Formation of a spatial memory trace : a behavioral, cellular and molecular study

THESIS ABSTRACTThis thesis project was aimed at studying the molecular mechanisms underlying learning and memory formation, in particular as they relate to the metabolic coupling between astrocytes and neurons. For that, changes in the metabolic activity of different mice brain regions after 1 or 9 days of training in an eight-arm radial maze were assessed by (14C) 2-deoxyglucose (2DG) autoradiography. Significant differences in the areas engaged during the behavioral task at day 1 (when animals are confronted for the first time to the learning task) and at day 9 (when animals are highly performing) have been identified. These areas include the hippocampus, the fornix, the parietal cortex, the laterodorsal thalamic nucleus and the mammillary bodies at day 1 ; and the anterior cingulate, the retrosplenial cortex and the dorsal striatum at day 9. Two of these cerebral regions (those presenting the greatest changes at day 1 and day 9: the hippocampus and the retrosplenial cortex, respectively) were microdissected by laser capture microscopy and selected genes related to neuron-glia metabolic coupling, glucose metabolism and synaptic plasticity were analyzed by RT-PCR. 2DG and gene expression analysis were performed at three different times: 1) immediately after the end of the behavioral paradigm, 2) 45 minutes and 3) 6 hours after training. The main goal of this study was the identification of the metabolic adaptations following the learning task. Gene expression results demonstrate that the learning task profoundly modulates the pattern of gene expression in time, meaning that these two cerebral regions with high 2DG signal (hippocampus and retrosplenial cortex) have adapted their metabolic molecular machinery in consequence. Almost all studied genes show a higher expression in the hippocampus at day 1 compared to day 9, while an increased expression was found in the retrosplenial cortex at day 9. We can observe these molecular adaptations with a short delay of 45 minutes after the end of the task. However, 6 hours after training a high gene expression was found at day 9 (compared to day 1) in both regions, suggesting that only one day of training is not sufficient to detect transcriptional modifications several hours after the task. Thus, gene expression data match 2DG results indicating a transfer of information in time (from day 1 to day 9) and in space (from the hippocampus to the retrosplenial cortex), and this at a cellular and a molecular level. Moreover, learning seems to modify the neuron-glia metabolic coupling, since several genes involved in this coupling are induced. These results also suggest a role of glia in neuronal plasticity.RESUME DU TRAVAIL DE THESECe projet de thèse a eu pour but l'étude des mécanismes moléculaires qui sont impliqués dans l'apprentissage et la mémoire et, en particulier, à les mettre en rapport avec le couplage métabolique existant entre les astrocytes et les neurones. Pour cela, des changements de l'activité métabolique dans différentes régions du cerveau des souris après 1 ou 9 jours d'entraînement dans un labyrinthe radial à huit-bras ont été évalués par autoradiographie au 2-désoxyglucose (2DG). Des différences significatives dans les régions engagées pendant la tâche comportementale au jour 1 (quand les animaux sont confrontés pour la première fois à la tâche) et au jour 9 (quand les animaux ont déjà appris) ont été identifiés. Ces régions incluent, au jour 1, l'hippocampe, le fornix, le cortex pariétal, le noyau thalamic laterodorsal et les corps mamillaires; et, au jour 9, le cingulaire antérieur, le cortex retrosplenial et le striatum dorsal. Deux de ces régions cérébrales (celles présentant les plus grands changements à jour 1 et à jour 9: l'hippocampe et le cortex retrosplenial, respectivement) ont été découpées par microdissection au laser et quelques gènes liés au couplage métabolique neurone-glie, au métabolisme du glucose et à la plasticité synaptique ont été analysées par RT-PCR. L'étude 2DG et l'analyse de l'expression de gènes ont été exécutés à trois temps différents: 1) juste après entraînement, 2) 45 minutes et 3) 6 heures après la fin de la tâche. L'objectif principal de cette étude était l'identification des adaptations métaboliques suivant la tâche d'apprentissage. Les résultats de l'expression de gènes démontrent que la tâche d'apprentissage module profondément le profile d'expression des gènes dans le temps, signifiant que ces deux régions cérébrales avec un signal 2DG élevé (l'hippocampe et le cortex retrosplenial) ont adapté leurs « machines moléculaires » en conséquence. Presque tous les gènes étudiés montrent une expression plus élevée dans l'hippocampe au jour 1 comparé au jour 9, alors qu'une expression accrue a été trouvée dans le cortex retrosplenial au jour 9. Nous pouvons observer ces adaptations moléculaires avec un retard court de 45 minutes après la fin de la tâche. Cependant, 6 heures après l'entraînement, une expression de gènes élevée a été trouvée au jour 9 (comparé à jour 1) dans les deux régions, suggérant que seulement un jour d'entraînement ne suffit pas pour détecter des modifications transcriptionelles plusieurs heures après la tâche. Ainsi, les données d'expression de gènes corroborent les résultats 2DG indiquant un transfert d'information dans le temps (de jour 1 à jour 9) et dans l'espace (de l'hippocampe au cortex retrosplenial), et ceci à un niveau cellulaire et moléculaire. D'ailleurs, la tâche d'apprentissage semble modifier le couplage métabolique neurone-glie, puisque de nombreux gènes impliqués dans ce couplage sont induits. Ces observations suggèrent un rôle important de la glie dans les mécanismes de plasticité du système nerveux.

