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Clustering algorithms and shape factor methods to discriminate among small GTPase phenotypes using DIC image analysis.Papaluca, Arturo 10 1900 (has links)
Naïvement perçu, le processus d’évolution est une succession d’événements de duplication et de mutations graduelles dans le génome qui mènent à des changements dans les fonctions et les interactions du protéome. La famille des hydrolases de guanosine triphosphate (GTPases) similaire à Ras constitue un bon modèle de travail afin de comprendre ce phénomène fondamental, car cette famille de protéines contient un nombre limité d’éléments qui diffèrent en fonctionnalité et en interactions. Globalement, nous désirons comprendre comment les mutations singulières au niveau des GTPases affectent la morphologie des cellules ainsi que leur degré d’impact sur les populations asynchrones.
Mon travail de maîtrise vise à classifier de manière significative différents phénotypes de la levure Saccaromyces cerevisiae via l’analyse de plusieurs critères morphologiques de souches exprimant des GTPases mutées et natives. Notre approche à base de microscopie et d’analyses bioinformatique des images DIC (microscopie d’interférence différentielle de contraste) permet de distinguer les phénotypes propres aux cellules natives et aux mutants. L’emploi de cette méthode a permis une détection automatisée et une caractérisation des phénotypes mutants associés à la sur-expression de GTPases constitutivement actives. Les mutants de GTPases constitutivement actifs Cdc42 Q61L, Rho5 Q91H, Ras1 Q68L et Rsr1 G12V ont été analysés avec succès.
En effet, l’implémentation de différents algorithmes de partitionnement, permet d’analyser des données qui combinent les mesures morphologiques de population native et mutantes. Nos résultats démontrent que l’algorithme Fuzzy C-Means performe un partitionnement efficace des cellules natives ou mutantes, où les différents types de cellules sont classifiés en fonction de plusieurs facteurs de formes cellulaires obtenus à partir des images DIC. Cette analyse démontre que les mutations Cdc42 Q61L, Rho5 Q91H, Ras1 Q68L et Rsr1 G12V induisent respectivement des phénotypes amorphe, allongé, rond et large qui sont représentés par des vecteurs de facteurs de forme distincts. Ces distinctions sont observées avec différentes proportions (morphologie mutante / morphologie native) dans les populations de mutants.
Le développement de nouvelles méthodes automatisées d’analyse morphologique des cellules natives et mutantes s’avère extrêmement utile pour l’étude de la famille des GTPases ainsi que des résidus spécifiques qui dictent leurs fonctions et réseau d’interaction. Nous pouvons maintenant envisager de produire des mutants de GTPases qui inversent leur fonction en ciblant des résidus divergents. La substitution fonctionnelle est ensuite détectée au niveau morphologique grâce à notre nouvelle stratégie quantitative. Ce type d’analyse peut également être transposé à d’autres familles de protéines et contribuer de manière significative au domaine de la biologie évolutive. / Evolution is a gradual process that gives rise to changes in the form of mutations that are reflected at the protein level. We propose that evolution of new pathways occurs by switching binding partners, hence creating new functions. The different functions encountered in a given family of related proteins have emerged from a common ancestor that has been duplicated and mutated to become implicated in new interactions and to gain new functions. In this study, we will use native and constitutive active mutant variants of the Ras-like family of small GTPases as working model, to explore such gene duplications, followed by neo / sub-functionalization. The reason for choosing this family resides in the fact that it is a defined set of proteins with well known functions that are mediated through multiple protein-protein interactions.
The aim of this master is to perform a classification of budding yeast phenotypes using different approaches in order to statistically determine at which level of the population these constitutively active mutations are capable to affect cell morphology. Working with a subset of the Ras-like small GTPases family, we recently developed an approach to catalogue and classify these proteins based on multiple physical and chemical criteria. Using microscopic and bioinformatics methods, we characterized phenotypes associated with over-expression of the native small GTPases of the budding yeast Saccharomyces cerevisiae, showing that an established classification is not very clear.
We are interested to investigate how point mutations in small GTPases can affect the cell morphology and their level of impact on asynchronous population. We want to establish a method to determine and quantify mutant and wild type-like phenotypes on these populations using Differential interference contrast microscopy (DIC) images only. As for the first aim of this study, we hypothesize that clustering algorithms can partition mutant cells from wild type cells based on cell shape factor measurements. To prove this hypothesis, we proposed to implement different clustering algorithms to analyze datasets which combines measurements from wild type and respective mutant populations.
