Molecular simulations and modeling of pharmaceutically relevant RNA-processing enzymes

The two-metal-ion architecture is a structural feature found in a variety of RNA processing metalloenzymes or ribozymes (RNA-based enzymes), which control the biogenesis and the metabolism of vital RNAs, including non-coding RNAs (ncRNAs). Notably, such ncRNAs are emerging as key players for the regulation of cellular homeostasis, and their altered expression has been often linked to the development of severe human pathologies, from cancer to mental disorders. Accordingly, understanding the biological processing of ncRNAs is foundational for the development of novel therapeutic strategies and tools. Here, we use state-of the-art molecular simulations, complemented with X-ray crystallography and biochemical experiments, to characterize the RNA processing cycle as catalyzed by two two-metal-ion enzymes: the group II intron ribozymes and the RNase H1. We show that multiple and diverse cations are strategically recruited at and timely released from the enzymes’ active site during catalysis. Such a controlled cations’ trafficking leads to the recursive formation and disruption of an extended two-metal ion architecture that is functional for RNA-hydrolysis – from substrate recruitment to product release. Importantly, we found that these cations’ binding sites are conserved among other RNA-processing machineries, including the human spliceosome and CRISPR-Cas systems, suggesting that an evolutionarily-converged catalytic strategy is adopted by these enzymes to process RNA molecules. Thus, our findings corroborate and sensibly extend the current knowledge of two-metal-ion enzymes, and support the design of novel drugs targeting RNA-processing metalloenzymes or ribozymes as well as the rational engineering of novel programmable gene-therapy tools.

CRISPR/Cas9-Mediated Gene Editing of ARG1 in Cell Lines and Primary Cells

Arginase 1 deficiency, a urea cycle disorder resulting from an inability of the body to convert arginine into urea, results in hyperargininemia and sporadic episodes of hyperammonemia. Arginase 1 deficiency can lead to a range of developmental disorders and progressive spastic diplegia in children, and current therapeutic options are limited. Clustered regularly interspaced short palindromic repeat (CRISPR) /CRISPR associated protein (Cas) 9 gene editing systems serve as a novel means of treating genetic disorders such as Arginase 1 (ARG1) deficiency, and must be thoroughly examined to determine their curative capabilities. In these experiments numerous guide RNAs and CRISPR/Cas9 systems targeting the ARG1 gene were designed and observed by heteroduplex assay for their targeting capabilities and cleavage efficiencies in multiple cell lines. The CRISPR/Cas9 system utilized in these experiments, along with a panel of guide RNAs targeting various locations in the arginase 1 gene, successfully produced targeted cleavage in HEK293, MCF7, A549, K562, HeLa, and HepG2 cells; however, targeted cleavage in human dermal fibroblasts, blood outgrowth endothelial cells, and induced pluripotent stem cells was not observed. Additionally, a CRISPR/Cas system involving partially inactivated Cas9 was capable of producing targeted DNA cleavage in intron 1 of ARG1, while a Cas protein termed Cpf1 was incapable of producing targeted cleavage. These results indicate a complex set of variables determining the CRISPR/Cas9 systems’ capabilities in the cell lines and primary cells tested. By examining epigenetic factors and alternative CRISPR/Cas9 gene targeting systems, the CRISPR/Cas9 system can be more thoroughly considered in its ability to act as a means towards editing the genome of arginase 1-deficient individuals.

The eukaryotic way to defend and edit genomes by sRNA-targeted DNA deletion

While there is currently burgeoning interest in the application of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) to genome editing, it is perhaps not widely appreciated that this is the second discovery of a small RNA (sRNA)-targeted DNA-deletion system. The first sRNA-targeted DNA-deletion system to be discovered, which we call IES/Ias (internal eliminated sequence/IES-associated genes) to contrast with CRISPR/Cas, is found in ciliates, and, like CRISPR/Cas, is thought to serve as a form of immune defense against invasive DNAs. The manner in which the ciliate IES/Ias system functions is distinct from that of the CRISPR/Cas system in archaea and bacteria, and arose independently through a synthesis of RNA interference-derived and DNA-specific molecular components. Despite the major differences between CRISPR/Cas and IES/Ias, both systems face similar conceptual challenges in targeting invasive DNAs. In this review, we focus on the discovery, effects, function, and evolutionary consequences of the IES/Ias system.

Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.

Towards modification of Medicago truncatula epigenome: genome editing with engineered nucleases

Periodic drought is the primary limitation of plant growth and crop yield. The rise of water demand caused by the increase in world population and climate change, leads to one of the biggest challenges of modern agriculture: to increase food and feed production. De novo DNA methylation is a process regulated by small interfering RNA (siRNAs), which play a role in plant response and adaptation to abiotic stress. In the particular case of water deficit, growing evidences suggest a link between the siRNA pathways and drought response in the model legume Medicago truncatula. As a first step to understand the role of DNA methylation under water stress, we have set up several bioinformatics and molecular methodologies allowing the design of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems and the assembly of TALENs (transcription activator-like effector nucleases), to target both dicer-like 3 (MtDCL3) and RNA-Dependent RNA polymerase (MtRDR2), enzymes of the RNA-directed DNA methylation pathway. TALENs efficiency was evaluated prior to plant transformation by a yeast-based assay using two different strategies to test TALENs activity: Polyacrylamide gel electrophoresis (PAGE) and Single strand conformation polymorphisms (SSCP). In this assay, yeast cells triple transformation emerged as good and rapid alternative to laborious yeast mating strategies. PAGE analysis might be a valuable tool to test TALENs efficacy in vivo if we could increase TALENs activity. SSCP-based approach proved to be ineffective due to the generation of several false positives. TALENs and CRISPR/Cas9 system constructed and designed in this work will in the future certainly enable the successful disruption of DCL3 and RDR2 genes and shed the light on the relationship between plant stress resistance and epigenetic regulation mediated by siRNAs in M.truncatula.

Imunidade celular: caracterização das proteínas celulares que inibem a infecção viral

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Integrated management systems as complex adaptive systems

It is a difficult task to avoid the “smart systems” topic when discussing smart prevention and, similarly, it is a difficult task to address smart systems without focusing their ability to learn. Following the same line of thought, in the current reality, it seems a Herculean task (or an irreparable omission) to approach the topic of certified occupational health and safety management systems (OHSMS) without discussing the integrated management systems (IMSs). The available data suggest that seldom are the OHSMS operating as the single management system (MS) in a company so, any statement concerning OHSMS should mainly be interpreted from an integrated perspective. A major distinction between generic systems can be drawn between those that learn, i.e., those systems that have “memory” and those that have not. These former systems are often depicted as adaptive since they take into account past events to deal with novel, similar and future events modifying their structure to enable success in its environment. Often, these systems, present a nonlinear behavior and a huge uncertainty related to the forecasting of some events. This paper seeks to portray, for the first time as we were able to find out, the IMSs as complex adaptive systems (CASs) by listing their properties and dissecting the features that enable them to evolve and self-organize in order to, holistically, fulfil the requirements from different stakeholders and thus thrive by assuring the successful sustainability of a company. Based on the revision of literature carried out, this is the first time that IMSs are pointed out as CASs which may develop fruitful synergies both for the MSs and for CASs communities. By performing a thorough revision of literature and based on some concepts embedded in the “DNA” of the subsystems implementation standards it is intended, specifically, to identify, determine and discuss the properties of a generic IMS that should be considered to classify it as a CAS.

Contribució de les varietats locals a la conservació de l'agrobiodiversitat: cas d'estudi en horts d'alta muntanya al Pallars Jussà, Pirineu català

Durant les últimes dècades, el paper dels horts familiars en la conservació de la agrobiodiversitat ha adquirit cada vegada més importància a nivell mundial. Les varietats locals que es cultiven en aquests horts tenen propietats intrínseques que les fan superiors a les comercials en molts aspectes, com han demostrat alguns dels estudis sobre aquests agroecosistemes duts a terme als Tròpics, i que posen en evidència l'actual sistema agrícola, alhora que representen una oportunitat per al desenvolupament sostenible de l´agricultura. En aquest treball d'investigació, dut a terme a la Vall Fosca (Pirineus Català d'Espanya) s'ha estudiat el paper les varietats locals dels horts familiars com a mecanisme de millora i manteniment d'aquests agroecosistemes, analitzant quins són els possibles factors sociodemogràfics i ecològics que condicionen el desenvolupament d'aquestes varietats als horts familiars.

