Despite the possible common histopathological features at diagnosis, cancer cells present within breast carcinomas are highly heterogeneous in their molecular signatures. This heterogeneity is responsible for disparate clinical behaviors, treatment responses and long-term outcomes in breast cancer patients. Although the few histopathological markers can partially describe the diversity of cells found in tumor tissue sections, the full molecular characterization of individual cancer cells is currently impossible in routine clinical practice. In this respect, Fourier transform infrared (FTIR) microspectroscopic imaging of histological sections allows obtaining, for each pixel of tissue images, hundreds of independent potential markers, which makes this technique a particularly powerful tool to distinguish cell types and subtypes. As a complement to the conventional clinicopathological evaluation, this spectroscopic approach has the potential to directly reveal molecular descriptors that should allow identifying different clonal lineages found within a single tumor and therefore provide knowledge relevant to diagnosis, prognosis and treatment personalization. Yet, interpretation of infrared (IR) spectra acquired on tissue sections requires a well-established calibration, which is currently missing. Conventionally, mammary epithelial cells are studied in vitro as adherent two-dimensional (2D) monolayers, which lead to the alteration of cell-microenvironmental interplay and consequently to the loss of tissue structure and function. A number of key in vivo-like interactions may be re-established with the use of three-dimensional (3D) laminin-rich extracellular matrix (lrECM)-based culture systems. The aim of this thesis is to investigate by FTIR imaging the influence of the in vitro growth environment (2D culture versus 3D lrECM culture and 3D monoculture versus 3D co-culture with fibroblasts) on a series of thirteen well-characterized human breast cancer cell lines and to determine culture conditions generating spectral phenotypes that are closer to the ones observed in malignant breast tissues. The reference cell lines cultured in a physiologically relevant basement membrane model and having undergone formalin fixation, paraffin embedding (FFPE), a routine treatment used to preserve clinical tissue specimens, could contribute to the construction of a spectral database. The latter could be ultimately employed as a valuable tool to interpret IR spectra of cells present in tumor tissue sections, particularly through the recognition of unique spectral markers.To achieve the goal, we developed and optimized, in a first step, the preparation of samples derived from traditional 2D and 3D lrECM cell cultures in order to preserve their morphological and molecular relevance for FTIR microspectroscopic analysis. We then highlighted the importance of the influence of the growth environment on the cellular phenotype by comparing spectra of 2D- and 3D-cultured breast cancer cell lines between them. A particular focus was placed to establish a correlation between FTIR spectral data and publicly available microarray-based gene expression patterns of the whole series of breast cancer cell lines grown in 2D and 3D lrECM cultures. Our results revealed that, although based on completely different principles, gene expression profiling and FTIR spectroscopy are similarly sensitive to both the cell line identity and the phenotypes induced by cell culture conditions. We also identified by FTIR imaging changes in the chemical content occurring in the microenvironment surrounding cell spheroids grown in 3D lrECM culture model. Finally, we illustrated the impact of the in vivo-like microenvironment on the IR spectra of breast cancer cell lines grown in 3D lrECM co-culture with fibroblasts and compared them with spectra of cell lines grown in 3D lrECM monoculture. Unsupervised statistical data analyses reported that cells grown in 3D co-cultures produce spectral phenotypes similar to the ones observed in FFPE tumor tissue sections from breast carcinoma patients. Altogether, our results suggest that FFPE samples prepared from 3D lrECM cultures of breast cancer cell lines and studied by FTIR microspectroscopic imaging provide reliable information that could be integrated in the setting up of a recognition model aiming to identify and interpret specific spectral signatures of cells present in breast tumor tissue sections. / Le cancer du sein est une maladie très hétérogène, tant au niveau clinique que biologique. Cette hétérogénéité rend impossible la caractérisation moléculaire complète des