We created constitutively active forms of these small GTPases and used Cdc42, Rho5, Ras1 and Rsr1 to validate our results. We observed that Cdc42 Q61L, Rho5 Q91H, Ras1 Q68L and Rsr1 G12V mutations induced characteristic amorphous, clumped/elongated, rounded and discrete large phenotypes respectively. This classification allowed us to define a phenotypical classification related to functions. Phenotype classification of the small GTPases has been confirmed using shape factor formulas accompanied with bioinformatics approaches. These approaches which involved different clustering methods allowed an automated quantitative characterization of the phenotypes of up to 7293 mutant cells.
Sequence alignment of Cdc42 and Rho5 showed 46.1% identity as well as 62.6% for Ras1 and Rsr1 allowing the identification of diverged residues potentially involved in specific functions and protein-protein interactions. Directed mutagenesis and substitution of these sites from one gene to another have been performed in some positions to test for specificity and involvement in morphology changes. In parallel, interactions observed for native and constitutively active mutants Cdc42 and Rho5 will be assayed with protein-fragment complementation assay (PCA). This will enable us to determine whether a high correlation exists between functions switches and binding partner’s switches.
We propose to expand this approach to the whole Ras-like small GTPases family and monitor protein-protein interactions and functions at a network scale. This research will confirm whether enrichment or depletion of residues in specific sites induces a switch of function due to switching binding partners. Understanding the mechanism underlying such correlation is important to gain insight in the biological mechanisms underlying the Ras-like small GTPases and other proteins evolution. Such knowledge is of fundamental importance in biomedical and pharmaceutical fields, since Ras-like small GTPases represent important targets for therapeutic interventions and for the evolutionary biology field.
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Cartographie du réseau d'interactions protéiques de la machinerie de transcription dans les cellules humainesRusu, Natalia 12 1900 (has links)
Les protéines sont les macromolécules les plus polyvalentes de la cellule. Elles jouent un rôle fondamental dans la majorité des processus biologiques à travers la formation de complexes multi-protéiques. Durant la transcription, une multitude de facteurs sont impliquées dans le contrôle de l’activité des complexes ARN polymérases.
Notre laboratoire s’est intéressé au réseau d’interaction de la machinerie de transcription des ARN polymérases nucléaires, dans le but de mieux comprendre leurs mécanismes de régulation. Pour ce faire, une procédure protéomique comprenant la purification de complexes protéiques par affinité couplée à la spectrométrie de masse et à l’analyse bioinformatique a été développée. La méthode de purification TAP (Tandem Affinity Purification) a été adaptée pour permettre la purification de complexes protéiques solubles assemblés in vivo à partir de cellules humaines.
L’objectif de mon projet de maîtrise était de purifier le complexe de l’ARN Pol I ainsi que de poursuivre l’expansion du réseau d’interactions protéine-protéine de la machinerie de transcription de l’ARN Pol II humaine. À l’aide des protéines POLR1E, TWISTNB, POLR2E, PFDN4, MBD2, XPA, CAND1 et PDCD5 étiquetées (TAP-tag) exprimées dans des lignées cellulaires ECR-293, plusieurs complexes protéiques solubles ont été purifiés et analysés par spectrométrie de masse. Les interactions protéiques ont été triées et validées bioinformatiquement pour donner en final une liste d’interactions ayant un haut degré de confiance à partir de laquelle des réseaux d’interactions protéine-protéine ont été créés.
Le réseau créé au cours de ce projet connecte plusieurs composantes de la machinerie transcriptionnelle tels que les ARN Pol I, II et III, les complexes RPAP3/R2TP/prefoldin-like, TRiC/CCT, Mi-2/NuRD et des facteurs de transcription et de réparation de l’ADN.
Ce type d’analyse nous a permis d’identifier et de caractériser de nouveaux régulateurs de la machinerie de transcription de l’ARN Pol I et II et de mieux comprendre son fonctionnement. / Proteins are the most versatile macromolecules of the cell. They play a fundamental role in the majority of biological processes through the formation of multiprotein complexes. During transcription, a multitude of factors are involved in the control of activity of RNA polymerases.
Our laboratory was interested in defining the nuclear RNA polymerases transcription machinery interaction network to better understand their regulatory mechanisms. To do so, a proteomic procedure that allows affinity purification of protein complexes coupled to mass spectrometry and computational data analysis was developed. The tandem affinity purification procedure has been adapted for the purification of soluble protein complexes as they likely exist in live mammalian cells.
The aim of my master project was to purify the RNA Pol I complex as well as to further pursue the expansion of the protein-protein interaction network of the human RNA Pol II transcription machinery. By using POLR1E, TWISTNB, POLR2E, PFDN4, MBD2, XPA, CAND1 and PDCD5 TAP – tagged proteins expressed in EcR-293 cell lines, multiple soluble protein complexes were purified and analyzed by mass spectrometry. Protein interactions have been sorted and validated computationally. High-confidence dataset of interactions were used to build the protein-protein interaction networks.
