Using matrix attachment regions to improve recombinant protein production.

Chinese hamster ovary (CHO) cells are the system of choice for the production of complex molecules, such as monoclonal antibodies. Despite significant progress in improving the yield from these cells, the process to the selection, identification, and maintenance of high-producing cell lines remains cumbersome, time consuming, and often of uncertain outcome. Matrix attachment regions (MARs) are DNA sequences that help generate and maintain an open chromatin domain that is favourable to transcription and may also facilitate the integration of several copies of the transgene. By incorporating MARs into expression vectors, an increase in the proportion of high-producer cells as well as an increase in protein production are seen, thereby reducing the number of clones to be screened and time to production by as much as 9 months. In this chapter, we describe how MARs can be used to increase transgene expression and provide protocols for the transfection of CHO cells in suspension and detection of high-producing antibody cell clones.

Anomalous crossover between thermal and shot noise in macroscopic diffusive conductors

We predict the existence of an anomalous crossover between thermal and shot noise in macroscopic diffusive conductors. We first show that, besides thermal noise, these systems may also exhibit shot noise due to fluctuations of the total number of carriers in the system. Then we show that at increasing currents the crossover between the two noise behaviors is anomalous, in the sense that the low-frequency current spectral density displays a region with a superlinear dependence on the current up to a cubic law. The anomaly is due to the nontrivial coupling in the presence of the long-range Coulomb interaction among the three time scales relevant to the phenomenon, namely, diffusion, transit, and dielectric relaxation time.

Shot noise anomalies in elastic nondegenerate diffusive conductors

We present a theoretical investigation of shot-noise properties in nondegenerate elastic diffusive conductors. Both Monte Carlo simulations and analytical approaches are used. Two interesting phenomena are found: (i) the display of enhanced shot noise for given energy dependences of the scattering time, and (ii) the recovery of full shot noise for asymptotic high applied bias. The first phenomenon is associated with the onset of negative differential conductivity in energy space that drives the system towards a dynamical electrical instability in excellent agreement with analytical predictions. The enhancement is found to be strongly amplified when the dimensionality in momentum space is lowered from three to two dimensions. The second phenomenon is due to the suppression of the effects of long-range Coulomb correlations that takes place when the transit time becomes the shortest time scale in the system, and is common to both elastic and inelastic nondegenerate diffusive conductors. These phenomena shed different light in the understanding of the anomalous behavior of shot noise in mesoscopic conductors, which is a signature of correlations among different current pulses.

Method for retinal gene repair in neonatal mouse.

Gene correction at the site of the mutation in the chromosome is the absolute way to really cure a genetic disease. The oligonucleotide (ODN)-mediated gene repair technology uses an ODN perfectly complementary to the genomic sequence except for a mismatch at the base that is mutated. The endogenous repair machinery of the targeted cell then mediates substitution of the desired base in the gene, resulting in a completely normal sequence. Theoretically, it avoids potential gene silencing or random integration associated with common viral gene augmentation approaches and allows an intact regulation of expression of the therapeutic protein. The eye is a particularly attractive target for gene repair because of its unique features (small organ, easily accessible, low diffusion into systemic circulation). Moreover therapeutic effects on visual impairment could be obtained with modest levels of repair. This chapter describes in details the optimized method to target active ODNs to the nuclei of photoreceptors in neonatal mouse using (1) an electric current application at the eye surface (saline transpalpebral iontophoresis), (2) combined with an intravitreous injection of ODNs, as well as the experimental methods for (3) the dissection of adult neural retinas, (4) their immuno-labelling, and (5) flat-mounting for direct observation of photoreceptor survival, a relevant criteria of treatment outcomes for retinal degeneration.

Potassium availability in a hapludalf soil under long term fertilization

In a system in which fertilization is recommended, diagnosis of soil K availability and the establishment of critical levels are made difficult by the possibility of a contribution of non-exchangeable forms of K for plant nutrition. Due to its magnitude, this contribution is well diagnosed in long term experiments and in those which compare fertilization systems with positive and negative balances in terms of replacement of the K extracted by plants. The objective of this study was to evaluate K availability in a Hapludalf under fertilization for sixteen years with the addition of K doses. The study was undertaken in an experiment set up in 1991 and carried out until 2007 in the experimental area of the Soil Department of the Federal University of Santa Maria (Universidade Federal de Santa Maria - UFSM), in Santa Maria (RS), Brazil. The soil was a Typic Hapludalf submitted to four doses of K (0, 60, 120 and 180 kg ha-1 K2O) and subdivided in the second year, when 60 kg ha-1 of K2O were reapplied in the subplots in 0, 1, 2 and 3 times. As of the fifth year, the procedure was repeated. Grain yield above ground dry matter and total K content contained in the plant tissue were evaluated. Soil samples were collected, oven dried, ground, passed through a sieve and submitted to exchangeable K analysis by the Mehlich-1 extractor; non-exchangeable K by boiling HNO3 1 mol L-1 and total K by HF digestion. Potassium fertilization guidelines should foresee the establishment of a critical level as of which the recommended dose should accompany crop needs, which coincides with the quantity exported by the grain, without there being the need for the creation of broad ranges of K availability to predict K fertilization. In adopting the K fertilization recommendations proposed in this manner, there will not be K translocation in the soil profile.