cellules cancéreuses individuelles dans la pratique clinique courante. Dans ce contexte, l’imagerie infrarouge à transformée de Fourier (FTIR) des coupes tissulaires permet d'obtenir pour chaque pixel d'une image de tissu des centaines de marqueurs potentiels indépendants, ce qui pourrait faire de cette technique un outil particulièrement puissant pour identifier des différents types et sous-types cellulaires. L'interprétation des spectres infrarouges (IR) enregistrés à partir des coupes histologiques nécessite cependant une calibration qui fait actuellement défaut. Cette calibration pourrait être obtenue à partir de lignées cellulaires tumorales bien caractérisées. Traditionnellement, les cellules épithéliales mammaires sont étudiées in vitro sous forme de monocouches adhérentes bidimensionnelles (2D), ce qui conduit à l'altération de la communication entre les cellules et leur environnement et, par conséquent, à la perte de l’architecture et de la fonction du tissu épithélial. Un certain nombre d'interactions physiologiques clés peuvent être rétablies en utilisant des systèmes de culture tridimensionnelle (3D) dans une matrice extracellulaire riche en laminine (lrECM). L'objectif de cette thèse consiste à étudier par imagerie FTIR l'influence du microenvironnement (via une comparaison entre les cultures 2D et 3D lrECM ou les cultures 3D lrECM en présence ou en l’absence de fibroblastes) sur une série de treize lignées de cellules tumorales mammaires humaines bien caractérisées et à déterminer les conditions de culture générant des phénotypes spectraux qui se rapprochent le plus de ceux observés dans les tissus tumoraux. Au cours de ce travail, nous avons mis au point la culture des lignées cellulaires dans un modèle 3D lrECM ainsi qu’une méthodologie de préparation des échantillons offrant la possibilité de les comparer de manière pertinente avec les cellules cancéreuses présentes dans les coupes histologiques. De même, nous avons étudié par imagerie FTIR les effets du microenvironnement sur les lignées de cellules tumorales et inversement. Pour les lignées investiguées, le passage d’une culture 2D à une culture 3D lrECM s’accompagne, en effet, de modifications du spectre IR étroitement corrélées aux modifications du transcriptome. Les marqueurs spectraux indiquent également que l’environnement 3D génère un phénotype cellulaire proche de celui trouvé dans les coupes histologiques. De manière intéressante, cette proximité est d’autant plus renforcée en présence de fibroblastes dans le milieu de culture. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Made available in DSpace on 2015-03-13T17:08:18Z (GMT). No. of bitstreams: 1 FabioEMR_Capa_ate_Cap3.pdf: 2956379 bytes, checksum: e1a356dd8141f0ac9ce3d477be9710dc (MD5) Previous issue date: 2009-02-27 / This study has as a main objective to make a detailed stratigraphic analysis of the Aptian-Albian interval in the east part of Araripe Basin, NE of Brazil which correspond, litostratigraphically, to Rio Da Batateira, Crato, Ipubi and Romualdo formations. The stratigraphic analysis was based on three different stages, the 1D, 2D and 3D analysis; these ones were adapted to the sequence stratigraphy concepts in order to create a chronostratigraphic framework for the study area within the basin. The database used in the present study contains field and well information, wells that belong to Santana Project, carried out by the Minist?rio de Minas e Energia- DNPM- CPRM from 1977 to 1978. The analysis 1D, which was done separately for each well and outcrop allowed the recognition of 13 sedimentary facies, mainly divided based on predominant litologies and sedimentary structures. Such facies are lithologically represented by pebble, sandstones, claystones, margas and evaporates; these facies are associated in order to characterize different depositional systems, that integrate from the continental environment (fluvial system and lacustre), paralic system (delta system and lagunar) to the marine environment (shelfenvironment). The first one, the fluvial system was divided into two subtypes: meandering fluvial system, characterized by fill channel and floodplain deposits; the facies of this system are associated vertically according to the textural thinning upward cycles (dirting-up trend pattern in well logs). Lacustrine environment is mainly related with the lithotypes of the Crato Formation, it shows a good distribution within the basin, been composed by green claystone deposits and calcareous laminated. Deltaic System represented by prodelta and delta front deposits which coarsening upward tendency. Lagunar system is