Early Changes in Soil Metabolic Diversity and Bacterial Community Structure in Sugarcane under Two Harvest Management Systems

Preharvest burning is widely used in Brazil for sugarcane cropping. However, due to environmental restrictions, harvest without burning is becoming the predominant option. Consequently, changes in the microbial community are expected from crop residue accumulation on the soil surface, as well as alterations in soil metabolic diversity as of the first harvest. Because biological properties respond quickly and can be used to monitor environmental changes, we evaluated soil metabolic diversity and bacterial community structure after the first harvest under sugarcane management without burning compared to management with preharvest burning. Soil samples were collected under three sugarcane varieties (SP813250, SP801842 and RB72454) and two harvest management systems (without and with preharvest burning). Microbial biomass C (MBC), carbon (C) substrate utilization profiles, bacterial community structure (based on profiles of 16S rRNA gene amplicons), and soil chemical properties were determined. MBC was not different among the treatments. C-substrate utilization and metabolic diversity were lower in soil without burning, except for the evenness index of C-substrate utilization. Soil samples under the variety SP801842 showed the greatest changes in substrate utilization and metabolic diversity, but showed no differences in bacterial community structure, regardless of the harvest management system. In conclusion, combined analysis of soil chemical and microbiological data can detect early changes in microbial metabolic capacity and diversity, with lower values in management without burning. However, after the first harvest, there were no changes in the soil bacterial community structure detected by PCR-DGGE under the sugarcane variety SP801842. Therefore, the metabolic profile is a more sensitive indicator of early changes in the soil microbial community caused by the harvest management system.

Changes in the Structure of a Nigerian Soil under Different Land Management Practices

Quantification of soil physical quality (SPQ) and pore size distribution (PSD) can assist understanding of how changes in land management practices influence dynamics of soil structure, and this understanding could greatly improve the predictability of soil physical behavior and crop yield. The objectives of this study were to measure the SPQ index under two different land management practices (the continuous arable cropping system and natural bush fallow system), and contrast the effects of these practices on the structure of PSD using soil water retention data. Soil water retention curves obtained from a pressure chamber were fitted to van Genuchten’s equation, setting m (= 1-1/n). Although values for soil bulk density were high, soils under the continuous arable cropping system had good SPQ, and maintained the capacity to support root development. However, soils under the natural bush fallow system had a worse structure than the continuous arable system, with restrictions in available water capacity. These two management systems had different PSDs. Results showed the inferiority of the natural bush fallow system with no traffic restriction (which is the common practice) in relation to the continuous arable cropping system in regard to physical quality and structure.

Occurrence and Structure of Arbuscular Mycorrhizal Fungal Communities in Cassava after Cultivation of Cover Crops as Observed by the “PCR-DGGE” Technique

ABSTRACT Cassava (Manihot esculenta Crantz) is a highly mycotrophic crop, and prior soil cover may affect the density of arbuscular mycorrhizal fungi (AMFs), as well as the composition of the AMFs community in the soil. The aim of this study was to evaluate the occurrence and the structure of AMFs communities in cassava grown after different cover crops, and the effect of the cover crop on mineral nutrition and cassava yield under an organic farming system. The occurrence and structure of the AMFs community was evaluated through polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A randomized block experimental design was used with four replications. Six different cover crop management systems before cassava were evaluated: black oats, vetch, oilseed radish, intercropped oats + vetch, intercropped oats + vetch + oilseed radish, plus a control (fallow) treatment mowed every 15 days. Oats as a single crop or oats intercropped with vetch or with oilseed radish increased AMFs inoculum potential in soil with a low number of propagules, thus benefiting mycorrhizal colonization of cassava root. The treatments did not affect the structure of AMFs communities in the soil since the AMFs communities were similar in cassava roots in succession to different cover crops. AMFs colonization was high despite high P availability in the soil. The cassava crop yield was above the regional average, and P levels in the leaves were adequate, regardless of which cover crop treatments were used. One cover crop cycle prior to the cassava crop was not enough to observe a significant response in variables, P in plant tissue, crop yield, and occurrence and structure of AMFs communities in the soil. In the cassava roots in succession, the plant developmental stage affected the groupings of the structure of the AMF community.

