Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

In vivo analysis of interactions between trans-acting factors and their target genes

The investigations presented in this thesis use various in vivo techniques to understand how trans-acting factors control gene expression. The first part addresses the transcriptional regulation of muscle creatine kinase (MCK). MCK expression is activated during the course of development and is found only in differentiated muscle. Several in vivo footprints are observed at the enhancer of this gene, but all of these interactions are limited to cell types that express MCK. This is interesting because two of the footprints appear to represent muscle specific use of general transcription factors, while the other two correspond to sites that can bind the myogenic regulator, MyoD1, in vitro. MyoD1 and these general factors are present in myoblasts, but can bind to the enhancer only in myocytes. This suggests that either the factors themselves are post-translationally modified (phosphorylation or protein:protein interactions), or the accessibility of the enhancer to the factors is limited (changes in chromatin structure). The in vivo footprinting study of MCK was performed with a new ligation mediated, single-sided PCR (polymerase chain reaction) technique that I have developed.

The second half of the thesis concerns the regulation of mouse metallothionein (MT). Metallothioneins are a family of highly conserved housekeeping genes whose expression can be induced by heavy metals, steroids, and other stresses. By adapting a primer extension method of genomic sequencing to in vivo footprinting, I've observed both metal inducible and noninducible interactions at the promoter of MT-I. From these results I've been able to limit the possible mechanisms by which metal responsive trans-acting factors induce transcription. These interpretations correlate with a second line of experiments involving the stable titration of positive acting factors necessary for induction of MT. I've amplified the promoter of MT to 10^2-10^3 copies per cell by fusing the 5' and 3' ends of the MT gene to the coding region of DHFR and selecting cells for methotrexate resistance. In these cells, there is a metal-specific titration effect, and although it acts at the level of transcription, it appears to be independent of direct DNA binding factors.

A fisheries management plan for the River Tweed in the Scottish Borders

The River Tweed is, in rod-catch terms, the second most important river in Britain for Atlantic salmon, with an annual rod catch of ca.10,000 fish. This article gives an outline of the second edition of the Tweed Fisheries Management Plan, which is defined as "The co-ordination of data collection and analyses with fundamental research into relevant topics to devise management actions that will beneficially affect the future state of fish stocks". Much of the work set down for the present is concerned with setting up recording and measuring systems for fish catches and exploitation rates, and for the operation of monitoring sites for juvenile densities and adult trout and salmon spawning runs. While surveys show the present state of affairs, the collection of records and regular monitoring of sites will mean that in the future, the past will be better known through a longer series of records. Two case histories are described. The first is concerned with setting aims for managing the brown trout of the Tweed and defining the present state of the fishery. The second is an investigation into the spring salmon of the Tweed.

Strong atom-light interactions along nanostructures: Transition from free-space to nanophotonic interfaces

An exciting frontier in quantum information science is the integration of otherwise "simple'' quantum elements into complex quantum networks. The laboratory realization of even small quantum networks enables the exploration of physical systems that have not heretofore existed in the natural world. Within this context, there is active research to achieve nanoscale quantum optical circuits, for which atoms are trapped near nano-scopic dielectric structures and "wired'' together by photons propagating through the circuit elements. Single atoms and atomic ensembles endow quantum functionality for otherwise linear optical circuits and thereby enable the capability of building quantum networks component by component. Toward these goals, we have experimentally investigated three different systems, from conventional to rather exotic systems : free-space atomic ensembles, optical nano fibers, and photonics crystal waveguides. First, we demonstrate measurement-induced quadripartite entanglement among four quantum memories. Next, following the landmark realization of a nanofiber trap, we demonstrate the implementation of a state-insensitive, compensated nanofiber trap. Finally, we reach more exotic systems based on photonics crystal devices. Beyond conventional topologies of resonators and waveguides, new opportunities emerge from the powerful capabilities of dispersion and modal engineering in photonic crystal waveguides. We have implemented an integrated optical circuit with a photonics crystal waveguide capable of both trapping and interfacing atoms with guided photons, and have observed the collective effect, superradiance, mediated by the guided photons. These advances provide an important capability for engineered light-matter interactions, enabling explorations of novel quantum transport and quantum many-body phenomena.