The network created for this project connects several components of the transcriptional machinery such as RNA Pol I, II and III, RPAP3/R2TP/prefoldin-like, TRiC/CTC, Mi-2/NuRD complexes and DNA repair and transcription factors.
This type of analysis allowed us to identify and characterize new regulators of RNA Pol I and II transcription machinery and to better understand its functioning.
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Des protéines et de leurs interactions aux principes évolutifs des systèmes biologiques / From proteins and their interactions to evolutionary principles of biological systemsCarvunis, Anne-Ruxandra 26 January 2011 (has links)
Darwin a révélé au monde que les espèces vivantes ne cessent jamais d’évoluer, mais les mécanismes moléculaires de cette évolution restent le sujet de recherches intenses. La biologie systémique propose que les relations entre génotype, environnement et phénotype soient sous-tendues par un ensemble de réseaux moléculaires dynamiques au sein de la cellule, mais l’organisation de ces réseaux demeure mystérieuse. En combinant des concepts établis en biologie évolutive et systémique avec la cartographie d’interactions protéiques et l’étude des méthodologies d’annotation de génomes, j’ai développé de nouvelles approches bioinformatiques qui ont en partie dévoilé la composition et l’organisation des systèmes cellulaires de trois organismes eucaryotes : la levure de boulanger, le nématode Caenorhabditis elegans et la plante Arabidopsis thaliana. L’analyse de ces systèmes m’a conduit à proposer des hypothèses sur les principes évolutifs des systèmes biologiques. En premier lieu, je propose une théorie selon laquelle la traduction fortuite de régions intergéniques produirait des peptides sur lesquels la sélection naturelle agirait pour aboutir occasionnellement à la création de protéines de novo. De plus, je montre que l’évolution de protéines apparues par duplication de gènes est corrélée avec celle de leurs profils d’interactions. Enfin, j’ai mis en évidence des signatures de la co-évolution ancestrale hôte-pathogène dans l’organisation topologique du réseau d‘interactions entre protéines de l’hôte. Mes travaux confortent l’hypothèse que les systèmes moléculaires évoluent, eux aussi, de manière darwinienne. / Darwin exposed to the world that living species continuously evolve. Yet the molecular mechanisms of evolution remain under intense research. Systems biology proposes that dynamic molecular networks underlie relationships between genotype, environment and phenotype, but the organization of these networks is mysterious. Combining established concepts from evolutionary and systems biology with protein interaction mapping and the study of genome annotation methodologies, I have developed new bioinformatics approaches that partially unveiled the composition and organization of cellular systems for three eukaryotic organisms: the baker’s yeast, the nematode Caenorhabditis elegans and the plant Arabidopsis thaliana. My analyses led to insights into the evolution of biological systems. First, I propose that the translation of peptides from intergenic regions could lead to de novo birth of new protein-coding genes. Second, I show that the evolution of proteins originating from gene duplications and of their physical interaction repertoires are tightly interrelated. Lastly, I uncover signatures of the ancestral host-pathogen co-evolution in the topology of a host protein interaction network. My PhD work supports the thesis that molecular systems also evolve in a Darwinian fashion.
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Clustering algorithms and shape factor methods to discriminate among small GTPase phenotypes using DIC image analysisPapaluca, Arturo 10 1900 (has links)
No description available.
