Dissertations / Theses on the topic 'Spatial hybridization'

Hybridization and introgression with non-native salmonids is one of the greatest factors threatening native cutthroat trout species. Westslope cutthroat trout (Oncorhynchus clarkii lewisi; WSCT) were recently listed under the Canadian Species at Risk Act (SARA) as "special concern" (British Columbia populations) and "threatened" (Alberta populations). I employed a 10 locus-microsatellite DNA assay to investigate levels of hybridization between westslope cutthroat trout and introduced rainbow trout (O. mykiss; RT) at 159 sampling locations in southwestern Alberta and parts of southeastern British Columbia. My results revealed that hybridization is extensive across the region sampled. Admixture levels (qwsct of 0 = pure rainbow trout, 1.0 = pure westslope cutthroat trout) at sampling locations ranged from 0.01 to 0.99. An average qwsct below 0.99 is a criterion that has been used in previous work to designate a population as "hybridized." Landscape genetic analysis using regression trees indicate that water temperature, elevation, distance to the nearest stocking site and distance to the nearest railway were significant components of a model that described 34% of the variation in qwsct across 58 sites for which habitat variables were available. Building on this finding, I explored the role of water temperature, the best predictor of hybridization levels amongst the variables tested, in limiting the spread of admixture by evaluating cold tolerance in both species using critical thermal methods (CTM). Analysis of variance revealed a statistically significant difference between the critical thermal minima (CTMin) of WSCT and RT acclimated to 15 °C (1.0 ± 0.8 °C and 1.4 ± 1.0 °C respectively). The heritability of cold tolerance observed in this study appears to be complex and does not seem to behave in a simple additive manner. The identification of water temperature as a major factor influencing admixture and subsequent test for physiological differences in cold tolerance provide evidence to support a hypothesis that cold water habitats act as a natural barrier to hybridization between WSCT and RT. This information provides insight into the evolutionary history of WSCT and RT and will be useful in assisting conservation efforts aimed at mitigating the wide-spread loss of WSCT to genomic extinction.

It has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this thesis was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems and structures involved. This work required the development of novel in situ hybridization methods for use with electron microscopy. This allowed resolution to visualize single mRNA molecules and individual filaments. The development of a silver enhancement methodology for both the light and electron microscopic detection of biotinated oligo-dT probes permitted a synoptic view of the intracellular distribution of poly(A) mRNA. At the light microscope, the distribution of poly(A) mRNA did not resemble the individual distribution patterns of microfilaments, intermediate filaments or microtubules. Ultrastructural examination revealed that poly(A) mRNA was not uniformly distributed along cytoskeletal filaments, but clustered at their intersections. The composition of these mRNA containing structures was investigated by both morphologic and in situ hybridization analysis using antibodies to cytoskeletal proteins. In thin sections, polysomes were observed attached to both microfilaments and intermediate filaments. To permit the simultaneous detection of oligo-dT hybridization and specific cytoskeletal proteins, a double labelling method using colloidal gold conjugated antibodies was developed. The majority of poly(A) mRNA was associated with the actin cytoskeleton, with 72% of the hybridization localized within 5nm of a labelled microfilament. Within the actin cytoskeleton, poly(A) mRNA was localized to intersections of orthogonal networks. Greater than 50% of poly(A) colocalized with the actin crosslinking proteins, filamin and α-actinin, but not vinculin. A significant amount of poly(A) mRNA was found to be associated with intermediate filaments. The double label gold analysis demonstrated that 33% of the hybridization signal localized within 5nm of labelled vimentin filaments. Prior disorganization of the actin cytoskeleton using cytochalasin did not disrupt the association of mRNA with vimentin. These observations are consistent with our morphologic results of polysome-intermediate filament associations, and indicate that microfilaments are not the only filament system to which mRNA is bound. Furthermore, a small amount of hybridization signal (12%) consistently was observed along microtubules, providing an additional cytoskeletal network to distribute mRNA. To further characterize the spatial organization of mRNA within the cytoskeleton, ultrastructural methods were developed to directly visualize individual mRNA molecules. First, oligonucleotide probes chemically modified with a single hapten and directly conjugated primary reagents were used to permit detection of an individual hybridized probe molecule by a single gold particle. Second, biotin and digoxigenin labelled oligonucleotide probes were used to simultaneously visualize the intermolecular and intramolecular relationships of two nucleic acid sequences. Third, reverse transcriptase was used to extend hybridized primers in situ which permitted visualization of the poly(A) sequence concomittant with the conformation of an mRNA molecule. These methods have permitted analysis of how single mRNA molecules may be positioned with respect to each other within the cytoskeleton. The ultrastructural visualization of mRNA within its structural environment has demonstrated heterogeneous interactions with the cytoskeleton. Future work will be needed to further characterize the mechanism of mRNA attachment. The proteins which bridge nucleic acid sequences to specific intersections can be identified. It will be interesting to learn how the identified mRNA-cytoskeletal interactions might be involved in the regulation of both mRNA translation and intracellular location. Lastly, and perhaps the most challenging goal, is to investigate whether the identified mRNA-cytoskeletal interactions are used by the cell to influence its own shape, polarity and architecture.