Efectos del crecimiento de las economías occidentales en la segunda mitad del siglo XX sobre la economia española tras el Plan de estabilización.

Mediante el análisis de la balanza comercial española desde la época posterior a la Guerra Civil hasta el Plan de Estabilización y Liberalización, se intenta explicar la evolución de la economía española. La evolución del comercio exterior fue la pieza clave para el estancamiento de la economía en su conjunto debido a las políticas autárquicas del gobierno de Franco, que poco a poco se fueron flexibilizando hasta lograr una total apertura hacia el exterior. Esta apertura vino como consecuencia del Plan de Estabilización y supuso el saneamiento de todos los sectores de la economía (agricultura, industria, hacienda pública, sector crediticio…), así como un cambio en las pautas del consumo de la sociedad española. Se crean nuevas necesidades que es preciso cubrir, y frecuentemente hay que acudir al comercio internacional para abastecerlas. Tras las medidas liberalizadoras de 1959, nace un sector que se convierte en el engranaje de la economía española: el turismo, ya que gracias a él se obtienen divisas que permiten a la economía entrar en el libre juego de mercado en el terreno internacional. El turismo, junto con las remesas de los emigrantes a sus familias, son los elementos que tiran de una economía desabastecida en muchos casos de productos básicos, y que contaba con una industria arcaica y desestructurada.

Giza baliabideetara zuzendutako eitb enpresaren plan estrategikoa

EiTBren Giza Baliabideen plan estrategiko honek, erakundearen langileen ibilbide-orriak finkatzen ditu, 2013-2016 plan estrategiko orokorra oinarritzat hartuta. Hausnarketa estrategiko honetan, etorkizunari buruzko gogoeta egiten da, eta etorkizuna giza baliabideen esfortzuarekin eraikitzen da.

Kutxabank, plan de marketing

El objetivo principal de este trabajo es la realización de un plan de marketing para el Grupo Kutxabank aplicando la metodología y procedimientos necesarios para la elaboración de este documento empresarial.

Normandya Reparaciones SL.Plan de marketing

CASTELLANO: El proyecto realizado es un plan de marketing para la empresa Normandya reparaciones S.L.. El plan de marketing es la herramienta básica de gestión que debe utilizar toda empresa orientada al mercado que quiera ser competitiva. En su puesta en marcha quedarán fijadas las diferentes actuaciones que deben realizarse en el área del marketing, para alcanzar los objetivos establecidos tras haber realizado un análisis previo de la situación en la que se encuentra la empresa.

Plan de marketing

Plan de marketin de la microcerveceria Basqueland Brewing Project

Plan de Marketing: Aixerrota Consulting

Para cualquier empresa sea cual sea su tamaño, sector y mercado al que pertenece es recomendable que se plantee la elaboración de un plan de marketing para su propio negocio. Dada la creciente competitividad existente hoy en día en cualquier sector, el hecho de conocer qué es lo que rodea a la empresa se antoja muy importante para el futuro de ésta. Y no sólo los factores que le rodean, sino que también resulta vital conocer los internos. El hecho de que cada organización pueda conocer sus propios puntos fuertes puede resultar una tarea sencilla, pero llevar a cabo un análisis interno a través de una óptica objetiva, permitirá mejorar los puntos débiles de cualquier organización, además de saber potenciar de la mejor manera posible los rasgos o características más fuertes. El objetivo principal de este plan de marketing no es otro que lo que se acaba de resumir en la reflexión anterior, lograr que la empresa objeto de estudio llegue a ser lo más competitiva posible en su sector, gracias al planteamiento de unos objetivos, estrategias y plan de acciones previa realización del análisis externo e interno ya citado. En definitiva, este documento pretenderá aportar una visión comercial de la empresa, centrando su atención sobre todo en el mercado de actuación, competidores, clientes, marketing mix… unido a aportar valor indirectamente a otras variables estratégicas, de tal forma que contribuya al crecimiento competitivo y corporativo de la citada organización.

Light-matter Interactions in Semiconductors and Metals: From Nitride Optoelectronics to Quantum Plasmonics

This thesis puts forth a theory-directed approach coupled with spectroscopy aimed at the discovery and understanding of light-matter interactions in semiconductors and metals.

