 non-sens tronquée au niveau du domaine NTF(E3043X) en la reproduisant chez une souris transgénique (Pkd1extra). Structurellement, cette mutation, qui mimique la forme Pc1deN, s’est également avérée causative de PKD. Le modèle Pkd1extra a permis entre autre de postuler l’existence d’une cross-interaction entre différentes formes de Pc1. De plus, nos deux modèles murins sont tous les deux associés à des niveaux altérés de c-Myc et Pc2, et soutiennent une implication réelle de ces derniers dans l’ADPKD tou comme une interaction fonctionnelle entre les polycystines. Finalement, nous avons démontré un chevauchement significatif entre l’ADPKD et le dommage rénal aigüe (ischémie/AKI) dont une expression augmentée de Pc1 et Pc2 mais aussi une stimulation de plusieurs facteurs cystogéniques tel que la tubérine, la β-caténine et l’oncogène c-Myc. Nos études ont donc apporté des évidences cruciales sur la contribution du gène dosage dans l’ADPKD. Nous avons développé deux modèles murins qui serviront d’outil pour l’analyse de la pathologie humaine ainsi que pour la validation préclinique ADPKD. L’identification d’une nouvelle forme de Pc1 ajoute un niveau de complexité supplémentaire expliquant en partie une capacité de régulation de plusieurs voies de signalisation par Pc1. Nos résultats nous amènent à proposer de nouvelles approches thérapeutiques: d’une part, le ciblage de CTF i.e., de style chaperonne, et d’autre part le ciblage de modulateurs intracellulaires (c-Myc, Pc2, Hif1α). Ensemble, nos travaux sont d’une importance primordiale du point de vue informatif et pratique pour un avancement vers une thérapie contre l’ADPKD. Le partage de voies communes entre AKI et ADPKD ouvre la voie aux approches thérapeutiques parallèles pour un traitement assurément beaucoup plus rapide.

Supramolecular polymers azo-containing : photo-responsive block copolymer elastomers and homopolymers

Beaucoup d'efforts dans le domaine des matériaux polymères sont déployés pour développer de nouveaux matériaux fonctionnels pour des applications spécifiques, souvent très sophistiquées, en employant des méthodes simplifiées de synthèse et de préparation. Cette thèse porte sur les polymères photosensibles – i.e. des matériaux fonctionnels qui répondent de diverses manières à la lumière – qui sont préparés à l'aide de la chimie supramoléculaire – i.e. une méthode de préparation qui repose sur l'auto-assemblage spontané de motifs moléculaires plus simples via des interactions non covalentes pour former le matériau final désiré. Deux types de matériaux photosensibles ont été ciblés, à savoir les élastomères thermoplastiques à base de copolymères à blocs (TPE) et les complexes d'homopolymères photosensibles. Les TPEs sont des matériaux bien connus, et même commercialisés, qui sont généralement composés d’un copolymère tribloc, avec un bloc central très flexible et des blocs terminaux rigides qui présentent une séparation de phase menant à des domaines durs isolés, composés des blocs terminaux rigides, dans une matrice molle formée du bloc central flexible, et ils ont l'avantage d'être recyclable. Pour la première fois, au meilleur de notre connaissance, nous avons préparé ces matériaux avec des propriétés photosensibles, basé sur la complexation supramoléculaire entre un copolymère tribloc simple parent et une petite molécule possédant une fonctionnalité photosensible via un groupe azobenzène. Plus précisément, il s’agit de la complexation ionique entre la forme quaternisée d'un copolymère à blocs, le poly(méthacrylate de diméthylaminoéthyle)-poly(acrylate de n-butyle)-poly(méthacrylate de diméthylaminoéthyle) (PDM-PnBA-PDM), synthétisé par polymérisation radicalaire par transfert d’atomes (ATRP), et l'orange de méthyle (MO), un composé azo disponible commercialement comportant un groupement SO3 -. Le PnBA possède une température de transition vitreuse en dessous de la température ambiante (-46 °C) et les blocs terminaux de PDM complexés avec le MO ont une température de transition vitreuse élevée (140-180 °C, en fonction de la masse molaire). Des tests simples d'élasticité montrent que les copolymères à blocs complexés avec des fractions massiques allant de 20 à 30% présentent un caractère élastomère. Des mesures d’AFM et de TEM (microscopie à force atomique et électronique à ii transmission) de films préparés à l’aide de la méthode de la tournette, montrent une corrélation entre le caractère élastomère et les morphologies où les blocs rigides forment une phase minoritaire dispersée (domaines sphériques ou cylindriques courts). Une phase dure continue (morphologie inversée) est observée pour une fraction massique en blocs rigides d'environ 37%, ce qui est beaucoup plus faible que celle observée pour les copolymères à blocs neutres, dû aux interactions ioniques. La réversibilité de la photoisomérisation a été démontrée pour ces matériaux, à la fois en solution et sous forme de film. La synthèse du copolymère à blocs PDM-PnBA-PDM a ensuite été optimisée en utilisant la technique d'échange d'halogène en ATRP, ainsi qu’en apportant d'autres modifications à la recette de polymérisation. Des produits monodisperses ont été obtenus à la fois pour la macroamorceur et le copolymère à blocs. À partir d'un seul copolymère à blocs parent, une série de copolymères à blocs partiellement/complètement quaternisés et complexés ont été préparés. Des tests préliminaires de traction sur les copolymères à blocs complexés avec le MO ont montré que leur élasticité est corrélée avec la fraction massique du bloc dur, qui peut être ajustée par le degré de quaternisation et de complexation. Finalement, une série de complexes d'homopolymères auto-assemblés à partir du PDM et de trois dérivés azobenzènes portant des groupes (OH, COOH et SO3) capables d'interactions directionnelles avec le groupement amino du PDM ont été préparés, où les dérivés azo sont associés avec le PDM, respectivement, via des interactions hydrogène, des liaisons ioniques combinées à une liaison hydrogène à travers un transfert de proton (acidebase), et des interactions purement ioniques. L'influence de la teneur en azo et du type de liaison sur la facilité d’inscription des réseaux de diffraction (SRG) a été étudiée. L’efficacité de diffraction des SRGs et la profondeur des réseaux inscrits à partir de films préparés à la méthode de la tournette montrent que la liaison ionique et une teneur élevée en azo conduit à une formation plus efficace des SRGs.