Influence of field slope and coffee plantation age on the physical properties of a red-yellow Latosol

In modern agriculture, several factors cause changes in the soil physical properties. The time of establishment of a crop (plantation age) and the slope are examples of factors that moderate the impact of mechanized operations on the soil structure. The objective of this study was to analyze the effect of machinery traffic on the physical properties of a Red-Yellow Latosol under coffee plantations with different ages (2, 7, 18, and 33 years) and slope positions (3, 9 and 15 %). Samples were collected from three positions between coffee rows (lower wheel track, inter-row and upper wheel track) and at two depths (surface layer and sub-surface). Changes in the total porosity, macroporosity, microporosity, organic matter, bulk density, and aggregate stability were investigated. Our results showed that the slope influenced the organic matter content, microporosity and aggregate stability. The soil samples under the inter-row were minimally damaged in their structure, compared to those from under the lower and upper wheel track, while the structure was better preserved under the lower than the upper track. The time since the establishment of the crop, i.e., the plantation age, was the main factor determining the extent of structural degradation in the coffee plantation.

Effects of traffic control on the soil physical quality and the cultivation of sugarcane

The cultivation of sugarcane with intensive use of machinery, especially for harvest, induces soil compaction, affecting the crop development. The control of agricultural traffic is an alternative of management in the sector, with a view to preserve the soil physical quality, resulting in increased sugarcane root growth, productivity and technological quality. The objective of this study was to evaluate the physical quality of an Oxisol with and without control traffic and the resulting effects on sugarcane root development, productivity and technological quality. The following managements were tested: no traffic control (NTC), traffic control consisting of an adjustment of the track width of the tractor and sugarcane trailer (TC1) and traffic control consisting of an adjustment of the track width of the tractor and trailer and use of an autopilot (TC2). Soil samples were collected (layers 0.00-0.10; 0.10-0.20 and 0.20-0.30 m) in the plant rows, inter-row center and seedbed region, 0.30 m away from the plant row. The productivity was measured with a specific weighing scale. The technological variables of sugarcane were measured in each plot. Soil cores were collected to analyze the root system. In TC2, the soil bulk density and compaction degree were lowest and total porosity and macroporosity highest in the plant row. Soil penetration resistance in the plant row, was less than 2 MPa in TC1 and TC2. Soil aggregation and total organic carbon did not differ between the management systems. The root surface and volume were increased in TC1 and TC2, with higher productivity and sugar yield than under NTC. The sugarcane variables did not differ between the managements. The soil physical quality in the plant row was preserved under management TC1 and TC2, with an improved root development and increases of 18.72 and 20.29 % in productivity and sugar yield, respectively.

