Les insectes phytophages forment un groupe d’organismes très diversifié et la plupart sont considérés comme spécialistes. Les patrons de spécialisation des insectes vis à vis de leurs plantes hôtes dépendent en partie de leur capacité à interagir avec les plantes (niche fondamentale) et aux facteurs environnementaux modulant ces interactions et aboutissant aux observations en milieu naturel (niche réalisée). La spécialisation fondamentale est déterminée par l'évolution conjointe de deux traits : la performance des larves et la préférence des femelles. Pour comprendre cette spécialisation, nous avons étudié une communauté de huit espèces de mouches des fruits (Diptera : Tephritidae) présentes à La Réunion. Dans un premier temps, nous avons déterminé la niche réalisée de chaque espèce et montré que ces niches étaient structurées par la phylogénie des plantes avec D. demmerezi, D. ciliatus et Z. cucurbitae spécialistes des Cucurbitaceae, N. cyanescensspécialiste des Solanaceae et C. catoirii, C. capitata et C. quilicii généralistes attaquant des plantes de différentes familles. Après l'invasion de B. zonata en 2000, C. capitata et C. quilicii ont subi une réduction de leur gamme d'hôtes. Dans un deuxième temps, nous avons déterminé la niche fondamentale de ces espèces (sauf D. ciliatus). Nous avons évalué les préférences des femelles en mesurant la fécondité de chacune d’entre elles sur une gamme de 29 fruits, puis nous avons testé l'existence d'une corrélation entre la préférence des femelles et la performance des larves (mother knows best hypothesis). Nous avons montré une corrélation positive chez spécialistes des Cucurbitaceae qui pondent sur les plantes pour lesquelles les larves survivent le mieux contrairement aux généralistes pondant et survivant sur une large gamme d'hôtes mais sans corrélation entre ces deux traits.Enfin, la sélection de l'hôte par les femelles se faisant principalement grâce aux composés organiques volatils (COVs) émis par les fruits, nous avons montré que les fruits infestés par les généralistes ont pour point commun l'émission de COVs responsables de la maturation des fruits. Au contraire, les fruits de plusieurs Solanaceae émettent des COVs spécifiques suggérant la détection de ceux-ci par les femelles de N. cyanescens. Les Cucurbitaceae émettent des COVs abondants peu présents dans les autres familles suggérant une détection d'un mélange spécifique de ces COVs par les Tephritidae spécialistes des Cucurbitaceae. / Phytophagous insects are a very diverse group of organisms and most of them are considered as specialized. Patterns of specialization regarding their host plants depend on their ability to interact with their hosts (fundamental niche) and on environmental factors which modulate these interactions leading to observed patterns in the field (realized niche). Fundamental specialization is determined by the joint evolution of two traits: larval performance and female preference. To understand this specialization, we studied a community of eight fruit fly species (Diptera: Tephritidae) present in La Réunion.First, we determined the realized niche of each species and showed that they were structured by plant phylogeny with D. demmerezi, D. ciliatus and Z. cucurbitae as Cucurbitaceae specialists, N. cyanescens as Solanaceae specialist and C. catoirii, C. capitata et C. quilicii as generalists feeding on plants belonging to different families. After the invasion of B. zonata in 2000, C. capitata et C. quilicii were subjected to a decrease of their host range.Then, we determined the fundamental niche of these species (except D. ciliatus). We assessed female preferences by measuring their fecundity on 29 fruits, then we tested the presence of a correlation between female preference and larval performance (mother knows best hypothesis). We showed a positive correlation for Cucurbitaceae specialists laying eggs on plants where larvae survive the best, at the opposite of generalist species laying eggs and surviving on many hosts without any correlation between these two traits.Finally, host selection by females being mostly done thanks to volatile organic compounds (VOCs) emitted by fruits, we showed that fruits infested by generalist species have common VOCs responsible for fruit maturation. On the contrary, the fruits of several Solanaceae emit specific VOCs suggesting their detection by N. cyanescens females. Cucurbitaceae species emit abundant VOCs rarely present in other families suggesting a detection of a specific blend of these VOCS by Cucurbitaceae specialists.