Biochemical and functional characterization of the tumor suppressors BRCA1 and BAP1

L’ubiquitination est une modification post-traductionnelle qui joue un rôle majeur dans la régulation d’une multitude de processus cellulaires. Dans cette thèse, je discuterai de la caractérisation de deux protéines, BRCA1 et BAP1, soit deux suppresseurs de tumeurs fonctionnellement reliés. BRCA1, une ubiquitine ligase qui catalyse la liaison de l’ubiquitine à une protéine cible, est mutée dans les cancers du sein et de l'ovaire. Il est bien établi que cette protéine aide à maintenir la stabilité génomique suite à un bris double brin de l’ADN (BDB), et ce, à l’aide d’un mécanisme de réparation bien caractérisé appelé recombinaison homologue. Cependant, les mécanismes de régulation de BRCA1 suite à des stresses génotoxiques n’impliquant pas directement un BDB ne sont pas pleinement élucidés. Nous avons démontré que BRCA1 est régulée par dégradation protéasomale suite à une exposition des cellules à deux agents génotoxiques reconnus pour ne pas directement générer des BDBs, soit les rayons UV, qui provoquent la distorsion de l’hélice d’ADN, et le méthyle méthanesulfonate (MMS), qui entraîne l’alkylation de l’ADN. La dégradation de BRCA1 est réversible et indépendante des kinases associées à la voie des PI3 kinase, soit ATM, ATR et DNA-PK, protéines qui sont rapidement activées par les dommages à l’ADN. Nous proposons que la dégradation de BRCA1 prévienne son recrutement intempestif, ainsi que celui des facteurs qui lui sont associés, à des sites de dommages d’ADN qui ne sont pas des BDBs, et que cette régulation coordonne la réparation de l’ADN. L’enzyme de déubiquitination BAP1 a initialement été identifiée comme une protéine capable d’interagir avec BRCA1 et de réguler sa fonction. Elle est également connue pour sa capacité à se lier avec les protéines du groupe Polycomb, ASXL1 et ASXL2. Cependant, l’importance de ces interactions n’a toujours pas été établie. Nous avons démontré que BAP1 forme deux complexes protéiques mutuellement exclusifs avec ASXL1 et ASXL2. Ces interactions sont critiques pour la liaison de BAP1 à l’ubiquitine ainsi que pour la stimulation de son activité enzymatique envers l’histone H2A. Nous avons également identifié des mutations de BAP1 dérivées de cancers qui empêchent à la fois son interaction avec ASXL1 et AXSL2, et son activité de déubiquitinase, ce qui fournit un lien mécanistique direct entre la déubiquitination de H2A et la tumorigenèse. Élucider les mécanismes de régulation de BRCA1 et BAP1 menera à une meilleure compréhension de leurs rôles de suppresseurs de tumeurs, permettant ainsi d’établir de nouvelles stratégies de diagnostic et traitement du cancer.

