The investigation of optimal bombardment parameters for transient and stable transgene expression in sorghum

This report outlines the development of optimized particle inflow gun (PIG) parameters for producing transgenic sorghum (Sorghum bicolor (L.) Moench). Both transient and stable expression were examined when determining these parameters. The uidA reporter gene (GUS) encoding beta -glucuronidase was used in transient experiments and the green fluorescent protein (GFP) used to monitor stable expression. Initially, optimization was conducted using leaf segments, as the generation of sorghum callus in sufficiently large quantities is time-consuming. Following leaf optimization, experiments were conducted using callus, identifying a high similarity between the two tissue types (r(s) = 0.83). High levels of GUS expression were observed in both leaf and callus material when most distant from the DNA expulsion point, and using a pressure greater than 1800 kPa. A higher level of expression was also observed when the aperture of the helium inlet valve was constricted. Using the optimized conditions (pressure of 2200 kPa, distance to target tissue of 15 cm from the expulsion point, and the aperture of the helium inlet valve at one full turn), three promoters (Ubiquitin, Actin1 and CaMV 35S) were evaluated over a 72-h period using GUS as the reporter gene. A significantly higher number of GUS foci were counted with the Ubiquitin construct over this period, compared to the Actin1 and CaMV 35S constructs. Stable callus sectors (on 2 mg l(-1) bialaphos) with GFP expression were visualized for as long as 6 wk post-bombardment. Using this optimized protocol, several plants were regenerated after having been bombarded with the pAHC20 construct (containing the bar gene), with molecular evidence confirming integration.

Transformação genética em cevada por bombardeamento de partículas

A presente Tese de Doutorado objetivou: (1) definir um método eficiente de transformação genética, por bombardeamento de partículas, para a obtenção de plantas transgênicas de cultivares brasileiras de cevada e (2) identificar gene(s) codificante(s) de quitinase(s) potencialmente capaz(es) de conferir resistência ao fungo patogênico de cevada Bipolaris sorokiniana. Culturas de calos obtidos a partir de escutelos imaturos das cultivares Brasileiras de cevada MN-599 e MN-698 (Cia. de Bebidas das Américas, AMBEV) foram bombardeadas com partículas de tungstênio e avaliadas quanto à expressão do gene repórter gusA através de ensaios histoquímicos de GUS e quanto ao efeito dos bombardeamentos na indução estruturas embriogênicas e regeneração de plantas. As condições de biobalística analisadas incluíram a região promotora regulando a expressão de gusA, tipo e pressão de gás hélio de dois aparelhos de bombardeamento, distância de migração das partículas, número de tiros e a realização de pré e pós-tratamento osmótico dos tecidos-alvo. No presente trabalho foram obtidos um número bastante alto de pontos azuis por calo, a indução de calos embriogênicos e embriões somáticos em uma freqüência de até 58,3% e a regeneração de 60 plantas, sendo 43 de calos bombardeados. As melhores condições observadas foram o promotor e primeiro íntron do gene Adh de milho (plasmídeo pNGI), o aparelho de bombardeamento “ Particle Inflow Gun” (PIG) utilizando-se a distância de migração de partículas de 14,8 cm, dois tiros disparados por placa e a realização de tratamento osmótico dos explantes com 0,2 M de manitol e 0,2 M de sorbitol 4-5 horas antes e 17-19 horas depois dos bombardeamentos. Das 43 plantas obtidas de calos bombardeadas, 3 apresentaram atividade de GUS na base das suas folhas. A utilização de primers sintéticos definidos a partir de genes de quitinases descritos na literatura em PCRs resultou na amplificação de dois fragmentos de aproximadamente 700 e 500 pb a partir de DNA total das cvs. MN-599 e MN-698 de cevada e um fragmento, com aproximadamente 500 pb, a partir do DNA total do isolado A4c de Trichoderma sp. Estes fragmentos foram purificados dos géis de agarose e diretamente seqüenciados de forma manual e automática. Os fragmentos de 700 e 500 pb amplificados do genoma da cultivar MN-599 foram identificados como genes de quitinases de cevada e o fragmento de 500 pb do isolado A4c de Trichoderma sp. não apresentou homologia com seqüências conhecidas de quitinases depositadas no EMBL/GenBank. A utilização de novos pares de primers, representando seqüências conservadas de quitinases do fungo Metarhizium anisopliae, resultou na amplificação de 3 fragmentos a partir do DNA total do isolado A4b de Trichoderma sp., que estão sendo purificados para realização de seqüenciamento.

