Optimization Of Promoter Sequences In Order To Mimic Physiological Pde6b Expression

Purpose: Many retinal degenerations result from defective retina-specific gene expressions. Thus, it is important to understand how the expression of a photoreceptor-specific gene is regulated in vivo in order to achieve successful gene therapy. The present study aims to design an AAV2/8 vector that can regulate the transcript level in a physiological manner to replace missing PDE6b in Rd1 and Rd10 mice. In previous studies (Ogieta, et al., 2000), the short 5' flanking sequence of the human PDE6b gene (350 bp) was shown to be photoreceptor-specific in transgenic mice. However, the efficiency and specificity of the 5' flanking region of the human PDE6b was not investigated in the context of gene therapy during retinal degeneration. In this study, two different sequences of the 5' flanking region of the human PDE6b gene were studied as promoter elements and their expression will be tested in wild type and diseased retinas (Rd 10 mice).Methods: Two 5' flanking fragments of the human PDE6b gene: (-93 to +53 (150 bp) and -297 to +53 (350 bp)) were cloned in different plasmids in order to check their expression in vitro and in vivo by constructing an AAV2/8 vector. These elements drove the activity of either luciferase (pGL3 plasmids) or EGFP. jetPEI transfection in Y 79 cells was used to evaluate gene expression through luciferase activity. Constructs encoding EGFP under the control of the two promoters were performed in AAV2.1-93 (or 297)-EGFP plasmids to produce AAV2/8 vectors.Results: When pGL3-93 (150 bp) or pGL3-297 (350 bp) were transfected in the Y-79 cells, the smaller fragment (150 bp) showed higher gene expression compared to the 350 bp element and to the SV40 control, as previously reported. The 350 bp drove similar levels of expression when compared to the SV40 promoter. In view of these results, the fragments (150 bp or 350 bp) were integrated into the AAV2.1-EGFP plasmid to produce AAV2/8 vector, and we are currently evaluating the efficiency and specificity of the produced constructs in vivo in normal and diseased retinas.Conclusions: Comparisons of these vectors with vectors bearing ubiquitous promoters should reveal which construct is the most suitable to drive efficient and specific gene expression in diseased retinas in order to restore a normal function on the long term.

Physiological responses of two tropical weeds to shade: I. Growth and biomass allocation

Ipomoea asarifolia (Desr.) Roem. & Schultz (Convolvulaceae) and Stachytarpheta cayennensis (Rich) Vahl. (Verbenaceae), two weeds found in pastures and crop areas in Brazilian Amazonia, were grown in controlled environment cabinets under high (800-1000 µmol m-² s-¹) and low (200-350 µmol m-² s-¹) light regimes during a 40-day period. For both species leaf dry mass and leaf area per total plant dry mass, and leaf area per leaf dry mass were higher for low-light plants, whereas root mass per total plant dry mass was higher for high-light plants. High-light S. cayennensis allocated significantly more biomass to reproductive tissue than low-light plants, suggesting a probably lower ability of this species to maintain itself under shaded conditions. Relative growth rate (RGR) in I. asarifolia was initially higher for high-light grown plants and after 20 days started decreasing, becoming similar to low-light plants at the last two harvests (at 30 and 40 days). In S. cayennensis, RGR was also higher for high-light plants; however, this trend was not significant at the first and last harvest dates (10 and 40 days). These results are discussed in relation to their ecological and weed management implications.

Physiological responses of two tropical weeds to shade: II. Leaf gas exchange and nitrogen content

Ipomoea asarifolia (Desr.) Roem. & Schultz (Convolvulaceae) and Stachytarpheta cayennensis (Rich) Vahl. (Verbenaceae), two weeds found in pastures and crop areas in the Brazilian Amazonia, Brazil, were grown in controlled environment cabinets under high (800-1000 µmol m-² s-¹) and low (200-350 µmol m-² s-¹) light regimes during a 40-day period. The objective was to determine the effect of shade on photosynthetic features and leaf nitrogen content of I. asarifolia and S. cayennensis. High-irradiance grown I. asarifolia leaves had significantly higher dark respiration and light saturated rates of photosynthesis than low-irradiance leaves. No significant differences for these traits, between treatments, were observed in S. cayennensis. Low-irradiance leaves of both species displayed higher CO2 assimilation rates under low irradiance. High-irradiance grown leaves of both species had less nitrogen per unit of weight. Low-irradiance S. cayennensis had more nitrogen per unit of leaf area than high-irradiance plants; however, I. asarifolia showed no consistent pattern for this variable through time. For S. cayennensis, leaf nitrogen content and CO2 assimilation were inversely correlated to the amount of biomass allocated to developing reproductive structures. These results are discussed in relation to their ecological and weed management implications.