Surface Complexation Modeling in Variable Charge Soils: Charge Characterization by Potentiometric Titration

ABSTRACT Intrinsic equilibrium constants of 17 representative Brazilian Oxisols were estimated from potentiometric titration measuring the adsorption of H+ and OH− on amphoteric surfaces in suspensions of varying ionic strength. Equilibrium constants were fitted to two surface complexation models: diffuse layer and constant capacitance. The former was fitted by calculating total site concentration from curve fitting estimates and pH-extrapolation of the intrinsic equilibrium constants to the PZNPC (hand calculation), considering one and two reactive sites, and by the FITEQL software. The latter was fitted only by FITEQL, with one reactive site. Soil chemical and physical properties were correlated to the intrinsic equilibrium constants. Both surface complexation models satisfactorily fit our experimental data, but for results at low ionic strength, optimization did not converge in FITEQL. Data were incorporated in Visual MINTEQ and they provide a modeling system that can predict protonation-dissociation reactions in the soil surface under changing environmental conditions.

Anisotropic fluid with SU(2) type structure in general relativity: A model of localized matter

A model of anisotropic fluid with three perfect fluid components in interaction is studied. Each fluid component obeys the stiff matter equation of state and is irrotational. The interaction is chosen to reproduce an integrable system of equations similar to the one associated to self-dual SU(2) gauge fields. An extension of the BelinskyZakharov version of the inverse scattering transform is presented and used to find soliton solutions to the coupled Einstein equations. A particular class of solutions that can be interpreted as lumps of matter propagating in empty space-time is examined.

Thermodynamic Characterization of Humic Acid-surfactant Interaction: New Insights into the Characteristics and Structure of Humic Acids

ABSTRACT Humic acids (HA) are a component of humic substances (HS), which are found in nearly all soils, sediments, and waters. They play a key role in many, if not most, chemical and physical properties in their environment. Despite the importance of HA, their high complexity makes them a poorly understood system. Therefore, understanding the physicochemical properties and interactions of HA is crucial for determining their fundamental role and obtaining structural details. Cationic surfactants are known to interact electrostatically and hydrophobically with HA. Because they are a very well-known and characterized system, they offer a good choice as molecular probes for studying HA. The objective of this study was to evaluate the interaction between cationic surfactants and HA through isothermal titration calorimetry in a thermodynamic manner, aiming to obtain information about the basic structure of HA, the nature of this interaction, and if HA from different origins show different basic structures. Contrary to what the supramolecular model asserts, HA structure is not loosely held, though it may separate depending on the conditions the HA are subjected to in their milieu. It did not show any division or conformational change when interacting with surfactants. The basic structure of the HA remains virtually the same regardless of the different sources and compositions of these HA.

Density-based hierarchical clustering of pyro-sequences on a large scale--the case of fungal ITS1.

MOTIVATION: Analysis of millions of pyro-sequences is currently playing a crucial role in the advance of environmental microbiology. Taxonomy-independent, i.e. unsupervised, clustering of these sequences is essential for the definition of Operational Taxonomic Units. For this application, reproducibility and robustness should be the most sought after qualities, but have thus far largely been overlooked. RESULTS: More than 1 million hyper-variable internal transcribed spacer 1 (ITS1) sequences of fungal origin have been analyzed. The ITS1 sequences were first properly extracted from 454 reads using generalized profiles. Then, otupipe, cd-hit-454, ESPRIT-Tree and DBC454, a new algorithm presented here, were used to analyze the sequences. A numerical assay was developed to measure the reproducibility and robustness of these algorithms. DBC454 was the most robust, closely followed by ESPRIT-Tree. DBC454 features density-based hierarchical clustering, which complements the other methods by providing insights into the structure of the data. AVAILABILITY: An executable is freely available for non-commercial users at ftp://ftp.vital-it.ch/tools/dbc454. It is designed to run under MPI on a cluster of 64-bit Linux machines running Red Hat 4.x, or on a multi-core OSX system. CONTACT: dbc454@vital-it.ch or nicolas.guex@isb-sib.ch.

