Ether Bridge Formation and Chemical Diversification in Loline Alkaloid Biosynthesis

Loline alkaloids, found in many grass-Epichloë symbiota, are toxic or feeding deterrent to invertebrates. The loline alkaloids all share a saturated pyrrolizidine ring with a 1-amine group and an ether bridge linking C2 and C7. The steps in biosynthesis of loline alkaloids are catalyzed by enzymes encoded by a gene cluster, designated LOL, in the Epichloë genome. This dissertation addresses the enzymatic, genetic and evolutionary basis for diversification of these alkaloids, focusing on ether bridge formation and the subsequent modifications of the 1-amine to form different loline alkaloids. Through gene complementation of a natural lolO mutant and comparison of LOL clusters in strains with different loline alkaloid profiles, I found that lolO, predicted to encode a 2-oxoglutarate-dependent nonheme iron (2OG/Fe) dioxygenase, is required in formation of the ether bridge. Through application of isotopically labeled compound to Epichloë uncinata culture, I established that exo-1-acetamidopyrrolizidine (AcAP) and N-acetylnorloline (NANL) are true pathway intermediates. Application of AcAP to yeast expressing lolO resulted in production of NANL, establishing that LolO is sufficient to catalyze this unusual oxygenation reaction. After ether formation, modifications on the 1-amino group give loline, N-methylloline (NML), N-formylloline (NFL) and N-acetylloline (NAL). A double knockout of lolN, predicted to encode an acetamidase, and lolM, predicted to encode a methyltransferase, produced only NANL. Complementation of the double knockout with wild-type lolN and lolM restored the loline alkaloid profile. These results indicate that LolN is involved in deacetylating NANL to produce norloline, which is then modified to form the other lolines. Crude protein extract of a yeast transformant expressing LolM converted norloline to loline and NML, and loline to NML, supporting the hypothesis that LolM functions as a methyltransferase in the loline-alkaloid biosynthesis pathway. The alkaloid NAL was observed in some but not all plants symbiotic with Epichloë siegelii, and when provided with exogenous loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylloline (NAL), indicating that a plant acetyltransferase converts loline to NAL. I further analyzed the basis for loline alkaloid diversity by comparing the LOL clusters in the Epichloë and Atkinsonella species with different loline alkaloid profiles, and found that LOL clusters changed position, orientation and gene content over their evolutionary history. Frequent, independent losses of some or all late pathway genes, lolO, lolN, lolM and lolP, resulted in diverse loline alkaloid profiles. In addition, phylogenetic analyses demonstrated transspecies polymorphism of the LOL clusters. Based on my findings, I established that in Epichloë and Atkinsonella species the ether bridge is formed on acetamidopyrrolizidine. My study of the loline alkaloid profile of Adenocarpus decorticans (Fabaceae) suggests that these plants probably use a similar strategy at least with respect to ether-bridge formation. Further diversification steps of loline alkaloids in grass-Clavicipitaceae symbiota are carried out by enzymes of both Epichloë species and the host plant. Finally, I present evidence that LOL clusters have evolved by balancing selection for chemical diversity

Vehicle re-routing strategies for congestion avoidance

Traffic congestion causes driver frustration and costs billions of dollars annually in lost time and fuel consumption. This dissertation introduces a cost-effective and easily deployable vehicular re-routing system that reduces the effects of traffic congestion. The system collects real-time traffic data from vehicles and road-side sensors, and computes proactive, individually tailored re-routing guidance, which is pushed to vehicles when signs of congestion are observed on their routes. Subsequently, this dissertation proposes and evaluates two classes of re-routing strategies designed to be incorporated into this system, namely, Single Shortest Path strategies and Multiple Shortest Paths Strategies. These strategies are firstly implemented in a centralized system, where a server receives traffic updates from cars, computes alternative routes, and pushes them as guidance to drivers. The extensive experimental results show that the proposed strategies are capable of reducing the travel time comparable to a state-of-the-art Dynamic Traffic Assignment (DTA) algorithm, while avoiding the issues that make DTA impractical, such as lack of scalability and robustness, and high computation time. Furthermore, the variety of proposed strategies allows the system to be tuned to different levels of trade-off between re-routing effectiveness and computational efficiency. Also, the proposed traffic guidance system is robust even if many drivers ignore the guidance, or if the system adoption rate is relatively low. The centralized system suffers from two intrinsic problems: the central server has to perform intensive computation and communication with the vehicles in real-time, which can make such solutions infeasible for large regions with many vehicles; and driver privacy is not protected since the drivers have to share their location as well as the origins and destinations of their trips with the server, which may prevent the adoption of such solutions. To address these problems, a hybrid vehicular re-routing system is presented in this dissertation. The system off-loads a large part of the re-routing computation at the vehicles, and thus, the re-routing process becomes practical in real-time. To make collaborative re-routing decisions, the vehicles exchange messages over vehicular ad hoc networks. The system is hybrid because it still uses a server to determine an accurate global view of the traffic. In addition, the user privacy is balanced with the re-routing effectiveness. The simulation results demonstrate that, compared with a centralized system, the proposed hybrid system increases the user privacy substantially, while the re-routing effectiveness is minimally impacted