Innovative Solutions for Slope Stability Reinforcement and Characterization: Vol. I, TR-489, 2005

Soil slope instability concerning highway infrastructure is an ongoing problem in Iowa, as slope failures endanger public safety and continue to result in costly repair work. Characterization of slope failures is complicated, because the factors affecting slope stability can be difficult to discern and measure, particularly soil shear strength parameters. While in the past extensive research has been conducted on slope stability investigations and analysis, this research consists of field investigations addressing both the characterization and reinforcement of such slope failures. The current research focuses on applying an infrequently-used testing technique comprised of the Borehole Shear Test (BST). This in-situ test rapidly provides effective (i.e., drained) shear strength parameter values of soil. Using the BST device, fifteen Iowa slopes (fourteen failures and one proposed slope) were investigated and documented. Particular attention was paid to highly weathered shale and glacial till soil deposits, which have both been associated with slope failures in the southern Iowa drift region. Conventional laboratory tests including direct shear tests, triaxial compression tests, and ring shear tests were also performed on undisturbed and reconstituted soil samples to supplement BST results. The shear strength measurements were incorporated into complete evaluations of slope stability using both limit equilibrium and probabilistic analyses. The research methods and findings of these investigations are summarized in Volume 1 of this report. Research details of the independent characterization and reinforcement investigations are provided in Volumes 2 and 3, respectively. Combined, the field investigations offer guidance on identifying the factors that affect slope stability at a particular location and also on designing slope reinforcement using pile elements for cases where remedial measures are necessary. The research findings are expected to benefit civil and geotechnical engineers of government transportation agencies, consultants, and contractors dealing with slope stability, slope remediation, and geotechnical testing in Iowa.

Innovative Solutions for Slope Stability Reinforcement and Characterization: Vol. II, TR-489, 2005

Soil slope instability concerning highway infrastructure is an ongoing problem in Iowa, as slope failures endanger public safety and continue to result in costly repair work. While in the past extensive research has been conducted on slope stability investigations and analysis, this current research study consists of field investigations addressing both the characterization and reinforcement of such slope failures. While Volume I summarizes the research methods and findings of this study, Volume II provides procedural details for incorporating an infrequently-used testing technique, borehole shear tests, into practice. Fifteen slopes along Iowa highways were investigated, including thirteen slides (failed slopes), one unfailed slope, and one proposed embankment slope (the Sugar Creek Project). The slopes are mainly comprised of either clay shale or glacial till, and are generally gentle and of small scale, with slope angle ranging from 11 deg to 23 deg and height ranging from 6 to 23 m. Extensive field investigations and laboratory tests were performed for each slope. Field investigations included survey of slope geometry, borehole drilling, soil sampling, in-situ Borehole Shear Testing (BST) and ground water table measurement. Laboratory investigations mainly comprised of ring shear tests, soil basic property tests (grain size analysis and Atterberg limits test), mineralogy analyses, soil classifications, and natural water contents and density measurements on the representative soil samples from each slope. Extensive direct shear tests and a few triaxial compression tests and unconfined compression tests were also performed on undisturbed soil samples for the Sugar Creek Project. Based on the results of field and lab investigations, slope stability analysis was performed on each of the slopes to determine the possible factors resulting in the slope failures or to evaluate the potential slope instabilities using limit equilibrium methods. Deterministic slope analyses were performed for all the slopes. Probabilistic slope analysis and sensitivity study were also performed for the slope of the Sugar Creek Project. Results indicate that while the in-situ test rapidly provides effective shear strength parameters of soils, some training may be required for effective and appropriate use of the BST. Also, it is primarily intended to test cohesive soils and can produce erroneous results in gravelly soils. Additionally, the quality of boreholes affects test results, and disturbance to borehole walls should be minimized before test performance. A final limitation of widespread borehole shear testing may be its limited availability, as only about four to six test devices are currently being used in Iowa. Based on the data gathered in the field testing, reinforcement investigations are continued in Volume III.