Développements et applications de méthodes computationnelles pour l'étude de l'agrégation des protéines amyloïdes

Les protéines sont au coeur de la vie. Ce sont d'incroyables nanomachines moléculaires spécialisées et améliorées par des millions d'années d'évolution pour des fonctions bien définies dans la cellule. La structure des protéines, c'est-à-dire l'arrangement tridimensionnel de leurs atomes, est intimement liée à leurs fonctions. L'absence apparente de structure pour certaines protéines est aussi de plus en plus reconnue comme étant tout aussi cruciale. Les protéines amyloïdes en sont un exemple marquant : elles adoptent un ensemble de structures variées difficilement observables expérimentalement qui sont associées à des maladies neurodégénératives. Cette thèse, dans un premier temps, porte sur l'étude structurelle des protéines amyloïdes bêta-amyloïde (Alzheimer) et huntingtine (Huntington) lors de leur processus de repliement et d'auto-assemblage. Les résultats obtenus permettent de décrire avec une résolution atomique les interactions des ensembles structurels de ces deux protéines. Concernant la protéine bêta-amyloïde (AB), nos résultats identifient des différences structurelles significatives entre trois de ses formes physiologiques durant ses premières étapes d'auto-assemblage en environnement aqueux. Nous avons ensuite comparé ces résultats avec ceux obtenus au cours des dernières années par d'autres groupes de recherche avec des protocoles expérimentaux et de simulations variés. Des tendances claires émergent de notre comparaison quant à l'influence de la forme physiologique de AB sur son ensemble structurel durant ses premières étapes d'auto-assemblage. L'identification des propriétés structurelles différentes rationalise l'origine de leurs propriétés d'agrégation distinctes. Par ailleurs, l'identification des propriétés structurelles communes offrent des cibles potentielles pour des agents thérapeutiques empêchant la formation des oligomères responsables de la neurotoxicité. Concernant la protéine huntingtine, nous avons élucidé l'ensemble structurel de sa région fonctionnelle située à son N-terminal en environnement aqueux et membranaire. En accord avec les données expérimentales disponibles, nos résultats sur son repliement en environnement aqueux révèlent les interactions dominantes ainsi que l'influence sur celles-ci des régions adjacentes à la région fonctionnelle. Nous avons aussi caractérisé la stabilité et la croissance de structures nanotubulaires qui sont des candidats potentiels aux chemins d'auto-assemblage de la région amyloïde de huntingtine. Par ailleurs, nous avons également élaboré, avec un groupe d'expérimentateurs, un modèle détaillé illustrant les principales interactions responsables du rôle d'ancre membranaire de la région N-terminal, qui sert à contrôler la localisation de huntingtine dans la cellule. Dans un deuxième temps, cette thèse porte sur le raffinement d'un modèle gros-grain (sOPEP) et sur le développement d'un nouveau modèle tout-atome (aaOPEP) qui sont tous deux basés sur le champ de force gros-grain OPEP, couramment utilisé pour l'étude du repliement des protéines et de l'agrégation des protéines amyloïdes. L'optimisation de ces modèles a été effectuée dans le but d'améliorer les prédictions de novo de la structure de peptides par la méthode PEP-FOLD. Par ailleurs, les modèles OPEP, sOPEP et aaOPEP ont été inclus dans un nouveau code de dynamique moléculaire très flexible afin de grandement simplifier leurs développements futurs.

Sol-Gel Alumina Nano Composites for Functional Applications

The thesis entitled "Sol-Gel Alumina Nano Composites for Functional Applications" investigate sol-gel methods of synthesis of alumina nanocomposites special reference to alumina-aluminium titanate and alumina-lanthanum phosphate composites. The functional properties such as thermal expansion coefficient and thermal shock resistance, machinability of composites as well as thermal protection are highlighted in addition to novel approach in synthesis of composites.A general introduction of alumina matrix composites materials, followed by brief coverage of alumina-aluminium titanate and alumina-lanthanum phosphate composites is highlight of the first chapter. The second chapter deals with the sol-gel synthesis of aluminium titanate and alumina-aluminium titanate composite. The synthesis of machinable substrate, based on alumina and lanthanum phosphate forms the basis of the third chapter. The fourth chapter describes the sol-gel coating of mullite on SiC substrate for the possible gas filtration application.

Design and study of self-assembled functional organic and hybrid systems for biological applications