La transition épithélio-mésenchymateuse (TEM) est un processus physiologique décrit dans le développement embryonnaire et chez l'adulte au cours de la cicatrisation. La TEM est également détournée dans le contexte pathologique au cours de l'invasion tumorale et les mécanismes moléculaires qui la contrôlent font à ce jour l'objet d'intenses investigations. Cette étude décrit le rôle du gène de la lignée germinale NANOS-3 dans la régulation de l'invasion tumorale broncho-pulmonaire associée à la TEM. Nous démontrons que l'expression de Nanos-3 est corrélée à l'agressivité des cancers bronchiques non à petites cellules (CBNPC) humains in vivo et qu'il est surexprimé pendant la TEM induite in vitro. De plus, la surexpression de Nanos-3 dans les lignées tumorales bronchiques augmente leurs capacités invasives in vitro en induisant la TEM alors que son inhibition induit l'effet opposé et promeut la transition mésenchymo-épithéliale (TME). Au cours de cette étude, nous rapportons également des mécanismes à la fois transcriptionnels et post-transcriptionnels de régulation des cibles de Nanos-3. Ainsi, nous montrons que Nanos-3 réprime la transcription du gène CADHERINE-E indépendamment des facteurs de transcription des familles Snail et ZEB. Nous décrivons également que la protéine Nanos-3 co-immunoprécipite avec certains ARNm de ses cibles et, plus particulièrement, qu'elle est capable de réguler la longueur de la queue poly-(A) du transcrit codant pour une de ses cibles majeures : la Vimentine. En parallèle, par des méthodes d'études in silico et in vitro, nous démontrons une localisation à la fois cytoplasmique et nucléaire de Nanos-3 ainsi que son accumulation nucléolaire. Enfin, nous mettons en évidence que la réexpression ectopique de Nanos-3 dans le contexte tumoral pourrait être attribuée à une dérégulation des mécanismes épigénétiques physiologiquement mis en place dans les cellules somatiques adultes. Ainsi, cette étude démontre le rôle de Nanos-3 dans l'acquisition d'un phénotype invasif par les cellules tumorales bronchiques et décrit un nouveau mécanisme de régulation de la TEM dépendant de la longueur de la queue poly-(A) de certains ARNm. / The Epithelial-Mesenchymal Transition (EMT) is a basic cellular process used by embryo to generate different tissues or in adult during wound healing. EMT is also misappropriated by cancer cells during the first step towards metastasis. Molecular mechanisms driving EMT during tumor progression are extensively studied and post-transcriptional regulations of EMT-associated genes emerge as major and promising field in oncology. Here we report a dual post-transcriptional and transcriptional regulation of EMT-associated genes by the NANOS-3 germline gene during lung tumor invasion. We show that the Nanos-3 expression in vivo correlates with aggressiveness of human non-small cell lung carcinomas (NSCLC) and that Nanos-3 is upregulated in cells which undergo an EMT in our in vitro EMT-inducible models. Moreover, Nanos-3 overexpression in human NSCLC cell lines enhances their invasive abilities by EMT regulation while its silencing induces the opposite effect leading to a Mesenchymal-Epithelial Transition (MET). Molecular investigations indicate that Nanos-3 controls its targets by either transcriptional or post-transcriptional mechanisms. We show that Nanos-3 represses E-CADHERIN transcription independently of Snail and ZEB transcription factor families. Moreover, we also find that mRNAs of post-transcriptionally regulated targets are co-immunoprecipitated with the Nanos-3 protein and that Nanos-3 regulates the length of the 3' poly-A tail of VIMENTIN mRNA. This dual mechanism of EMT regulation by Nanos-3 is to be related to the specific subcellular localization of Nanos-3 in both cytoplasm and nucleus associated with a nucleolus accumulation as shown by in vitro and in silico experiments. Finally, we demonstrate an epigenetic regulation of NANOS-3 gene expression in lung cell lines, thus supporting that its ectopic expression could be attributed to an epigenetic machinery deregulation in cancer cells.Thus, here we demonstrate a new innovative role for Nanos-3 in the acquisition of an invasive phenotype by lung tumor cells and we describe a novel mechanism of post-transcriptional regulation of EMT via the control of the mRNA poly-A tail length.