Water-Stable Aggregates and Associated Carbon in a Subtropical Rice Soil Under Variable Tillage

ABSTRACT Tillage systems can influence C sequestration by changing aggregate formation and C distribution within the aggregate. This study was undertaken to explore the impact of no-tillage without straw (NT-S) and with straw (NT+S), and moldboard plow without straw (MP-S) and with straw (MP+S), on soil aggregation and aggregate-associated C after six years of double rice planting in a Hydragric Anthrosol in Guangxi, southwest of China. Soil samples of 0.00-0.05, 0.05-0.20 and 0.20-0.30 m layers were wet-sieved and divided into four aggregate-size classes, >2 mm, 2.00-0.25 mm, 0.25-0.053 and <0.053 mm, respectively, for measuring aggregate associated C and humic and fulvic acids. Results showed that the soil organic carbon (SOC) stock in bulk soil was 40.2-51.1 % higher in the 0.00-0.05 m layer and 11.3-17.0 % lower in the 0.05-0.20 m layer in NT system (NT+S and NT-S) compared to the MP system (MP+S and MP-S), respectively. However, no statistical difference was found across the whole 0.00-0.30 m layer. The NT system increased the proportion of >2 mm aggregate fraction and reduced the proportion of <0.053 mm aggregates in both 0.00-0.05 and 0.05-0.20 m layers. The SOC concentration, SOC stock and humic and fulvic acids within the >0.25 mm macroaggregate fraction also significantly increased in the 0.00-0.5 m layer in NT system. However, those within the 2.00-0.25 mm aggregate fraction were significantly reduced in the 0.05-0.200 m layer under NT system. Straw incorporation increased not only the SOC stock in bulk soil, but also the proportion of macroaggregate, aggregate associated with SOC and humic and fulvic acids concentration within the aggregate. The effect of straw on C sequestration might be dependent on the location of straw incorporation. In conclusion, the NT system increased the total SOC accumulation and humic and fulvic acids within macroaggregates, thus contributing to C sequestration in the 0.00-0.05 m layer.

Do Rates and Splitting of Phosphogypsum Applications Influence the Soil and Annual Crops in a No-Tillage System?

ABSTRACT Applications of phosphogypsum (PG) provide nutrients to the soil and reduce Al3+ activity, favoring soil fertility and root growth, but allow Mg2+ mobilization through the soil profile, resulting in variations in the PG rate required to achieve the optimum crop yield. This study evaluated the effect of application rates and splitting of PG on soil fertility of a Typic Hapludox, as well as the influence on annual crops under no-tillage. Using a (4 × 3) + 1 factorial structure, the treatments consisted of four PG rates (3, 6, 9, and 12 Mg ha-1) and three split applications (P1 = 100 % in 2009; P2 = 50+50 % in 2009 and 2010; P3 = 33+33+33 % in 2009, 2010 and 2011), plus a control without PG. The soil was sampled six months after the last PG application, in stratified layers to a depth of 0.8 m. Corn, wheat and soybean were sown between November 2011 and December 2012, and leaf samples were collected for analysis when at least 50 % of the plants showed reproductive structures. The application of PG increased Ca2+ concentrations in all sampled soil layers and the soil pH between 0.2 and 0.8 m, and reduced the concentrations of Al3+ in all layers and of Mg2+ to a depth of 0.6 m, without any effect of splitting the applications. The soil Ca/Mg ratio increased linearly to a depth of 0.6 m with the rates and were found to be higher in the 0.0-0.1 m layer of the P2 and P3 treatments than without splitting (P1). Sulfur concentrations increased linearly by application rates to a depth of 0.8 m, decreasing in the order P3>P2>P1 to a depth of 0.4 m and were higher in the treatments P3 and P2 than P1 between 0.4-0.6 m, whereas no differences were observed in the 0.6-0.8 m layer. No effect was recorded for K, P and potential acidity (H+Al). The leaf Ca and S concentration increased, while Mg decreased for all crops treated with PG, and there was no effect of splitting the application. The yield response of corn to PG rates was quadratic, with the maximum technical efficiency achieved at 6.38 Mg ha-1 of PG, while wheat yield increased linearly in a growing season with a drought period. Soybean yield was not affected by the PG rate, and splitting had no effect on the yield of any of the crops. Phosphogypsum improved soil fertility in the profile, however, Mg2+ migrated downwards, regardless of application splitting. Splitting the PG application induced a higher Ca/Mg ratio in the 0.0-0.1 m layer and less S leaching, but did not affect the crop yield. The application rates had no effect on soybean yield, but were beneficial for corn and, especially, for wheat, which was affected by a drought period during growth.

Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis oocytes system.

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.

Temporal and spatial dynamics of benthic invertebrate communities in an alpine glacier-fed alluvial system

Summary The present thesis work focused on the ecology of benthic invertebrates in the proglacial floodplain of the Rhone in the Swiss Alps. The main glacial Rhone River and a smaller glacial tributary, the Mutt River, joined and entered a braiding multi-thread area. A first part concentrated on the disruption of the longitudinal patterns of environmental conditions and benthic invertebrate fauna in the Rhone by its tributary the Mutt. The Mutt had less harsh environmental conditions, higher taxonomic richness and more abundant zoobenthos compared to the Rhone upstream of the confluence. Although the habitat conditions in the main stream were little modified by the tributary, the fauna was richer and more diverse below the confluence. Colonisation from the Mutt induced the occurrence of faunal elements uncommon of glacial streams in the upper Rhone, where water temperature remains below 4°C. Although the glacial Rhone dominated the system with regard to hydrology and certain environmental conditions, the Mutt tributary has to be seen as the faunal driver of the system. The second part of the study concerned the spatio-temporal differentiation of the habitats and the benthic communities along and across the flood plain. No longitudinal differentiation was found. The spatial transversal differentiation of three habitat types with different environmental characteristics was successfully reflected in the spatial variability of benthic assemblages. This typology separated marginal sites of the flood plain, left bank sites under the influence of the Mutt, and the right bank sites under the influence of the Rh6ne. Faunistic spatial differences were emphasized by the quantitative structure of the fauna, richness, abundances and Simpson index of diversity. Seasonal environmental variability was positively related with Simpson index of diversity and the total richness per site. Low flow conditions were the most favourable season for the fauna and November was characterized by low spatial environmental heterogeneity, high spatial heterogeneity of faunal assemblage, maximum taxonomic richness, a particular taxonomic composition, highest abundances, as well as the highest primary food resources. The third part studied the egg development of three species of Ephemeroptera in the laboratory at 1.5 to 7°C and the ecological implications in the field. Species revealed very contrasting development strategies. Baetis alpinus has a synchronous and efficient egg development, which is faster in warmer habitats, enabling it to exploit short periods of favourable conditions in the floodplain. Ecdyonurus picteti has a very long development time slightly decreasing in warmer conditions. The high degree of individual variation suggests a genetic determination of the degree-days demand. Combined with the glacial local conditions, this strategy leads to an extreme delay of hatching and allows it to develop in very unpredictable habitats. Rhithrogena nivata is the second cold adapted species in Ephemeroptera. The incubation duration is long and success largely depends on the timing of hatching and the discharge conditions. This species is able to exploit extremely unstable and cold habitats where other species are limited by low water temperatures. The fourth part dealt with larval development in different habitats of the floodplain. Addition of data on egg development allowed the description of the life histories of the species from oviposition until emergence. Rhithrogena nivata and loyolaea generally have a two-year development, with the first winter passed as eggs and the second one as larvae. Development of Ecdyonurus picteti is difficult to document but appears to be efficient in a harsh and unpredictable environment. Baetis alpinus was studied separately in four habitats of the floodplain system with contrasting thermal regimes. Differences in success and duration of larval development and in growth rates are emphasised. Subvention mechanisms between habitats by migration of young or grown larvae were demonstrated. Development success and persistence of the populations in the system were thus increased. Emergence was synchronised to the detriment of the optimisation of the adult's size and fecundity. These very different development strategies induce a spatial and temporal distribution in the use of food resources and ecological niches. The last part of this work aimed at the synthesis of the characteristics and the ecological features of three distinct compartments of the system that are the upper Rhone, the Mutt and the floodplain. Their particular role as well as their inter-dependence concerning the structure and the dynamics of the benthic communities was emphasised. Résumé Ce travail de thèse est consacré à l'écologie des