Limnology of Macrobrachium amazonicum grow-out ponds subject to high inflow of nutrient-rich water and different stocking and harvest management

We evaluated the water characteristics and particle sedimentation in Macrobrachium amazonicum (Heller 1862) grow-out ponds supplied with a high inflow of nutrient-rich water. Prawns were subject to different stocking and harvesting strategies: upper-graded juveniles, lower-graded juveniles, non-graded juveniles + selective harvesting and traditional farming (non-grading juveniles and total harvest only). Dissolved oxygen, afternoon N-ammonia and N-nitrate and soluble orthophosphate were lower in the ponds in comparison with inflow water through the rearing cycle. Ponds stocked with the upper population fraction of graded prawns showed higher turbidity, total suspended solids and total Kjeldahl nitrogen than the remaining treatments. An increase in the chemical oxygen demand:biochemical oxygen demand ratio from inlet (4.9) to pond (7.1-8.0) waters indicated a non-readily biodegradable fraction enhancement in ponds. The sedimentation mean rate ranged from 0.08 to 0.16 mm day(-1) and sediment contained >80% of organic matter. The major factors affecting pond ecosystem dynamic were the organic load (due to primary production and feed addition) and bioturbation caused by stocking larger animals. Data suggest that M. amazonicum grow-out in ponds subjected to a high inflow of nutrient-rich water produce changes in the water properties, huge accumulation of organic sediment at the pond bottom and non-readily biodegradable material in the water column. However, the water quality remains suitable for aquaculture purposes. Therefore, nutrient-rich waters, when available, may represent a source of unpaid nutrients, which may be incorporated into economically valued biomass if managed properly.

Danger assessment of a spring-gun device (muzzleloader) loaded only with black powder

We report a case involving a spring-gun device (muzzleloader) loaded solely with gunpowder, installed next to shoes to prevent the neighbors' puppy from removing them. The booby trap was triggered by the 15-year-old dog-owners son when he tried to put the shoes out of the reach of the puppy. The boy suffered second degree superficial burns located mainly at the dorsal side of the right hand and fingers. To estimate the danger of the used weapon, several tests were undertaken on soap blocks from different distances and with different loads of black powder. The particle density per mm and the depth of black powder tattooing in the soap was compared with the boy's injuries, and found conclusive with the gun-owners statement regarding the loading of the weapon. Furthermore, our experiments indicated that the gunpowder load involved was not able to inflict permanent damage, not even to the eyes, at the here estimated firing distance.

Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors.

Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 +/- 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with IL-12 cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional IL-12 gene transfections, could significantly inhibit systemic metastases, resulting in extended survival of test mice. These results suggest that gene gun-mediated in vivo delivery of IL-12 cDNA should be further developed for potential clinical testing as an approach for human cancer gene therapy.

Numerical analysis of gas and micro-particle interactions in a hand-held shock-tube device

A unique hand-held gene gun is employed for ballistically delivering biomolecules to key cells in the skin and mucosa in the treatment of the major diseases. One of these types of devices, called the Contoured Shock Tube (CST), delivers powdered micro-particles to the skin with a narrow and highly controllable velocity distribution and a nominally uniform spatial distribution. In this paper, we apply a numerical approach to gain new insights in to the behavior of the CST prototype device. The drag correlations proposed by Henderson (1976), Igra and Takayama (1993) and Kurian and Das (1997) were applied to predict the micro-particle transport in a numerically simulated gas flow. Simulated pressure histories agree well with the corresponding static and Pitot pressure measurements, validating the CFD approach. The calculated velocity distributions show a good agreement, with the best prediction from Igra & Takayama correlation (maximum discrepancy of 5%). Key features of the gas dynamics and gas-particle interaction are discussed. Statistic analyses show a tight free-jet particle velocity distribution is achieved (570 +/- 14.7 m/s) for polystyrene particles (39 +/- 1 mu m), representative of a drug payload.

