Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus

El virus de la tristeza de los cítricos (Citrus tristeza virus; CTV) es el agente causal de unas de las enfermedades virales de los árboles cítricos más devastadoras en el mundo. CTV está restringido al floema en su huésped cítrico natural, y ha desarrollado tres proteínas supresoras de silenciamiento que actúan a nivel intra-(p23 y p20) e intercelular (p20 y p25) para superar la fuerte defensa antiviral del huésped. La interferencia de RNA, una aproximación basada en el uso de dsRNA para desencadenar el silenciamiento de RNA, ha sido utilizada ampliamente para generar plantas transgénicas resistentes a virus. Considerando el importante papel de p23, p20 y p25 en la patogénesis de CTV, hemos transformado plantas de lima Mexicana con un vector intrón-horquilla que porta la secuencia completa en versión no traducible de los genes p25, p20, p23 y el extremo 3¿-UTR de la cepa T36 de CTV, para intentar silenciar su expresión en células infectadas. Se ha observado resistencia completa a la infección viral en tres líneas transgénicas, manteniéndose todas sus propagaciones asintomáticas y libres de virus tras ser inoculadas mediante injerto con CTV-T36, tanto en el portainjertos no transgénico como directamente sobre la variedad transgénica. La acumulación de siRNA derivados del transgén fue necesaria pero no suficiente para lograr resistencia frente a CTV en las plantas. Al inocular propagaciones de las líneas transgénicas inmunes con una cepa de CTV divergente, la resistencia fue parcialmente superada, destacando la importancia de la identidad de secuencia en el mecanismo subyacente a la interferencia de RNA. Este trabajo es el primero en que se consigue resistencia completa a CTV en un huésped cítrico muy sensible, actuando simultáneamente sobre los tres supresores virales de silenciamiento mediante interferencia de RNA. La proteína p23 codificada por el virus es además un importante factor de patogenicidad. La expresión ectópica de p23 en plantas de cítricos induce aberraciones fenológicas semejantes a síntomas de CTV. Para estudiar en más detalle el papel de p23 en la patogénesis de CTV, se ha sobre-expresado en lima Mexicana el gen p23 de CTV T36 y tres versiones truncadas del mismo bajo el control del promotor 35S del virus del mosaico de la coliflor (Cauliflower mosaic virus). Solo la versión truncada, que expresa los aminoácidos del 1 al 157 (p23-¿157) indujo síntomas similares a los producidos por CTV, aunque más suaves que los inducidos por la expresión de la proteína p23 entera (209 aminoácidos), permitiendo delimitar la región responsable de la patogénesis de p23 en cítricos a un fragmento de 157 aminoácidos que incluye el dedo de zinc y los motivos básicos flanqueantes de la proteína. La actividad de p23 como supresor de silenciamiento de RNA en N. benthamiana se perdía en todos los mutantes de p23 probados, lo cual indica que la supresión de silenciamiento implica a la mayoría de las regiones de la proteína. Para profundizar más en el papel de p23 en la patogénesis, en un siguiente paso hemos restringido la expresión de transgenes derivados de p23 a células asociadas al floema de lima Mexicana mediante el uso del promotor especifico de floema del virus del moteado amarillo de la comelina (Commelina yellow mottle virus, CoYMV). Se transformó lima Mexicana con construcciones que portaban el gen p23 completo, ya sea de la cepa agresiva de CTV T36 o de la suave T317, o con un fragmento que comprende el dedo de zinc y los motivos básicos flanqueantes de la primera, todas ellas bajo el control bien del promotor de CoYMV o bien del promotor constitutivo 35S. La expresión de estas construcciones en el floema dio lugar a aberraciones semejantes a los síntomas específicos de CTV, pero no a los síntomas inespecíficos observados cuando se expresaba p23 de forma constitutiva. Por otra parte, la apariencia e intensidad de las aberraciones fenotípicas más notorias similares a síntomas inducidos por CTV generadas por la expresión específica en floema del gen p23 se relacionó positivamente con la agresividad de la cepa origen utilizada. Además, la expresión en tejidos floemáticos del fragmento de p23 que comprende el dominio de dedo de zinc y los motivos básicos flanqueantes fue suficiente para inducir síntomas semejantes a los producidos por la infección con CTV, confirmando así que la región N-terminal delimitada por los aminoácidos 1 y 157 podría determinar, al menos en parte, la patogénesis de CTV en lima Mexicana.Citrus tristeza virus (CTV) is the causal agent of one of the most devastating viral diseases of citrus trees in the world. CTV is phloem-restricted in natural citrus hosts, and has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and inter-cellular level (p20 and p25) to overcome strong host antiviral defense in citrus. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has been widely used for generating transgenic plants resistant against viruses. Considering the important role of p23, p20 and p25 in CTV pathogenesis, we have transformed Mexican lime plants with an intron-hairpin vector carrying full untranslatable versions of genes p25, p20, p23 and the 3¿-UTR from the CTV strain T36, to attempt silencing their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36, either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Challenging immune transformants with a divergent CTV strain resulted in partial breakage of the resistance, stressing the importance of sequence identity in the underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi. The p23 protein encoded by the virus is additionally an important pathogenicity factor. Ectopic expression of p23 in transgenic citrus plants induces developmental aberrations resembling CTV symptoms. To explore in more detail the role of p23 in CTV pathogenesis, the p23 gene from CTV T36 and three truncated versions thereof under the control of the Cauliflower mosaic virus 35S promoter were used to transform Mexican lime. Only the truncated version expressing amino acids 1 to 157 (p23¿158-209) elicited CTV-like symptoms, similar to, albeit milder than, those incited by expressing the whole p23 protein (209 amino acids), thus delimiting the region responsible for p23 pathogenesis in citrus to a 157 amino acid fragment including the Zn finger and flanking basic motifs of the protein. RNA silencing suppressor activity of p23 in N. benthamiana was abolished by all mutants tested, indicating that silencing suppression involves most p23 regions. To better define the role of p23 in CTV pathogenesis, we next restricted the expression of p23-derived transgenes to phloem-associated cells in Mexican lime plants by means of using the phloem-specific promoter from Commelina yellow mottle virus (CoYMV). Constructions carrying the complete gene p23 from either the severe T36 or the mild T317 CTV strains, or a fragment comprising the zinc-finger and flanking basic motifs from the former, either under the control of the CoYMV promoter or the constitutive 35S promoter were used for genetic transformation of Mexican lime. Expression of these constructs in the phloem incited aberrations resembling CTV-specific symptoms, but not the unspecific symptoms observed when p23 was constitutively expressed. Moreover, appearance and intensity of the most notorious CTV-like phenotypic aberrations induced by the phloem-specific expression of the p23 gene were positively related with the aggressiveness of the source CTV strain used. Additionally, expression in phloem-tissues of the p23 fragment comprising the zinc-finger domain and flanking basic motifs was sufficient to induce CTV-like symptoms, corroborating that the N-terminal region (delimited by amino acids 1 and 157) determines, at least in part, CTV pathogenesis in Mexican lime.Soler Calvo, N. (2013). Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31631TESI