Algebraic properties of isochronous centers of polynomial quadratic-like Hamiltonian systems

Projecte de recerca elaborat a partir d’una estada a la School of Mathematics and Statistics de la University of Plymouth, United Kingdom, entre abril juliol del 2007.Aquesta investigació és encara oberta i la memòria que presento constitueix un informe de la recerca que estem duent a terme actualment. En aquesta nota estudiem els centres isòcrons dels sistemes Hamiltonians analítics, parant especial atenció en el cas polinomial. Ens centrem en els anomenats quadratic-like Hamiltonian systems. Diverses propietats dels centres isòcrons d'aquest tipus de sistemes van ser donades a [A. Cima, F. Mañosas and J. Villadelprat, Isochronicity for several classes of Hamiltonian systems, J. Di®erential Equations 157 (1999) 373{413]. Aquell article estava centrat principalment en el cas en que A; B i C fossin funcions analítiques. El nostre objectiu amb l'estudi que estem duent a terme és investigar el cas en el que aquestes funcions són polinomis. En aquesta nota formulem una conjectura concreta sobre les propietats algebraiques que venen forçades per la isocronia del centre i provem alguns resultats parcials.

Anàlisi del metabolisme ambiental de nuclis turístics litorals: el cas de Menorca

L’objectiu del present estudi és l’anàlisi del metabolisme energètic associat a nuclis turístics litorals de l’illa de Menorca (Mediterrani occidental) i el grau d’autosuficiència a partir d’energies renovables. La caracterització dels nuclis i la definició del perfil del turista s’ha realitzat mitjançant SIG i informació de qualitat a partir d’enquestes. Els principals resultats mostren que els nuclis turístics de Menorca tenen unes emissions associades entre 213 i 318 kg de CO2 per estada. De mitjana, el recorregut del turista fins a la illa (mobilitat externa) és de 1334 km (representant el 80% de les emissions), mentre que la mobilitat interna durant l’estada és de 22 km. A diari, cada turista consumeix entre 8 i 26 kWh d’electricitat, consum que es podria satisfer en un 100% amb la instal·lació de sistemes fotovoltaics a les cobertes del nucli.

Llenguatges de consulta en l'àmbit de la Web Semàntica : Cas d'estudi SPARQL

El projecte es basa en estudiar i avaluar diferents sistemes gestors de bases de dades (SGBD) per desar informació dins del context de la Web Semàntica., tal com es veurà en el capítol 4. La Web Semàntica permet dotar de significat al contingut textual de la web, permetent que sigui interpretable per una màquina.

Llenguatges de consulta a l'àmbit de la Web Semàntica : Cas d'estudi SPARQL

El projecte es basa en estudiar i avaluar diferents sistemes gestors de bases de dades (SGBDs) per desar informació dins el context de la Web Semàntica. Els SGBDs hauran de tractar, emmagatzemar i gestionar la informació classificada segons uns criteris semàntics i interrelacionada amb conceptes afins, alhora que permetin la comunicació entre sistemes de manera transparent a l'usuari.