The methods presented in this study are aimed at the identification of subpopulations (clones) of genetically similar cells within tissue samples through measurement of loci-specific Fluorescence in-situ hybridization (FISH) spot signals for each nucleus and analyzing cell spatial distributions by way of Voronoi tessellation and Delaunay triangulation to robustly define cell neighbourhoods. The motivation for the system is to examine lung cancer patient for subpopulations of Non-Small Cell Lung Cancer (NSCLC) cells with biologically meaningful gene copy-number profiles: patterns of genetic alterations statistically associated with resistance to cis-platinum/vinorelbine doublet chemotherapy treatment. Current technologies for gene-copy number profiling rely on large amount of cellular material, which is not always available and suffers from limited sensitivity to only the most dominant clone in often heterogeneous samples. Thus, through the use of FISH, the detection of gene copy-numbers is possible in unprocessed tissues, allowing identification of specific tumour clones with biologically relevant patterns of genetic aberrations. The tissue-wide characterization of multiplexed loci-specific FISH signals, described herein, is achieved through a fully automated, multicolour fluorescence imaging microscope and object segmentation algorithms to identify cell nuclei and FISH spots within. Related tumour clones are identified through analysis of robustly defined cell neighbourhoods and cell-to-cell connections for regions of cells with homogenous and highly interconnected FISH spot signal characteristics. This study presents experiments which demonstrate the system’s ability to accurately quantify FISH spot signals in various tumour tissues and in up to 5 colours simultaneously or more through multiple rounds of FISH staining. Furthermore, the system’s FISH-based cell classification performance is evaluated at a sensitivity of 84% and specificity 81% and clonal identification algorithm results are determined to be comparable to clone delineation by a human-observer. Additionally, guidelines and procedures to perform anticipated, routine analysis experiments are established.

La numérisation a déclenché une révolution dans l'étude de la Géographie, marquant un tournant décisif. La nécessité de comprendre les relations complexes entre la géographie et les technologies numériques devient de plus en plus évidente. Les nouvelles technologies ont transformé l'espace géographique, affectant en particulier la Géographie Humaine. L'avancement des Technologies de l'Information et de la Communication (TIC) a placé le numérique au centre de l'attention, devenant, comme l'indique Gillian Rose, à la fois objet et sujet de recherche, modifiant ainsi la pratique géographique et déclenchant un débat continu autour du "virage numérique".Ce virage a fourni de nouveaux outils à la Géographie pour relever les défis contemporains, transformant notre conception de l'espace en une entité qui fusionne des éléments tangibles et intangibles. La numérisation a créé des réalités hybrides où le physique et le numérique se mêlent, se connectent et se floutent, remettant en question les notions traditionnelles de territorialité et d'échelle. Cela oblige à revoir et à mettre à jour les approches géographiques, en développant de nouvelles méthodologies pour comprendre ces territoires hybrides et comment les étudier.La numérisation a ouvert de nombreuses possibilités et interrogations pour la science géographique, qui nécessitent des réponses d'une perspective intégrale et collaborative, en intégrant plusieurs disciplines. Tout d'abord, une étude exhaustive de l'état de l'art est réalisée, justifiant l'inclusion des concepts classiques de la Géographie et leur évolution dans le cadre des révolutions méthodologiques actuelles. Une "Généalogie des Géographies Numériques" est présentée, différenciant les concepts techniques liés à l'espace numérique et réfléchissant sur l'hybridation entre les espaces physique et numérique, ainsi que la redéfinition du rôle de la distance dans ce nouveau contexte.La thèse se concentre également sur l'application pratique de quatre études de cas de Géographie Humaine dans des environnements numériques, montrant comment le "virage numérique" a impacté la discipline. Ces recherches sont classées selon une taxonomie tripartite qui couvre les géographies générées, produites et propres au numérique. La Géographie Quantitative est reconnue comme précurseur de ces nouvelles formes d'analyse spatiale, adoptant des technologies numériques pour explorer l'espace.L'espace numérique est présenté comme une extension de l'espace relationnel, soulignant l'importance de la dualité spatiale et la redéfinition du concept de distance à l'ère numérique. L'immersion dans le numérique peut conduire à une fusion totale entre les espaces physique et numérique, où les individus expérimentent une perception sensorielle complète du lieu numérique. En s'intégrant dans les éléments numériques, les individus acquièrent un sentiment d'identité et d'appartenance, interagissent socialement, établissent des relations et ressentent des émotions dans cet espace hybride
Digitalization has triggered a revolution in the study of Geography, marking a decisive turning point. The need to understand the complex relationships between geography and digital technologies is increasingly evident. New technologies have transformed geographical space, particularly affecting Human Geography. The advancement of Information and Communication Technologies (ICT) has placed digital technology at the forefront, becoming, as Gillian Rose points out, both the object and subject of research, thus altering geographical practice and sparking a continuous debate around the "digital turn."This turn has provided Geography with new tools to address contemporary challenges, transforming our conception of space into an entity that merges tangible and intangible elements. Digitalization has created hybrid realities where the physical and digital intertwine, connect, and blur, challenging traditional notions of territoriality and scale. This necessitates a revision and update of geographical approaches, developing new