Modeling Techniques for the High-Resolution Interpretation of Cryo-Electron Microscopy Reconstructions

Essential biological processes are governed by organized, dynamic interactions between multiple biomolecular systems. Complexes are thus formed to enable the biological function and get dissembled as the process is completed. Examples of such processes include the translation of the messenger RNA into protein by the ribosome, the folding of proteins by chaperonins or the entry of viruses in host cells. Understanding these fundamental processes by characterizing the molecular mechanisms that enable then, would allow the (better) design of therapies and drugs. Such molecular mechanisms may be revealed trough the structural elucidation of the biomolecular assemblies at the core of these processes. Various experimental techniques may be applied to investigate the molecular architecture of biomolecular assemblies. High-resolution techniques, such as X-ray crystallography, may solve the atomic structure of the system, but are typically constrained to biomolecules of reduced flexibility and dimensions. In particular, X-ray crystallography requires the sample to form a three dimensional (3D) crystal lattice which is technically di‑cult, if not impossible, to obtain, especially for large, dynamic systems. Often these techniques solve the structure of the different constituent components within the assembly, but encounter difficulties when investigating the entire system. On the other hand, imaging techniques, such as cryo-electron microscopy (cryo-EM), are able to depict large systems in near-native environment, without requiring the formation of crystals. The structures solved by cryo-EM cover a wide range of resolutions, from very low level of detail where only the overall shape of the system is visible, to high-resolution that approach, but not yet reach, atomic level of detail. In this dissertation, several modeling methods are introduced to either integrate cryo-EM datasets with structural data from X-ray crystallography, or to directly interpret the cryo-EM reconstruction. Such computational techniques were developed with the goal of creating an atomic model for the cryo-EM data. The low-resolution reconstructions lack the level of detail to permit a direct atomic interpretation, i.e. one cannot reliably locate the atoms or amino-acid residues within the structure obtained by cryo-EM. Thereby one needs to consider additional information, for example, structural data from other sources such as X-ray crystallography, in order to enable such a high-resolution interpretation. Modeling techniques are thus developed to integrate the structural data from the different biophysical sources, examples including the work described in the manuscript I and II of this dissertation. At intermediate and high-resolution, cryo-EM reconstructions depict consistent 3D folds such as tubular features which in general correspond to alpha-helices. Such features can be annotated and later on used to build the atomic model of the system, see manuscript III as alternative. Three manuscripts are presented as part of the PhD dissertation, each introducing a computational technique that facilitates the interpretation of cryo-EM reconstructions. The first manuscript is an application paper that describes a heuristics to generate the atomic model for the protein envelope of the Rift Valley fever virus. The second manuscript introduces the evolutionary tabu search strategies to enable the integration of multiple component atomic structures with the cryo-EM map of their assembly. Finally, the third manuscript develops further the latter technique and apply it to annotate consistent 3D patterns in intermediate-resolution cryo-EM reconstructions. The first manuscript, titled An assembly model for Rift Valley fever virus, was submitted for publication in the Journal of Molecular Biology. The cryo-EM structure of the Rift Valley fever virus was previously solved at 27Å-resolution by Dr. Freiberg and collaborators. Such reconstruction shows the overall shape of the virus envelope, yet the reduced level of detail prevents the direct atomic interpretation. High-resolution structures are not yet available for the entire virus nor for the two different component glycoproteins that form its envelope. However, homology models may be generated for these glycoproteins based on similar structures that are available at atomic resolutions. The manuscript presents the steps required to identify an atomic model of the entire virus envelope, based on the low-resolution cryo-EM map of the envelope and the homology models of the two glycoproteins. Starting with the results of the exhaustive search to place the two glycoproteins, the model is built iterative by running multiple multi-body refinements to hierarchically generate models for the different regions of the envelope. The generated atomic model is supported by prior knowledge regarding virus biology and contains valuable information about the molecular architecture of the system. It provides the basis for further investigations seeking to reveal different processes in which the virus is involved such as assembly or fusion. The second manuscript was recently published in the of Journal of Structural Biology (doi:10.1016/j.jsb.2009.12.028) under the title Evolutionary tabu search strategies for the simultaneous registration of multiple atomic structures in cryo-EM reconstructions. This manuscript introduces the evolutionary tabu search strategies applied to enable a multi-body registration. This technique is a hybrid approach that combines a genetic algorithm with a tabu search strategy to promote the proper exploration of the high-dimensional search space. Similar to the Rift Valley fever virus, it is common that the structure of a large multi-component assembly is available at low-resolution from cryo-EM, while high-resolution structures are solved for the different components but lack for the entire system. Evolutionary tabu search strategies enable the building of an atomic model for the entire system by considering simultaneously the different components. Such registration indirectly introduces spatial constrains as all components need to be placed within the assembly, enabling the proper docked in the low-resolution map of the entire assembly. Along with the method description, the manuscript covers the validation, presenting the benefit of the technique in both synthetic and experimental test cases. Such approach successfully docked multiple components up to resolutions of 40Å. The third manuscript is entitled Evolutionary Bidirectional Expansion for the Annotation of Alpha Helices in Electron Cryo-Microscopy Reconstructions and was submitted for publication in the Journal of Structural Biology. The modeling approach described in this manuscript applies the evolutionary tabu search strategies in combination with the bidirectional expansion to annotate secondary structure elements in intermediate resolution cryo-EM reconstructions. In particular, secondary structure elements such as alpha helices show consistent patterns in cryo-EM data, and are visible as rod-like patterns of high density. The evolutionary tabu search strategy is applied to identify the placement of the different alpha helices, while the bidirectional expansion characterizes their length and curvature. The manuscript presents the validation of the approach at resolutions ranging between 6 and 14Å, a level of detail where alpha helices are visible. Up to resolution of 12 Å, the method measures sensitivities between 70-100% as estimated in experimental test cases, i.e. 70-100% of the alpha-helices were correctly predicted in an automatic manner in the experimental data. The three manuscripts presented in this PhD dissertation cover different computation methods for the integration and interpretation of cryo-EM reconstructions. The methods were developed in the molecular modeling software Sculptor (http://sculptor.biomachina.org) and are available for the scientific community interested in the multi-resolution modeling of cryo-EM data. The work spans a wide range of resolution covering multi-body refinement and registration at low-resolution along with annotation of consistent patterns at high-resolution. Such methods are essential for the modeling of cryo-EM data, and may be applied in other fields where similar spatial problems are encountered, such as medical imaging.