Perennial grasses for recovery of the aggregation capacity of a reconstructed soil in a coal mining area in southern Brazil

The construction of a soil after surface coal mining involves heavy machinery traffic during the topographic regeneration of the area, resulting in compaction of the relocated soil layers. This leads to problems with water infiltration and redistribution along the new profile, causing water erosion and consequently hampering the revegetation of the reconstructed soil. The planting of species useful in the process of soil decompaction is a promising strategy for the recovery of the soil structural quality. This study investigated the influence of different perennial grasses on the recovery of reconstructed soil aggregation in a coal mining area of the Companhia Riograndense de Mineração, located in Candiota-RS, which were planted in September/October 2007. The treatments consisted of planting: T1- Cynodon dactylon cv vaquero; T2 - Urochloa brizantha; T3 - Panicum maximun; T4 - Urochloa humidicola; T5 - Hemarthria altissima; T6 - Cynodon dactylon cv tifton 85. Bare reconstructed soil, adjacent to the experimental area, was used as control treatment (T7) and natural soil adjacent to the mining area covered with native vegetation was used as reference area (T8). Disturbed and undisturbed soil samples were collected in October/2009 (layers 0.00-0.05 and 0.10-0.15 m) to determine the percentage of macro- and microaggregates, mean weight diameter (MWD) of aggregates, organic matter content, bulk density, and macro- and microporosity. The lower values of macroaggregates and MWD in the surface than in the subsurface layer of the reconstructed soil resulted from the high degree of compaction caused by the traffic of heavy machinery on the clay material. After 24 months, all experimental grass treatments showed improvements in soil aggregation compared to the bare reconstructed soil (control), mainly in the 0.00-0.05 m layer, particularly in the two Urochloa treatments (T2 and T4) and Hemarthria altissima (T5). However, the great differences between the treatments with grasses and natural soil (reference) indicate that the recovery of the pre-mining soil structure could take decades.

Least limiting water range in assessing compaction in a Brazilian Cerrado latosol growing sugarcane

In the south-central region of Brazil, there is a trend toward reducing the sugarcane inter-harvest period and increasing traffic of heavy harvesting machinery on soil with high water content, which may intensify the compaction process. In this study, we assessed the structural changes of a distroferric Red Latosol (Oxisol) by monitoring soil water content as a function of the Least Limiting Water Range (LLWR) and quantified its effects on the crop yield and industrial quality of the first ratoon crop of sugarcane cultivars with different maturation cycles. Three cultivars (RB 83-5054, RB 84-5210 and RB 86-7515) were subjected to four levels of soil compaction brought about by a differing number of passes of a farm tractor (T0 = soil not trafficked, T2 = 2 passes, T10 = 10 passes, and T20 = 20 passes of the tractor in the same place) in a 3 × 4 factorial arrangement with three replications. The deleterious effects on the soil structure from the farm machinery traffic were limited to the surface layer (0-10 cm) of the inter-row area of the ratoon crop. The LLWR dropped to nearly zero after 20 tractor passes between the cane rows. We detected differences among the cultivars studied; cultivar RB 86-7515 stood out for its industrial processing quality, regardless of the level of soil compaction. Monitoring of soil moisture in the crop showed exposure to water stress conditions, although soil compaction did not affect the production variables of the sugarcane cultivars. We thus conclude that the absence of traffic on the plant row maintained suitable soil conditions for plant development and may have offset the harmful effects of soil compaction shown by the high values for bulk density between the rows of the sugarcane cultivars.