The first part of the thesis presents the discovery and development of Zn-IV nitride materials.The commercial prominence in the optoelectronics industry of tunable semiconductor alloy materials based on nitride semiconductor devices, specifically InGaN, motivates the search for earth-abundant alternatives for use in efficient, high-quality optoelectronic devices. II-IV-N2 compounds, which are closely related to the wurtzite-structured III-N semiconductors, have similar electronic and optical properties to InGaN namely direct band gaps, high quantum efficiencies and large optical absorption coefficients. The choice of different group II and group IV elements provides chemical diversity that can be exploited to tune the structural and electronic properties through the series of alloys. The first theoretical and experimental investigation of the ZnSnxGe1−xN2 series as a replacement for III-nitrides is discussed here.

The second half of the thesis shows ab−initio calculations for surface plasmons and plasmonic hot carrier dynamics. Surface plasmons, electromagnetic modes confined to the surface of a conductor-dielectric interface, have sparked renewed interest because of their quantum nature and their broad range of applications. The decay of surface plasmons is usually a detriment in the field of plasmonics, but the possibility to capture the energy normally lost to heat would open new opportunities in photon sensors, energy conversion devices and switching. A theoretical understanding of plasmon-driven hot carrier generation and relaxation dynamics in the ultrafast regime is presented here. Additionally calculations for plasmon-mediated upconversion as well as an energy-dependent transport model for these non-equilibrium carriers are shown.

Finally, this thesis gives an outlook on the potential of non-equilibrium phenomena in metals and semiconductors for future light-based technologies.

Plan de marketing de una PYME : Abereen Klinikak

Ante el reciente aumento del dinamismo y la globalización en los mercados, los análisis, estrategias y planes de acción están adquiriendo una mayor importancia para todas las empresas. En una economía cambiante, anticiparse a los cambios y estar preparados para nuevas realidades es un objetivo prioritario de las empresas. El plan de marketing es un medio más para conseguir anticiparse a las nuevas características de los mercados. Cada empresa tiene características diferentes, por lo que es necesario que el plan se adapte a la organización y no al revés. Por ejemplo, en las grandes empresas se ha mostrado como un arma comercial determinante, pero en las PYMES puede convertirse incluso en un sustitutivo de la planificación estratégica. En éste trabajo se realizará un plan de marketing en la práctica para “Abereen Klinikak”, una PYME del sector veterinario, aplicando el método analítico sintético. Se realizará un análisis previo del mercado y un diagnóstico de la situación de la empresa, se marcarán los objetivos, estrategias y acciones a seguir, y se implantará un plan de control y seguimiento de ellos. Todo ello desde una forma estructurada y sistematizada, con el fin de seguir la mejor estrategia comercial posible para la empresa. Abstract Due to the recent increase of dynamism and market globalization, analyzes, strategies and action plans are becoming increasingly important for all businesses. In a changing economy, to anticipate changes and be prepared for new realities is a priority for companies. The marketing plan is another way of being able to anticipate the new market characteristics. Each company has different characteristics, so it is necessary that the marketing plan suits the organization and not vice versa. For example, in large companies it has been shown as a key commercial weapon, but for SMEs it could even become a substitute for strategic planning. In this project a marketing plan for "Abereen Klinikak", an enterprise which belongs to the SME sector, will be implemented. The marketing plan will be put into practice using the synthetic analytical method. To do so, preliminary market analysis and an assessment of the company’s situation will be done. Furthermore, goals, strategies and action steps will be scored, and a control and monitoring plan will be implemented. The plan will be put into practice in a structured and systematic way, in order to follow the best business strategy for the company.

Plan de marketing de una PYME: Eder Rodamientos

El objetivo del presente documento es realizar un Plan de Marketing para una PYME. La empresa analizada es Rodamientos Eder SA que se dedica a la producción de rodamientos para otras empresas industriales. Fabrican rodamientos para muchos sectores diferentes, pero me voy a circunscribir en la línea de rodamientos para mamparas de baño.

Plan de marketing El Ganso

El principal objetivo de este trabajo es la adecuada realización de un plan de marketing para una empresa real, aplicando la metodología y los procedimientos pertinentes en la elaboración de este tipo de documentos.