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Redes de interação entre morcegos frugívoros e plantas = variação geográfica e conservação de nicho / Interaction networks between frugivorous bats and plants : geographical variation and niche conservatismMarquitti, Flávia Maria Darcie 18 August 2018 (has links)
Orientador: Paulo Roberto Guimarães Júnior / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T07:48:51Z (GMT). No. of bitstreams: 1
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Previous issue date: 2011 / Resumo: Os mutualismos são interações importantes do ponto de vista ecológico e evolutivo, uma vez que influenciam a sobrevivência de diversos organismos, podendo determinar a organização da comunidade. Dada a importância ecológica e evolutiva das interações mutualísticas, é importante estudar quais fatores influenciam a sua organização. Como organismos aparentados compartilham características fenotípicas similares, é comum que espécies filogeneticamente próximas interajam com um conjunto de espécies similar, levando a um padrão conhecido por conservação filogenética do nicho. Apesar de este padrão ocorrer com frequencia, condições bióticas e abióticas locais podem levar à variação geográfica nas interações. A presença ou ausência de potenciais parceiros mutualísticos também podem alterar o padrão local de interações de uma espécie. Estudando interações entre morcegos frugívoros e plantas, investiguei como os padrões de interação de espécies em dez redes mutualísticas variavam conforme a distribuição geográfica. Nesse sentido, duas hipóteses principais guiaram este trabalho: a hipótese da conservação de nicho das interações e a hipótese da variação geográfica das interações. Caracterizei o padrão de interação das espécies de morcegos nas redes por meio de diferentes métricas usadas no estudo de redes ecológicas. Analisei a conservação de nicho das interações e a variação geográfica de espécies de morcego que ocorreram em um maior número de redes. Espécies filogeneticamente próximas não tenderam a apresentar padrões de interação mais parecidos entre si. Algumas das espécies mais frequentes apresentaram características dos padrões de interação em gradiente ou mosaico ao longo de sua distribuição geográfica. Estes resultados evidenciam que algumas características do padrão de interação de morcegos frugívoros, como grau de generalismo e o grau padronizado dentro do módulo são conservadas pela filogenia. No entanto algumas espécies apresentaram o grau de generalismo e o papel em relação aos módulos da rede variando ao longo da geografia, seguindo previsões da teoria do mosaico geográfico coevolutivo / Abstract: Mutualisms are important interactions to ecology and evolution because they affect the survivorship of many organisms and may shape community organization. Therefore, it is important to study which factors affect their organization. As related organisms share similar phenotypic traits, it is common that close phylogenetic species interact with a similar set of species, resulting in a pattern known as phylogenetic niche conservatism. Although this pattern frequently occurs, biotic and abiotic local conditions may lead to geographic variation of the interactions. The presence or absence of potential mutualistic partners may also change local interactions of a species. I studied interactions between frugivorous bats and plants and I investigated how species interactions patterns varied geographically across ten mutualistic networks. Two main hypotheses guided this work: the interaction niche conservatism hypothesis and the geographical variation hypothesis. I delineated the interaction patterns of bat species in the networks through different metrics used in ecological network studies. I analyzed the niche conservatism and geographical variation of bat species which occurred frequently in the networks. Related species did not tend to have similar patterns of interaction. Within their geographical range, some species vary markedly in their patterns of interaction and this variation often occurs as mosaics or, less frequently, as gradients. These results point out that some patterns of interaction of frugivorous bats, as the specialization degree and the standardized within-module degree, are conserved by phylogeny. However, some species showed variations through geography both in the specialization degree and in their role regarding the network modules, leading to a geographic mosaic of coevolution with potential implications to ecology and coevolution / Mestrado / Ecologia / Mestre em Ecologia
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A fragmentação florestal e a interação entre formigas e diásporos carnosos na floresta Atlântica / Forest fragmentation and the interaction between ants and fleshy diaspores in the Atlantic forestBieber, Ana Gabriela Delgado, 1981- 02 October 2012 (has links)
Orientador: Paulo Sérgio Moreira Carvalho de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T12:16:35Z (GMT). No. of bitstreams: 1
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Previous issue date: 2012 / Resumo: Em florestas tropicais, formigas de folhiço são freqüentemente vistas em interação com diásporos vegetais (frutos e sementes). Em alguns casos, esta interação oportunista resulta em um mutualismo. Enquanto formigas se beneficiam ao alimentar-se de partes carnosas nutritivas (i.e., polpa, arilo), elas podem beneficiar a semente, por exemplo, ao aumentar sua chance de germinação ou ao dispersá-la para micro-sítios ricos em nutrientes. Portanto, para algumas espécies de plantas, a perda de interações formiga-diásporo pode implicar numa redução do recrutamento de novos indivíduos. Nesta tese, estudamos os padrões de interação entre formigas e diásporos em dois tipos contrastantes de florestas, representativos do que resta da Floresta Atlântica no Estado de São Paulo: florestas contínuas (CFs) e fragmentos de floresta com vegetação secundária (FFs) (quatro áreas cada). Durante este estudo, quatro abordagens complementares foram adotadas. Primeiramente, registramos as interações entre formigas e diásporos em cada uma das oito áreas durante um ano. As principais famílias de plantas assim como os principais gêneros de formigas registrados correspondem a grupos importantes já