characterised by the presence of anhydrite and gypsum deposits besides the black claystone deposits with vegetal fragments which do not contain a fauna typically marine. The marine platform system is composed by successions of black and gray claystone with fossiliferous fauna of Dinoflagellates (Spiniferites Mantell, Subtilisphaera Jain e Subtilisphaera Millipied genre) typical of this kind of depositional system. The sedimentary facies described are vertically arranged in cycles with progradational patterns which form textural coersening upward cycles and retrogradational, represented by textural thinning dowward cycles. Based in these cycles, in their stack pattern and the vertical change between these patterns, the systems tracks and the depositional sequences were recognized. The Low System Track (LST) and High System Track (HST) are composed by cycles with progradational stack pattern, whereas the Trangessive System Track (TST) is composed by retrogradational stack pattern cycles. The 2D stratigraphic analysis was done through the carrying out of two stratigraphic sections. For the selection of the datum the deepest maximum flooding surface was chosen, inside the Sequence 1, the execution of these sections allowed to understand the behaviour of six depositional systems along the study area, which were interpreted as cycles of second order or supercycles (cycles between 3 and 10 Ma), according to the Vail, et al (1977) classification. The Sequence 1, the oldest of the six identified is composed by the low, transgressive and high systems tracks. The first two system tracks are formed exclusively by fluvial deposits of the Rio da Batateira Formation whereas the third one includes deltaic and lacustrine deposits of the Crato Formation. The sequences 2 and 3 are formed by the transgressive systems tracks (lake spreading phase) and the highstand system track (lake backward phase). The TST of these sequences are formed by lacustrine deposits whereas HST contains deltaic deposits, indicating high rates of sedimentary supply at the time of it s deposition. The sequence 4 is composed by LST, TST and HST, The TST4 shows a significant fall of the lake base level, this track was developed in conditions of low relation between the creation rate of space of accommodation and the sedimentary influx. The TST4 marks the third phase of expansion of the lacustrine system in the section after the basin?s rift, the lacustrine system established in the previous track starts a backward phase in conditions that the sedimentary supply rate exceeds the creation rate of space accommodation. The sequence 5 was developed in two different phases, the first one is related with the latest expansion stage of the lake, (TST5), the basal track of this sequence. In this phase the base level of the lake rose considerably. The second phase (related to the TST5) indicates the end of the lacustrine domain in the Araripe Basin and the change to lagunar system ant tidal flat, with great portions in the supratidal. These systems were formed by restricted lagoons, with shallow level of water and with intermittent connections with the sea. This, was the phase when the Araripe Basin recorded the most several arid conditions of the whole interval studied, Aptian Albian, conditions that allow the formation of evaporitic deposits. The sequence 6 began its deposition after a significant fall of the sea (LST6). The sequence 6 is without any doubtlessly, the sequence that has deposits that prove the effective entrance of the sea into the Araripe Basin. The TST6, end of this sequence, represents the moment which the sea reaches its maximum level during the Aptian Albian time. The stratigraphic analysis of the Aptian Albian interval made possible the understanding that the main control in the development of the depositional sequences recognized in the Araripe Basin were the variations of the local base level, which are controlled itself by the climate changes / O presente trabalho tem como objetivo principal realizar uma an?lise estratigr?fica detalhada do intervalo Aptiano ao Albiano na parte leste da Bacia do Araripe, NE do Brasil, o qual corresponde, litoestratigraficamente, ?s forma??es Rio da Batateira, Crato, Ipub? e Romualdo. A an?lise estratigr?fica envolveu tr?s etapas distintas, a an?lise 1D, 2D e 3D, nas quais foram adaptados os conceitos da estratigrafia de seq??ncias visando elaborar um arcabou?o de car?ter cronoestratigr?fico para a por??o estudada da bacia. Tal estudo contou com uma base de dados integrada por informa??o de campo e de po?os pertencentes ao projeto Santana, realizado pelo Minist?rio