invertébrés benthiques dans la zone alluviale proglaciaire du Rhône dans les Alpes suisses. Le Rhône, torrent glaciaire principal, reçoit les eaux de la Mutt, affluent glaciaire secondaire, puis pénètre dans une zone de tressage formée de plusieurs bras. La première partie de l'étude se concentre sur la disruption par la Mutt des processus longitudinaux, tant environnementaux que faunistiques, existants dans le Rhône. Les conditions environnementales régnant dans la Mutt sont moins rudes, la richesse taxonomique plus élevée et le zoobenthos plus abondant que dans le Rhône en amont de la confluence. Bien que les conditions environnementales dans le torrent principal soient peu modifiées par l'affluent, la faune s'avère être plus riche et plus diversifiée en aval de la confluence. La colonisation depuis la Mutt permet l'occurrence de taxons inhabituels dans le Rhône en amont de la confluence, où la température de l'eau se maintient en dessous de 4°C. Bien que le Rhône, torrent glaciaire principal, domine le système du point de vu de l'hydrologie et de certains paramètres environnementaux, l'affluent Mutt doit être considéré comme l'élément structurant la faune dans le système. La deuxième partie concerne la différentiation spatiale et temporelle des habitats et des communautés benthiques à travers la plaine alluviale. Aucune différentiation longitudinale n'a été mise en évidence. La différentiation transversale de trois types d'habitats sur la base des caractéristiques environnementales a été confirmée par la variabilité spatiale de la faune. Cette typologie sépare les sites marginaux de la plaine alluviale, ceux sous l'influence de la Mutt (en rive gauche) et ceux sous l'influence du Rhône amont (en rive droite). Les différences spatiales de la faune sont mises en évidence par la structure quantitative de la faune, la richesse, les abondances et l'indice de diversité de Simpson. La variabilité saisonnière du milieu est positivement liée avec l'indice de diversité de Simpson et la richesse totale par site. L'étiage correspond à la période la plus favorable pour la faune et novembre réunit des conditions de faible hétérogénéité spatiale du milieu, de forte hétérogénéité spatiale de la faune, une richesse taxonomique maximale, une composition faunistique particulière, les abondances ainsi que les ressources primaires les plus élevées. La troisième partie est consacrée à l'étude du développement des oeufs de trois espèces d'Ephémères au laboratoire à des températures de 1.5 à 7°C, ainsi qu'aux implications écologiques sur le terrain. Ces espèces présentent des stratégies de développement très contrastées. Baetis alpinus a un développement synchrone et efficace, plus rapide en milieu plus chaud et lui permettant d'exploiter les courtes périodes de conditions favorables. Ecdyonurus picteti présente une durée de développement très longue, diminuant légèrement dans des conditions plus chaudes. L'importante variation interindividuelle suggère un déterminisme génétique de la durée de développement. Cette stratégie, associée aux conditions locales, conduit à un décalage extrême des éclosions et permet à l'espèce de se développer dans des habitats imprévisibles. Rhithrogena nivata est la seconde espèce d'Ephémères présentant une adaptation au froid. L'incubation des oeufs est longue et son succès dépend de la période des éclosions et des conditions hydrologiques. Cette espèce est capable d'exploiter des habitats extrêmement instables et froids, où la température est facteur limitant pour d'autres espèces. La quatrième partie traite du développement larvaire dans différents habitats de la plaine alluviale. Le développement complet est décrit pour les espèces étudiées de la ponte jusqu'à l'émergence. Rhithrogena nivata et loyolaea atteignent généralement le stade adulte en deux ans, le premier hiver étant passé sous forme d'oeuf et le second sous forme de larve. Le développement de Ecdyonurus picteti est difficile à documenter, mais s'avère cependant efficace dans un environnement rude et imprévisible. Baetis alpinus a été étudié séparément dans quatre habitats de la plaine ayant des régimes thermiques contrastés. La réussite et la durée du développement embryonnaire ainsi que les taux de croissance y sont variables. Des mécanismes de subvention entre habitats sont possibles par la migration de larves juvéniles ou plus développées, augmentant ainsi la réussite du développement et le maintien des populations dans le système. L'émergence devient synchrone, au détriment de l'optimisation de la taille et de la fécondité des adultes. Ces stratégies très différentes induisent une distribution spatiale et temporelle dans l'usage des ressources et des niches écologiques. La dernière partie synthétise les caractéristiques écologiques des trois compartiments du système que sont le Rhône amont, la Mutt et la zone alluviale. Leurs rôles particuliers et leurs interdépendances du point de vue de la structure et de la dynamique des communautés benthiques sont mis en avant.