GENOME-WIDE PROFILING UNVEILS CRITICIAL FUNCTIONS OF p53 IN HUMAN EMBRYONIC STEM CELLS

Embryonic stem cells (ESCs) possess two unique characteristics: infinite self-renewal and the potential to differentiate into almost every cell type (pluripotency). Recently, global expression analyses of metastatic breast and lung cancers revealed an ESC-like expression program or signature, specifically for cancers that are mutant for p53 function. Surprisingly, although p53 is widely recognized as the guardian of the genome, due to its roles in cell cycle checkpoints, programmed cell death or senescence, relatively little is known about p53 functions in normal cells, especially in ESCs. My hypothesis is that p53 has specific transcription regulatory functions in human ESCs (hESCs) that a) oppose pluripotency and b) protect the stem cell genome in response to DNA damage and stress signaling. In mouse ESCs, these roles are believed to coincide, as p53 promotes differentiation in response to DNA damage, but this is unexplored in hESCs. To determine the biological roles of p53, specifically in hESCs, we mapped genome-wide chromatin interactions of p53 by chromatin immunoprecipitation and massively parallel tag sequencing (ChIP-Seq), and did so under three VIdifferent conditions of hESC status: pluripotency, differentiation-initiated and DNA-damage-induced. ChIP-Seq showed that p53 is enriched at distinct, induction-specific gene loci during each of these different conditions. Microarray gene expression analysis and functional annotation of the distinct p53-target genes revealed that p53 regulates specific genes encoding developmental regulators, which are expressed in differentiation-initiated but not DNA- damaged hESCs. We further discovered that, in response to differentiation signaling, p53 binds regions of chromatin that are repressed but also poised for rapid activation by core pluripotency factors OCT4 and NANOG in pluripotent hESCs. In response to DNA damage, genes associated with migration and motility are targeted by p53; whereas, the prime targets of p53 in control of cell death are conserved for p53 regulation in both differentiation and DNA damage. Our genome-wide profiling and bioinformatics analyses show that p53 occupies a special set of developmental regulatory genes during early differentiation of hESCs and functions in an induction-specific manner. In conclusion, our research unveiled previously unknown functions of p53 in ESC biology, which augments our understanding of one of the most deregulated proteins in human cancers.