methodologies to understand these hybrid territories and how to study them.Digitalization has opened many possibilities and questions for geographical science, requiring responses from an integral and collaborative perspective, incorporating multiple disciplines. Firstly, an exhaustive study of the state of the art is conducted, justifying the inclusion of classical geographical concepts and their evolution within the framework of current methodological revolutions. A "Genealogy of Digital Geographies" is presented, differentiating technical concepts related to digital space and reflecting on the hybridization between physical and digital spaces, as well as the redefinition of the role of distance in this new context. The thesis also focuses on the practical application of four case studies in Human Geography in digital environments, showing how the "digital turn" has impacted the discipline. These studies are classified according to a tripartite taxonomy that covers geographies generated, produced, and native to the digital realm. Quantitative Geography is recognized as a precursor to these new forms of spatial analysis, adopting digital technologies to explore space.Digital space is presented as an extension of relational space, highlighting the importance of spatial duality and the redefinition of the concept of distance in the digital age. Immersion in the digital realm can lead to a complete fusion between physical and digital spaces, where individuals experience a full sensory perception of the digital place. By integrating into digital elements, individuals gain a sense of identity and belonging, interact socially, establish relationships, and experience emotions within this hybrid space

Dans cette thèse, nous nous intéressons à l’amélioration du schéma de Yee pour traiter demanière plus efficace et pertinente les problèmes industriels auxquels nous sommes confrontés à l’heureactuelle. Pour cela, nous cherchons avant tout à diminuer les erreurs numériques de dispersion et àaméliorer les modélisations des géométries courbes ainsi que des réseaux de câbles. Pour répondre àces besoins, une solution basée sur un schéma Galerkin discontinu pourrait être envisagée. Toutefois,l’utilisation d’une telle technique sur la totalité du volume de calcul est relativement coûteuse. De plus,la prise en compte de structures filaires sur un tel schéma n’est pas encore opérationnelle. C’est pourquoi,dans l’optique d’avoir un outil industriel, et après une étude bibliographique, nous nous sommes plutôtorientés sur l’étude d’un schéma éléments finis (FEM) sur maillage cartésien qui possède toutes lesbonnes propriétés du schéma de Yee. Notamment, à l’ordre d’approximation spatiale égal à 0 ce schémaFEM est exactement le schéma de Yee, et, pour des ordres supérieurs, il permet de réduire fortementl’erreur de dispersion numérique de ce dernier. Dans le travail de cette thèse, pour ce schéma, nous avons notamment donné un critère de stabilité théorique, étudié sa convergence théorique et fait une analyse de l’erreur de dispersion. Pour tenircompte des possibilités d’ordre d’approximation spatiale variable par direction, nous avons mis en placeune stratégie d’affectation des ordres suivant le maillage donné. Ceci nous a permis d’obtenir un pas detemps optimal pour une précision souhaitée tout en réduisant les coûts de calcul. Après avoir porté ceschéma sur des machines de production, différents problèmes de CEM, antennes, IEM ou foudre ont ététraités afin de montrer les avantages et le potentiel de celui-ci. En conclusion de ces expérimentationsnumériques, il s’avère que la méthode est limitée par le manque de précision pour prendre en comptedes géométries courbes. Afin d’améliorer cela, nous avons proposé une hybridation entre ce schéma et leschéma GD que l’on peut étendre aux autres schémas comme les méthodes différences finies (FDTD) etvolumes finis (FVTD). Nous avons montré que la technique d’hybridation proposée conserve l’énergie etest stable sous une condition que nous avons évaluée de manière théorique. Des exemples de validationont ensuite été montrés. Enfin, pour tenir compte des réseaux de câbles, un modèle de fils minces d’ordred’approximation spatiale élevé a été proposé. Malheureusement, celui-ci ne peut pas couvrir l’ensembledes cas industriels et pour remédier à cela, nous avons proposé une hybridation de notre approche avecune équation de ligne de transmission. L’intérêt de cette hybridation a été montré sur un certain nombred’exemples, que nous n’aurions pas pu traiter par un modèle de structure filaire simple
In this thesis, we study the improvement of the Yee’s scheme to treat efficiently and in arelevant way the industrial issues we are facing nowadays. For that, we first of all try to reduce thenumerical errors of dispersion and then to improve the modeling of the curved surfaces and of theharness networks. To answer these needs, a solution based on a Galerkin Discontinuous (GD) methodhas been first considered. However, the use of such method on the entire modeling volume is quite costly ;moreover the wires are not taken into account in this method. That is the reason why, with the objectiveof an industrial tool and after a large bibliographic research, we headed for the study of finite elementsscheme (FEM) on a Cartesian mesh which has all the good properties of the Yee’s scheme. Especially,this scheme is exactly the Yee’s scheme when the spatial order of approximation is set to zero. Forthe higher orders, this new scheme allows to greatly reduce the numerical error of dispersion. In theframe of this thesis and for this scheme, we give a theoretical criterion of stability, study its theoreticalconvergence and we perform an analysis of the error of dispersion. To take into account the possibilityof the variable spatial orders of approximation in each direction, we put in place a strategy of orderaffectation according to the given mesh. This strategy allows to obtain an optimal time step for a givenselected precision while reducing the cost of the calculations. Once this new scheme has been adaptedto large industrial computing means, different EMC, antennas, NEMP or lightning problems are treatedto demonstrate the advantages and the potential