listados em estudos prévios realizados neste bioma. Apesar da abundância similar de diásporos considerados atrativos entre os dois tipos de floresta, houve um maior número de interações na floresta contínua. Esta diferença é provavelmente devida à comunidade vegetal depauperada dos fragmentos, composta por espécies menos atrativas para as formigas. Com base no mesmo conjunto de dados, comparamos se a topologia das redes de interação formiga-fruto diferiu entre os dois tipos de floresta. Três das sete métricas calculadas foram afetadas pelo tipo de floresta. A diminuição do número de espécies de plantas nos fragmentos florestais parece ser um fator-chave para justificar os resultados obtidos. Na terceira abordagem, oferecemos um diásporo sintético rico em lipídios em estações de remoção para comparar a visitação e o comportamento das formigas entre os dois tipos florestais. Em geral, um maior número de espécies de formigas foi atraído às estações das CFs. A freqüência de grandes poneríneas (em especial, Pachycondyla striata) em interação com diásporos sintéticos foi mais elevada em CFs, estando relacionada com uma maior remoção de diásporos nestas áreas. Por fim, avaliamos experimentalmente se a manipulação prévia por aves (uma condição mais comum em florestas contínuas) afeta a atratividade de diásporos caídos em relação a formigas. Para este procedimento, utilizamos o arbusto Psychotria suterella (Rubiaceae), comum no subosque de nossa área de estudo e cujos diásporos são dispersos por aves. Frutos que já tiveram contato com vertebrados apresentaram uma maior chance de interagir com formigas. Este resultado sugere que espécies vegetais em florestas fragmentadas, comumente sujeitas à perda de seus dispersores primários, ainda são afetadas pela diminuição da atratividade de frutos intactos para formigas (dispersores secundários), a despeito da abundância destes insetos no chão da floresta. De forma geral, os resultados desta tese indicam que a fragmentação da Floresta Atlântica afeta negativamente as interações formiga-diásporo. Portanto, é possível que em fragmentos florestais haja uma diminuição dos potenciais benefícios a sementes e plântulas derivados da interação com formigas, podendo repercutir negativamente no recrutamento das plantas / Abstract: In tropical rainforests, ground-dwelling ants opportunistically interact with fleshy diaspores and, in some cases, this interaction can be classified as mutualistic. While ants gain from eating the nutritious fleshy parts, they may benefit the seed in two ways: (1) greater germination success; and (2) directed dispersal to nutrient-rich microsites where seedlings grow better. Thus, for some plant species, the loss of ant-diaspore interactions may negatively affect plant recruitment. Here, we explore ant-diaspore interaction patterns in two divergent forest types, representing what has remained from the Brazilian Atlantic Forest of São Paulo State: continuous old-growth forests (CFs) and secondary forest fragments (FFs) (four sites each). During this study, four complementary approaches were adopted. First, we surveyed ant-diaspore interactions in all forest sites during one year. Main plant families and ant genera registered during our survey correspond to previous studies performed in this biome. In spite of the similar abundance of ant-attractive diaspores on the forest ground, there were striking differences on ant-diaspore interactions between continuous and fragmented forests. Differences are most certainly attributed to the species-poor plant community, composed by less attractive species, found in the fragments. Based on the same dataset, we investigated whether the topology of ant-diaspore interaction networks differed between our two studied forest types. Three out of seven network-level metrics calculated diverged between fragments and continuous forests. The decrease in the number of interacting plant species observed in the fragmented forests appears to be a key-factor for explaining the observed results. Third, we offered a lipid-rich synthetic diaspore in experimental removal stations to compare ant attendance and behavior between forest types. Again, the experiment evidenced differences between the opposing forest types. In general, a higher number of ant species was recorded in CFs. The frequency of large ponerines (mainly Pachycondyla striata) was higher in CFs, corresponding to the higher frequency of diaspore removal and the higher removal distances observed in these sites. Finally, we experimentally evaluated if previous handling by birds (a condition more frequent in continuous forests) would affect ant attendance to fallen fruits. For this approach, we used as a model the bird-dispersed species Psychotria suterella (Rubiaceae), a common treelet in the understory of our forest sites. As expected, "feces-embedded" and "mandibulated" P. suterella fruits had increased frequencies of ant attendance than "intact" fruits. This result suggests that plant species in fragmented forests, together with the pervasive loss of their primary seed dispersers, may also be affected by the decreased attractiveness of unhandled fruits to ants (secondary dispersers), in spite of the abundance of these insects on the forest floor. Altogether, our results indicate that Atlantic Forest fragmentation negatively affects ant-diaspore interactions with respect to most of the studied parameters. Therefore, we expect that fragments will present a decrease on ant-derived potential benefits to seeds and seedlings / Doutorado / Ecologia / Doutor em Ecologia
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Unraveling the Structure and Assessing the Quality of Protein Interaction Networks with Power Graph AnalysisRoyer, Loic 12 December 2017 (has links) (PDF)
Molecular biology has entered an era of systematic and automated experimentation. High-throughput techniques have moved biology from small-scale experiments focused on specific genes and proteins to genome and proteome-wide screens. One result of this endeavor is the compilation of complex networks of interacting proteins. Molecular biologists hope to understand life's complex molecular machines by studying these networks. This thesis addresses tree open problems centered upon their analysis and quality assessment.