De los registros a los objetos : Semántica y comportamiento de los documentos: el desafío de la Web 3.0

Se presenta un panorama y los interrogantes fundamentales de la etapa de la Web 3.0. Se analizan las características actuales de los sistemas bibliográficos estructurados con el modelo entidad-relación. Se definen los niveles conceptual, lógico y físico en los sistemas informáticos; consecuentemente se presentan las características de los FRBR y se obervan las relaciones entre obra y documento en el modelo conceptual FRBR. Se describen los FRBRoo como una interpretación con una lógica de objetos de los mismos requerimientos funcionales. Finalmente se plantean las tendencias a futuro, tales como pasar de las modelizaciones de entidad-relación a la de objetos, la explicitación con anotación semántica consistente, el mapeo de bases bibliográficas existentes y el desarrollo de ontologías para que los sistemas documentales se integren en la Web Semática

De los registros a los objetos : Semántica y comportamiento de los documentos: el desafío de la Web 3.0

Se presenta un panorama y los interrogantes fundamentales de la etapa de la Web 3.0. Se analizan las características actuales de los sistemas bibliográficos estructurados con el modelo entidad-relación. Se definen los niveles conceptual, lógico y físico en los sistemas informáticos; consecuentemente se presentan las características de los FRBR y se obervan las relaciones entre obra y documento en el modelo conceptual FRBR. Se describen los FRBRoo como una interpretación con una lógica de objetos de los mismos requerimientos funcionales. Finalmente se plantean las tendencias a futuro, tales como pasar de las modelizaciones de entidad-relación a la de objetos, la explicitación con anotación semántica consistente, el mapeo de bases bibliográficas existentes y el desarrollo de ontologías para que los sistemas documentales se integren en la Web Semática

De los registros a los objetos : Semántica y comportamiento de los documentos: el desafío de la Web 3.0

Se presenta un panorama y los interrogantes fundamentales de la etapa de la Web 3.0. Se analizan las características actuales de los sistemas bibliográficos estructurados con el modelo entidad-relación. Se definen los niveles conceptual, lógico y físico en los sistemas informáticos; consecuentemente se presentan las características de los FRBR y se obervan las relaciones entre obra y documento en el modelo conceptual FRBR. Se describen los FRBRoo como una interpretación con una lógica de objetos de los mismos requerimientos funcionales. Finalmente se plantean las tendencias a futuro, tales como pasar de las modelizaciones de entidad-relación a la de objetos, la explicitación con anotación semántica consistente, el mapeo de bases bibliográficas existentes y el desarrollo de ontologías para que los sistemas documentales se integren en la Web Semática

Experiment on marine fungus Microascus brevicaulis strain

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularide A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing besides higher production levels faster growth and differences in pellet formation. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of this fungus and its mutant. For this purpose, an optimised protein extraction protocol was established. Here, we show the first proteome study of a marine fungus. In total, 4759 proteins were identified. The central metabolic pathway of LF580 could be mapped by using KEGG pathway analysis and GO annotation. Using iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to a limited nutrient availability in wild type strain due to a strong pellet formation. This information can be applied to optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.

Plant protein-coding gene families: emerging bioinformatics approaches

Protein-coding gene families are sets of similar genes with a shared evolutionary origin and, generally, with similar biological functions. In plants, the size and role of gene families has been only partially addressed. However, suitable bioinformatics tools are being developed to cluster the enormous number of sequences currently available in databases. Specifically, comparative genomic databases promise to become powerful tools for gene family annotation in plant clades. In this review, I evaluate the data retrieved from various gene family databases, the ease with which they can be extracted and how useful the extracted information is.

DBpedia spotlight: shedding light on the web of documents

Interlinking text documents with Linked Open Data enables the Web of Data to be used as background knowledge within document-oriented applications such as search and faceted browsing. As a step towards interconnecting the Web of Documents with the Web of Data, we developed DBpedia Spotlight, a system for automatically annotating text documents with DBpedia URIs. DBpedia Spotlight allows users to congure the annotations to their specic needs through the DBpedia Ontology and quality measures such as prominence, topical pertinence, contextual ambiguity and disambiguation condence. We compare our approach with the state of the art in disambiguation, and evaluate our results in light of three baselines and six publicly available annotation systems, demonstrating the competitiveness of our system. DBpedia Spotlight is shared as open source and deployed as a Web Service freely available for public use.