of this scheme. As a conclusion of these numericalsimulations we demonstrate that this method is limited by a lack of precision when taking into accountcurved geometries. To improve the treatment of the curved surfaces, we propose an hybridization between this scheme andthe GD scheme. This hybridization can also be applied to other methods such as Finite Differences(FDTD) or Finite Volumes (FVTD). We demonstrate that the technique of hybridization proposed,allows to conserve the energy and is stable under a condition that we study theoretically. Some examplesare presented for validation. Finally and to take into account the cables, a thin wire model with a highorder of spatial approximation is proposed. Unfortunately, this model does not allow to cover all theindustrial cases. To solve this issue we propose an hybridization with a transmission line method. Theadvantage of this hybridization is demonstrated thanks to different cases which would not have beenfeasible with a more simple thin wire method

Axolotl uniquely generates blastema cells as a pool of progenitor/stem cells to restore an entire limb, a particular property that other organisms, such as humans, do not have. What underlies these differences? Is the main difference that cells residing at the amputation plane (in the stump) undergo reprogramming processes to re-enter the embryonic program, which allows developmental patterning to start, or are there fundamental differences? There is also a significant debate about whether regeneration occurs via stem cell differentiation or by dedifferentiation of mature limb tissue. The aim of my thesis was to address following questions: Are the cells in the blastema reprogrammed or differentiated to regenerate? Are the blastema cells genetically reactivated de novo during regeneration? How does the amputated limb exactly know which part of the limb needs to be regenerate? Using a novel technique of long-term genetic fate mapping, my team demonstrated that dedifferentiation in regenerated axolotl muscle tissue does not occur. Instead, PAX7+ satellite cells indeed play an important role during muscle regeneration in the axolotl limb. Surprisingly, this is in contrast to the newt, which regenerates muscle cells through a dedifferentiation process. Therefore, there is a fundamental difference that underlies the regenerative mechanism ((Sandoval-Guzman et al., 2014) [KR1]). This demonstrates that there is an unexpected diversity and flexibility of cellular mechanims used during limb regeneration, even among two closely related species. Finally, if one salamander species uses a mammalian regenerative strategy (Cornelison and Wold, 1997; Collins et al., 2005) involving stem cells and another uses a dedifferentiative strategy, this raises the question of whether there are other fundamental aspects of regeneration that could also be anomalous. This hypothesis is promising since there could be more than one possible mechanism to induce mammalian regeneration. The process of limb regeneration in principle seems to be more similar to those of limb development as historically assumed. We showed molecularly that embryonic players are reused during regeneration by reactivating the position- and tissue-specific developmental gene programs by using the newly isolated Twist sequences as early blastema cell markers ((Kragl et al., 2013) [KR2]). To gain insights into the molecular mechanisms of the P/D limb patterning in general, it was crucial to study the early patterning events of the resident progenitor/stem cells by using the specific blastema cell marker HoxA as a positional marker along the proximo-distal axis. Our HOXA protein analysis using high molecular and cellular resolution as well as transplantation assays demonstrated for the first time that axolotl limb blastema cells acquire their positional identity in a proximal to distal sequence. We found a hierarchy of cellular restrictions in positional identities. Amputation at the level of the upper arm showed that the blastema harbors cells, which convert to lower arm and hand. We observed ((Roensch et al., 2013) [KR3]) for the first time that intercalation- the intermediate element (lower arm) arises later from an interaction between the proximal and distal cells identities- does not occur. Intercalation, which has been an accepted model for a long time, is not the patterning mechanism underlying normal (without any manipulation) limb regeneration that is unique to axolotl. We further demonstrated, using the Hox genes as markers that positional identity is cell-type specific since their effects were confirmed to be present in the lateral plate mesoderm- derived cells of the limb. As our knowledge about limb blastemas expands concerning cell composition and molecular events controlling patterning, the similarity to development is becoming more and more clear. My work has resolved many ambiguities surrounding the molecularly identification of different types of blastema cells and how P/D limb patterning occurs during regeneration in comparison to development. It has highlighted the importance of combining high-resolution methods, such as in situ hybridizations, single-cell PCR (sc-PCR) of individual dissociated blastema cells and genetic labeling methods with grafting experiments to map cell fates in vivo. In addition to understanding the processes of regeneration, another long-term goal in the regenerative medicine field is to identify key molecules that trigger the regeneration of tissues. Recently, my colleague Takuji Sugiura (Sugiura et al., 2016) observed that an early event of blastema formation is the secretion of molecules like MLP (MARCKS-like protein), which induces wound-associated cell cycle re-entry. Such findings further increase the enthusiasm of biologists to understand the underlying principles of regeneration. By building our knowledge of the molecules and pathways that are involved in tissue regeneration, we increase the possibility of identifying a way to ‘activate’ regenerative processes in humans and thus reach the final goal of regenerative medicine, which is to use the concepts of cellular reprogramming, stem cell biology and tissue engineering to repair complex body structures.