Portabilitat d'aplicacions de PC a PDA mitjançant un cas d'estudi : ScummVM

L’objectiu del treball és estudiar la portabilitat dels programes que han estat originalment dissenyats per a un PC a undispositiu mòbil, en concret a un PDA amb el sistema operatiu Palm OS. Per al nostre treball, hem escollit una aplicaciócom a cas d’estudi que ens permet donar solucions a les limitacions que presenta el dispositiu amb Palm OS. Aquestaaplicació s’anomena ScummVM, i es tracta d’una implementació de codi lliure del conegut motor de videojocs SCUMM, creat i utilitzat per LucasArts en les seves aventures gràfiques. Donarem possibles solucions a la problemàtica que genera el fet que la pantalla del dispositiu mòbil utilitzat tingui una resolució de 160x160 a l’hora de mostrar imatges i textos renderitzats per una resolució superior. També solucionarem el problema que representa el fet de no tenir ratolí, ni altres dispositius d’entrada i sortida tradicionals. La intenció és explicar també el procés que s’hauria de seguir per poder portar aplicacions de PC a Palm OS i les eines que s’utilitzarien. Tot això es farà sempre de la manera menys invasiva possible, és a dir, els canvis al codi font del’aplicació original seran els mínims per garantir el correcte funcionament del programa en la nova plataforma

Lire l'oral : pour une typologie linguistique des représentations écrites de l'oralité (le cas du français)

Cette thèse propose de passer en revue les modalités de la représentation écrite de l'oralité en français. La pratique littéraire constitue le matériau et l'horizon de la théorisation. La problématique - comment l'écrit représente-t-il l'oral ? - est d'abord située et reformulée dans le cadre de la linguistique de la parole (I). Les rapports entre oralité et scripturalité sont ensuite étudiés sous trois angles. L'angle biotechnologique compare la matérialité et l'affordance des signaux graphiques et des signaux acoustiques (II 1). L'examen sémiotique reconnaît dans le français écrit un système dit phonographique dont la fonction est de représenter l'expression des signes du français oral. Sont analysées alors les relations entre les systèmes de signes impliqués, la diversité des actualisations possibles du système phonographique (effets d'écoute), ainsi que diverses sémiotiques analogiques (II 2). On étudie ensuite le rôle de la prosodie dans la lecture. La position adoptée est la suivante : bien qu'elle soit facultative dans l'activité de lecture, la prosodie est spécialement sollicitée par des écrits qu'on peut caractériser linguistiquement. L'interprétation prosodique apporte à ces écrits un surcroît de signification en même temps qu'il produit un mode spécifique de représentation de l'oral appelé effet prosodique (II 3). L'angle sémantique est esquissé finalement : il conduit à dégager deux modalités de représentation supplémentaire. Pour la première, l'oral se situe sur le plan sémantico-référentiel de l'expression écrite (écrire à propos d'oral) ; pour la seconde, l'oral est un extérieur discursif modalisant le dire écrit : l'écrit est reconnu comme énoncé à la manière de l'oral (effet de style oral). - This PhD thesis attempts to review the modalities of orality in written representation. Literary writings act as the material for theorization. First of all, the thesis statement - how does writing represent oral - is situated and then, reformulated within the frame of linguistique de la parole (the linguistic field of speech) (I). The connections between orality and writing are then studied under three angles. The biotechnological angle compares the materiality and the affordance of graphic signs and acoustic signals (II 1). A semiotic examination acknowledges, in French, a phonographical system whose function is to represent the expression of French oral signs. Thus, the relationships between the systems of implicated signs, the diversity of possible actualisations of the phonographic system (voice effects), as well as various analogical semiotics are analysed (II 2). Furthermore, the role of prosody is studied within reading. The stand taken is the following : even though it is optional during a reading activity, prosody is especially sought-after by linguistically characterised writings. The prosodie interpretation brings to these writings a surge of signification while producing a specific mode of oral representation called the prosodie effects (II 3). The semantic angle is finally drawn : it leads to two additional modalities of representation. For the first part, speech is located on the semantic and referential plan of the written expression (writing about speech); as for the second part, spoken language is a discursive exteriority : writing is recognised as an oral-like utterance {oral-like effect).