The focus of self-assembly as a strategy for the synthesis has been confined largely to molecules, because of the importance of manipulating the structure of matter at the molecular scale. We have investigated the influence of temperature and pH, in addition to the concentration of the capping agent used for the formation of the nano-bio conjugates. For example, the formation of the narrower size distribution of the nanoparticles was observed with the increase in the concentration of the protein, which supports the fact that γ-globulin acts both as a controller of nucleation as well as stabiliser. As analyzed through various photophysical, biophysical and microscopic techniques such as TEM, AFM, C-AFM, SEM, DLS, OPM, CD and FTIR, we observed that the initial photoactivation of γ-globulin at pH 12 for 3 h resulted in small protein fibres of ca. Further irradiation for 24 h, led to the formation of selfassembled long fibres of the protein of ca. 5-6 nm and observation of surface plasmon resonance band at around 520 nm with the concomitant quenching of luminescence intensity at 680 nm. The observation of light triggered self-assembly of the protein and its effect on controlling the fate of the anchored nanoparticles can be compared with the naturally occurring process such as photomorphogenesis.Furthermore,our approach offers a way to understand the role played by the self-assembly of the protein in ordering and knock out of the metal nanoparticles and also in the design of nano-biohybrid materials for medicinal and optoelectronic applications. Investigation of the potential applications of NIR absorbing and water soluble squaraine dyes 1-3 for protein labeling and anti-amyloid agents forms the subject matter of the third chapter of the thesis. The study of their interactions with various proteins revealed that 1-3 showed unique interactions towards serum albumins as well as lysozyme. 69%, 71% and 49% in the absorption spectra as well as significant quenching in the fluorescence intensity of the dyes 1-3, respectively. Half-reciprocal analysis of the absorption data and isothermal titration calorimetric (ITC) analysis of the titration experiments gave a 1:1 stoichiometry for the complexes formed between the lysozyme and squaraine dyes with association constants (Kass) in the range 104-105 M-1. We have determined the changes in the free energy (ΔG) for the complex formation and the values are found to be -30.78, -32.31 and -28.58 kJmol-1, respectively for the dyes 1, 2 and 3. Furthermore, we have observed a strong induced CD (ICD) signal corresponding to the squaraine chromophore in the case of the halogenated squaraine dyes 2 and 3 at 636 and 637 nm confirming the complex formation in these cases. To understand the nature of interaction of the squaraine dyes 1-3 with lysozyme, we have investigated the interaction of dyes 1-3 with different amino acids. These results indicated that the dyes 1-3 showed significant interactions with cysteine and glutamic acid which are present in the side chains of lysozyme. In addition the temperature dependent studies have revealed that the interaction of the dye and the lysozyme are irreversible. Furthermore, we have investigated the interactions of these NIR dyes 1-3 with β- amyloid fibres derived from lysozyme to evaluate their potential as inhibitors of this biologically important protein aggregation. These β-amyloid fibrils were insoluble protein aggregates that have been associated with a range of neurodegenerative diseases, including Huntington, Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob diseases. We have synthesized amyloid fibres from lysozyme through its incubation in acidic solution below pH 4 and by allowing to form amyloid fibres at elevated temperature. To quantify the binding affinities of the squaraine dyes 1-3 with β-amyloids, we have carried out the isothermal titration calorimetric (ITC) measurements. The association constants were determined and are found to be 1.2 × 105, 3.6× 105 and 3.2 × 105 M-1 for the dyes, 1-3, respectively. To gain more insights into the amyloid inhibiting nature of the squaraine dyes under investigations, we have carried out thioflavin assay, CD, isothermal titration calorimetry and microscopic analysis. The addition of the dyes 1-3 (5μM) led to the complete quenching in the apparent thioflavin fluorescence, thereby indicating the destabilization of β-amyloid fibres in the presence of the squaraine dyes. Further, the inhibition of the amyloid fibres by the squaraine dyes 1-3, has been evidenced though the DLS, TEM AFM and SAED, wherein we observed the complete destabilization of the amyloid fibre and transformation of the fibre into spherical particles of ca. These results demonstrate the fact that the squaraine dyes 1-3 can act as protein labeling agents as well as the inhibitors of the protein amyloidogenesis. The last chapter of the thesis describes the synthesis and investigation of selfassembly as well as bio-imaging aspects of a few novel tetraphenylethene conjugates 4-6.Expectedly, these conjugates showed significant solvatochromism and exhibited a hypsochromic shift (negative solvatochromism) as the solvent polarity increased, and these observations were justified though theoretical studies employing the B3LYP/6-31g method. We have investigated the self-assembly properties of these D-A conjugates though variation in the percentage of water in acetonitrile solution due to the formation of nanoaggregates. Further the contour map of the observed fluorescence intensity as a function of the fluorescence excitation and emission wavelength confirmed the formation of J-type aggregates in these cases. To have a better understanding of the type of self-assemblies formed from the TPE conjugates 4-6, we have carried out the morphological analysis through various microscopic techniques such as DLS, SEM and TEM. 70%, we observed rod shape architectures having ~ 780 nm in diameter and ~ 12 μM in length as evidenced through TEM and SEM analysis. We have made similar observations with the dodecyl conjugate 5 at ca. 70% and 50% water/acetonitrile mixtures, the aggregates formed from 4 and 5 were found to be highly crystalline and such structures were transformed to amorphous nature as the water fraction was increased to 99%. To evaluate the potential of the conjugate as bio-imaging agents, we have carried out their in vitro cytotoxicity and cellular uptake studies though MTT assay, flow cytometric and confocal laser scanning microscopic techniques. Thus nanoparticle of these conjugates which exhibited efficient emission, large stoke shift, good stability, biocompatibility and excellent cellular imaging properties can have potential applications for tracking cells as well as in cell-based therapies. In summary we have synthesized novel functional organic chromophores and have studied systematic investigation of self-assembly of these synthetic and biological building blocks under a variety of conditions. The investigation of interaction of water soluble NIR squaraine dyes with lysozyme indicates that these dyes can act as the protein labeling agents and the efficiency of inhibition of β-amyloid indicate, thereby their potential as anti-amyloid agents.