de Minas e Energia- DNPM- CPRM nos anos de 1977 e 1978. A an?lise 1D, por meio da an?lise individual de cada afloramento e po?o, permitiu o reconhecimento de 13 f?cies sedimentares, individualizadas com base principalmente nas litologias predominantes e nas estruturas sedimentares. Tais f?cies s?o representadas litologicamente por conglomerados, arenitos, pelitos, calc?rios, margas e evaporitos. As mesmas se associam de forma a caracterizar diferentes sistemas deposicionais, que integram desde o ambiente continental (sistemas fluvial e lacustre), ambiente par?lico (sistemas deltaico e lagunar) at? o ambiente marinho (sistema plataformal). O primeiro deles, o sistema fluvial, foi dividido em dois sub-tipos: o sistema fluvial meandrante, caracterizado por dep?sitos de preenchimento de canal e dep?sitos de plan?cie de inunda??o, e o sistema fluvial entrela?ado formado principalmente por dep?sitos de preenchimento de canal; as f?cies deste sistema se associam verticalmente segundo ciclos de afinamento textural para o topo (padr?o em sino nos perfis geof?sicos). O sistema lacustre, relacionado principalmente aos lit?tipos da Forma??o Crato, apresenta uma boa distribui??o na bacia, sendo composto por dep?sitos de pelitos verdes e calc?rios laminados. O sistema deltaico, representado por dep?sitos de prodelta e de frente deltaica, os quais se disp?em verticalmente em ciclos com granocresc?ncia para o topo (padr?o em sino invertido nos perfis geof?sicos). O sistema lagunar caracteriza-se pela presen?a de dep?sitos de anidrita e gipsita, al?m dos dep?sitos de pelitos negros com restos vegetais, os quais n?o cont?m uma fauna tipicamente marinha. O sistema de plataforma marinha ? composto por sucess?es de pelitos pretos e cinza com faunas fossiliferas de Dinoflagelados (g?neros Spiniferites Mantell, Subtilisphaera Jain e Subtilisphaera Millipied), t?picas de este tipo de sistema deposicional. As f?cies sedimentares descritas se arranjam verticalmente de forma a compor ciclos com padr?es de empilhamento progradacional, os quais formam ciclos com engrossamento textural para o topo, e retrogradacional, representados por ciclos com afinamento textural para o topo. Com base nestes ciclos, em seus padr?es de empilhamento e na mudan?a verticais entre estes padr?es, foram reconhecidos os tratos de sistemas e as seq??ncias deposicionais. Os tratos de sistemas de N?vel Baixo e o de N?vel Alto s?o compostos por ciclos com padr?o de empilhamento progradacional; o Trato de Sistemas Transgressivo, por sua vez, ? formado por ciclos com padr?o de empilhamento retrogradacional. A an?lise estratigr?fica 2D foi elaborada por meio da realiza??o de duas se??es estratigr?ficas. Para a escolha do datum foi privilegiada a superf?cie de m?xima inunda??o mais basal, interna ? Seq??ncia 1. A elabora??o destas se??es possibilitou compreender o comportamento de seis seq??ncias deposicionais ao longo da ?rea de estudo, as quais foram interpretadas como ciclos de segunda ordem ou superciclos (ciclos com dura??es entre 3 e 10 Ma), segundo a classifica??o de Vail, et al (1977). A Seq??ncia 1, mais antiga das seis seq??ncias identificadas, ? composta pelos tratos de sistemas de N?vel Baixo, Transgressivo e de N?vel Alto. Os dois primeiros tratos s?o formados exclusivamente pelos dep?sitos fluviais da Forma??o Rio da Batateira, ao passo que o terceiro inclui dep?sitos deltaicos e lacustres da Forma??o Crato. As seq??ncias 2 e 3 s?o formadas pelos tratos de sistemas Transgressivo (TST; fase de expans?o do lago) e de N?vel Alto (TSNA; fase retra??o do lago). Os TST s destas seq??ncias s?o formados por dep?sitos lacustres, ao passo que os TSNA s cont?m dep?sitos deltaicos, indicando assim condi??es de alta taxa de suprimento sedimentar na ?poca da deposi??o deste. A seq??ncia 4 ? formada pelos tratos de sistemas de N?vel Baixo (TSNA), Transgressivo e de N?vel Alto. O TSNB registra uma queda importante do n?vel base do lago; este trato se desenvolveu em condi??es de baixa raz?o entre a taxa de cria??o de espa?o de acomoda??o e a taxa de influxo sedimentar. O TST marca a terceira fase de expans?o do sistema lacustre na se??o p?s rifte da bacia; o sistema lacustre implantado no trato anterior inicia uma fase de retra??o em condi??es em que a taxa de aporte sedimentar passa a suplantar a de cria??o de espa?o de acomoda??o. A seq??ncia 5 desenvolveu-se em duas fases distintas. A primeira relaciona-se com a ?ltima etapa de expans?o do lago, (TST), trato basal desta seq??ncia. Nesta fase, o n?vel base do lago subiu consideravelmente. A segunda fase, relacionada ao TSNA), indica o final do dom?nio lacustre na Bacia