DBpedia Spotlight: Shedding Light on the Web of Documents

Interlinking text documents with Linked Open Data enables the Web of Data to be used as background knowledge within document-oriented applications such as search and faceted browsing. As a step towards interconnecting the Web of Documents with the Web of Data, we developed DBpedia Spotlight, a system for automatically annotating text documents with DBpedia URIs. DBpedia Spotlight allows users to configure the annotations to their specific needs through the DBpedia Ontology and quality measures such as prominence, topical pertinence, contextual ambiguity and disambiguation confidence. We compare our approach with the state of the art in disambiguation, and evaluate our results in light of three baselines and six publicly available annotation systems, demonstrating the competitiveness of our system. DBpedia Spotlight is shared as open source and deployed as a Web Service freely available for public use.

Using SCXML to integrate semantic sensor information into context-aware user interfaces

This paper describes a novel architecture to introduce automatic annotation and processing of semantic sensor data within context-aware applications. Based on the well-known state-charts technologies, and represented using W3C SCXML language combined with Semantic Web technologies, our architecture is able to provide enriched higher-level semantic representations of user’s context. This capability to detect and model relevant user situations allows a seamless modeling of the actual interaction situation, which can be integrated during the design of multimodal user interfaces (also based on SCXML) for them to be adequately adapted. Therefore, the final result of this contribution can be described as a flexible context-aware SCXML-based architecture, suitable for both designing a wide range of multimodal context-aware user interfaces, and implementing the automatic enrichment of sensor data, making it available to the entire Semantic Sensor Web

Automatic compile-time parallelization of logic programs for restricted, goal-level, independent and-parallelism.

A framework for the automatic parallelization of (constraint) logic programs is proposed and proved correct. Intuitively, the parallelization process replaces conjunctions of literals with parallel expressions. Such expressions trigger at run-time the exploitation of restricted, goal-level, independent and-parallelism. The parallelization process performs two steps. The first one builds a conditional dependency graph (which can be implified using compile-time analysis information), while the second transforms the resulting graph into linear conditional expressions, the parallel expressions of the &-Prolog language. Several heuristic algorithms for the latter ("annotation") process are proposed and proved correct. Algorithms are also given which determine if there is any loss of parallelism in the linearization process with respect to a proposed notion of maximal parallelism. Finally, a system is presented which implements the proposed approach. The performance of the different annotation algorithms is compared experimentally in this system by studying the time spent in parallelization and the effectiveness of the results in terms of speedups.

Extracting non-strict independent and-parallelism using sharing and freeness information

Logic programming systems which exploit and-parallelism among non-deterministic goals rely on notions of independence among those goals in order to ensure certain efficiency properties. "Non-strict" independence (NSI) is a more relaxed notion than the traditional notion of "strict" independence (SI) which still ensures the relevant efficiency properties and can allow considerable more parallelism than SI. However, all compilation technology developed to date has been based on SI, because of the intrinsic complexity of exploiting NSI. This is related to the fact that NSI cannot be determined "a priori" as SI. This paper filis this gap by developing a technique for compile-time detection and annotation of NSI. It also proposes algorithms for combined compiletime/ run-time detection, presenting novel run-time checks for this type of parallelism. Also, a transformation procedure to eliminate shared variables among parallel goals is presented, aimed at performing as much work as possible at compile-time. The approach is based on the knowledge of certain properties regarding the run-time instantiations of program variables —sharing and freeness— for which compile-time technology is available, with new approaches being currently proposed. Thus, the paper does not deal with the analysis itself, but rather with how the analysis results can be used to parallelize programs.

The CDG, UDG, and MEL methods for automatic compile-time parallelization of logic programs for independent and-parallelism

There has been significant interest in parallel execution models for logic programs which exploit Independent And-Parallelism (IAP). In these models, it is necessary to determine which goals are independent and therefore eligible for parallel execution and which goals have to wait for which others during execution. Although this can be done at run-time, it can imply a very heavy overhead. In this paper, we present three algorithms for automatic compiletime parallelization of logic programs using IAP. This is done by converting a clause into a graph-based computational form and then transforming this graph into linear expressions based on &-Prolog, a language for IAP. We also present an algorithm which, given a clause, determines if there is any loss of parallelism due to linearization, for the case in which only unconditional parallelism is desired. Finally, the performance of these annotation algorithms is discussed for some benchmark programs.