L'infection des baies de raisin par le champignon Botrytis cinerea, responsable de la pourriture grise, est fréquente et occasionne des dégâts importants. Pourtant, il semble que la baie dispose de moyens de défense parmi lesquels des protéines PR "Pathogenesis-Related". Chez le Pinot Noir, une chitinase (CHI4D) et une thaumatin-like (TL3) s'accumulent naturellement en grande quantité à partir de la véraison et présentent une activité antifongique contre B. cinerea in vitro. L'objectif de ce travail est de comprendre comment B. cinerea peut se développer sur des baies censées disposer de défenses suffisantes. Pour cela, l'expression spatio-temporelle des ARNm et des protéines CHI4D et TL3 a été suivie respectivement par hybridation in situ et immunohistolocalisation dans les baies, au cours de la maturation, à la suite d'un stress abiotique (UV-C) ou d'un stress biotique (B. cinerea). Dans des baies avant véraison (vertes), n'exprimant naturellement que très faiblement CHI4D et TL3, les ARNm et les protéines s'accumulent en grande quantité après application d'un stress abiotique (UV-C) ou biotique (infection artificielle par B. cinerea). Les protéines CHI4D et TL3 sont localisées au niveau des faisceaux conducteurs ainsi qu'au niveau des tissus proches des sites d'exposition aux UV-C (exocarpe) ou au niveau des sites d'inoculation de B. cinerea, suggérant qu'elles sont impliquées dans la défense de la baie avant véraison. Après véraison, les ARNm et les protéines sont naturellement accumulés au niveau de l'exocarpe et des faisceaux conducteurs qui correspondent à des sites potentiels d'entrée ou de propagation des agents pathogènes. Alors que l'application d'un stress UV-C sur ces baies ne provoque qu'un effet mineur sur l'expression de CHI4D et de TL3, au cours de l'infection par B. cinerea, les quantités de transcrits et de protéines diminuent. A un stade précoce d'infection, la diminution de la quantité des deux protéines est observée en avant du front de propagation du champignon, suggérant une dégradation par des protéases sécrétées par B. cinerea. A un stade d'infection plus avancé, cette diminution s'étend à l'ensemble de la baie. La production hétérologue des protéines CHI4D et TL3 nous a permis de confirmer que CHI4D pouvait être dégradée par des protéases aspartiques sécrétées par B. cinerea. La dégradation de TL3 n'a pas pu être reproduite in vitro. Des tests antigerminatifs effectués in vitro avec les protéines hétérologues n'ont pas permis de mettre en évidence d'effet antifongique malgré la présence d'une activité chitinase pour CHI4D et β-1,3,-glucanase pour TL3. Il est donc possible que ces protéines possèdent des fonctions autres que celles impliquées dans la défense
Grape berries infection by the phytopathogenic fungus Botrytis cinerea, the causal agent of gray mold, is quite common and causes significant damage. However, it seems that berries have a mechanism of defense, among which are pathogenesis related proteins. In Pinot Noir grape berries, a chitinase (CHI4D) and a thaumatin-like (TL3) protein naturally accrue in large amounts from véraison and show in vitro an antifungal effect against B. cinerea. The aim of this work was to understand how B. cinerea can develop on grape berries that seem to have sufficient defense mechanisms. To do so, the spatio-temporal expression of CHI4D and TL3 mRNAs and proteins in berries was studied respectively by in situ hybridization and immunohistolocalization during maturation, after an abiotic stress (UV-C) or a biotic stress (B. cinerea). Before véraison (green berries) while the expression of CHI4D and TL3 is naturally low, mRNAs and proteins have accumulated in large amounts in berries after UV-C exposition or artificial infection with B. cinerea. CHI4D and TL3 proteins have accumulated around vascular bundles as well as near the sites of UV-C exposition (exocarp) or B. cinerea inoculation, suggesting that before véraison these proteins could be involved in the berry defense. After veraison, mRNAs and proteins naturally accumulate in the exocarp and around vascular bundles that correspond to potential sites of penetration or propagation of pathogenic agents. While the application of UV-C stress on these berries causes only a minor effect on the expression of CHI4D and TL3, during infection by B. cinerea, the amounts of mRNA and proteins decreased. At an early stage of infection, the less amounts of both proteins were observed around the fungus propagation area, suggesting that these proteins could be degraded by B. cinerea secreted proteases. At a more advanced stage of infection, the decrease extended to the entire berry.Production of heterologous CHI4D and TL3 proteins allowed us to confirm that CHI4D could be degraded by aspartic proteases secreted by B. cinerea whereas no degradation of TL3 could be observed in vitro. Both heterologous proteins showed no antifungal effect while a chitinase and a β-1,3-glucanase activities were observed respectively for CHI4D and TL3. It is therefore possible that these proteins have other functions than those involved in the defense