Vacancy-driven ordering in a two-dimensional binary alloy

Domain growth in a system with nonconserved order parameter is studied. We simulate the usual Ising model for binary alloys with concentration 0.5 on a two-dimensional square lattice by Monte Carlo techniques. Measurements of the energy, jump-acceptance ratio, and order parameters are performed. Dynamics based on the diffusion of a single vacancy in the system gives a growth law faster than the usual Allen-Cahn law. Allowing vacancy jumps to next-nearest-neighbor sites is essential to prevent vacancy trapping in the ordered regions. By measuring local order parameters we show that the vacancy prefers to be in the disordered regions (domain boundaries). This naturally concentrates the atomic jumps in the domain boundaries, accelerating the growth compared with the usual exchange mechanism that causes jumps to be homogeneously distributed on the lattice.

Dissipation in quasistatically driven disordered systems

This work presents an analysis of hysteresis and dissipation in quasistatically driven disordered systems. The study is based on the random field Ising model with fluctuationless dynamics. It enables us to sort out the fraction of the energy input by the driving field stored in the system and the fraction dissipated in every step of the transformation. The dissipation is directly related to the occurrence of avalanches, and does not scale with the size of Barkhausen magnetization jumps. In addition, the change in magnetic field between avalanches provides a measure of the energy barriers between consecutive metastable states

Social Capital and Sport Governance in Europe

Although there is significant interest in the social role of sport in fostering civil society from both policymakers and academics, there is a lack of evidence of the specific role of sport federations in this system. This book critically presents the mechanisms and structures in a selection of sport federations within a variety of European countries that illuminate the varied relationships between not-for-profit sport federations, their members, governments and the citizens they represent. The contributors explore the contrasts and synergies between core social capital theoretical perspectives, and how these may be informed by and/or shape the realities of governance from different perspectives within the sport system.

In vitro whole-genome analysis identifies a susceptibility locus for HIV-1.

Advances in large-scale analysis of human genomic variability provide unprecedented opportunities to study the genetic basis of susceptibility to infectious agents. We report here the use of an in vitro system for the identification of a locus on HSA8q24.3 associated with cellular susceptibility to HIV-1. This locus was mapped through quantitative linkage analysis using cell lines from multigeneration families, validated in vitro, and followed up by two independent association studies in HIV-positive individuals. Single nucleotide polymorphism rs2572886, which is associated with cellular susceptibility to HIV-1 in lymphoblastoid B cells and in primary T cells, was also associated with accelerated disease progression in one of two cohorts of HIV-1-infected patients. Biological analysis suggests a role of the rs2572886 region in the regulation of the LY6 family of glycosyl-phosphatidyl-inositol (GPI)-anchored proteins. Genetic analysis of in vitro cellular phenotypes provides an attractive approach for the discovery of susceptibility loci to infectious agents.

Nitrogen dynamics in soil management systems. I - flux of inorganic nitrogen (NH4+ and NO3-)

In agricultural systems the N-NH4+ and N-NO3- contents is significantly affected by soil management. This study investigated the dynamics of inorganic nitrogen (N; NH4+ and NO3-) in an experimental evaluation of soil management systems (SMSs) adopted in 1988 at the experimental station of the ABC Foundation in Ponta Grossa, in the Central South region of the State of Paraná. The objective of this study was to evaluate the changes in N-NH4+ and N-NO3- flux in the surface layer of a Red Latosol arising from SMSs over a 12-month period. The experiment was arranged in a completely randomized block design in split plots, in three replications. The plots consisted of the following SMSs: 1) conventional tillage (CT); 2) minimum tillage (MT); 3) no-tillage with chisel plow every three years (NT CH); and 4) continuous no-tillage (CNT). To evaluate the dynamics of inorganic N, the subplots represented samplings (11 sampling times, T1 - T11). The ammonium N (N-NH4+) and nitric N (N-NO3-) contents were higher in systems with reduced tillage (MT and NT CH) and without tillage (CNT) than in the CT system. In the period from October 2003 to February 2004, the N-NH4+ was higher than the N-NO3- soil content. Conversely, in the period from May 2004 to July 2004, the N-NO3- was higher than the N-NH4+ content. The greatest fluctuation in the N-NH4+ and N-NO3- contents occurred in the 0-2.5 cm layer, and the highest peak in the N-NH4+ and N-NO3- concentrations occurred after the surface application of N. Both N-NH4+ and N-NO3- were strongly correlated with the soil organic C content, which indicated that these properties vary together in the system.