A study on the performance of flexible pavements on mature soil subgrades

The country has witnessed tremendous increase in the vehicle population and increased axle loading pattern during the last decade, leaving its road network overstressed and leading to premature failure. The type of deterioration present in the pavement should be considered for determining whether it has a functional or structural deficiency, so that appropriate overlay type and design can be developed. Structural failure arises from the conditions that adversely affect the load carrying capability of the pavement structure. Inadequate thickness, cracking, distortion and disintegration cause structural deficiency. Functional deficiency arises when the pavement does not provide a smooth riding surface and comfort to the user. This can be due to poor surface friction and texture, hydro planning and splash from wheel path, rutting and excess surface distortion such as potholes, corrugation, faulting, blow up, settlement, heaves etc. Functional condition determines the level of service provided by the facility to its users at a particular time and also the Vehicle Operating Costs (VOC), thus influencing the national economy. Prediction of the pavement deterioration is helpful to assess the remaining effective service life (RSL) of the pavement structure on the basis of reduction in performance levels, and apply various alternative designs and rehabilitation strategies with a long range funding requirement for pavement preservation. In addition, they can predict the impact of treatment on the condition of the sections. The infrastructure prediction models can thus be classified into four groups, namely primary response models, structural performance models, functional performance models and damage models. The factors affecting the deterioration of the roads are very complex in nature and vary from place to place. Hence there is need to have a thorough study of the deterioration mechanism under varied climatic zones and soil conditions before arriving at a definite strategy of road improvement. Realizing the need for a detailed study involving all types of roads in the state with varying traffic and soil conditions, the present study has been attempted. This study attempts to identify the parameters that affect the performance of roads and to develop performance models suitable to Kerala conditions. A critical review of the various factors that contribute to the pavement performance has been presented based on the data collected from selected road stretches and also from five corporations of Kerala. These roads represent the urban conditions as well as National Highways, State Highways and Major District Roads in the sub urban and rural conditions. This research work is a pursuit towards a study of the road condition of Kerala with respect to varying soil, traffic and climatic conditions, periodic performance evaluation of selected roads of representative types and development of distress prediction models for roads of Kerala. In order to achieve this aim, the study is focused into 2 parts. The first part deals with the study of the pavement condition and subgrade soil properties of urban roads distributed in 5 Corporations of Kerala; namely Thiruvananthapuram, Kollam, Kochi, Thrissur and Kozhikode. From selected 44 roads, 68 homogeneous sections were studied. The data collected on the functional and structural condition of the surface include pavement distress in terms of cracks, potholes, rutting, raveling and pothole patching. The structural strength of the pavement was measured as rebound deflection using Benkelman Beam deflection studies. In order to collect the details of the pavement layers and find out the subgrade soil properties, trial pits were dug and the in-situ field density was found using the Sand Replacement Method. Laboratory investigations were carried out to find out the subgrade soil properties, soil classification, Atterberg limits, Optimum Moisture Content, Field Moisture Content and 4 days soaked CBR. The relative compaction in the field was also determined. The traffic details were also collected by conducting traffic volume count survey and axle load survey. From the data thus collected, the strength of the pavement was calculated which is a function of the layer coefficient and thickness and is represented as Structural Number (SN). This was further related to the CBR value of the soil and the Modified Structural Number (MSN) was found out. The condition of the pavement was represented in terms of the Pavement Condition Index (PCI) which is a function of the distress of the surface at the time of the investigation and calculated in the present study using deduct value method developed by U S Army Corps of Engineers. The influence of subgrade soil type and pavement condition on the relationship between MSN and rebound deflection was studied using appropriate plots for predominant types of soil and for classified value of Pavement Condition Index. The relationship will be helpful for practicing engineers to design the overlay thickness required for the pavement, without conducting the BBD test. Regression analysis using SPSS was done with various trials to find out the best fit relationship between the rebound deflection and CBR, and other soil properties for Gravel, Sand, Silt & Clay fractions. The second part of the study deals with periodic performance evaluation of selected road stretches representing National Highway (NH), State Highway (SH) and Major District Road (MDR), located in different geographical conditions and with varying traffic. 8 road sections divided into 15 homogeneous sections were selected for the study and 6 sets of continuous periodic data were collected. The periodic data collected include the functional and structural condition in terms of distress (pothole, pothole patch, cracks, rutting and raveling), skid resistance using a portable skid resistance pendulum, surface unevenness using Bump Integrator, texture depth using sand patch method and rebound deflection using Benkelman Beam. Baseline data of the study stretches were collected as one time data. Pavement history was obtained as secondary data. Pavement drainage characteristics were collected in terms of camber or cross slope using camber board (slope meter) for the carriage way and shoulders, availability of longitudinal side drain, presence of valley, terrain condition, soil moisture content, water table data, High Flood Level, rainfall data, land use and cross slope of the adjoining land. These data were used for finding out the drainage condition of the study stretches. Traffic studies were conducted, including classified volume count and axle load studies. From the field data thus collected, the progression of each parameter was plotted for all the study roads; and validated for their accuracy. Structural Number (SN) and Modified Structural Number (MSN) were calculated for the study stretches. Progression of the deflection, distress, unevenness, skid resistance and macro texture of the study roads were evaluated. Since the deterioration of the pavement is a complex phenomena contributed by all the above factors, pavement deterioration models were developed as non linear regression models, using SPSS with the periodic data collected for all the above road stretches. General models were developed for cracking progression, raveling progression, pothole progression and roughness progression using SPSS. A model for construction quality was also developed. Calibration of HDM–4 pavement deterioration models for local conditions was done using the data for Cracking, Raveling, Pothole and Roughness. Validation was done using the data collected in 2013. The application of HDM-4 to compare different maintenance and rehabilitation options were studied considering the deterioration parameters like cracking, pothole and raveling. The alternatives considered for analysis were base alternative with crack sealing and patching, overlay with 40 mm BC using ordinary bitumen, overlay with 40 mm BC using Natural Rubber Modified Bitumen and an overlay of Ultra Thin White Topping. Economic analysis of these options was done considering the Life Cycle Cost (LCC). The average speed that can be obtained by applying these options were also compared. The results were in favour of Ultra Thin White Topping over flexible pavements. Hence, Design Charts were also plotted for estimation of maximum wheel load stresses for different slab thickness under different soil conditions. The design charts showed the maximum stress for a particular slab thickness and different soil conditions incorporating different k values. These charts can be handy for a design engineer. Fuzzy rule based models developed for site specific conditions were compared with regression models developed using SPSS. The Riding Comfort Index (RCI) was calculated and correlated with unevenness to develop a relationship. Relationships were developed between Skid Number and Macro Texture of the pavement. The effort made through this research work will be helpful to highway engineers in understanding the behaviour of flexible pavements in Kerala conditions and for arriving at suitable maintenance and rehabilitation strategies. Key Words: Flexible Pavements – Performance Evaluation – Urban Roads – NH – SH and other roads – Performance Models – Deflection – Riding Comfort Index – Skid Resistance – Texture Depth – Unevenness – Ultra Thin White Topping

