A revision of the polychaete family Nephtyidae, based on morphological and molecular data

A família Nephtyidae é uma das mais frequentes em habitats costeiros e marinhos de todo o mundo. São organismos errantes típicos de sedimentos arenosos e lodosos, ocorrendo frequentemente no domínio costeiro até 100 m de profundidade, e mais raramente em profundidades batiais e abissais. As primeiras espécies descritas foram o Nephtys caeca (Fabricius, 1780) e o N. ciliata (O. F. Müller, 1789), ambas atribuídas inicialmente ao género Nereis e posteriormente transferidas para o género Nephtys por Savigny, em 1818. A família Nephtyidae foi criada em 1851 por Grube para o género Nephtys Cuvier, 1817. No âmbito desta tese é feito um estudo taxonómico e filogenético da família Nephtyidae. O estudo filogenético inclui dados morfológicos e moleculares de 24 taxa representantes dos cinco géneros da família, Nephtys Cuvier, 1817, Aglaophamus Kinberg, 1866, Micronephthys (Friedrich, 1939), Inermonephtys Fauchald, 1967 e Dentinephtys Imajima e Takeda, 1987. A análise evidenciou dois grandes grupos correspondentes aos dois principais géneros, Aglaophamus e Nephtys. Duas espécies do género Nephtys (N. pulchra e N. australiensis) são transferidas para o género Aglaophamus, e consequentemente são propostas novas diagnoses para os géneros. O género Dentinephtys é sinonimizado com Nephtys e um novo género, Bipalponephtys, é descrito para acomodar as espécies Nephtys cornuta, N. danida e Micronephthys neotena. As relações filogenéticas entre os géneros são discutidas. Do estudo taxonómico resultou a revisão da família Nephtyidae para o Sul da Europa (entre o Canal da Mancha e o Mediterrâneo), com a descrição de uma nova espécie, Inermonephtys foretmontardoi. A espécie Micronephthys maryae é sinonimizada com M. stammeri. Para cada espécie são incluídas notas sobre a sua ecologia bem como a distribuição geográfica e batimétrica. São propostas novas diagnoses para os géneros do Sul da Europa bem como uma chave de identificação taxonómica para as espécies desta região. Após uma exaustiva revisão bibliográfica da família, e da observação de material museológico relativo a 44 espécies, foi compilada uma lista completa para a família de 128 espécies, distribuídas por cinco géneros (57 Nephtys, 53 Aglaophamus, sete Micronephthys, oito Inermonephtys e três Bipalponephtys), na qual são incluídas sinonímias e considerações taxonómicas para cada espécie. A espécie Nephtys serrata é sinonimizada com N. serratifolia. São apresentados as distribuições geográficas e batimétricas das diferentes espécies e notas sobre o seu habitat. São também incluídas tabelas de identificação com as principais características taxonómicas das espécies. O valor diagnóstico dos caracteres morfológicos é discutido. Vários problemas taxonómicos são realçados, indicando a necessidade de revisões adicionais para 23 espécies. Este trabalho realça a existência de vários problemas taxonómicos e filogenéticos dentro da família Nephtyidae, podendo ser considerado como a base para estudos futuros. Análises filogenéticas adicionais incluindo dados morfológicos e moleculares de um maior número de espécies vão certamente conduzir a uma melhor avaliação do estatuto e relações entre os géneros dentro da família.

Molecular cloning, full-length sequence and preliminary characterization of a 56-kDa protein induced by human interferons

Among the various proteins which are induced when human cells are are treatened with interferon, a predominant protein of unknown function, with molecular mass 56 kDa, has been observed. With the aim of exploring the molecular basis of the regulation of this protein and of its mRNA, in order to understand its biological functionand its possible contribution to the various antiviral and non-antiviral actions exerted by interferons.

Molecular characterization of Citrus tristeza virus isolates from Cyprus on the basis of the coat protein gene

Within the context of a program in Cyprus for the control of Citrus tristeza virus (CTV), the coat protein (CP) genes of 12 local isolates of the virus that induced different symptoms on host trees, were compared to those of known isolates. The CP genes were reverse-transcribed (RT) and amplified by polymerase chain reaction (PCR) and the resulting amplicons were cloned and sequenced. Nucleotide sequence analysis revealed no signs of geographic speciation. All the sequences obtained clustered close to those of previously known isolates of worldwide origin that are in five distinct groups. The nucleotide diversity was high compared to that found using a worldwide database of CP gene sequences. These data support the existence of different CTV introductions into Cyprus or an introduction from a location in which CTV is relatively diverse. Some of the isolates induced stem pitting on branches of grapefruit and sweet orange. Such isolates have not been noted often in the Mediterranean basin. They were close in CP sequence to isolate B249 from Venezuela, which induces stem pitting, and are of particular concern for the whole region.