Analysis of emerging barriers for e-learning models : an empirical study

The diffusion of the e-learning model during the last decade has contributed to solve many problems concerned with education at the workplace. Technological progress has significantly contributed to eliminating most limitations of the traditional education model, through improved access, diffusion of information, and the adaptation to individual needs. However, e-learning is an instrument, which does not ensure the automatic achievement of intended goals. Some studies have demonstrated that the mere conversion of course materials into an e-learning course may reduce motivation and thus, the level of effective learning. In addition, appropriate design of course materials for e-learning programs might not be sufficient to achieve optimal results. In order to maximize the benefits of implementing an e-learning program, it is if fact necessary to take into account organizational issues, such as the existence of incentives, the distribution of time for learning at the workplace, or the management system being used, for example. This paper analyzes some organizational features, which are linked to learning in organizations and might help to better explain success of an e-learning program. In particular, we attempt to assess the impact of both incentives and the assignment of a specific period of time for education to personal satisfaction and the level of learning. To this end, we proposed a specific survey to users of e-learning courses at a consulting company, which includes questions about satisfaction, self-assessed learning and monetary or long-term (career) incentives, among others. The impact of different organizational characteristics on satisfaction and learning has been then estimated by a bivariate ordered probit model, which allows for considering both the nature of dependent variables and the possible correlation between equations. The analysis sheds light on key practical organizational features, which should be taken into account in order to improving results of e-learning programs The main goal of this analysis has been to provide evidences to support policies aimed to increase effectiveness of e-learning models. We have based on the e-learning experience of a consulting company, evaluating the effectiveness of this new model in terms of two variables: learning perceived and level of satisfaction of participants. In order to analyze the determinants of both learning and satisfaction, we collected data from participants of e-learning programs. We found out that self-assessed satisfaction and perceived learning are likely to be affected by the same set of variables. On considering these relationships, and the nature of available data, both a simultaneous bivariate ordered probit model and a simultaneous bivariate probit model have been used in order to assess the impact of different factors on both satisfaction and perceived learning.peer-reviewe

Analysis of the growth of the e-learning industry through sustainable business model archetypes: A case study.