Stable alpha-synuclein oligomers strongly inhibit chaperone activity of the Hsp70 system by weak interactions with J-domain co-chaperones.

α-Synuclein aggregation and accumulation in Lewy bodies are implicated in progressive loss of dopaminergic neurons in Parkinson disease and related disorders. In neurons, the Hsp70s and their Hsp40-like J-domain co-chaperones are the only known components of chaperone network that can use ATP to convert cytotoxic protein aggregates into harmless natively refolded polypeptides. Here we developed a protocol for preparing a homogeneous population of highly stable β-sheet enriched toroid-shaped α-Syn oligomers with a diameter typical of toxic pore-forming oligomers. These oligomers were partially resistant to in vitro unfolding by the bacterial Hsp70 chaperone system (DnaK, DnaJ, GrpE). Moreover, both bacterial and human Hsp70/Hsp40 unfolding/refolding activities of model chaperone substrates were strongly inhibited by the oligomers but, remarkably, not by unstructured α-Syn monomers even in large excess. The oligomers acted as a specific competitive inhibitor of the J-domain co-chaperones, indicating that J-domain co-chaperones may preferably bind to exposed bulky misfolded structures in misfolded proteins and, thus, complement Hsp70s that bind to extended segments. Together, our findings suggest that inhibition of the Hsp70/Hsp40 chaperone system by α-Syn oligomers may contribute to the disruption of protein homeostasis in dopaminergic neurons, leading to apoptosis and tissue loss in Parkinson disease and related neurodegenerative diseases.

Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure.

The recently discovered epithelial sodium channel (ENaC)/degenerin (DEG) gene family encodes sodium channels involved in various cell functions in metazoans. Subfamilies found in invertebrates or mammals are functionally distinct. The degenerins in Caenorhabditis elegans participate in mechanotransduction in neuronal cells, FaNaC in snails is a ligand-gated channel activated by neuropeptides, and the Drosophila subfamily is expressed in gonads and neurons. In mammals, ENaC mediates Na+ transport in epithelia and is essential for sodium homeostasis. The ASIC genes encode proton-gated cation channels in both the central and peripheral nervous system that could be involved in pain transduction. This review summarizes the physiological roles of the different channels belonging to this family, their biophysical and pharmacological characteristics, and the emerging knowledge of their molecular structure. Although functionally different, the ENaC/DEG family members share functional domains that are involved in the control of channel activity and in the formation of the pore. The functional heterogeneity among the members of the ENaC/DEG channel family provides a unique opportunity to address the molecular basis of basic channel functions such as activation by ligands, mechanotransduction, ionic selectivity, or block by pharmacological ligands.

The mammalian circadian timing system: from gene expression to physiology.

Many physiological processes in organisms from bacteria to man are rhythmic, and some of these are controlled by self-sustained oscillators that persist in the absence of external time cues. Circadian clocks are perhaps the best characterized biological oscillators and they exist in virtually all light-sensitive organisms. In mammals, they influence nearly all aspects of physiology and behavior, including sleep-wake cycles, cardiovascular activity, endocrinology, body temperature, renal activity, physiology of the gastro-intestinal tract, and hepatic metabolism. The master pacemaker is located in the suprachiasmatic nuclei, two small groups of neurons in the ventral part of the hypothalamus. However, most peripheral body cells contain self-sustained circadian oscillators with a molecular makeup similar to that of SCN (suprachiasmatic nucleus) neurons. This organization implies that the SCN must synchronize countless subsidiary oscillators in peripheral tissues, in order to coordinate cyclic physiology. In this review, we will discuss some recent studies on the structure and putative functions of the mammalian circadian timing system, but we will also point out some apparent inconsistencies in the currently publicized model for rhythm generation.