Molecular characterization of Gla-rich protein (GRP) gene expression and function

Gla-rich protein (GRP) is a vitamin K-dependent protein related to bone and cartilage recently described. This protein is characterized by a large number of Gla (γ-carboxyglutamic acid) residues being the protein with the highest Gla content of any known protein. It was found in a widely variety of tissues but highest levels was found in skeletal and cartilaginous tissues. This small secreted protein was also expressed and accumulated in soft tissues and it was clearly associated with calcification pathologies in the same tissues. Although the biological importance of GRP remains to be elucidated, it was suggested a physiological role in cartilage development and calcification process during vertebrate skeleton formation. Using zebrafish, an accepted model to study skeletal development, we have described two grp paralog genes, grp1 and grp2, which exhibited distinct patterns of expression, suggesting different regulatory pathways for each gene. Gene synteny analysis showed that grp2 gene is more closely related to tetrapod grp, although grp1 gene was proposed to be the vertebrate ortholog by sequence comparison. In addition, we identified a functional promoter of grp2 gene and using a functional approach we confirmed the involvement of transcription factors from Sox family (Sox9b and Sox10) in the regulation of grp2 expression. In an effort to provide more information about the function of grp isoforms, we generated two zebrafish transgenic lines capable to overexpress conditionally grp genes and possible roles in the skeleton development were studied. To better understand GRP function a mammalian system was used and the analysis of knockout mice showed that GRP is involved in chondrocyte maturation and the absence of GRP is associated to proteoglycans loss in calcified articular cartilage. In addition, we detected differences in chondrogenesis markers in articular chondrocyte primary culture. Overall, our data suggest a main role for GRP on chondrocyte differentiation.

Protein interaction data curation: the International Molecular Exchange (IMEx) consortium.

The International Molecular Exchange (IMEx) consortium is an international collaboration between major public interaction data providers to share literature-curation efforts and make a nonredundant set of protein interactions available in a single search interface on a common website (http://www.imexconsortium.org/). Common curation rules have been developed, and a central registry is used to manage the selection of articles to enter into the dataset. We discuss the advantages of such a service to the user, our quality-control measures and our data-distribution practices.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

Molecular interactions of arbuscular mycorrhizal fungi with mycotoxin-producing fungi and their role in plant defense responses