Semantic technologies in idea management systems: a model for interoperability, linking and filtering

Abstract Idea Management Systems are web applications that implement the notion of open innovation though crowdsourcing. Typically, organizations use those kind of systems to connect to large communities in order to gather ideas for improvement of products or services. Originating from simple suggestion boxes, Idea Management Systems advanced beyond collecting ideas and aspire to be a knowledge management solution capable to select best ideas via collaborative as well as expert assessment methods. In practice, however, the contemporary systems still face a number of problems usually related to information overflow and recognizing questionable quality of submissions with reasonable time and effort allocation. This thesis focuses on idea assessment problem area and contributes a number of solutions that allow to filter, compare and evaluate ideas submitted into an Idea Management System. With respect to Idea Management System interoperability the thesis proposes theoretical model of Idea Life Cycle and formalizes it as the Gi2MO ontology which enables to go beyond the boundaries of a single system to compare and assess innovation in an organization wide or market wide context. Furthermore, based on the ontology, the thesis builds a number of solutions for improving idea assessment via: community opinion analysis (MARL), annotation of idea characteristics (Gi2MO Types) and study of idea relationships (Gi2MO Links). The main achievements of the thesis are: application of theoretical innovation models for practice of Idea Management to successfully recognize the differentiation between communities, opinion metrics and their recognition as a new tool for idea assessment, discovery of new relationship types between ideas and their impact on idea clustering. Finally, the thesis outcome is establishment of Gi2MO Project that serves as an incubator for Idea Management solutions and mature open-source software alternatives for the widely available commercial suites. From the academic point of view the project delivers resources to undertake experiments in the Idea Management Systems area and managed to become a forum that gathered a number of academic and industrial partners. Resumen Los Sistemas de Gestión de Ideas son aplicaciones Web que implementan el concepto de innovación abierta con técnicas de crowdsourcing. Típicamente, las organizaciones utilizan ese tipo de sistemas para conectar con comunidades grandes y así recoger ideas sobre cómo mejorar productos o servicios. Los Sistemas de Gestión de Ideas lian avanzado más allá de recoger simplemente ideas de buzones de sugerencias y ahora aspiran ser una solución de gestión de conocimiento capaz de seleccionar las mejores ideas por medio de técnicas colaborativas, así como métodos de evaluación llevados a cabo por expertos. Sin embargo, en la práctica, los sistemas contemporáneos todavía se enfrentan a una serie de problemas, que, por lo general, están relacionados con la sobrecarga de información y el reconocimiento de las ideas de dudosa calidad con la asignación de un tiempo y un esfuerzo razonables. Esta tesis se centra en el área de la evaluación de ideas y aporta una serie de soluciones que permiten filtrar, comparar y evaluar las ideas publicadas en un Sistema de Gestión de Ideas. Con respecto a la interoperabilidad de los Sistemas de Gestión de Ideas, la tesis propone un modelo teórico del Ciclo de Vida de la Idea y lo formaliza como la ontología Gi2MO que permite ir más allá de los límites de un sistema único para comparar y evaluar la innovación en un contexto amplio dentro de cualquier organización o mercado. Por otra parte, basado en la ontología, la tesis desarrolla una serie de soluciones para mejorar la evaluación de las ideas a través de: análisis de las opiniones de la comunidad (MARL), la anotación de las características de las ideas (Gi2MO Types) y el estudio de las relaciones de las ideas (Gi2MO Links). Los logros principales de la tesis son: la aplicación de los modelos teóricos de innovación para la práctica de Sistemas de Gestión de Ideas para reconocer las diferenciasentre comu¬nidades, métricas de opiniones de comunidad y su reconocimiento como una nueva herramienta para la evaluación de ideas, el descubrimiento de nuevos tipos de relaciones entre ideas y su impacto en la agrupación de estas. Por último, el resultado de tesis es el establecimiento de proyecto Gi2MO que sirve como incubadora de soluciones para Gestión de Ideas y herramientas de código abierto ya maduras como alternativas a otros sistemas comerciales. Desde el punto de vista académico, el proyecto ha provisto de recursos a ciertos experimentos en el área de Sistemas de Gestión de Ideas y logró convertirse en un foro que reunión para un número de socios tanto académicos como industriales.

Automatic unrestricted independent and-parallelism in logic programs

We present new algorithms which perform automatic parallelization via source-to-source transformations. The objective is to exploit goal-level, unrestricted independent andparallelism. The proposed algorithms use as targets new parallel execution primitives which are simpler and more flexible than the well-known &/2 parallel operator, which makes it possible to generate better parallel expressions by exposing more potential parallelism among the literals of a clause than is possible with &/2. The main differences between the algorithms stem from whether the order of the solutions obtained is preserved or not, and on the use of determinacy information. We briefly describe the environment where the algorithms have been implemented and the runtime platform in which the parallelized programs are executed. We also report on an evaluation of an implementation of our approach. We compare the performance obtained to that of previous annotation algorithms and show that relevant improvements can be obtained.