The structure of nervous tissue and of Gram-positive bacteria studied by cryo-electron microscopy of vitreous sections

Résumé La structure, ou l'architecture, des êtres vivants définit le cadre dans lequel la physique de la vie s'accomplit. La connaissance de cette structure dans ses moindres détails est un but essentiel de la biologie. Son étude est toutefois entravée par des limitations techniques. Malgré son potentiel théorique, la microscopie électronique n'atteint pas une résolution atomique lorsqu'elle est appliquée ä la matièxe biologique. Cela est dû en grande partie au fait qu'elle contient beaucoup d'eau qui ne résiste pas au vide du microscope. Elle doit donc être déshydratée avant d'être introduite dans un microscope conventionnel. Des artéfacts d'agrégation en découlent inévitablement. La cryo-microscopie électronique des sections vitreuses (CEMOVIS) a ëté développée afin de résoudre cela. Les spécimens sont vitrifiés, c.-à-d. que leur eau est immobilisée sans cristalliser par le froid. Ils sont ensuite coupés en sections ultrafines et celles-ci sont observées à basse température. Les spécimens sont donc observés sous forme hydratée et non fixée; ils sont proches de leur état natif. Durant longtemps, CEMOVIS était très difficile à exécuter mais ce n'est plus le cas. Durant cette thèse, CEMOVIS a été appliqué à différents spécimens. La synapse du système nerveux central a été étudiée. La présence dans la fente synaptique d'une forte densité de molécules organisées de manière périodique a été démontrée. Des particules luminales ont été trouvées dans Ies microtubules cérébraux. Les microtubules ont servi d'objets-test et ont permis de démontrer que des détails moléculaires de l'ordre du nm sont préservés. La compréhension de la structure de l'enveloppe cellulaire des bactéries Grampositives aété améliorée. Nos observations ont abouti à l'élaboration d'un nouveau modèle hypothétique de la synthèse de la paroi. Nous avons aussi focalisé notre attention sur le nucléoïde bactérien et cela a suscité un modèle de la fonction des différents états structuraux du nucléoïde. En conclusion, cette thèse a démontré que CEMOVIS est une excellente méthode poux étudier la structure d'échantillons biologiques à haute résolution. L'étude de la structure de divers aspects des êtres vivants a évoqué des hypothèses quant à la compréhension de leur fonctionnement. Summary The structure, or the architecture, of living beings defines the framework in which the physics of life takes place. Understanding it in its finest details is an essential goal of biology. Its study is however hampered by technical limitations. Despite its theoretical potential, electron microscopy cannot resolve individual atoms in biological matter. This is in great part due to the fact. that it contains a lot of water that cannot stand the vacuum of the microscope. It must therefore be dehydrated before being introduced in a conventional mìcroscope. Aggregation artefacts unavoidably happen. Cryo-electron microscopy of vitreous sections (CEMOVIS) has been developed to solve this problem. Specimens are vitrified, i.e. they are rapidly cooled and their water is immobilised without crystallising by the cold. They are then. sectioned in ultrathin slices, which are observed at low temperatures. Specimens are therefore observed in hydrated and unfixed form; they are close to their native state. For a long time, CEMOVIS was extremely tedious but this is not the case anymore. During this thesis, CEMOVIS was applied to different specimens. Synapse of central nervous system was studied. A high density of periodically-organised molecules was shown in the synaptic cleft. Luminal particles were found in brain microtubules. Microtubules, used as test specimen, permitted to demonstrate that molecular details of the order of nm .are preserved. The understanding of the structure of cell envelope of Gram-positive bacteria was improved. Our observations led to the elaboration of a new hypothetic model of cell wall synthesis. We also focused our attention on bacterial nucleoids and this also gave rise to a functional model of nucleoid structural states. In conclusion, this thesis demonstrated that CEMOVIS is an excellent method for studying the structure of bìologìcal specimens at high resolution. The study of the structure of various aspects of living beings evoked hypothesis for their functioning.