First, we introduce power graph analysis as a novel approach to the representation and visualization of biological networks. Power graphs are a graph theoretic approach to lossless and compact representation of complex networks. It groups edges into cliques and bicliques, and nodes into a neighborhood hierarchy. We demonstrate power graph analysis on five examples, and show its advantages over traditional network representations. Moreover, we evaluate the algorithm performance on a benchmark, test the robustness of the algorithm to noise, and measure its empirical time complexity at O (e1.71)- sub-quadratic in the number of edges e.
Second, we tackle the difficult and controversial problem of data quality in protein interaction networks. We propose a novel measure for accuracy and completeness of genome-wide protein interaction networks based on network compressibility. We validate this new measure by i) verifying the detrimental effect of false positives and false negatives, ii) showing that gold standard networks are highly compressible, iii) showing that authors' choice of confidence thresholds is consistent with high network compressibility, iv) presenting evidence that compressibility is correlated with co-expression, co-localization and shared function, v) showing that complete and accurate networks of complex systems in other domains exhibit similar levels of compressibility than current high quality interactomes.
Third, we apply power graph analysis to networks derived from text-mining as well to gene expression microarray data. In particular, we present i) the network-based analysis of genome-wide expression profiles of the neuroectodermal conversion of mesenchymal stem cells. ii) the analysis of regulatory modules in a rare mitochondrial cytopathy: emph{Mitochondrial Encephalomyopathy, Lactic acidosis, and Stroke-like episodes} (MELAS), and iii) we investigate the biochemical causes behind the enhanced biocompatibility of tantalum compared with titanium.
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Genetic architecture of the interactions between English oak (Quercus robur L.) and the microbial community of its phyllosphere / Architecture génétique des interactions entre le chêne pédonculé (Quercus robur L.) et les communautés microbienne de sa phyllosphèreJakuschkin, Boris 15 December 2015 (has links)
De nombreux et divers micro-organismes vivent dans les tissus interne et externe desfeuilles des plantes, la phyllosphère. Ils influencent de nombreux traits, les interactions biotiques,le flux d’énergie, la tolérance au stress de leur hôte et en fin de compte la valeur sélectivede leurs hôtes. Il a été montré que plusieurs traits quantitatifs de plantes structurentla communauté microbienne de la phyllosphère. Ainsi des Loci de ces traits quantitatifs(Quantitative Trait Loci QTL) liés à la structure de cette communauté étaient attendus.L’objectif principal de ce travail était de rechercher des régions génomiques chez le chêne(Quercus robur L.), dont l’effet se prolonge jusqu’au niveau de la communauté, influençantainsi le microbiote de la phyllosphère. Tout d’abord, nous avons étudié la composition etle réseau d’interactions du microbiote de la phyllosphère partant un intérêt particulier àErysiphe alphitoides, un agent pathogène majeur pour les chênes. Nous avons montré quel’infection par E. alphitoides est accompagnée par des changements dans la composition dela communauté fongique foliaire, mais pas dans le composition de la communauté bactérienne.Nous avons souligné certains partenaires d’interaction d’E. alphitoides et nous avonsmontré que le réseau d’interactions microbiennes, contrairement aux résultats précédents,été dominé par des interactions positives. Ensuite nous avons effectué une analyse QTLde descripteurs de la communauté microbienne dans une population de pleins frères. Nousavons trouvé 8 QTL correspondant à des traits de la communauté microbienne: compositionfongique et bactérienne, diversité fongique, pourcentage de Erysiphe alphitoides. Troisd’entre eux sont colocalisés avec un QTL de sensibilité à E. alphitoides, suggèrent un fortdéterminisme génétique de la résistance à l’oïdium chez Q. robur. Enfin, nous présentonsles résultats préliminaires d’une étude d’association génétique et discutons nos résultatsavec une perspective évolutive. / Numerous and various microorganisms inhabit inner and outer tissues of plant leaves, thephyllosphere. They influence many plant traits, biotic interactions, energy flux, host stresstolerance and ultimately the fitness of their hosts. Many plant quantitative traits wereshown to structure the phyllosphere microbial community. Hence quantitative trait loci(QTLs) linked to the structure of this community were expected. The main objective ofthis work was to search for genomic regions in oak (Quercus robur L.), whose effect extendsto the community level, influencing the phyllosphere microbiota. First, we studied thecomposition and the interaction network of oak phyllosphere microbiota with specific focuson Erysiphe alphitoides, a major biotrophic pathogen of oak. We showed that infection byE. alphitoides is accompanied by changes in the foliar fungal community composition butnot in the bacterial community composition. We highlighted likely interaction partners ofE. alphitoides and we showed that the complex microbial interaction network, in contrastto previous findings, was dominated by positive interactions. Next we performed QTLanalysis of microbial community descriptors in a full-sib mapping population of oak. Wefound 8 QTLs for microbial community traits: fungal and bacterial composition, fungaldiversity, and percentage of Erysiphe alphitoides reads. Three of these QTLs colocalizedwith a QTL for powdery mildew sensibility, suggesting for strong genetic determinism ofpowdery mildew resistance in Q. robur. Finally we present preliminary results of a geneticassociation study and discuss our findings within an evolutionary perspective.