Functional studies of the deubiquitinating enzymes USP19, USP4 and UCH-L1

El marcaje de proteínas con ubiquitina, conocido como ubiquitinación, cumple diferentes funciones que incluyen la regulación de varios procesos celulares, tales como: la degradación de proteínas por medio del proteosoma, la reparación del ADN, la señalización mediada por receptores de membrana, y la endocitosis, entre otras (1). Las moléculas de ubiquitina pueden ser removidas de sus sustratos gracias a la acción de un gran grupo de proteasas, llamadas enzimas deubiquitinizantes (DUBs) (2). Las DUBs son esenciales para la manutención de la homeostasis de la ubiquitina y para la regulación del estado de ubiquitinación de diferentes sustratos. El gran número y la diversidad de DUBs descritas refleja tanto su especificidad como su utilización para regular un amplio espectro de sustratos y vías celulares. Aunque muchas DUBs han sido estudiadas a profundidad, actualmente se desconocen los sustratos y las funciones biológicas de la mayoría de ellas. En este trabajo se investigaron las funciones de las DUBs: USP19, USP4 y UCH-L1. Utilizando varias técnicas de biología molecular y celular se encontró que: i) USP19 es regulada por las ubiquitin ligasas SIAH1 y SIAH2 ii) USP19 es importante para regular HIF-1α, un factor de transcripción clave en la respuesta celular a hipoxia, iii) USP4 interactúa con el proteosoma, iv) La quimera mCherry-UCH-L1 reproduce parcialmente los fenotipos que nuestro grupo ha descrito previamente al usar otros constructos de la misma enzima, y v) UCH-L1 promueve la internalización de la bacteria Yersinia pseudotuberculosis.

Designing flexible procurement systems

The systems used for the procurement of buildings are organizational systems. They involve people in a series of strategic decisions, and a pattern of roles, responsibilities and relationships that combine to form the organizational structure of the project. To ensure effectiveness of the building team, this organizational structure needs to be contingent upon the environment within which the construction project takes place. In addition, a changing environment means that the organizational structure within a project needs to be responsive, and dynamic. These needs are often not satisfied in the construction industry, due to the lack of analytical tools with which to analyse the environment and to design appropriate temporary organizations. This paper presents two techniques. First is the technique of "Environmental Complexity Analysis", which identifies the key variables in the environment of the construction project. These are classified as Financial, Legal, Technological, Aesthetic and Policy. It is proposed that their identification will set the parameters within which the project has to be managed. This provides a basis for the project managers to define the relevant set of decision points that will be required for the project. The Environmental Complexity Analysis also identifies the project's requirements for control systems concerning Budget, Contractual, Functional, Quality and Time control. The process of environmental scanning needs to be done at regular points during the procurement process to ensure that the organizational structure is adaptive to the changing environment. The second technique introduced is the technique of "3R analysis", being a graphical technique for describing and modelling Roles, Responsibilities and Relationships. A list of steps is introduced that explains the procedure recommended for setting up a flexible organizational structure that is responsive to the environment of the project. This is by contrast with the current trend towards predetermined procurement paths that may not always be in the best interests of the client.

