Effects of forage type on diversity in bacterial pellets isolated from liquid and solid phases of the rumen content in sheep and goats.

Two sheep and two goats, fitted with a ruminal cannula, received two diets composed of 30% concentrate and 70% of either alfalfa hay (AL) or grass hay (GR) as forage in a two-period crossover design. Solid and liquid phases of the rumen were sampled from each animal immediately before feeding and 4 h post-feeding. Pellets containing solid associated bacteria (SAB) and liquid associated bacteria (LAB) were isolated from the corresponding ruminal phase and composited by time to obtain 2 pellets per animal (one SAB and one LAB) before DNA extraction. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal DNA was used to analyze bacterial diversity. A total of 78 and 77 bands were detected in the DGGE gel from sheep and goats samples, respectively. There were 18 bands only found in the pellets from sheep fed AL-fed sheep and 7 found exclusively in samples from sheep fed the GR diet. In goats, 21 bands were found only in animals fed the AL diet and 17 were found exclusively in GR-fed ones. In all animals, feeding AL diet tended (P < 0.10) to promote greater NB and SI in LAB and SAB pellets compared with the GR diet. The dendrogram generated by the cluster analysis showed that in both animal species all samples can be included in two major clusters. The four SAB pellets within each animal species clustered together and the four LAB pellets grouped in a different cluster. Moreover, SAB and LAB clusters contained two clear subclusters according to forage type. Results show that in all animals bacterial diversity was more markedly affected by the ruminal phase (solid vs. liquid) than by the type of forage in the diet.

Integral development of an Agricultural Training Center to ensure food security in Glory Special Needs Primary School. Kitgum, Uganda. DANIEL

El presente proyecto, titulado “Integral development of an Agricultural Training Center to ensure food security in Glory Special Needs Primary School. Kitgum, Uganda” fue llevado a cabo de Julio de 2011 a Febrero de 2012 en la escuela primaria Glory Special Needs, dedicada a la atención y educación de jóvenes discapacitados, en Kitgum, Uganda. El proyecto se realizó con la colaboración del grupo de cooperación de la ETSI Agrónomos, AgSystems, la Fundación AmigoSolidarios y la ONG local NUCBACD. Su objetivo principal fue el desarrollo y puesta en marcha de un centro de capacitación Agrícola para dotar de igualdad de oportunidades a los jóvenes con discapacidad de Kitgum, potenciando y favoreciendo su integración en la comunidad, y garantizar así la seguridad alimentaria de los 137 alumnos internos en la escuela Glory Special Needs. El trabajo realizado supuso una acción relevante en Kitgum, superando la visión de una economía familiar basada en las actividades agrícolas, para centrarse en la profesionalización de la Agricultura como motor económico de la región. Este documento presenta una descripción de las principales actividades que se desarrollaron con el fin de alcanzar el objetivo planteado, desde un punto de vista educativo, sostenible e inclusivo. Para conseguirlo, se plantearon tres lineas de trabajo: - Programa productivo. - Programa educativo. - Programa organizativo.

Effect of cage type on the behaviour pattern of rabbit does at different physiological stages

Interest in commercially farmed rabbit welfare has increased in recent years. As a result, new alternative housing systems have been developed, although they require evaluation in order to demonstrate their potential for improving welfare. The aim of this trial was to study the behavioural traits of rabbit does housed in 2 different types of cage (TC): conventional vs. alternative with an elevated platform, at different physiological stages (PS); lactation and gestation. Behavioural observations were carried out on 12 rabbit commercial does using continuous 24 h video recording. Independently of PS and TC, rabbit does spent most of their time on foot mats (on av. 57.7%). However, due to the use of platforms (on av. 23.0% of time), lactating does spent 36.6% less time on foot mats (P<0.001) and gestating does spent 27.0% less time on wire mesh (P<0.001) in alternative cages than in conventional cages. Alternative cages allowed for standing posture, but this behaviour was only observed in gestating does (on av. 4.6 times a day). Frequency of drinking was higher in conventional than in alternative cages (24.6 vs. 19.1 times a day; P<0.05). Gestating does housed in conventional cages reached the highest duration and frequency of interacting with neighbours (276 s/d and 4.6 times/d; P<0.05). The frequency of interacting with kits was lower in alternative than in conventional cages (2.4 vs. 8.6 times a day; P<0.01). Doe behaviour was influenced by the time of day, with less activity during the midday hours. During dark hours, rabbit does more frequently performed restless behaviour such as hyperactivity or nursing, matching the time at which rabbit does spent more time on the platform. The platform was frequently used by rabbit does, regardless of their physiological stage, and during late lactation phase, when mothers were not receptive to nursing, does housed in alternative cages used the platform as a mean to flee from kits trying to suckle. Use of the platform might lead to hygienic problems due to retained faeces on the platform and faeces and urine falling onto animals located in the lower part of the cage. The absence of stereotypies in rabbit does of this trial, suggested that animal welfare was not compromised by the type of housing (conventional or alternative cages).