Particle positions of numerical experiments 1 to 4

In mixed sediment beds, erosion resistance can change relative to that of beds composed of a uniform sediment because of varying textural and/or other grain-size parameters, with effects on pore water flow that are difficult to quantify by means of analogue techniques. To overcome this difficulty, a three-dimensional numerical model was developed using a finite difference method (FDM) flow model coupled with a distinct element method (DEM) particle model. The main aim was to investigate, at a high spatial resolution, the physical processes occurring during the initiation of motion of single grains at the sediment-water interface and in the shallow subsurface of simplified sediment beds under different flow velocities. Increasing proportions of very fine sand (D50=0.08 mm) were mixed into a coarse sand matrix (D50=0.6 mm) to simulate mixed sediment beds, starting with a pure coarse sand bed in experiment 1 (0 wt% fines), and proceeding through experiment 2 (6.5 wt% fines), experiment 3 (10.5 wt% fines), and experiment 4 (28.7 wt% fines). All mixed beds were tested for their erosion behavior at predefined flow velocities varying in the range of U 1-5=10-30 cm/s. The experiments show that, with increasing fine content, the smaller particles increasingly fill the spaces between the larger particles. As a consequence, pore water inflow into the sediment is increasingly blocked, i.e., there is a decrease in pore water flow velocity and, hence, in the flow momentum available to entrain particles. These findings are portrayed in a new conceptual model of enhanced sediment bed stabilization.

Limnology of Macrobrachium amazonicum grow-out ponds subject to high inflow of nutrient-rich water and different stocking and harvest management

We evaluated the water characteristics and particle sedimentation in Macrobrachium amazonicum (Heller 1862) grow-out ponds supplied with a high inflow of nutrient-rich water. Prawns were subject to different stocking and harvesting strategies: upper-graded juveniles, lower-graded juveniles, non-graded juveniles + selective harvesting and traditional farming (non-grading juveniles and total harvest only). Dissolved oxygen, afternoon N-ammonia and N-nitrate and soluble orthophosphate were lower in the ponds in comparison with inflow water through the rearing cycle. Ponds stocked with the upper population fraction of graded prawns showed higher turbidity, total suspended solids and total Kjeldahl nitrogen than the remaining treatments. An increase in the chemical oxygen demand:biochemical oxygen demand ratio from inlet (4.9) to pond (7.1-8.0) waters indicated a non-readily biodegradable fraction enhancement in ponds. The sedimentation mean rate ranged from 0.08 to 0.16 mm day(-1) and sediment contained >80% of organic matter. The major factors affecting pond ecosystem dynamic were the organic load (due to primary production and feed addition) and bioturbation caused by stocking larger animals. Data suggest that M. amazonicum grow-out in ponds subjected to a high inflow of nutrient-rich water produce changes in the water properties, huge accumulation of organic sediment at the pond bottom and non-readily biodegradable material in the water column. However, the water quality remains suitable for aquaculture purposes. Therefore, nutrient-rich waters, when available, may represent a source of unpaid nutrients, which may be incorporated into economically valued biomass if managed properly.

Reference Values For High-density Lipoprotein Particle Size And Volume By Dynamic Light Scattering In A Brazilian Population Sample And Their Relationships With Metabolic Parameters.

Current data indicate that the size of high-density lipoprotein (HDL) may be considered an important marker for cardiovascular disease risk. We established reference values of mean HDL size and volume in an asymptomatic representative Brazilian population sample (n=590) and their associations with metabolic parameters by gender. Size and volume were determined in HDL isolated from plasma by polyethyleneglycol precipitation of apoB-containing lipoproteins and measured using the dynamic light scattering (DLS) technique. Although the gender and age distributions agreed with other studies, the mean HDL size reference value was slightly lower than in some other populations. Both HDL size and volume were influenced by gender and varied according to age. HDL size was associated with age and HDL-C (total population); non- white ethnicity and CETP inversely (females); HDL-C and PLTP mass (males). On the other hand, HDL volume was determined only by HDL-C (total population and in both genders) and by PLTP mass (males). The reference values for mean HDL size and volume using the DLS technique were established in an asymptomatic and representative Brazilian population sample, as well as their related metabolic factors. HDL-C was a major determinant of HDL size and volume, which were differently modulated in females and in males.