Compared performance of penetrometers and effect of soil water content on penetration resistance measurements

Modern agriculture techniques have a great impact on crops and soil quality, especially by the increased machinery traffic and weight. Several devices have been developed for determining soil properties in the field, aimed at managing compacted areas. Penetrometry is a widely used technique; however, there are several types of penetrometers, which have different action modes that can affect the soil resistance measurement. The objective of this study was to compare the functionality of two penetrometry methods (manual and automated mode) in the field identification of compacted, highly mechanized sugarcane areas, considering the influence of soil water volumetric content (θ) on soil penetration resistance (PR). Three sugarcane fields on a Rhodic Eutrudrox were chosen, under a sequence of harvest systems: one manual harvest (1ManH), one mechanized harvest (1MH) and three mechanized harvests (3MH). The different degrees of mechanization were associated to cumulative compaction processes. An electronic penetrometer was used on PR measurements, so that the rod was introduced into the soil by hand (Manual) and by an electromechanical motor (Auto). The θ was measured in the field with a soil moisture sensor. Results showed an effect of θ on PR measurements and that regression models must be used to correct data before comparing harvesting systems. The rod introduction modes resulted in different mean PR values, where the "Manual" overestimated PR compared to the "Auto" mode at low θ.

Physical-hydraulic properties of a sandy loam typic paleudalf soil under organic cultivation of 'montenegrina' mandarin (Citrus deliciosa Tenore)¹

Citrus plants are the most important fruit species in the world, with emphasis to oranges, mandarins and lemons. In Rio Grande do Sul, Brazil, most fruit production is found on small properties under organic cultivation. Soil compaction is one of the factors limiting production and due to the fixed row placement of this crop, compaction can arise in various manners in the interrows of the orchard. The aim of this study was to evaluate soil physical properties and water infiltration capacity in response to interrow management in an orchard of mandarin (Citrus deliciosa Tenore 'Montenegrina') under organic cultivation. Interrow management was performed through harrowing, logs in em "V", mowing, and cutting/knocking down plants with a knife roller. Soil physical properties were evaluated in the wheel tracks of the tractor (WT), between the wheel tracks (BWT), and in the area under the line projection of the canopy (CLP), with undisturbed soil samples collected in the 0.00-0.15, 0.15-0.30, 0.30-0.45, and 0.45-0.60 m layers, with four replicates. The soil water infiltration test was performed using the concentric cylinder method, with a maximum time of 90 min for each test. In general, soil analysis showed a variation in the physical-hydraulic properties of the Argissolo Vermelho-Amarelo distrófico arênico (sandy loam Typic Paleudalf) in the three sampling sites in all layers, regardless of the management procedure in the interrows. Machinery traffic leads to heterogeneity in the soil physical-hydraulic properties in the interrows of the orchard. Soil porosity and bulk density are affected especially in the wheel tracks of the tractor (WT), which causes a reduction in the constant rate of infiltration and in the accumulated infiltration of water in this sampling site. The use of the disk harrow and mower leads to greater harmful effects on the soil, which can interfere with mandarin production.

Primordial neurosecretory apparatus identified in the choanoflagellate Monosiga brevicollis.

SNARE protein-driven secretion of neurotransmitters from synaptic vesicles is at the center of neuronal communication. In the absence of the cytosolic protein Munc18-1, synaptic secretion comes to a halt. Although it is believed that Munc18-1 orchestrates SNARE complexes, its mode of action is still a matter of debate. In particular, it has been challenging to clarify the role of a tight Munc18/syntaxin 1 complex, because this interaction interferes strongly with syntaxin's ability to form a SNARE complex. In this complex, two regions of syntaxin, the N-peptide and the remainder in closed conformation, bind to Munc18 simultaneously. Until now, this binary complex has been reported for neuronal tissues only, leading to the hypothesis that it might be a specialization of the neuronal secretion apparatus. Here we aimed, by comparing the core secretion machinery of the unicellular choanoflagellate Monosiga brevicollis with that of animals, to reconstruct the ancestral function of the Munc18/syntaxin1 complex. We found that the Munc18/syntaxin 1 complex from M. brevicollis is structurally and functionally highly similar to the vertebrate complex, suggesting that it constitutes a fundamental step in the reaction pathway toward SNARE assembly. We thus propose that the primordial secretion machinery of the common ancestor of choanoflagellates and animals has been co-opted for synaptic roles during the rise of animals.

Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae.

The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

MODELING OF SOIL LOAD-BEARING CAPACITY AS A FUNCTION OF SOIL MECHANICAL RESISTANCE TO PENETRATION

Estimation of soil load-bearing capacity from mathematical models that relate preconsolidation pressure (σp) to mechanical resistance to penetration (PR) and gravimetric soil water content (U) is important for defining strategies to prevent compaction of agricultural soils. Our objective was therefore to model the σp and compression index (CI) according to the PR (with an impact penetrometer in the field and a static penetrometer inserted at a constant rate in the laboratory) and U in a Rhodic Eutrudox. The experiment consisted of six treatments: no-tillage system (NT); NT with chiseling; and NT with additional compaction by combine traffic (passing 4, 8, 10, and 20 times). Soil bulk density, total porosity, PR (in field and laboratory measurements), U, σp, and CI values were determined in the 5.5-10.5 cm and 13.5-18.5 cm layers. Preconsolidation pressure (σp) and CI were modeled according to PR in different U. The σp increased and the CI decreased linearly with increases in the PR values. The correlations between σp and PR and PR and CI are influenced by U. From these correlations, the soil load-bearing capacity and compaction susceptibility can be estimated by PR readings evaluated in different U.

Scaffold attachment factor B1 directly interacts with nuclear receptors in living cells and represses transcriptional activity.

Transcriptional activity relies on coregulators that modify the chromatin structure and serve as bridging factors between transcription factors and the basal transcription machinery. Using the DE domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) as bait in a yeast two-hybrid screen of a human adipose tissue library, we isolated the scaffold attachment factor B1 (SAFB1/HET/HAP), which was previously shown to be a corepressor of estrogen receptor alpha. We show here that SAFB1 has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis. SAFB1 interacts in pull-down assays not only with PPARgamma but also with all nuclear receptors tested so far, albeit with different affinities. The association of SAFB1 and PPARgamma in vivo is further demonstrated by fluorescence resonance energy transfer (FRET) experiments in living cells. We finally show that SAFB1 is a rather general corepressor for nuclear receptors. Its change in expression during the early phases of adipocyte and enterocyte differentiation suggests that SAFB1 potentially influences cell proliferation and differentiation decisions.

SWEET SORGHUM PERFORMANCE AFFECTED BY SOIL COMPACTION AND SOWING TIME AS A SECOND CROP IN THE BRAZILIAN CERRADO

ABSTRACT Increasing attention has recently been given to sweet sorghum as a renewable raw material for ethanol production, mainly because its cultivation can be fully mechanized. However, the intensive use of agricultural machinery causes soil structural degradation, especially when performed under inadequate conditions of soil moisture. The aims of this study were to evaluate the physical quality of aLatossolo Vermelho Distroférrico (Oxisol) under compaction and its components on sweet sorghum yield forsecond cropsowing in the Brazilian Cerrado (Brazilian tropical savanna). The experiment was conducted in a randomized block design, in a split plot arrangement, with four replications. Five levels of soil compaction were tested from the passing of a tractor at the following traffic intensities: 0 (absence of additional compaction), 1, 2, 7, and 15 passes over the same spot. The subplots consisted of three different sowing times of sweet sorghum during the off-season of 2013 (20/01, 17/02, and 16/03). Soil physical quality was measured through the least limiting water range (LLWR) and soil water limitation; crop yield and technological parameters were also measured. Monitoring of soil water contents indicated a reduction in the frequency of water content in the soil within the limits of the LLWR (Fwithin) as agricultural traffic increased (T0 = T1 = T2>T7>T15), and crop yield is directly associated with soil water content. The crop sown in January had higher industrial quality; however, there was stalk yield reduction when bulk density was greater than 1.26 Mg m-3, with a maximum yield of 50 Mg ha-1 in this sowing time. Cultivation of sweet sorghum as a second crop is a promising alternative, but care should be taken in cultivation under conditions of pronounced climatic risks, due to low stalk yield.