[Abstract]: Information and communication technologies applied to education through e-learning innovative solutions emerge as a relevant driver of change in the publishing industry, involving not only the supply of new educational resources, but also a reduction of paper consumption used in the traditional book edition. The dual goal of this paper is, firstly, to identify sustainable business model archetypes that illustrate the behavior and potential growth of firms in the e-learning industry and, secondly, to provide evidence of the existence of these archetypes through the study of strategic actions and managerial perceptions in a case study of rapid internationalization in this industry. The analysis follows a two-stage method. First, the main behaviors of e-learning firms are identified and grouped in sustainable business archetypes and the research proposals defined. Then these proposals are applied to a case study of the e-learning industry. This analysis furnishes evidence to conclude that the alliance of software firms with publishers becomes a driver that allows e-learning firms to overcome some of the limits of their growth model and maintain sustainable business models

From the Triple Helix model to the Global Open Innovation model a case study based on international cooperation for innovation in Dominican Republic

[Abstract]: This study presents a case study research that sets out the process of designing the Dominican Republic’s RDI strategy during the period 2001-2007 and the role played by international cooperation in that process. We discuss the Triple Helix model as framework, and the use of a new approach that can be transferred to other countries. The results have validated some of the model’s assumptions, but they have also confirmed the existence of certain explanatory limitations in it. In order to rectify them, a new model –the Global Open Innovation model– has been proposed as alternative approach to the innovation transfer

Internationalization as process of value distribution through innovation: Polyhedral diagnosis of a ‘born global’ firm

[Abstract]: Purpose: Technology applied to learning is blurring the traditional outlines of the relations between publishing and technology industries, moving the internationalization strategy away from a gradual perspective towards accelerated internationalization. This paper provides a conceptual model of polyhedral diagnosis of market entry strategy (PODMES model) for ‘born global’ firms involved in e-learning industries. Design/methodology/approach: The use of the case study methodology allowed apply PODMES model to a ‘born global’ firm of the e-learning industry. Findings: Results confirm that `born global´ firms in the e-learning industry can adopt behaviors contrary to those expected in the literature. These behaviors imply new entry patterns by creating a new strategic partner-supplier-client-competitor relations that extends the traditional analysis of the internal value chain to a process of value distribution through product and technology alliances. Originality/value: This research provides two main contributions: First, an advance upon previous research into ‘born global’ firms, through deep analysis of a case study that offers new findings regarding the phenomenon studied. It permits to align this research with previous analysis, following a strict case study methodology approach. Second, this study offers the application of a new methodology (PODMES model) that integrates the most relevant theories of internationalization (Factorial Dodecagon) within the five contingencies of entry market and the design of a strategic profile of internationalizatio