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Écologie et conservation des abeilles sauvages le long d'un gradient d'urbanisation / Ecology and conservation of wild bees along an urbanization gradientFortel, Laura 01 October 2014 (has links)
Depuis des années, on observe un déclin des insectes pollinisateurs. La perte d’habitats naturels, en partie liée àl’urbanisation, est considérée comme l’une des causes majeures de ce déclin. Des populations d’abeilles(Hymenoptera : Anthophila) se maintiennent cependant en milieux urbains. La structure de leurs communautés,ainsi que leurs comportements de butinage et de nidification peuvent être affectés par les perturbations liées àl’urbanisation. Notre objectif était d’évaluer l’ampleur de ces modifications et de comprendre leurs mécanismes enutilisant 24 sites dans le Grand Lyon localisés selon un gradient d’urbanisation croissante (mesurée par laproportion de surfaces imperméables). Nous avons analysé les réseaux d’interactions pour étudier les relationsflore-abeilles et nous avons mis en place des aménagements pour la nidification (carrés de sol et hôtels à abeilles)sur 16 sites urbains ou périurbains pour étudier la dynamique de nidification et son impact sur les populations.Nous avons capturé 16352 spécimens appartenant à 293 espèces, soit près du tiers de la faune françaised’abeilles. Les sites périurbains (avec environ 50% de surface imperméable) avaient la plus grande richessespécifique. Les abeilles à langue longue et les abeilles cavicoles étaient plus présentes dans les milieux urbanisés,mais la spécialisation des interactions plantes-abeilles est restée stable le long du gradient d’urbanisation. Ladiversité spécifique des abeilles était associée de façon positive avec la diversité florale, la durée de floraison desespèces végétales et/ou leur floribondité. Les communautés d’abeilles étaient dépendantes de ces trois facteurs etaussi des plantes spontanées (natives ou naturalisées) plus que des plantes horticoles (ornementales ou exotiques).Enfin, les aménagements pour la nidification ont été colonisés par une faune d’abeilles diversifiée. Hormis Osmiabicornis, les abeilles ne présentaient aucune préférence quant aux substrats dans lesquels elles nidifiaient. Même sil’urbanisation change la structure des communautés d’abeilles, nos résultats confirment qu’une diversité importanted’abeilles sauvages peut perdurer dans des milieux moyennement, mais aussi fortement urbanisés.Dans un contexte d’urbanisation croissante et de déclin des abeilles, il semble indispensable de mettre en placedes plans de gestion en faveur de ces insectes pollinisateurs sauvages en agissant sur l’étendue des surfaces fleuries,les continuités écologiques entre ces surfaces, et une gestion plus appropriée des parcs, jardins et espaces vertsurbains. La présence d’une grande diversité d’espèces y compris dans des milieux très anthropisés fait vraiment desabeilles un groupe phare pour sensibiliser les citoyens à la biodiversité et aux services écosystémiques / Evidence has been accumulating for years that pollinator populations are declining. The loss of natural habitats,in part linked to urbanization, is considered to be one of the major causes of this decline. Some bee populationspersist nevertheless in urban environments. The structure of their communities, as well as their foraging and nestingbehaviors can be affected by urbanization. Our objective was to assess the magnitude of these changes and tounderstand their mechanisms by using 24 sites located in the Grand Lyon along a gradient of urbanization(measured by the proportion of impervious surface). We analyzed interaction networks to investigate plant-beerelations, and man-made nesting structures on urban sites (squares of soil and bee hotels) to study nesting dynamicsand its relevance for bee populations.We collected 16,352 specimens belonging to 293 species. Periurban environments (i.e., with ca. 50% ofimpervious surface) had the greatest diversity of bees. Long-tongued bees and cavity nesting bees were moreprevalent in urban environments, but the specialization of plant-bee interactions remained stable along the gradientof urbanization. The species richness of the bee community was positively associated with floral diversity, theduration of flowering of plant species and/or their floribundity. Bee communities depended on these three factorsand also spontaneous (native and naturalized) plant species more than on horticultural plants (ornamentals andexotics). Finally, man-made nesting sites were colonized by a diverse bee fauna. Apart from Osmia bicornis, beesshowed no preference for the substrates in which they nested. Our work confirms that, even if urbanization changesthe structure of the bee communities, an important diversity of bee species can persist in periurban, but also urbanenvironments.In a context of increasing urbanization and declining bee populations, it appears essential to create managementplans for these wild pollinators by acting on the surface of flowering areas, the ecological network linking them,and the appropriate management of parks, gardens, and recreational areas. The presence of a diverse array of beespecies even in the most urbanized area makes these pollinators worthy of being a flagship group to raise theawareness of citizens about biodiversity and ecosystemic services