Inulin: a prebiotic functional food ingredient

The human gut microbiota is increasingly recognized as playing a central role in human health and disease. This dichotomous relationship with the host forms a central theme in this review, which addresses how we may divert the gut microbiota away from some of its more harmful activities towards beneficial interactions with the human host. We describe the concept of prebiotics, which use specific dietary carbohydrates to increase the numbers of what are seen as beneficial bacteria within the colon, in a selective manner. Specifically, the use of β(2-1) fructans or inulin in general, and certain of its fractions in particular as prebiotics, will be described. Prebiotic fructans constitute efficacious functional foods and there is strong evidence supporting the selectivity of their fermentation within the human gut microbiota, resulting in an increase in the relative numbers of Bifidobacterium spp. There is also considerable evidence, mainly from animal studies but also in humans, that dietary supplementation with prebiotic fructans, through modulation of the microbiota, plays a protective role in colon cancer, heart disease and bone health. However, the mechanisms by which this prebiotic microbiota modulation mediates such diverse health outcomes remain unclear. The future challenge facing the field of prebiotic functional foods will be the elucidation of these mechanisms of action. Recent high resolution bioomics technologies, especially metabonomics, provide the tools necessary to define the metabolic consequences of prebiotic microbiota modulation.

The neural response to emotional prosody, as revealed by functional magnetic resonance imaging

Prosody is an important feature of language, comprising intonation, loudness, and tempo. Emotional prosodic processing forms an integral part of our social interactions. The main aim of this study was to use bold contrast fMRI to clarify the normal functional neuroanatomy of emotional prosody, in passive and active contexts. Subjects performed six separate scanning studies, within which two different conditions were contrasted: (1) "pure" emotional prosody versus rest; (2) congruent emotional prosody versus 'neutral' sentences; (3) congruent emotional prosody versus rest; (4) incongruent emotional prosody versus rest; (5) congruent versus incongruent emotional prosody; and (6) an active experiment in which subjects were instructed to either attend to the emotion conveyed by semantic content or that conveyed by tone of voice. Data resulting from these contrasts were analysed using SPM99. Passive listening to emotional prosody consistently activated the lateral temporal lobe (superior and/or middle temporal gyri). This temporal lobe response was relatively right-lateralised with or without semantic information. Both the separate and direct comparisons of congruent and incongruent emotional prosody revealed that subjects used fewer brain regions to process incongruent emotional prosody than congruent. The neural response to attention to semantics, was left lateralised, and recruited an extensive network not activated by attention to emotional prosody. Attention to emotional prosody modulated the response to speech, and induced right-lateralised activity, including the middle temporal gyrus. In confirming the results of lesion and neuropsychological studies, the current study emphasises the importance of the right hemisphere in the processing of emotional prosody, specifically the lateral temporal lobes. (C) 2003 Elsevier Science Ltd. All rights reserved.

Syntheses, x-ray structures, photochemistry, redox properties, and DFT calculations of interconvertible fac- and mer-[Mn(SPS)(CO)3] isomers containing a flexible SPS-based pincer ligand

The lithium salt of the anionic SPS pincer ligand composed of a central hypervalent lambda(4)-phosphinine ring bearing two ortho-positioned diphenylphosphine sulfide side arms reacts with [Mn(CO)(5)Br] to give fac-[Mn(SPS)(CO)(3)], This isomer can be converted photochemicaily to mer-[Mn(SPS)(CO)(3)], with a very high quantum yield (0.80 +/- 0.05). The thermal backreaction is slow (taking ca. 8 h at room temperature), in contrast to rapid electrodecatalyzed mer-to-fac isomerization triggered by electrochemical reduction of mer-[Mn(SPS)(CO)(3)]. Both geometric isomers of [Mn(SPS)(CO)(3)] have been characterized by X-ray crystallography. Both isomers show luminescence from a low-lying (IL)-I-3 (SPS-based) excited state. The light emission of fac-[Mn(SPS)(CO)(3)] is largely quenched by the efficient photoisomerization occurring probably from a low-lying Mn-CO dissociative excited state. Density functional theory (DFT) and time-dependent DFT calculations describe the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of fac- and mer-[Mn(CO)(3)(SPS)] as ligand-centered orbitals, largely localized on the phosphinine ring of the SPS pincer ligand. In line with the ligand nature of its frontier orbitals, fac-[Mn(SPS)(CO)(3)] is electrochemically reversibly oxidized and reduced to the corresponding radical cation and anion, respectively. The spectroscopic (electron paramagnetic resonance, IR, and UV-vis) characterization of the radical species provides other evidence for the localization of the redox steps on the SIPS ligand. The smaller HOMO-LUMO energy difference in the case of mer-[Mn(CO)(3)(SPS)], reflected in the electronic absorption and emission spectra, corresponds with its lower oxidation potential compared to that of the fac isomer. The thermodynamic instability of mer-[Mn(CO)(3)(SPS)], confirmed by the DFT calculations, increases upon one-electron reduction and oxidation of the complex.