Synthesis of novel aminomethylphosphine complexes

A new series of aminomethylphosphine ligands incorporating a PCN backbone and a pendant amine were synthesised using a phosphorus Mannich condensation reaction. Their coordination capabilities were investigated with late transition metal centres. Following a procedure well established within our research group, several phosphines were obtained using {P(CH2OH)4}Cl (THPC) in two steps. Firstly, THPC was reacted with various ortho and para anilines to give phosphonium salts P{CH2N(H)R}4Cl (R = C6H5, o–MeC6H4, o– i PrC6H4, o–t BuC6H4, o–FC6H4, o–CF3C6H4, o–{C(Me)=CH2}C6H4, p–MeC6H4, p–i PrC6H4, p–FC6H4 and p–EtC6H4). Secondly, these new salts were reacted with Et3N to obtain the cyclic RN(H)CH2P{(CH2)3(NR)2} phosphines (R = para–substituted anilines) or with KOt Bu to obtain acyclic P{CH2N(H)R}3 phosphines (R = ortho-substituted anilines) and cyclic P{CH2N(R)CH2}2P diphosphines (R = ortho–substituted anilines). Double condensation was observed in the 31P{1 H} NMR of cyclic phosphines RN(H)CH2P{(CH2)3(NR)2} to form {(CH2)3(NR)2}PCH2N(R)CH2P{(CH2)3(NR)2} diphosphines. Some cyclic phosphines were investigated under a wide range of conditions with Ph2PCH2OH giving asymmetric Ph2PCH2N(R)CH2P{(CH2)3(NR)2} diphosphines along with symmetric diphosphines counterparts and other phosphorus coproducts according to 31P{1 H} NMR and MS. A novel bicyclic OP{CH2N(R)CH2}3PO diphosphine (R = p–MeC6H4) was obtained as crystalline solid from the filtrate of the reaction between RN(H)CH2P{(CH2)3(NR)2} and Ph2PCH2OH and it was characterised by X–ray spectroscopy. Attempts to synthesise this diphosphine from P(CH2OH)3 and p–MeC6H4 were unsuccessful. The family of aminomethylphosphine ligands was extended by treating Ph2PCH2OH or CgPCH2OH (Cg = 1,3,5,7–tetramethyl–2,4,8–trioxa–6–phosphaadamantane) with various primary amines to afford R(H)NCH2PR’2 (R’ = Cg or Ph). Symmetric Ph2PCH2N(R)CH2Ph2 diphosphines were observed in the 31P{1 H} NMR spectra indicating that phosphines with Ph2P– moiety were more susceptible to undertake a second condensation. Novel asymmetric o–{Ph2PCH2C(H)(CH3)}C6H4N(H)CH2PPh2 diphosphine was synthesised by treatment of o-{C(Me)=CH2}C6H4NH2 with Ph2PH to afford o– {Ph2PCH2C(H)(CH3)}C6H4NH2 followed by condensation of the amino group with Ph2PCH2OH. Analogous o–{CgPCH2C(H)(CH3)}C6H4NH2 phosphine was also obtained. The coordination capabilities of selected compounds was studied with late transition metal precursor such as Pt(II), Pd(II) and Ru(II). Monophosphine compounds acted as Pmonodentate ligands to form square planar cis and trans MCl2L2 (M = Pt and Pd) complexes. However, whereas cyclic ligands RN(H)CH2P{(CH2)3(NR)2} afforded cisMCl2L2, acyclic ligands P{CH2N(H)R}3 afforded trans-MCl2L2 along with cis-PtCl2L2 in some cases. Those phosphines which conducted double condensation adopted a P,Pchelate mode to form a 6–membered M–P–C–N–C–P to afford cis-MCl2L (M = Pt and Pd). More interesting is the rarely observed P,P-bridging mode exhibited by P{CH2N(R)CH2}2P ligands to form homobimetallic cis–(PtCl2L)2 complexes and {RhCl2(η5 –Cp*)}2L complexes. These metal compounds were obtained and characterised by in situ NMR however their structures were further supported by simulated 31P{1 H} NMR and X–ray studies. Asymmetric Ph2PCH2N(R)CH2P{(CH2)3(NR)2} diphosphines coordinated to Pt(II) in a P,P–chelate fashion affording a mixture of cis and trans bis(chelate) (PtL2)Cl2. Complex in trans geometry was isolated and the structure was further supported by X–ray crystallography. The coordination capabilities of the ligands were investigated with Ru(II) metal centre revealing “piano–stool” structure of the type RuCl2(η6 –p–cymene)L where the monophosphines adopted a P-monodentate mode and {RuCl2(η6 –p–cymene)}2L where the diphosphines adopted a P,P-bridging mode. Preliminary studies with selected Ru complexes were carried out to investigate their potential catalytic activity to trap CO2 by its insertion into the Ru–Cl bond which suggested that the chloride needs to be substituted by a hydride prior the CO2 insertion. Compounds were characterised by spectroscopic techniques and the structure of some phosphines was further supported by X–ray crystallography

Analysis of the interface systems as mediating agents in university/industry relations : proposal of the “relationship promoter” as a strategic role in the R&D transference

A regional, national or international innovation system’s capacity to be innovative requires a commitment to collaborate between universities and industry. To facilitate this commitment, public institutions have championed the creation of interface organisations to be responsible for developing a network of university/industry partnerships. What has scarcely been acknowledged, however, is the role that professionals within these organisations should play to ensure that relationships are as productive as possible. To address this shortfall, an analysis has been proposed, which, based on the findings of a panel of international experts in innovation transfer, identifies the professional profile of the “relationship promoter” as a catalyst for university/industry relations at transregional and transnational level, and examines the strengths and weaknesses of university/industry exchange, from this approach. Finally, and based on the above analysis, the design of a strategic framework has been proposed to facilitate the commitment to collaborate between the various actors in the innovation system. This examination has enabled us to consider the value of the “relationship promoter” in the strategic analysis of the innovation system, and their role in facilitating the commitment to collaborate between actors within the system, from a dynamic perspective.peer-reviewe