Cette thèse s'inscrit dans le cadre de l'optimisation d'agencement, une étape importante dans la conception de systèmes multidisciplinaires complexes tels que les véhicules aérospatiaux. Les problèmes d'agencement optimal (OLP) consistent à trouver la meilleure disposition d'un ensemble de composants dans un système ou un espace, afin d'atteindre certains objectifs (réduction des coûts, amélioration des performances, etc.) tout en satisfaisant diverses contraintes (géométriques, fonctionnelles, etc.). Le traitement des OLP est encore un défi aujourd'hui, tant en termes de formulation que de résolution. En effet, les OLP sont souvent très contraints et impliquent de nombreuses variables de décision (continues, discrètes, catégorielles), qui peuvent être fixes ou conditionnelles. Les variables conditionnelles sont utiles pour définir différents choix de conception qui doivent être faits en même temps que l'optimisation de l'agencement des composants. Ainsi, la résolution des OLP nécessite l'utilisation d'algorithmes d'optimisation avancés combinant différentes catégories de méthodes, comme par exemple les métaheuristiques et l'optimisation bayésienne.L'objectif global de la thèse est d'étudier les OLP, leur formulation dans différents contextes, leur résolution à l'aide de diverses méthodes d'optimisation et hybridations, ainsi que la validation de ces méthodes dans le cadre de la conception de véhicules aérospatiaux. Les contributions de la thèse sont organisées en deux parties correspondant à deux types d'OLP. Dans la première (respectivement deuxième) partie, la liste de composants à agencer est fixe (resp. variable), impliquant des OLP à espace de recherche fixe ou FSS-OLP, (resp. des OLP à espace de recherche conditionnel ou CSS-OLP). Dans les deux cas, le système/l'espace dans lequel les composants sont agencés est considéré comme mono ou multi-contenant.Dans la première partie, une étude des FSS-OLP est proposée, incluant leurs formulations génériques, leurs applications et méthodes de résolution, avec un focus particulier sur les méthodes quasi-physiques et les métaheuristiques. Basés sur un système de force virtuelle (VF), les algorithmes quasi-physiques simulent les lois de la dynamique et traitent efficacement les problèmes fortement contraints. Une variante (nommée CSO-VF) de de ces algorithmes est développée afin de résoudre les FSS-OLP à un seul contenant. Dans CSO-VF, la position et l'orientation des composants évoluent grâce au VF. Pour traiter les systèmes multi-contenants, CSO-VF est hybridé à un algorithme génétique (GA) dans un algorithme à deux étages qui affecte les composants aux contenants puis optimise leur disposition dans chacun des contenants. Ces deux algorithmes sont évalués grâce à des problèmes d'agencement de satellites.Dans la deuxième partie, une étude des CSS-OLP est proposée avec la même approche que dans la première partie. Les variables conditionnelles engendrent des OLP plus complexes. Par exemple, dans le contexte de la conception aérospatiale, une quantité donnée de carburant peut être incluse dans le système, soit dans un grand réservoir, soit dans deux plus petits. Par conséquent, le nombre de composants à positionner n'est pas le même dans les deux cas et le nombre de variables de conception et de contraintes varie donc au cours du processus d'optimisation. Deux approches ont été développées pour traiter les CSS-OLP à un seul contenant : la première est un GA modifié pour introduire des variables cachées dans les chromosomes. La seconde est une approche bi-niveaux combinant optimisation bayésienne et l'algorithme CSO-VF. L'optimisation bayésienne sélectionne les composants et CSO-VF optimise leur agencement. Cette dernière approche a été hybridée avec un GA dans un algorithme tri-niveaux afin de traiter les CSS-OLP multi-contenants. Enfin, tous les algorithmes sont évalués et comparés grâce à des problèmes d'agencement de satellites
This thesis falls within the scope of layout optimization, which is an important stage in the design of complex multidisciplinary engineering systems such as aerospace vehicles. Optimal layout problems (OLPs) involve finding the best arrangement of a set of components within a single- or multi-container system or space to meet specific objectives (cost reduction, performance enhancement, etc.) while satisfying various constraints (geometrical, functional, etc.). Dealing with OLPs is challenging both in terms of their formulation and their efficient and effective resolution. Actually, OLPs are often highly constrained and involve many mixed decision variables (continuous, discrete/categorial) which may be fixed or conditional. Conditional variables are highly useful to define different design choices when the set of components to be arranged is variable and dynamic. Consequently, their resolution requires the use of advanced optimization algorithms combining different classes of (mixed-variable) methods including metaheuristics and Bayesian optimization.The overall objective of the thesis is to investigate OLPs, their formulation in different contexts, their resolution using various optimization methods and their hybridization, and their validation within the framework of aerospace vehicle design. The contributions of the thesis are organized in two parts corresponding to two types of OLPs. In the first (resp. second) part, the set of components to be arranged is fixed (variable or conditional) involving fixed search space OLPs or FSS-OLPs (resp. conditional search space OLPs or CSS-OLPs). In both cases, the system/space in which the components are arranged is considered single- or multi-container.In the first part, a survey of constrained mixed-variable FSS-OLPs is proposed including their generic formulations, applications and resolution methods with a particular focus on quasi-physical methods and population-based metaheuristics. Based on a virtual force system (VF) quasi-physical algorithms emulate the principle of physical laws in system dynamics and deal efficiently with highly constrained problems. A variant (namely CSO-VF) of these algorithms is devised for solving single-container FSS-OLPs. In CSO-VF, the positions and orientations of the components are evolved using VF. To deal with multi-container systems, CSO-VF is combined with a Genetic Algorithm (GA) in a two-stage algorithm that assigns the components to the containers and optimizes their layout. These single- and multi-container algorithms are assessed considering satellite module FSS-OLPs that are representative benchmarks.In the second part, a survey of constrained mixed-variable CSS-OLPs is proposed in the same way than in the first part. Conditional variables involve more complex OLPs. Actually, for instance, in the context of aerospace concept design, a given amount of fuel could be included in a container in either one large tank or two smaller ones. Therefore, as the number of components to position is not the same in both cases the number of design variables as well as constraint functions vary during the optimization process. To deal with single-container CSS-OLPs, two approaches have been investigated: the first one is a GA revisited considering hidden variables, leading to variable-geometry OLPs (in objective and constraint functions). The second approach is a two-stage surrogate guided-CSO-VF algorithm combining Bayesian Optimization with CSO-VF. Bayesian Optimization selects the components with are considered by CSO-VF for layout optimization. This latter approach has been extended with a GA in a three-stage algorithm to tackle multi-container CSS-OLPs. Finally, all the algorithms are evaluated and compared based on their application to CSS variants of satellite module OLPs

Studies of coral-­associated bacterial communities have repeatedly demonstrated that the microbial assemblages of the coral host are highly specific and complex. In particular, bacterial community surveys of scleractinian and soft corals from geographically diverse reefs continually uncover a high abundance of sequences affiliated with the Gammaproteobacteria genus Endozoicomonas. The role of these bacteria within the complex coral holobiont is currently unknown. In order to localize these cells and gain an understanding of their potential interactions within the coral, we developed a fluorescence in situ hybridization(FISH) approach for reef-­building coral tissues. Using a custom small-­subunit ribosomal RNA gene database, we developed two Endozoicomonas-­specific probes that cover almost all known coral-­associated Endozoicomonas sequences. Probe hybridization conditions were quantitatively evaluated against target and non-­target bacterial cultures using fluorescence microscopy. Using these experimentally tested conditions, probes were then hybridized to the branching coral Stylophora pistillata, obtained from the Red Sea, using whole mount and paraffin embedding techniques. This study allowed preliminary spatial exploration and characterization of Endozoicomonas in coral, which has provided insight into their functional role and interactions within the coral holobiont.

Guppies exhibit color based sexual dimorphism and females generally prefer the most colorful males. It has also recently been found that guppies possess a large opsin repertoire. As opsins are the receptors responsible for color vision, this ten gene repertoire might have contributed to the evolution of extravagant male coloration in this species. My study starts by characterizing the opsin repertoire of Jenynsia onca, a noncolorful relative of the guppy belonging to the family Anablepidae (sister group to Poeciliidae, of which the guppy is a member). A PCR based survey indicated that J. onca had a very similar opsin repertoire to the guppy; J. onca had nine genes including orthologs of all but one of the guppy opsins. To gain further insight into the origin of the guppy repertoire, a bioinformatics based survey of ray-finned fish opsins was undertaken. This revealed that large opsin repertoires are common in ray-finned fish and are the product of gene duplication events, spanning the age of the taxon Teleostei. Given that the large opsin repertoire of the guppy did not appear to be perfectly correlated with the evolution of color based sexual selection in this lineage, I turned to investigating the expression of this opsin repertoire. In situ hybridization was used to characterize the pattern of opsin expression across the surface of the retina of adult male and female guppies. In situ hybridization demonstrated that most opsin genes had distinct expression profiles. These expression patterns also indicated that sensitivity and discrimination in the dorsal retina might differ from the ventral retina; the ventral retina appears to be tuned to middle-wavelength light (green), while the dorsal retina is predicted to have exceptional wavelength discriminatory ability and broad spectral sensitivity. This expression data was then used to evaluate models of sexual selection in the context of the predicted visual capacity of the guppy.
Graduate

No
Spatial genome organization in the cell nucleus plays a crucial role in the control of genome functions. Our knowledge about spatial genome organization is relying on the advances in gene imaging technologies and the biochemical approaches based on the spatial dependent ligation of the genomic regions. Fluorescent in situ hybridization using specific fluorescent DNA and RNA probes in cells and tissues with the spatially preserved nuclear and genome architecture (3D-FISH) provides a powerful tool for the further advancement of our knowledge about genome structure and functions. Here we describe the 3D-FISH protocols allowing for such an analysis in mammalian tissue in situ including in the skin. These protocols include DNA probe amplification and labeling; tissue fixation; preservation and preparation for hybridization; hybridization of the DNA probes with genomic DNA in the tissue; and post-hybridization tissue sample processing.