Innovative Solutions for Slope Stability Reinforcement and Characterization: Vol. III, TR-489, 2005

Soil slope instability concerning highway infrastructure is an ongoing problem in Iowa, as slope failures endanger public safety and continue to result in costly repair work. Volume I of this current study summarizes research methods and findings, while Volume II provides procedural details for incorporating into practice an infrequently-used testing technique–borehole shear tests. Volume III of this study of field investigation of fifteen slopes in Iowa demonstrates through further experimental testing how lateral forces develop along stabilizing piles to resist slope movements. Results establish the feasibility of an alternative stabilization approach utilizing small-diameter pile elements. Also, a step-by-step procedure that can be used by both state and county transportation agencies to design slope reinforcement using slender piles is documented. Initial evidence of the efficiency and cost-effectiveness of stabilizing nuisance slope failures with grouted micropiles is presented. Employment of the remediation alternative is deemed more appropriate for stabilizing shallow slope failures. Overall, work accomplished in this research study included completing a comprehensive literature review on the state of the knowledge of slope stability and slope stabilization, the preparation and performance of fourteen full-scale pile load tests, the analysis of load test results, and the documentation of a design methodology for implementing the technology into current practices of slope stabilization. Recommendations for further research include monitoring pilot studies of slope reinforcement with grouted micropiles, supplementary experimental studies, and advanced numerical studies.

Phenotypic stability and genetic gains in six-year girth growth of Hevea clones

Rubber tree [Hevea brasiliensis (Willd. ex Adr. de Juss.) Müell. Arg.] budgrafts of seven clones were evaluated on five contrasting sites in the plateau region of the São Paulo State, Brazil. The objective of this work was to study the phenotypic stability for girth growth. The experimental design was a randomized block design with three replications and seven treatments. Analysis of variance of girth at six-year plant growth indicated a highly significant clone x site interaction. Only linear sites and clone x site components of clone x year interaction were significant, indicating that the performance of clones over sites for this trait could be predicted. The clones GT 1 and PB 235 showed the greatest stability in relation to girth growth, with foreseen responses to change, introduced in the sites. The clones PB 235 and IAN 873 showed significative difference in relation to regression coefficient, representing clones with specific adaptability on favorable and unfavorable sites respectively. The clone GT 1 became the most promissory one in the study of stability and adaptability even showing low girth growth. Expected genetic gains from planting sites, along with estimates of clonal variance and repeatability of clonal means are generally greatest or close to the greatest when selection is done at the same site.