Genetics of normal and pathological sleep in humans.

The complexity of sleep-wake regulation, in addition to the many environmental influences, includes genetic predisposing factors, which begin to be discovered. Most of the current progress in the study of sleep genetics comes from animal models (dogs, mice, and drosophila). Multiple approaches using both animal models and different genetic techniques are needed to follow the segregation and ultimately to identify 'sleep genes' and molecular bases of sleep disorders. Recent progress in molecular genetics and the development of detailed human genome map have already led to the identification of genetic factors in several complex disorders. Only a few genes are known for which a mutation causes a sleep disorder. However, single gene disorders are rare and most common disorders are complex in terms of their genetic susceptibility, environmental factors, gene-gene, and gene-environment interactions. We review here the current progress in the genetics of normal and pathological sleep and suggest a few future perspectives.

Neurogenic subventricular zone stem/progenitor cells are notch1-dependent in their active but not quiescent state.

The adult mammalian forebrain contains neural stem/progenitor cells (NSCs) that generate neurons throughout life. As in other somatic stem cell systems, NSCs are proposed to be predominantly quiescent and proliferate only sporadically to produce more committed progeny. However, quiescence has recently been shown not to be an essential criterion for stem cells. It is not known whether NSCs show differences in molecular dependence based on their proliferation state. The subventricular zone (SVZ) of the adult mouse brain has a remarkable capacity for repair by activation of NSCs. The molecular interplay controlling adult NSCs during neurogenesis or regeneration is not clear but resolving these interactions is critical in order to understand brain homeostasis and repair. Using conditional genetics and fate mapping, we show that Notch signaling is essential for neurogenesis in the SVZ. By mosaic analysis, we uncovered a surprising difference in Notch dependence between active neurogenic and regenerative NSCs. While both active and regenerative NSCs depend upon canonical Notch signaling, Notch1-deletion results in a selective loss of active NSCs (aNSCs). In sharp contrast, quiescent NSCs (qNSCs) remain after Notch1 ablation until induced during regeneration or aging, whereupon they become Notch1-dependent and fail to fully reinstate neurogenesis. Our results suggest that Notch1 is a key component of the adult SVZ niche, promoting maintenance of aNSCs, and that this function is compensated in qNSCs. Therefore, we confirm the importance of Notch signaling for maintaining NSCs and neurogenesis in the adult SVZ and reveal that NSCs display a selective reliance on Notch1 that may be dictated by mitotic state.

Competitive evaporation in arrays of droplets

We consider the evaporation of periodic arrays of initially equal droplets in two-dimensional systems with open (absorbing) boundaries. Our study is based on the numerical solution of the Cahn-Hilliard equation. We show that due to cooperative effects the droplets which are further from the boundary may evaporate earlier than those in the boundary¿s vicinity. The time evolution of the overall amount of matter in the system is also studied.

Effect of External Noise Correlation in Optical Coherence Resonance

Coherence resonance occurring in semiconductor lasers with optical feedback is studied via the Lang-Kobayashi model with external nonwhite noise in the pumping current. The temporal correlation and the amplitude of the noise have a highly relevant influence in the system, leading to an optimal coherent response for suitable values of both the noise amplitude and correlation time. This phenomenon is quantitatively characterized by means of several statistical measures.

Fluctuations in Saffman-Taylor fingers with quenched disorder

We make an experimental characterization of the effect that static disorder has on the shape of a normal Saffman-Taylor finger. We find that static noise induces a small amplitude and long wavelength instability on the sides of the finger. Fluctuations on the finger sides have a dominant wavelength, indicating that the system acts as a selective amplifier of static noise. The dominant wavelength does not seem to be very sensitive to the intensity of static noise present in the system. On the other hand, at a given flow rate, rms fluctuations of the finger width, decrease with decreasing intensity of static noise. This might explain why the sides of the fingers are flat for typical Saffman-Taylor experiments. Comparison with previous numerical studies of the effect that temporal noise has on the Saffman-Taylor finger, leads to conclude that the effect of temporal noise and static noise are similar. The behavior of fluctuations of the finger width found in our experiments, is qualitatively similar to one recently reported, in the sense that, the magnitude of the width fluctuations decays as a power law of the capillary number, at low flow rates, and increases with capillary number for larger flow rates.