Strongly nucleophilic RhI centre in square-planar complexes with terdentate (κ3) 2,2′:6′,2′′-terpyridine ligands: crystallographic, electrochemical and density functional theoretical studies

Rh-I-terpyridine complexes have been unambiguously formed for the first time. The 2,21:6',2"-terpyridine (tpy), 4'-chloro-2,2':6',2"-terpyridine (4'-Cl-tpy) and 4'-(tert-butyldimethylsilyl-ortho-carboranyl)-2,2':6',2"-terpyridine (carboranyl-tpy) ligands were used for successful syntheses and characterisation of the corresponding Rh-I complexes with halide coligands, [Rh(X)(4'-Y-terpyridine)] (X = Cl, Y = H, Cl, carboranyl; X = Br, Y = H). All four neutral Rh-tpy complexes are square planar, with Rh-X bonds in the plane of the 4'-Y-terpyridine ligands. Full characterisation of these dark blue, highly air-sensitive compounds was hampered by their poor solubility in various organic solvents. This is mainly due to the formation of pi-stacked aggregates, as evidenced by the crystal structure of [Rh(Cl)(tpy)]; in addition, [Rh(Cl)(carboranyl-tpy)] merely forms discrete dimers. The (bonding) properties of the novel Rh-I-terpyridine complexes have been studied with single-crystal X-ray diffraction, (time-dependent) density functional theoretical (DFT) calculations, far-infrared spectroscopy, electronic absorption spectroscopy and cyclic voltammetry. From DFT calculations, the HOMO of the studied Rh-I-terpyridine complexes involves predominantly the metal centre, while the LUMO resides on the terpyridine ligand. Absorption bands of the studied complexes in the visible region (400-900 nm) can be assigned to MLCT and MLCT/XLCT transitions. The relatively low oxidation potentials of [Rh(X)(tpy)] (X = Cl, Br) point to a high electron density on the metal centre. This makes the Rh-I-terpyridine complexes strongly nucleophilic and (potentially) highly reactive towards various (small) substrate molecules containing carbon-halide bonds.

Towards an improved and more flexible representation of water stress in coupled photosynthesis–stomatal conductance models

Coupled photosynthesis–stomatal conductance (A–gs) models are commonly used in ecosystem models to represent the exchange rate of CO2 and H2O between vegetation and the atmosphere. The ways these models account for water stress differ greatly among modelling schemes. This study provides insight into the impact of contrasting model configurations of water stress on the simulated leaf-level values of net photosynthesis (A), stomatal conductance (gs), the functional relationship among them and their ratio, the intrinsic water use efficiency (A/gs), as soil dries. A simple, yet versatile, normalized soil moisture dependent function was used to account for the effects of water stress on gs, on mesophyll conductance (gm) and on the biochemical capacity. Model output was compared to leaf-level values obtained from the literature. The sensitivity analyses emphasized the necessity to combine both stomatal and non-stomatal limitations of A in coupled A–gs models to accurately capture the observed functional relationships A vs. gs and A/gsvs. gs in response to drought. Accounting for water stress in coupled A–gs models by imposing either stomatal or biochemical limitations of A, as commonly practiced in most ecosystem models, failed to reproduce the observed functional relationship between key leaf gas exchange attributes. A quantitative limitation analysis revealed that the general pattern of C3 photosynthetic response to water stress may be well represented in coupled A–gs models by imposing the highest limitation strength to gm, then to gs and finally to the biochemical capacity.

Ecophysiological and bioclimatic foundations for a global plant functional classification

Question: What plant properties might define plant functional types (PFTs) for the analysis of global vegetation responses to climate change, and what aspects of the physical environment might be expected to predict the distributions of PFTs? Methods: We review principles to explain the distribution of key plant traits as a function of bioclimatic variables. We focus on those whole-plant and leaf traits that are commonly used to define biomes and PFTs in global maps and models. Results: Raunkiær's plant life forms (underlying most later classifications) describe different adaptive strategies for surviving low temperature or drought, while satisfying requirements for reproduction and growth. Simple conceptual models and published observations are used to quantify the adaptive significance of leaf size for temperature regulation, leaf consistency for maintaining transpiration under drought, and phenology for the optimization of annual carbon balance. A new compilation of experimental data supports the functional definition of tropical, warm-temperate, temperate and boreal phanerophytes based on mechanisms for withstanding low temperature extremes. Chilling requirements are less well quantified, but are a necessary adjunct to cold tolerance. Functional traits generally confer both advantages and restrictions; the existence of trade-offs contributes to the diversity of plants along bioclimatic gradients. Conclusions: Quantitative analysis of plant trait distributions against bioclimatic variables is becoming possible; this opens up new opportunities for PFT classification. A PFT classification based on bioclimatic responses will need to be enhanced by information on traits related to competition, successional dynamics and disturbance.