WNT Signaling in Drosophila Neuromuscular Junctions

The Wnt -Wingless (Wg) in Drosophila- signaling is an evolutionary conserved, fundamental signal transduction pathway in animals, having a crucial role in early developmental processes. In the adult animal the Wnt cascade is mainly shut off; aberrant activation leads to cancer. One physiological exception in the adult animal is the activation of Wnt signaling in the nervous system. In the present work, we investigated Wg signaling in the Drosophila neuromuscular junctions (NMJs). The fly NMJs closely resemble the glutamatergic synapses in the mammalian central nervous system and serves as a model system to investigate the mechanism of synapse formation and stability. We demonstrate that the trimeric G-protein Go has a fundamental role in the presynaptic cell in the NMJ. It is implicated in the presynaptic Wg pathway, acting downstream of the ligand Wg and its receptor Frizzled2 (Fz2). Furthermore, we prove that the presynaptic Wg-Fz2-Gαo pathway is essential for correct NMJ formation. The neuronal protein Ankyrin2 (Ank2) localizes to the NMJ and has so far been considered to be a static player in NMJ formation, linking the plasma membrane to the cytoskeleton. We identify Ank2 as a direct target of Gαo. The physical and genetic interaction of Gαo with Ank2 represents a novel branch of the presynaptic Wg pathway, regulating the microtubule cytoskeleton in NMJ formation, jointly with the previously established Futsch-dependent branch, which controls microtubule stability downstream of the kinase Sgg (the homolog of GSK3ß). We moreover demonstrate that the Gαo-Ankyrin interaction to regulate the cytoskeleton is conserved in mammalian neuronal cells. Our findings therefore provide a novel, universally valid regulation of the cytoskeleton in the nervous system. Aberrant inactivation of the neuronal Wnt pathway is believed to be involved in the pathogenesis of the Aß peptide in Alzheimer's disease (AD). We modeled AD in Drosophila by expressing Aß42 in the nervous system and in the eye. Neuronal expression drastically shortens the life span of the flies. We prove that this effect depends on the expression specifically in glutamatergic neurons. However, Aß42 does not induce any morphological changes in the NMJ; therefore this synapse is not suitable to study the mechanism of Aß42 induced neurotoxicity. We furthermore demonstrate that genetic activation of the Wnt pathway does not rescue the Aß42 induced phenotypes - in opposition to the dominating view in the field. These results advice caution when interpreting data on the potential interaction of Wnt signaling and AD in other models. -- La voie de signalisation Wnt (Wingless (Wg) chez la drosophile) est conservée dans l'évolution et fondamentale pour le développement des animaux. Cette signalisation est normalement inactive chez l'animal adulte; une activation anormale peut provoquer le cancer. Or, ceci n'est pas le cas dans le système nerveux des adultes. La présente thèse avait pour but d'analyser le rôle de la voie de signalisation Wingless dans la plaque motrice de Drosophila melanogaster. En effet, cette plaque ressemble fortement aux synapses glutaminergiques du système nerveux central des mammifères et procure ainsi un bon modèle pour l'étude des mécanismes impliqués dans la formation et la stabilisation des synapses. Nos résultats montrent que la protéine trimérique Go joue un rôle fondamental dans la fonction de la cellule présynaptique de la plaque motrice. Go est en effet impliqué dans la voie de signalisation Wg, opérant en aval du ligand Wg et de son récepteur Frizzled2. Nous avons pu démontrer que cette voie de signalisation Wg-Fz2-Gαo est essentielle pour le bon développement et le fonctionnement de la plaque motrice. Fait intéressant, nous avons montré que la protéine neuronale Ankyrin2 (Ank2), qui est connue pour jouer un rôle statique en liant la membrane plasmique au cytosquelette dans la plaque motrice, est une cible directe de Gαo. L'interaction physique et génétique entre Gαo et Ank2 constitue ainsi une bifurcation de la voie de signalisation présynaptique Wg. Cette voie régule le cytosquelette des microtubules en coopération avec la branche liée à la protéine Futsch. Cette protéine est l'homologue de la protéine liant les microtubules MAP1B des mammifères et contrôle la stabilité des microtubules opérant en aval de la kinase Sgg (l'homologue de GSK3ß). De plus, la régulation du cytosquelette par l'interaction entre Gαo et Ankyrin est conservée chez les mammifères. Dans leur ensemble, nos résultats ont permis d'identifier un nouveau mode de régulation du cytosquelette dans le système nerveux, probablement valable de manière universelle. La voie de signalisation Wnt est soupçonnée d'être impliquée dans la toxicité provoquée par le peptide Aß dans le cadre de la maladie d'Alzheimer. Nous avons tenté de modéliser la maladie chez la drosophile en exprimant Aß42 spécifiquement dans le cerveau. Cette expérience a montré que l'expression neuronale d'Aß42 réduit la durée de vie des mouches de manière significative par un mécanisme impliquant les cellules glutamatergiques. Par contre, aucune modification morphologique n'est provoquée par Aß42 dans les plaques motrices glutamatergiques. Ces résultats montrent que ce modèle de Drosophile n'est pas adéquat pour l'étude de la maladie d'Alzheimer. De plus, l'activation génétique de la voie de signalisation Wg n'a pas réussi à restaurer les phénotypes de survie ou ceux des yeux causés par Aß42. Ces résultats indiquent que l'implication de la voie de signalisation Wg dans la maladie d'Alzheimer doit être considérée avec prudence.