Les trichothécènes de Fusarium appartiennent au groupe des sesquiterpènes qui sont des inhibiteurs la synthèse des protéines des eucaryotes. Les trichothécènes causent d’une part de sérieux problèmes de santé aux humains et aux animaux qui ont consommé des aliments infectés par le champignon et de l’autre part, elles sont des facteurs importants de la virulence chez plantes. Dans cette étude, nous avons isolé et caractérisé seize isolats de Fusarium de la pomme de terre infectée naturellement dans un champs. Les tests de pathogénicité ont été réalisés pour évaluer la virulence des isolats sur la pomme de terre ainsi que leur capacité à produire des trichothécènes. Nous avons choisi F. sambucinum souche T5 comme un modèle pour cette étude parce qu’il était le plus agressif sur la pomme de terre en serre en induisant un flétrissement rapide, un jaunissement suivi de la mort des plantes. Cette souche produit le 4,15-diacétoxyscirpénol (4,15-DAS) lorsqu’elle est cultivée en milieu liquide. Nous avons amplifié et caractérisé cinq gènes de biosynthèse trichothécènes (TRI5, TRI4, TRI3, TRI11, et TRI101) impliqués dans la production du 4,15-DAS. La comparaison des séquences avec les bases de données a montré 98% et 97% d'identité de séquence avec les gènes de la biosynthèse des trichothécènes chez F. sporotrichioides et Gibberella zeae, respectivement. Nous avons confrenté F. sambucinum avec le champignon mycorhizien à arbuscule Glomus irregulare en culture in vitro. Les racines de carotte et F. sambucinum seul, ont été utilisés comme témoins. Nous avons observé que la croissance de F. sambucinum a été significativement réduite avec la présence de G. irregulare par rapport aux témoins. Nous avons remarqué que l'inhibition de la croissance F. sambucinum a été associée avec des changements morphologiques, qui ont été observés lorsque les hyphes de G. irregulare ont atteint le mycélium de F. sambucinum. Ceci suggère que G. irregulare pourrait produire des composés qui inhibent la croissance de F. sambucinum. Nous avons étudié les patrons d’expression des gènes de biosynthèse de trichothécènes de F. sambucinum en présence ou non de G. irregulare, en utilisant le PCR en temps-réel. Nous avons observé que TRI5 et TRI6 étaient sur-exprimés, tandis que TRI4, TRI13 et TRI101 étaient en sous-exprimés en présence de G. irregulare. Des analyses par chromatographie en phase-gazeuse (GC-MS) montrent clairement que la présence de G. irregulare réduit significativement la production des trichothécènes par F. sambucinum. Le dosage du 4,15-DAS a été réduit à 39 μg/ml milieu GYEP par G. irregulare, comparativement à 144 μg/ml milieu GYEP quand F. sambucinum est cultivé sans G. irregulare. Nous avons testé la capacité de G. irregulare à induire la défense des plants de pomme de terre contre l'infection de F. sambucinum. Des essais en chambre de croissance montrent que G. irregulare réduit significativement l’incidence de la maladie causée par F. sambucinum. Nous avons aussi observé que G. irregulare augmente la biomasse des racines, des feuilles et des tubercules. En utilisant le PCR en temps-réel, nous avons étudié les niveaux d’expression des gènes impliqué dans la défense des plants de pommes de terre tels que : chitinase class II (ChtA3), 1,3-β-glucanase (Glub), peroxidase (CEVI16), osmotin-like protéin (OSM-8e) et pathogenèses-related protein (PR-1). Nous avons observé que G. irregulare a induit une sur-expression de tous ces gènes dans les racines après 72 heures de l'infection avec F. sambucinum. Nous avons également trové que la baisse provoquée par F. sambucinum des gènes Glub et CEVI16 dans les feuilles pourrait etre bloquée par le traitement AMF. Ceci montre que l’inoculation avec G. irregulare constitut un bio-inducteur systémique même dans les parties non infectées par F. sambucinum. En conclusion, cette étude apporte de nouvelles connaissances importantes sur les interactions entre les plants et les microbes, d’une part sur les effets directs des champignons mycorhiziens sur l’inhibition de la croissance et la diminution de la production des mycotoxines chez Fusarium et d’autre part, l’atténuation de la sévérité de la maladie dans des plantes par stimulation leur défense. Les données présentées ouvrent de nouvelles perspectives de bio-contrôle contre les pathogènes mycotoxinogènes des plantes.

Mean Squared Residue Based Biclustering Algorithms for the Analysis of Gene Expression Data

Computational Biology is the research are that contributes to the analysis of biological data through the development of algorithms which will address significant research problems.The data from molecular biology includes DNA,RNA ,Protein and Gene expression data.Gene Expression Data provides the expression level of genes under different conditions.Gene expression is the process of transcribing the DNA sequence of a gene into mRNA sequences which in turn are later translated into proteins.The number of copies of mRNA produced is called the expression level of a gene.Gene expression data is organized in the form of a matrix. Rows in the matrix represent genes and columns in the matrix represent experimental conditions.Experimental conditions can be different tissue types or time points.Entries in the gene expression matrix are real values.Through the analysis of gene expression data it is possible to determine the behavioral patterns of genes such as similarity of their behavior,nature of their interaction,their respective contribution to the same pathways and so on. Similar expression patterns are exhibited by the genes participating in the same biological process.These patterns have immense relevance and application in bioinformatics and clinical research.Theses patterns are used in the medical domain for aid in more accurate diagnosis,prognosis,treatment planning.drug discovery and protein network analysis.To identify various patterns from gene expression data,data mining techniques are essential.Clustering is an important data mining technique for the analysis of gene expression data.To overcome the problems associated with clustering,biclustering is introduced.Biclustering refers to simultaneous clustering of both rows and columns of a data matrix. Clustering is a global whereas biclustering is a local model.Discovering local expression patterns is essential for identfying many genetic pathways that are not apparent otherwise.It is therefore necessary to move beyond the clustering paradigm towards developing approaches which are capable of discovering local patterns in gene expression data.A biclusters is a submatrix of the gene expression data matrix.The rows and columns in the submatrix need not be contiguous as in the gene expression data matrix.Biclusters are not disjoint.Computation of biclusters is costly because one will have to consider all the combinations of columans and rows in order to find out all the biclusters.The search space for the biclustering problem is 2 m+n where m and n are the number of genes and conditions respectively.Usually m+n is more than 3000.The biclustering problem is NP-hard.Biclustering is a powerful analytical tool for the biologist.The research reported in this thesis addresses the problem of biclustering.Ten algorithms are developed for the identification of coherent biclusters from gene expression data.All these algorithms are making use of a measure called mean squared residue to search for biclusters.The objective here is to identify the biclusters of maximum size with the mean squared residue lower than a given threshold. All these algorithms begin the search from tightly coregulated submatrices called the seeds.These seeds are generated by K-Means clustering algorithm.The algorithms developed can be classified as constraint based,greedy and metaheuristic.Constarint based algorithms uses one or more of the various constaints namely the MSR threshold and the MSR difference threshold.The greedy approach makes a locally optimal choice at each stage with the objective of finding the global optimum.In metaheuristic approaches particle Swarm Optimization(PSO) and variants of Greedy Randomized Adaptive Search Procedure(GRASP) are used for the identification of biclusters.These algorithms are implemented on the Yeast and Lymphoma datasets.Biologically relevant and statistically significant biclusters are identified by all these algorithms which are validated by Gene Ontology database.All these algorithms are compared with some other biclustering algorithms.Algorithms developed in this work overcome some of the problems associated with the already existing algorithms.With the help of some of the algorithms which are developed in this work biclusters with very high row variance,which is higher than the row variance of any other algorithm using mean squared residue, are identified from both Yeast and Lymphoma data sets.Such biclusters which make significant change in the expression level are highly relevant biologically.