do Araripe e a mudan?a para sistemas lagunares e de plan?cie de mar?, com grande desenvolvimento das por??es de supramar?. Estes sistemas eram formados por lagunas restritas, com l?mina de ?gua rasa, e com conex?o intermitente com o mar. Esta foi a fase em que a Bacia do Araripe registrou as mais severas condi??es de aridez de todo intervalo estudado, Aptiano ao Albiano, condi??es estas que propiciaram a forma??o de dep?sitos evapor?ticos. A seq??ncia 6 iniciou sua deposi??o ap?s uma queda significativa do incipiente mar (TSNB). Esta seq??ncia ?, indubitavelmente, a que cont?m os dep?sitos que comprovam a efetiva entrada do mar na Bacia do Araripe. O TST, trato final desta seq??ncia, representa o momento em que o n?vel do mar atingiu o seu m?ximo durante todo o intervalo Aptiano ao Albiano. A an?lise estratigr?fica do intervalo Aptiano ao Albiano permitiu compreender que o controle principal no desenvolvimento das seq??ncias deposicionais reconhecidas na Bacia do Araripe foram ?s varia??es do n?vel de base local, as quais s?o controladas, por sua vez, pelas mudan?as clim?ticas

Nous développons dans ce travail de thèse un outil de simulation numérique pour les matériaux à changement de phase (MCP), en tenant compte du phénomène de convection naturelle dans la phase liquide, pour des configurations en deux et trois dimensions. Les équations de Navier-Stokes incompressible avec le modèle de Boussinesq pour la prise en compte des forces de flottabilité liées aux effets thermiques, couplées avec une formulation de l’équation d’énergie suivant la méthode d’enthalpie, sont résolues par une méthode d’éléments finis adaptatifs. Une approche mono-domaine, consistant à résoudre les mêmes systèmes d’équations dans les phases solide et liquide, est utilisée. La vitesse est ramenée à zéro dans la phase solide, en introduisant un terme de pénalisation dans l’équation de quantité de mouvement, suivant le modèle de Carman-Kozeny, consistant à freiner la vitesse à travers un milieu poreux. Une discrétisation spatiale des équations utilisant des éléments finis de Taylor-Hood, éléments finis P2 pour la vitesse et éléments finis P1 pour la pression, est appliquée, avec un schéma d’intégration en temps implicite d’ordre deux (GEAR). Le système d’équations non-linéaires est résolu par un algorithme de Newton. Les méthodes numériques sont implémentées avec le logiciel libre FreeFem++ (www.freefem.org), disponible pour tout système d’exploitation. Les programmes sont distribués sous forme de logiciel libre, sous la forme d’une forme de toolbox simple d’utilisation, permettant à l’utilisateur de rajouter d’autres configurations numériques pour des problèmes avecchangement de phase. Nous présentons dans ce manuscrit des cas de validation du code de calcul, en simulant des cas tests bien connus, présentés par ordre de difficulté croissant : convection naturelle de l’air, fusion d’un MCP, le cycle complet fusion-solidification, chauffage par le bas d’un MCP, et enfin, la solidification de l’eau. / In this thesis we develop a numerical simulation tool for computing two and three-dimensional liquid-solid phase-change systems involving natural convection. It consists of solving the incompressible Navier-Stokes equations with Boussinesq approximation for thermal effects combined with an enthalpy-porosity method for the phase-change modeling, using a finite elements method with mesh adaptivity. A single-domain approach is applied by solving the same set of equations over the whole domain. A Carman-Kozeny-type penalty term is added to the momentum equation to bring to zero the velocity in the solid phase through an artificial mushy region. Model equations are discretized using Galerkin triangular finite elements. Piecewise quadratic (P2) finite-elements are used for the velocity and piecewise linear (P1) for the pressure. The coupled system of equations is integrated in time using a second-order Gear scheme. Non-linearities are treated implicitly and the resulting discrete equations are solved using a Newton algorithm. The numerical method is implemented with the finite elements software FreeFem++ (www.freefem.org), available for all existing operating systems. The programs are written and distributed as an easy-to-use open-source toolbox, allowing the user to code new numerical algorithms for similar problems with phase-change. We present several validations, by simulating classical benchmark cases of increasing difficulty: natural convection of air, melting of a phase-change material, a melting-solidification cycle, a basal melting of a phase-change material, and finally, a water freezing case.