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Structure et interactions de la lysyl oxydase et de fragments de la matrice extracellulaire / Structure and interactions of lysyl oxidase and extracellular matrix fragmentsVallet, Sylvain D. 14 December 2018 (has links)
La matrice extracellulaire est un réseau tridimensionnel complexe qui joue le rôle de support aux cellules ainsi que de réservoir de molécules bioactives régulant le comportement cellulaire. Elle est composée de 1027 protéines chez l’Homme (Naba et al., Matrix Biol. 2016), 274 protéines constituant le matrisome et 753 associées (facteurs de croissance et protéines régulatrices de la matrice extracellulaire) et de 6 glycosaminoglycanes dont 5 sulfatés. La matrice extracellulaire est impliquée dans de nombreuses pathologies (Bonnans et al., Nat. Rev. Mol. Cell Biol. 2014). La lysyl oxydase, responsable de la réticulation des collagènes et de l’élastine est impliquée dans de nombreux cancers. La matrice extracellulaire est un réservoir de fragments bioactifs, nommés matricryptines, qui sont libérés par protéolyse des biomolécules de la matrice et régulent de nombreux processus biologiques tels que l’angiogenèse et l’adipogenèse (Ricard-Blum et Vallet Matrix Biol. 2017). Nous avons exprimé en cellules humaines plusieurs matricryptines dont les ectodomaines des collagènes membranaires XIII, XVII, XXIII et XXV et identifié leurs partenaires extracellulaires. Nous avons caractérisé le propeptide de la lysyl oxydase par SEC-MALS, diffusion dynamique de la lumière et par SAXS et avons modélisé à partir des données de SAXS sa structure tridimensionnelle. Nous avons identifié 17 nouveaux partenaires de ce fragment et analysé le mutant Arg158Gln dépourvu d’activité biologique. Cette mutation identifiée chez l’Homme inhibe les activités anti-prolifératives du propeptide et est associée à un risque accru de cancer du sein (Min et al., Cancer Res. 2009). Nous avons exprimé la lysyl oxydase mature et modélisé sa structure tridimensionnelle en utilisant toutes les données disponibles. Les interactions identifiées au cours de ce travail ont été associées à celles obtenues par curation manuelle de la littérature pour construire la première version de l’interactome extracellulaire humain / The extracellular matrix is an intricate tridimensional network supporting cells and a bioactive molecule reservoir involved in the regulation of cell behavior. It is composed of 1027 proteins in humans (Naba et al., Matrix Biol. 2016), including 274 of the core matrisome and 753 associated proteins (growth factors and extracellular matrix regulators) and 6 glycosaminoglycans including 5 sulfated. The extracellular matrix is altered in numerous pathologies (Bonnans et al., Nat. Rev. Mol. Cell Biol. 2014). The lysyl oxidase is responsible for the cross-linking of collagens and elastin and is involved in many cancers. The extracellular matrix is a reservoir of bioactive fragments named matricryptins which are released by proteolysis of extracellular matrix proteins and regulate numerous biological processes like angiogenesis and adipogenesis (Ricard-Blum et Vallet, Matrix Biol. 2017). We have expressed under a recombinant form in human cells some matricryptins including the ectodomains of the membrane collagens XIII, XVII, XXIII and XXV and have identified their extracellular partners. We have characterized the propeptide of lysyl oxidase by SEC-MALS, dynamic light scattering, and SAXS and have built a coarse-grained 3D model by SAXS-derived constraints. We have identified 17 new partners of this fragment and analyzed the mutant Arg158Gln which has no biological activity. This mutation has been identified in humans and inhibits the propeptide anti-proliferative properties. It is associated to an increased risk of breast cancer (Min et al., Cancer Res. 2009). We have expressed the mature lysyl oxidase and modelled its tridimensional structure using available data. All the interactions identified in this study were associated to manually curated interactions described in the literature to build the first version of the human extracellular interactions network
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