ZNF217 est un oncogène potentiel codant pour un facteur de transcription Krüppel-like. Cette étude vise à explorer le rôle délétère et la valeur pronostique de ZNF217 dans le cancer du sein. Nos résultats ont montré que : (i) des niveaux d'expression élevés de ZNF217 (tant au niveau de l'ARNm qu'au niveau protéique) sont associés à un mauvais pronostic chez les patientes atteintes d'un cancer du sein, et plus particulièrement dans les cancers du sein de type ER+/Luminaux/Luminaux A ; (ii) ZNF217 induit la transition épithélio mésenchymateuse (EMT) dans les cellules épithéliales mammaires humaines via la voie de signalisation du TGF-beta ; (iii) ZNF217 induit un phénotype agressif dans les cellules cancéreuses mammaires se traduisant in vitro par la stimulation de la croissance indépendante de l'ancrage, de la migration et de l'invasion cellulaire ; (iv) ZNF217 stimule la croissance tumorale et le développement spontané de métastases chez la souris ; (v) ZNF217 se lie à ERalpha et augmente l'activité transcriptionnelle ligand-dépendante de ce dernier en favorisant le recrutement d'ERalpha sur les éléments de réponse aux oestrogènes (EREs) ; (vi) ZNF217 stimule la formation de mammosphères dans des lignées cellulaires de cancer du sein ER– ou ER+ ; (vii) ZNF217 induit la résistance à l'hormonothérapie (tamoxifène) dans des cellules cancéreuses mammaires ER+ ; (viii) des niveaux élevés d'expression de ZNF217 sont associés à un mauvais pronostic en terme de survie sans récidive chez les patientes atteintes d'un cancer du sein et traitées par hormonothérapie uniquement. Nos résultats suggèrent que l'expression de ZNF217 représente un nouveau et puissant biomarqueur pronostique dans le cancer du sein ER+/Luminal/Luminal A, permettant la re-stratification de ces cancers du sein dits « de bon pronostic », pour lesquels il n'existe pas à l'heure actuelle de biomarqueurs permettant de les identifier. En conclusion, ZNF217 représente une nouvelle cible thérapeutique pour le traitement personnalisé des patientes atteintes d'un cancer du sein et exprimant de forts niveaux d'expression de ZNF217, en particulier les patientes ER+/ZNF217+ / ZNF217 is a candidate oncogene encoding for a Krüppel-like transcription factor. This study aims at exploring deeper insights on deleterious roles of ZNF217 and the prognostic significance of ZNF217 expression in breast cancers. We found that: (i) high levels of ZNF217 expression (at both mRNA and protein levels) are associated with poor prognosis in breast cancer patients, more particularly in ER+/Luminal/Luminal A breast cancers; (ii) ZNF217 induces epithelial-mesenchymal transition (EMT) in human mammary epithelial cells via the TGF-beta-activated Smad signaling pathway; (iii) in vitro ZNF217 stimulates several aggressive phenotypes in breast cancer cells, including anchorage-independent growth, cell migration and invasion; (iv) ZNF217 stimulates tumor growth and promotes the development of metastases in vivo; (v) ZNF217 binds with ERalpha and enhances 17beta- estradiol (E2)-induced ERalpha transactivation by increasing the recruitment of ERalpha to estrogen-responsive elements (EREs); (vi) ZNF217 increases mammosphere formation in ER– or ER+ breast cancer cell lines; (vii) ZNF217 confers resistance to endocrine therapy (tamoxifen) in ER+ breast cancer cells, and (viii) high levels of ZNF217 expression are associated with shorter relapse-free survival (RFS) in breast cancer patients treated with endocrine therapy only. Our findings suggest that ZNF217 expression represents a novel and powerful prognostic biomarker in ER+/Luminal/Luminal A breast cancers, allowing the re-stratification of these “good prognosis” breast cancers, which are currently not further classified by any other biomarkers available. In conclusion, ZNF217 could be a potential therapeutic target for a personalized treatment strategy in patients overexpressing ZNF217, in particular in ER+/ZNF217+ patients