A methodology to analyze, design and implement very fast and robust controls of Buck-type converters

La electrónica digital moderna presenta un desafío a los diseñadores de sistemas de potencia. El creciente alto rendimiento de microprocesadores, FPGAs y ASICs necesitan sistemas de alimentación que cumplan con requirimientos dinámicos y estáticos muy estrictos. Específicamente, estas alimentaciones son convertidores DC-DC de baja tensión y alta corriente que necesitan ser diseñados para tener un pequeño rizado de tensión y una pequeña desviación de tensión de salida bajo transitorios de carga de una alta pendiente. Además, dependiendo de la aplicación, se necesita cumplir con otros requerimientos tal y como proveer a la carga con ”Escalado dinámico de tensión”, donde el convertidor necesitar cambiar su tensión de salida tan rápidamente posible sin sobreoscilaciones, o ”Posicionado Adaptativo de la Tensión” donde la tensión de salida se reduce ligeramente cuanto más grande sea la potencia de salida. Por supuesto, desde el punto de vista de la industria, las figuras de mérito de estos convertidores son el coste, la eficiencia y el tamaño/peso. Idealmente, la industria necesita un convertidor que es más barato, más eficiente, más pequeño y que aún así cumpla con los requerimienos dinámicos de la aplicación. En este contexto, varios enfoques para mejorar la figuras de mérito de estos convertidores se han seguido por la industria y la academia tales como mejorar la topología del convertidor, mejorar la tecnología de semiconducores y mejorar el control. En efecto, el control es una parte fundamental en estas aplicaciones ya que un control muy rápido hace que sea más fácil que una determinada topología cumpla con los estrictos requerimientos dinámicos y, consecuentemente, le da al diseñador un margen de libertar más amplio para mejorar el coste, la eficiencia y/o el tamaño del sistema de potencia. En esta tesis, se investiga cómo diseñar e implementar controles muy rápidos para el convertidor tipo Buck. En esta tesis se demuestra que medir la tensión de salida es todo lo que se necesita para lograr una respuesta casi óptima y se propone una guía de diseño unificada para controles que sólo miden la tensión de salida Luego, para asegurar robustez en controles muy rápidos, se proponen un modelado y un análisis de estabilidad muy precisos de convertidores DC-DC que tienen en cuenta circuitería para sensado y elementos parásitos críticos. También, usando este modelado, se propone una algoritmo de optimización que tiene en cuenta las tolerancias de los componentes y sensados distorsionados. Us ando este algoritmo, se comparan controles muy rápidos del estado del arte y su capacidad para lograr una rápida respuesta dinámica se posiciona según el condensador de salida utilizado. Además, se propone una técnica para mejorar la respuesta dinámica de los controladores. Todas las propuestas se han corroborado por extensas simulaciones y prototipos experimentales. Con todo, esta tesis sirve como una metodología para ingenieros para diseñar e implementar controles rápidos y robustos de convertidores tipo Buck. ABSTRACT Modern digital electronics present a challenge to designers of power systems. The increasingly high-performance of microprocessors, FPGAs (Field Programmable Gate Array) and ASICs (Application-Specific Integrated Circuit) require power supplies to comply with very demanding static and dynamic requirements. Specifically, these power supplies are low-voltage/high-current DC-DC converters that need to be designed to exhibit low voltage ripple and low voltage deviation under high slew-rate load transients. Additionally, depending on the application, other requirements need to be met such as to provide to the load ”Dynamic Voltage Scaling” (DVS), where the converter needs to change the output voltage as fast as possible without underdamping, or ”Adaptive Voltage Positioning” (AVP) where the output voltage is slightly reduced the greater the output power. Of course, from the point of view of the industry, the figures of merit of these converters are the cost, efficiency and size/weight. Ideally, the industry needs a converter that is cheaper, more efficient, smaller and that can still meet the dynamic requirements of the application. In this context, several approaches to improve the figures of merit of these power supplies are followed in the industry and academia such as improving the topology of the converter, improving the semiconductor technology and improving the control. Indeed, the control is a fundamental part in these applications as a very fast control makes it easier for the topology to comply with the strict dynamic requirements and, consequently, gives the designer a larger margin of freedom to improve the cost, efficiency and/or size of the power supply. In this thesis, how to design and implement very fast controls for the Buck converter is investigated. This thesis proves that sensing the output voltage is all that is needed to achieve an almost time-optimal response and a unified design guideline for controls that only sense the output voltage is proposed. Then, in order to assure robustness in very fast controls, a very accurate modeling and stability analysis of DC-DC converters is proposed that takes into account sensing networks and critical parasitic elements. Also, using this modeling approach, an optimization algorithm that takes into account tolerances of components and distorted measurements is proposed. With the use of the algorithm, very fast analog controls of the state-of-the-art are compared and their capabilities to achieve a fast dynamic response are positioned de pending on the output capacitor. Additionally, a technique to improve the dynamic response of controllers is also proposed. All the proposals are corroborated by extensive simulations and experimental prototypes. Overall, this thesis serves as a methodology for engineers to design and implement fast and robust controls for Buck-type converters.

Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment

Novel anti-neoplastic agents such as gene targeting vectors and encapsulated carriers are quite large (approximately 100–300 nm in diameter). An understanding of the functional size and physiological regulation of transvascular pathways is necessary to optimize delivery of these agents. Here we analyze the functional limits of transvascular transport and its modulation by the microenvironment. One human and five murine tumors including mammary and colorectal carcinomas, hepatoma, glioma, and sarcoma were implanted in the dorsal skin-fold chamber or cranial window, and the pore cutoff size, a functional measure of transvascular gap size, was determined. The microenvironment was modulated: (i) spatially, by growing tumors in subcutaneous or cranial locations and (ii) temporally, by inducing vascular regression in hormone-dependent tumors. Tumors grown subcutaneously exhibited a characteristic pore cutoff size ranging from 200 nm to 1.2 μm. This pore cutoff size was reduced in tumors grown in the cranium or in regressing tumors after hormone withdrawal. Vessels induced in basic fibroblast growth factor-containing gels had a pore cutoff size of 200 nm. Albumin permeability was independent of pore cutoff size. These results have three major implications for the delivery of therapeutic agents: (i) delivery may be less efficient in cranial tumors than in subcutaneous tumors, (ii) delivery may be reduced during tumor regression induced by hormonal ablation, and (iii) permeability to a molecule is independent of pore cutoff size as long as the diameter of the molecule is much less than the pore diameter.

Unique regulatory properties of the type 2a Ca2+ channel β subunit caused by palmitoylation

β subunits of voltage-gated Ca2+ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for β2a, which differs in that it does not support prepulse facilitation of α1C Ca2+ channels, inhibits voltage-induced inactivation of neuronal α1E Ca2+ channels, and is more effective in blocking inhibition of α1E channels by G protein-coupled receptors. We show that the distinguishing properties of β2a, rather than interaction with a distinct site of α1, are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of β2a were lost in a mutant that could not be palmitoylated [β2a(Cys3,4Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three β2 splice variants differing only in their N-termini.

The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.

Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection

A genetic defect in a CC-chemokine receptor (CCR)-5, the principal coreceptor for the macrophage-tropic HIV type 1 (HIV-1), recently was found to naturally protect CCR-5-defective, but healthy, individuals from HIV-1 infection. In this study, we mimic the natural resistance of the CCR-5-defective individuals by designing a strategy to phenotypically knock out CCR-5. The inactivation of the CCR-5 coreceptor is accomplished by targeting a modified CC-chemokine to the endoplasmic reticulum to block the surface expression of newly synthesized CCR-5. The lymphocytes transduced to express the intracellular chemokine, termed “intrakine,” were found to be viable and resistant to macrophage-tropic HIV-1 infection. Thus, this gene-based intrakine strategy targeted at the conserved cellular receptor for the prevention of HIV-1 entry should have significant advantages over currently described approaches for HIV-1 therapy.

Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N

SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.

Quantitative insight into proliferation and differentiation of oligodendrocyte type 2 astrocyte progenitor cells in vitro

As part of our attempts at understanding fundamental principles that underlie the generation of nondividing terminally differentiated progeny from dividing precursor cells, we have developed approaches to a quantitative analysis of proliferation and differentiation of oligodendrocyte type 2 astrocyte (O-2A) progenitor cells at the clonal level. Owing to extensive previous studies of clonal differentiation in this lineage, O-2A progenitor cells represent an excellent system for such an analysis. Previous studies have resulted in two competing hypotheses; one of them suggests that progenitor cell differentiation is symmetric, the other hypothesis introduces an asymmetric process of differentiation. We propose a general model that incorporates both such extreme hypotheses as special cases. Our analysis of experimental data has shown, however, that neither of these extreme cases completely explains the observed kinetics of O-2A progenitor cell proliferation and oligodendrocyte generation in vitro. Instead, our results indicate that O-2A progenitor cells become competent for differentiation after they complete a certain number of critical mitotic cycles that represent a period of symmetric development. This number varies from clone to clone and may be thought of as a random variable; its probability distribution was estimated from experimental data. Those O-2A cells that have undergone the critical divisions then may differentiate into an oligodendrocyte in each of the subsequent mitotic cycles with a certain probability, thereby exhibiting the asymmetric type of differentiation.

Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+ channels

Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1B subunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release. Here we identify a voltage-dependent, but calcium-independent, enhancement of transmitter release that is elicited by trains of action potentials in the presence of a hyperosmotic extracellular concentration of sucrose. This enhancement of transmitter release requires interaction of SNARE proteins with the synprint site. Our results provide evidence for a voltage-dependent signal that is transmitted by protein–protein interactions from the N-type calcium channel to the SNARE proteins and enhances neurotransmitter release by altering SNARE protein function.

Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin I

N-type Ca2+ channels mediate Ca2+ influx, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1B subunits. Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission. Here we report a direct interaction of the synprint peptide from N-type Ca2+ channels with synaptotagmin I, an important Ca2+ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay. This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca2+. Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin. This interaction is Ca2+-dependent because of the Ca2+ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca2+ channels and synaptotagmin, and suggest that N-type Ca2+ channels may undergo a complex series of Ca2+-dependent interactions with multiple presynaptic proteins during neurotransmission.

Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli

Enterohemorrhagic Escherichia coli O157:H7 and enteropathogenic E. coli cause a characteristic histopathology in intestinal cells known as attaching and effacing. The attaching and effacing lesion is encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells. Using lacZ reporter gene fusions, we show that expression of the LEE operons encoding the type III secretion system, translocated intimin receptor, and intimin is regulated by quorum sensing in both enterohemorrhagic E. coli and enteropathogenic E. coli. The luxS gene recently shown to be responsible for production of autoinducer in the Vibrio harveyi and E. coli quorum-sensing systems is responsible for regulation of the LEE operons, as shown by the mutation and complementation of the luxS gene. Regulation of intestinal colonization factors by quorum sensing could play an important role in the pathogenesis of disease caused by these organisms. These results suggest that intestinal colonization by E. coli O157:H7, which has an unusually low infectious dose, could be induced by quorum sensing of signals produced by nonpathogenic E. coli of the normal intestinal flora.

On representations of finite type

Representations of the (infinite) canonical anticommutation relations and the associated operator algebra, the fermion algebra, are studied. A “coupling constant” (in (0,1]) is defined for primary states of “finite type” of that algebra. Primary, faithful states of finite type with arbitrary coupling are constructed and classified. Their physical significance for quantum thermodynamical systems at high temperatures is discussed. The scope of this study is broadened to include a large class of operator algebras sharing some of the structural properties of the fermion algebra.

Thermodynamic stability of wild-type and mutant p53 core domain

Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn2+ ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal = 4.18 kJ)/mol at 25°C and 9.8 kcal/mol at 10°C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal/mol, respectively. Under certain denaturing conditions, the wild-type domain forms an aggregate that is relatively highly fluorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this fluorescence under milder denaturation conditions.