Apprentissage automatique de la morphologie : le cas des structures racine-schème

RÉSUMÉ Cette thèse porte sur le développement de méthodes algorithmiques pour découvrir automatiquement la structure morphologique des mots d'un corpus. On considère en particulier le cas des langues s'approchant du type introflexionnel, comme l'arabe ou l'hébreu. La tradition linguistique décrit la morphologie de ces langues en termes d'unités discontinues : les racines consonantiques et les schèmes vocaliques. Ce genre de structure constitue un défi pour les systèmes actuels d'apprentissage automatique, qui opèrent généralement avec des unités continues. La stratégie adoptée ici consiste à traiter le problème comme une séquence de deux sous-problèmes. Le premier est d'ordre phonologique : il s'agit de diviser les symboles (phonèmes, lettres) du corpus en deux groupes correspondant autant que possible aux consonnes et voyelles phonétiques. Le second est de nature morphologique et repose sur les résultats du premier : il s'agit d'établir l'inventaire des racines et schèmes du corpus et de déterminer leurs règles de combinaison. On examine la portée et les limites d'une approche basée sur deux hypothèses : (i) la distinction entre consonnes et voyelles peut être inférée sur la base de leur tendance à alterner dans la chaîne parlée; (ii) les racines et les schèmes peuvent être identifiés respectivement aux séquences de consonnes et voyelles découvertes précédemment. L'algorithme proposé utilise une méthode purement distributionnelle pour partitionner les symboles du corpus. Puis il applique des principes analogiques pour identifier un ensemble de candidats sérieux au titre de racine ou de schème, et pour élargir progressivement cet ensemble. Cette extension est soumise à une procédure d'évaluation basée sur le principe de la longueur de description minimale, dans- l'esprit de LINGUISTICA (Goldsmith, 2001). L'algorithme est implémenté sous la forme d'un programme informatique nommé ARABICA, et évalué sur un corpus de noms arabes, du point de vue de sa capacité à décrire le système du pluriel. Cette étude montre que des structures linguistiques complexes peuvent être découvertes en ne faisant qu'un minimum d'hypothèses a priori sur les phénomènes considérés. Elle illustre la synergie possible entre des mécanismes d'apprentissage portant sur des niveaux de description linguistique distincts, et cherche à déterminer quand et pourquoi cette coopération échoue. Elle conclut que la tension entre l'universalité de la distinction consonnes-voyelles et la spécificité de la structuration racine-schème est cruciale pour expliquer les forces et les faiblesses d'une telle approche. ABSTRACT This dissertation is concerned with the development of algorithmic methods for the unsupervised learning of natural language morphology, using a symbolically transcribed wordlist. It focuses on the case of languages approaching the introflectional type, such as Arabic or Hebrew. The morphology of such languages is traditionally described in terms of discontinuous units: consonantal roots and vocalic patterns. Inferring this kind of structure is a challenging task for current unsupervised learning systems, which generally operate with continuous units. In this study, the problem of learning root-and-pattern morphology is divided into a phonological and a morphological subproblem. The phonological component of the analysis seeks to partition the symbols of a corpus (phonemes, letters) into two subsets that correspond well with the phonetic definition of consonants and vowels; building around this result, the morphological component attempts to establish the list of roots and patterns in the corpus, and to infer the rules that govern their combinations. We assess the extent to which this can be done on the basis of two hypotheses: (i) the distinction between consonants and vowels can be learned by observing their tendency to alternate in speech; (ii) roots and patterns can be identified as sequences of the previously discovered consonants and vowels respectively. The proposed algorithm uses a purely distributional method for partitioning symbols. Then it applies analogical principles to identify a preliminary set of reliable roots and patterns, and gradually enlarge it. This extension process is guided by an evaluation procedure based on the minimum description length principle, in line with the approach to morphological learning embodied in LINGUISTICA (Goldsmith, 2001). The algorithm is implemented as a computer program named ARABICA; it is evaluated with regard to its ability to account for the system of plural formation in a corpus of Arabic nouns. This thesis shows that complex linguistic structures can be discovered without recourse to a rich set of a priori hypotheses about the phenomena under consideration. It illustrates the possible synergy between learning mechanisms operating at distinct levels of linguistic description, and attempts to determine where and why such a cooperation fails. It concludes that the tension between the universality of the consonant-vowel distinction and the specificity of root-and-pattern structure is crucial for understanding the advantages and weaknesses of this approach.