Estudio térmico del edificio de la EPSEB (UPC) mediante certificación con Calener y estudio de eficiencia energética mediante Lider

La intención de este proyecto es realizar un estudio energético, para ello se ha elegido el edificio de la EPSEB. El edificio que pertenece a la UPC está situado en C/ Doctor Marañón de Barcelona. Fue construido a principio de los años 60 y tiene una planta semisótano, planta baja y cuatro plantas piso. En primer lugar, se ha realizado un trabajo experimental consistente en la determinación de las transmisiones de flujo de calor y las temperaturas superficiales en las diferentes fachadas, así como los registros de temperatura en las diferentes estancias del edificio. Estas mediciones nos han permitido conocer la variabilidad de flujo según influyen los factores ambientales. Gracias a los diversos ensayos realizados en las fachadas del edificio hemos podido ver la gran influencia que tienen tanto la radiación solar como la velocidad del viento. Cuando la radiación solar incide directamente sobre el vidrio hace que el flujo varíe, pudiendo incluso llegar a haber aportación de calor del exterior al interior. Se analizó también la diferencia de flujo entre una ventana con la persiana subida y otra con la persiana bajada, y se corroboró la importancia de hacer un buen uso de las protecciones solares para intentar reducir el gasto energético ya sea en calefactar o refrigerar las estancias. Durante el invierno se debería aprovechar la radiación solar dejando las persianas subidas durante las horas que incida el sol y después bajarlas para conservar el calor. En verano se recomienda tenerlas bajas para que no incida la radiación solar y aumente desproporcionadamente la temperatura interior de las aulas En cuanto a la velocidad del viento se han realizado ensayos que nos llevan a pensar que en las fachadas a barlovento la resistividad exterior del aire coincide con los datos que se indican en el CTE (Rse=0,04 m2K/W), mientras que para fachadas a sotavento la resistividad exterior es mayor, alcanzando los 0,08 m2K/W. El Código Técnico de la Edificación siempre se queda con el valor más desfavorable. Se debería tener en cuenta este dato a la hora de hacer medidas in situ con el fin de poder conocer los valores reales. Después se ha realizado un estudio del estado energético de la piel del edificio de la EPSEB (UPC). Para realizar este estudio se han utilizado una serie de programas informáticos especializados: LIDER y CALENER. Los registros de temperatura interior permiten evaluar el grado de confort en el interior del edificio y correlacionarlo con el coste energético. En general la temperatura interior media en invierno no alcanza los 20ºC y puesto que el coste energético es inferior al que demanda el LIDER podríamos pensar que en el edificio se pasa frío. Estos programas se utilizan actualmente para hacer la calificación energética de los edificios y para su adecuación al Código Técnico de la Edificación. Por último, aprovechando que durante el desarrollo de este proyecto se realizó el cambio de carpinterías del edificio, se aprovecho para comprobar la mejora que generaba en la certificación energética. Hemos visto que en cuanto a la perdida de flujo con las nuevas carpinterías hay una mejora considerable, ya que se pierden 20KWh/m2 menos. Aunque ésta es una buena intervención puede ser que no sea suficiente para mejorar el rendimiento energético del edificio. Según los datos extraídos del CALENER la instalación que mas CO2 emite es la iluminación, más del 60% del total. Por tanto, se debería plantear alguna mejora en dicha instalación como podría ser la sustitución de las luminarias por lámparas fluorescentes compactas de la clase A o por otros sistemas eficientes que permitan optimizar la instalación

NETEX and the pinnacle of E-LEARNING: in search of the American dream

It is a decisional business case study. The decision is about the process of internationalization of NETEX, an organization that develops e-learning solutions.The case shows the steps taken by the team led by Carlos Ezquerro, general director of NETEX, from the creation of the company in 1997 until the summer of 2014. NETEX's growth model is based on internal growth, at the pace of other investors and financial support, which was limited by the economic crisis of 2008.The case is described from the point of view of Carlos Ezquerro, founder and director of NETEX, along with some other comments from Ricardo Álvarez, director of internationalization of the firm.The learning objective for the students is to analyse the decisions to be taken by considering the history of a company that is very clear about its objectives and its positioning strategy, but that has several alternatives in its internationalization model