Fine-Tuning Translation Kinetics Selection as the Driving Force of Codon Usage Bias in the Hepatitis A Virus Capsid

Hepatitis A virus (HAV), the prototype of genus Hepatovirus, has several unique biological characteristics that distinguish it from other members of the Picornaviridae family. Among these, the need for an intact eIF4G factor for the initiation of translation results in an inability to shut down host protein synthesis by a mechanism similar to that of other picornaviruses. Consequently, HAV must inefficiently compete for the cellular translational machinery and this may explain its poor growth in cell culture. In this context of virus/cell competition, HAV has strategically adopted a naturally highly deoptimized codon usage with respect to that of its cellular host. With the aim to optimize its codon usage the virus was adapted to propagate in cells with impaired protein synthesis, in order to make tRNA pools more available for the virus. A significant loss of fitness was the immediate response to the adaptation process that was, however, later on recovered and more associated to a re-deoptimization rather than to an optimization of the codon usage specifically in the capsid coding region. These results exclude translation selection and instead suggest fine-tuning translation kinetics selection as the underlying mechanism of the codon usage bias in this specific genome region. Additionally, the results provide clear evidence of the Red Queen dynamics of evolution since the virus has very much evolved to re-adapt its codon usage to the environmental cellular changing conditions in order to recover the original fitness.

Effects of lactation upon circulating plasma metabolites in cafeteria-fed rats

1. The effects of "cafeteria feeding" on primiparous Wistar rats during lactation have been studied by measuring circulating levels of glucose, amino acids, lactate, urea and ammonia as well as glycogen levels in liver and muscle. 2. No significant changes in glucose levels were observed despite alterations in blood glucose compartmentation. 3. Compared with controls, the dams given the cafeteria diet had higher liver glycogen stores which were more easily mobilized at the peak of lactation. 4. Rats given the cafeteria diet showed a lower amino acid utilization than controls and adequately maintained circulating levels, as determined by the lower circulating levels of ammonia and urea. 5. No significant differences in body-weight were observed in the period studied despite increasing dam weight after weaning in the cafeteria-fed group. 6. The size of pups of cafeteria-fed dams was greater than that of controls, and the differences were marked after weaning, when the metabolic machinery of the cafeteria pup maintained high protein accretion and body build-up using fat as the main energy substrate characteristic of the preweaning stage. The controls, however, changed to greater utilization of amino acids as an energy substrate and adapted to high-protein (lowbiological-quality) diets with a significantly different pattern of circulating nitrogen distribution.

The First privatization : selling SOEs and privatizing public monopolies in fascist Italy, 1922-1925

[cat] El primer govern feixista d’Itàlia va aplicar una política de privatització a gran escala entre 1922 i 1925. El govern va privatitzar el monopoli estatal de llumins, el monopoli estatal d’assegurances de vida, va vendre la major part de la xarxa i serveis de telefònica pública a empreses privades, va reprivatitzar el major productor de productes metàl·lics, i va atorgar concessions a empreses privades per construir i explotar autopistes de peatge. Tot i que algunes consideracions ideològiques van poder tenir alguna influència, la privatització va ser usada sobre tot com un instrument polític per construir confiança amb els grans industrials i per augmentar el suport al govern i al Partito Nazionale Fascista. La privatització també va contribuir a equilibrar el pressupost públic, qüestió aquesta que va ser el principal objectiu de la primera fase de la política econòmica feixista.