Physiological changes during postharvest senescence of broccoli

The objective of this work was to determine the early physiological changes throughout shelf life of fresh broccoli (Brassica oleracea L. var. italica) cv. Piracicaba Precoce at 25ºC and relative humidity of 96% in the dark until complete senescence. Head inflorescences showed lack of turgidity and commercial value when weight loss reached up to 5%, coinciding with 48 hour after harvest. Chlorophyll content was stable until 24 hours after harvesting; afterwards, an intense degradation phase took place. At 72 hours, total head yellowing was observed when chlorophyll content dropped to 30% of its initial content. Peroxidase activity increased by 1.4 fold during the first six hours, dropping to its lowest level approximately 24 hours after harvesting. However, from this time on, an increment of activity was observed until 72 hours. At 24 hours after harvesting, respiration was reduced by 50%. At later stages of senescence, respiration of florets was stable, but in a lower level than that determined at harvest. Sharp reduction of starch and reducing sugars was observed within 24 hours after harvesting, followed by continuous period of decline in starch and non-reducing sugars.

Preliminary description and slope stability analyses of the 2008 Little Salmon lake and 2007 Mt. Steele landslides, Yukon

In August 2008, reactivation of the Little Salmon Lake landslide occurred. During this event, hundreds of conical mounds of variable size and composition formed in the deposition zone. The characteristics of these landforms are described and a potential mechanism for their formation is proposed. A preliminary slope stability analysis of the 2007 Mount Steele rock and ice avalanche was also undertaken. The orientation of very high persistence (>20 m long) structural planes (e.g., faults, joints and bedding) within bedrock in the source zone was obtained using an airborne-LiDAR digital elevation model and the software COLTOP-3D. Using these discontinuity orientation measurements, kinematic, surface wedge and simple three-dimensional distinct element slope stability analyses were performed.

Stability-dependent behavioural and electro-cortical reorganizations during intentional switching between bimanual tapping modes

This study investigated behavioural and electro-cortical reorganizations accompanying intentional switching between two distinct bimanual coordination tapping modes (In-phase and Anti-phase) that differ in stability when produced at the same movement rate. We expected that switching to a less stable tapping mode (In-to-Anti switching) would lead to larger behavioural perturbations and require supplementary neural resources than switching to a more stable tapping mode (Anti-to-In switching). Behavioural results confirmed that the In-to-Anti switching lasted longer than the Anti-to-In switching. A general increase in attention-related neural activity was found at the moment of switching for both conditions. Additionally, two condition-dependent EEG reorganizations were observed. First, a specific increase in cortico-cortical coherence appeared exclusively during the In-to-Anti switching. This result may reflect a strengthening in inter-regional communication in order to engage in the subsequent, less stable, tapping mode. Second, a decrease in motor-related neural activity (increased beta spectral power) was found for the Anti-to-In switching only. The latter effect may reflect the interruption of the previous, less stable, tapping mode. Given that previous results on spontaneous Anti-to-In switching revealing an inverse pattern of EEG reorganization (decreased beta spectral power), present findings give new insight on the stability-dependent neural correlates of intentional motor switching. © 2010 Elsevier Ireland Ltd. All rights reserved

Physiological responses of maize and cowpea to intercropping

The effect of intercropping on plant water status, gas exchange and productivity of maize (Zea mays L.) cv. Centralmex, and cowpea (Vigna unguiculata L. (Walp)) cv. Pitiuba were evaluated under semi-arid conditions at the Embrapa-Centro de Pesquisa Agropecuária do Trópico Semi-Árido (CPATSA) at Petrolina, PE, Brazil. The treatments were: maize and cowpea as sole crops, at a population of 40,000 plants ha-1, and intercropped at a population of 20,000 plants ha-1. The results obtained in this paper appear to be related to the degree of competition experienced by the components, mainly for water and light. Maize intercropped had higher values of leaf water potential, stomatal conductance, transpiration and photosynthesis than as sole crop. Intercropped cowpea had higher values of leaf water potential but lower stomatal conductance, transpiration and photosynthesis than sole cowpea. Maize productivity increased 18% in relation to sole crop whereas a 5% decrease was observed with cowpea. Despite these facts the Land Equivalent Ratio obtained was 1.13 indicating intercropping advantage over the sole system. The higher partial Land Equivalent Ratio observed for maize suggests that this specie was the main component influencing the final productivity of the intercropping system studied.