This thesis is based on a qualitative research which draws from three case-studies of different households of alternative buildings. I am focused on those processes which are actively shaping these places regarding mutual human and non-human interactions. The movements of households' material elements are shown through spatial and temporal trajectories. By doing so I refer to the active meaning of materiality which was neglected by social sciences for a long time. The study demonstrates the importance of materiality not only as a significant driver of household's material reality but also of social practices overlapping societal phenomena. Key words semi-structural interview; alternative architecture; household; materiality; quasi- technologies; hybridization; spatial and temporal trajectories

Axolotl uniquely generates blastema cells as a pool of progenitor/stem cells to restore an entire limb, a particular property that other organisms, such as humans, do not have. What underlies these differences? Is the main difference that cells residing at the amputation plane (in the stump) undergo reprogramming processes to re-enter the embryonic program, which allows developmental patterning to start, or are there fundamental differences? There is also a significant debate about whether regeneration occurs via stem cell differentiation or by dedifferentiation of mature limb tissue. The aim of my thesis was to address following questions: Are the cells in the blastema reprogrammed or differentiated to regenerate? Are the blastema cells genetically reactivated de novo during regeneration? How does the amputated limb exactly know which part of the limb needs to be regenerate? Using a novel technique of long-term genetic fate mapping, my team demonstrated that dedifferentiation in regenerated axolotl muscle tissue does not occur. Instead, PAX7+ satellite cells indeed play an important role during muscle regeneration in the axolotl limb. Surprisingly, this is in contrast to the newt, which regenerates muscle cells through a dedifferentiation process. Therefore, there is a fundamental difference that underlies the regenerative mechanism ((Sandoval-Guzman et al., 2014) [KR1]). This demonstrates that there is an unexpected diversity and flexibility of cellular mechanims used during limb regeneration, even among two closely related species. Finally, if one salamander species uses a mammalian regenerative strategy (Cornelison and Wold, 1997; Collins et al., 2005) involving stem cells and another uses a dedifferentiative strategy, this raises the question of whether there are other fundamental aspects of regeneration that could also be anomalous. This hypothesis is promising since there could be more than one possible mechanism to induce mammalian regeneration. The process of limb regeneration in principle seems to be more similar to those of limb development as historically assumed. We showed molecularly that embryonic players are reused during regeneration by reactivating the position- and tissue-specific developmental gene programs by using the newly isolated Twist sequences as early blastema cell markers ((Kragl et al., 2013) [KR2]). To gain insights into the molecular mechanisms of the P/D limb patterning in general, it was crucial to study the early patterning events of the resident progenitor/stem cells by using the specific blastema cell marker HoxA as a positional marker along the proximo-distal axis. Our HOXA protein analysis using high molecular and cellular resolution as well as transplantation assays demonstrated for the first time that axolotl limb blastema cells acquire their positional identity in a proximal to distal sequence. We found a hierarchy of cellular restrictions in positional identities. Amputation at the level of the upper arm showed that the blastema harbors cells, which convert to lower arm and hand. We observed ((Roensch et al., 2013) [KR3]) for the first time that intercalation- the intermediate element (lower arm) arises later from an interaction between the proximal and distal cells identities- does not occur. Intercalation, which has been an accepted model for a long time, is not the patterning mechanism underlying normal (without any manipulation) limb regeneration that is unique to axolotl. We further demonstrated, using the Hox genes as markers that positional identity is cell-type specific since their effects were confirmed to be present in the lateral plate mesoderm- derived cells of the limb. As our knowledge about limb blastemas expands concerning cell composition and molecular events controlling patterning, the similarity to development is becoming more and more clear. My work has resolved many ambiguities surrounding the molecularly identification of different types of blastema cells and how P/D limb patterning occurs during regeneration in comparison to development. It has highlighted the importance of combining high-resolution methods, such as in situ hybridizations, single-cell PCR (sc-PCR) of individual dissociated blastema cells and genetic labeling methods with grafting experiments to map cell fates in vivo. In addition to understanding the processes of regeneration, another long-term goal in the regenerative medicine field is to identify key molecules that trigger the regeneration of tissues. Recently, my colleague Takuji Sugiura (Sugiura et al., 2016) observed that an early event of blastema formation is the secretion of molecules like MLP (MARCKS-like protein), which induces wound-associated cell cycle re-entry. Such findings further increase the enthusiasm of biologists to understand the underlying principles of regeneration. By building our knowledge of the molecules and pathways that are involved in tissue regeneration, we increase the possibility of identifying a way to ‘activate’ regenerative processes in humans and thus reach the final goal of regenerative medicine, which is to use the concepts of cellular reprogramming, stem cell biology and tissue engineering to repair complex body structures.