Metamorfose planar via métodos Level Set e Particle Level Set para a reconstrução de superfícies tridimensionais

Evolving interfaces were initially focused on solutions to scientific problems in Fluid Dynamics. With the advent of the more robust modeling provided by Level Set method, their original boundaries of applicability were extended. Specifically to the Geometric Modeling area, works published until then, relating Level Set to tridimensional surface reconstruction, centered themselves on reconstruction from a data cloud dispersed in space; the approach based on parallel planar slices transversal to the object to be reconstructed is still incipient. Based on this fact, the present work proposes to analyse the feasibility of Level Set to tridimensional reconstruction, offering a methodology that simultaneously integrates the proved efficient ideas already published about such approximation and the proposals to process the inherent limitations of the method not satisfactorily treated yet, in particular the excessive smoothing of fine characteristics of contours evolving under Level Set. In relation to this, the application of the variant Particle Level Set is suggested as a solution, for its intrinsic proved capability to preserve mass of dynamic fronts. At the end, synthetic and real data sets are used to evaluate the presented tridimensional surface reconstruction methodology qualitatively.

Metamorfose planar via métodos Level Set e Particle Level Set para a reconstrução de superfícies tridimensionais

Evolving interfaces were initially focused on solutions to scientific problems in Fluid Dynamics. With the advent of the more robust modeling provided by Level Set method, their original boundaries of applicability were extended. Specifically to the Geometric Modeling area, works published until then, relating Level Set to tridimensional surface reconstruction, centered themselves on reconstruction from a data cloud dispersed in space; the approach based on parallel planar slices transversal to the object to be reconstructed is still incipient. Based on this fact, the present work proposes to analyse the feasibility of Level Set to tridimensional reconstruction, offering a methodology that simultaneously integrates the proved efficient ideas already published about such approximation and the proposals to process the inherent limitations of the method not satisfactorily treated yet, in particular the excessive smoothing of fine characteristics of contours evolving under Level Set. In relation to this, the application of the variant Particle Level Set is suggested as a solution, for its intrinsic proved capability to preserve mass of dynamic fronts. At the end, synthetic and real data sets are used to evaluate the presented tridimensional surface reconstruction methodology qualitatively.

Particle competition for complex network community detection

In many real situations, randomness is considered to be uncertainty or even confusion which impedes human beings from making a correct decision. Here we study the combined role of randomness and determinism in particle dynamics for complex network community detection. In the proposed model, particles walk in the network and compete with each other in such a way that each of them tries to possess as many nodes as possible. Moreover, we introduce a rule to adjust the level of randomness of particle walking in the network, and we have found that a portion of randomness can largely improve the community detection rate. Computer simulations show that the model has good community detection performance and at the same time presents low computational complexity. (C) 2008 American Institute of Physics.

Reduction of chaotic particle transport driven by drift waves in sheared flows

Investigations of chaotic particle transport by drift waves propagating in the edge plasma of tokamaks with poloidal zonal flow are described. For large aspect ratio tokamaks, the influence of radial electric field profiles on convective cells and transport barriers, created by the nonlinear interaction between the poloidal flow and resonant waves, is investigated. For equilibria with edge shear flow, particle transport is seen to be reduced when the electric field shear is reversed. The transport reduction is attributed to the robust invariant tori that occur in nontwist Hamiltonian systems. This mechanism is proposed as an explanation for the transport reduction in Tokamak Chauffage Alfven Bresilien [R. M. O. Galvao , Plasma Phys. Controlled Fusion 43, 1181 (2001)] for discharges with a biased electrode at the plasma edge.