Pascual Carrión: política agraria e ingeniería social

This article approaches the personal profile and the writings of Pascual Carrión. In the first place it studies the course of Iife of this agricultural engineer between his years as a student of engineering and his death in 1984, revealing the strong relationship between his professional career, his political undertakings and his publications. The classification of the latter according to chronological and thematic criteria is the second step of the article. Thirdly it deals with Carrión's texts on the Spanish agrarian structures, their reform, and with those devoted to the wine sector. The article finishes with a global evaluation of Carrión's works as a source and as a model to approach agrarian and, more generally, political economic problems for contemporary readers

La administración agraria en España, 1847-1907

En este trabajo se examina la génesis y el desarrollo de la Administración de Fomento en su vertiente agraria entre 1847 y 1907, con particular atención a sus niveles provinciales. Tras una presentación, cronológicamente ordenada, del despliegue de las instituciones públicas agrarias y los conflictos a que dio lugar, se acomete un balance de su composición y competencias.This paper analyses the creation and development of the agricultural aspects of public administration within the “Ministerio de Fomento”, between 1847 and 1907, with particular reference to its involvement at provincial level. After a chronological account of the implementation of agricultural public institutions, and the conflicts which that generated, an initial attempt is made to identify the composition, and the real functions, of these institutions

El Estado según los altos funcionarios de la Hacienda española, 1881-1936

Este artículo presenta los elementos comunes de las visiones del Estado entre los cuerpos especiales y las clases superiores del cuerpo general de Hacienda entre las décadas finales del siglo xix y la Guerra Civil. Se organiza en tres partes. Las dos primeras describen, respectivamente, los grandes rasgos del modelo administrativo que se impuso en Hacienda tras las reformas de Bravo Murillo y la evolución de este modelo a partir de 1881, gracias a la construcción de cuerpos especiales, llamados a convertirse en la referencia de la evolución del Ministerio. La tercera parte enumera los elementos centrales de las visiones de Estado que se desprenden de diversos discursos públicos de estos altos funcionarios hacendísticos. Se destacan en este último ámbito tres rasgos que se hallaban conectados con la evolución anterior de la propia burocracia hacendística: la ecuación del Estado con la Administración; la conversión del conocimiento técnico especializado en el fundamento mismo de la carrera funcionarial; y una visión organicista de la Administración (entendida como esencia del Estado) que remite el buen funcionamiento de esta al de sus partes componentesThis article presents the common elements of the visions of the State among the special corps and superior classes of the general corps of the Ministry of Finance between the final decades of the 19th century and the Civil War. It is organised in three parts. The two first parts describe the general traits of the administrative model that became hegemonic in the Ministry after the reforms of Bravo Murillo and the evolution of this model after 1881, thanks to the multiplication of special corps, which were to become the reference of the evolution of the Ministry. The third part sums up the central elements in the comprehension of the State, which can be derived from the different public discourses of the civil servants who worked for the Ministry of Finance. Three features, which were linked to the previous evolution of the tax administration, are underlined in the third section. Firstly, the understanding of the State as Administration. In the second place, the generalised assumption of technical and specialised knowledge as the very foundation of the bureaucratic career. And, finally, an organicist vision of the Administration (understood as the essence of the State) which referred its good working to the good working of its component part

El proceso de transformación de las industrias de cantos tallados como base para establecer un sistema clasificatorio

Los cantos tallados que sirvieron para este estudio se encuentran esparcidos, en grandes cantidades, en las costas de Galicia (España). La principal característica de la tecnología es que se constata una dinámica evolutiva de transformación en dos esferas diferentes: (1) Un proceso diacrónico corto, en el que el soporte se transforma en una fase concreta. (2) Un proceso diacrónico largo, en el que el soporte se transforma en dos etapas interrumpidas por un proceso natural de erosión. La transformación diacrónica corta es la base del sistema clasificatorio y es la que comporta las cadenas operatorias.Les galets amenagés qu 'ils servirons pour cette étude se trouvent respandus en grands cantités sur les côtes de Galice (Espagne). On constate une dinamique evolutive de deux façon diferente: (1) Un procès diachronique court, dans le quel le soport est transformé dans une phase concrète. (2) Un procès diachronique long, en ce cas le soport est transformé dans deux etapes anthropiques qu'ils sont interrumpues par un procèdus naturel d'erosion. La transformation diachronique courte c'est la base du système clasificateur et c'est la que comporte les chaînes opératoires