Studies on molecular structure and properties using microwave and infrared spectral data

The major objective of the thesis is essentially to evolve and apply certain computational procedures to evaluate the structure and properties of some simple polyatomic molecules making use of spectroscopic data available from the literature. It must be said that though there is dwindling interest in recent times in such analyses, there exists tremendous scope and utility for attempting such calculations as the precision and reliability of'experimental techniques in spectroscopy have increased vastly due to enormous sophistication of the instruments used for these measurements. In the present thesis an attempt is made to extract maximum amount of information regarding the geometrical structure and interatmic forces of simple molecules from the experimental data on microwave and infrared spectra of these molecules

Studies in molecular mechanics using spectroscopic data exact force field and bond in homogeneity

This thesis deals with some studies in molecular mechanic using spectroscopic data. It includes an improvement in the parameter technique for the evaluation of exact force fields, the introduction of a new and simple algebraic method for the force field calculation and a study of asymmetric variation of bonding forces along a bond.

Phylogenomics of eukaryotes: Impact of missing data on large alignments

Resolving the relationships between Metazoa and other eukaryotic groups as well as between metazoan phyla is central to the understanding of the origin and evolution of animals. The current view is based on limited data sets, either a single gene with many species (e.g., ribosomal RNA) or many genes but with only a few species. Because a reliable phylogenetic inference simultaneously requires numerous genes and numerous species, we assembled a very large data set containing 129 orthologous proteins (similar to30,000 aligned amino acid positions) for 36 eukaryotic species. Included in the alignments are data from the choanoflagellate Monosiga ovata, obtained through the sequencing of about 1,000 cDNAs. We provide conclusive support for choanoflagellates as the closest relative of animals and for fungi as the second closest. The monophyly of Plantae and chromalveolates was recovered but without strong statistical support. Within animals, in contrast to the monophyly of Coelomata observed in several recent large-scale analyses, we recovered a paraphyletic Coelamata, with nematodes and platyhelminths nested within. To include a diverse sample of organisms, data from EST projects were used for several species, resulting in a large amount of missing data in our alignment (about 25%). By using different approaches, we verify that the inferred phylogeny is not sensitive to these missing data. Therefore, this large data set provides a reliable phylogenetic framework for studying eukaryotic and animal evolution and will be easily extendable when large amounts of sequence information become available from a broader taxonomic range.

Crystal and molecular structure of the thioether-analogue of PEEK from X-ray powder data and diffraction-modelling

Analysis of X-ray powder data for the melt-crystallisable aromatic poly(thioether thioether ketone) [-S-Ar-S-Ar-CO-Ar](n), ('PTTK', Ar= 1,4-phenylene), reveals that it adopts a crystal structure very different from that established for its ether-analogue PEEK. Molecular modelling and diffraction-simulation studies of PTTK show that the structure of this polymer is analogous to that of melt-crystallised poly(thioetherketone) [-SAr-CO-Ar](n) in which the carbonyl linkages in symmetry-related chains are aligned anti-parallel to one another. and that these bridging units are crystallographically interchangeable. The final model for the crystal structure of PTTK is thus disordered, in the monoclinic space group 121a (two chains per unit cell), with cell dimensions a = 7.83, b = 6.06, c = 10.35 angstrom, beta = 93.47 degrees. (c) 2005 Elsevier Ltd. All rights reserved.