Les propriétés invasives des cellules cancéreuses sont liées à des modulations importantes de l’expression de gènes. Des protéases doivent être exprimées afin de permettre la dégradation de la matrice extracellulaire (MEC), l’activation protéolytique de protéines matricielles et la libération de facteurs de croissance, de cytokines, de récepteurs et de molécules d’adhérence. Parmi ces protéases, les métalloprotéinases matricielles (MMPs) jouent un rôle crucial dans la dégradation de la MEC et dans le remodelage tissulaire observéau cours de l’invasion tumorale. L’émergence de thérapeutiques anticancéreuses basées sur des stratégies épigénétiques nécessitent d’évaluer leurs effets sur les propriétés des cellules tumorales. Ce travail a pour objectif d’analyser les effets de modulateurs épigénétiques (un agent hypométhylant de l’ADN et des inhibiteurs d’histone désacétylases (inhibiteursd’HDACs ou HDIs)) sur l’expression des MMP-1, -2 et -9 dans la lignée cellulaire de fibrosarcome humain HT1080. Dans un premier temps, il apparaît que l’agenthypométhylant de l’ADN, la 5-aza-2’désoxycytidine (5-azadC), augmente l’expressiongénique et protéique des MMP-1, -2 et -9. Ces modifications de l’expression sont associées à (i) une déméthylation globale de l’ADN et (ii) des modifications de la supra-organisation chromatinienne correspondant globalement à une chromatine moins condensée. De plus, la5-azadC est capable d’accroître les propriétés invasives des cellules par l’intermédiaire,notamment, d’une augmentation de l’expression de la MMP-1 par un mécanisme transcriptionnel. Cette augmentation de la transcription implique le recrutement du facteurSp1 et un remodelage chromatinien au niveau du promoteur du gène de la MMP-1.Néanmoins, une déméthylation totale de ce promoteur n’est pas nécessaire à cette induction. De manière complémentaire, le traitement des cellules HT1080 par différents HDIs révèle le rôle potentiel d’HDACs dans la régulation de l’expression de la MMP-1. Un HDIà large spectre, la trichostatine A (TSA), est capable de moduler l’expression de la MMP-1 et la texture nucléaire, mais uniquement après déméthylation préalable de l’ADN par la 5-azadC. Par contre, l’HDI spécifique des HDACs de classe I, le MS-275, est capable d’induire, à lui seul, l’expression génique et protéique de la MMP-1. Cette expression génique requiert un remodelage de la chromatine et le recrutement de l’histone acétyltransférase p300 au niveau du promoteur du gène de la MMP-1. L’ensemble de ces résultats suggèrent que des mécanismes épigénétiques jouent un rôle crucial dans le contrôle de l’expression de laMMP-1 dans les cellules HT1080, influençant ainsi les propriétés invasives de ces cellules. / Invasive properties of cancer cells require critical changes in gene expression. Proteasesmust be expressed for the degradation of the extracellular matrix (ECM), the proteolyticactivation of matrix proteins and the release of bioactive molecules such as growth factors,cytokines, receptors and adhesion molecules. Among these proteases, the matrixmetalloproteinase (MMP) family members play a crucial role in the ECM breakdown andremodeling of tissues during tumor invasion. The introduction of epigenetic strategies in thetherapeutic arsenal against cancer led to the need to evaluate the effects of suchtherapeutic approaches on cell behavior. Here we focused our attention on the effects ofepigenetic modulators, a DNA hypomethylating agent and histone deacetylase inhibitors(HDAC inhibitors or HDI), on the expressions of MMP-1,-2, and -9 in the human HT1080fibrosarcoma cell line. First, we showed that the DNA hypomethylating drug 5-aza-2’deoxycytidine (5-azadC) increases MMP-1, -2, -9 expressions both at the mRNA andprotein levels. These changes in gene expression are associated with (i) a global DNAdemethylation and with (ii) modifications in chromatin supra-organization which globally correspond to a more decondensed chromatin. Moreover, 5-azadC is able to increase theinvasive properties capability of the HT1080 cells mainly via MMP-1 transcription-dependent expression. This enhancement of transcription occurs through (i) Sp1 recruitment, (ii)chromatin remodeling and (iii) in absence of full demethylation on the MMP-1 genepromoter. Using different HDIs reveals that HDACs could potentially play a role in MMP-1expression. The pan-HDI trichostatin A (TSA) act in synergy with 5-azadC and is able tomodulate MMP-1 expression and nuclear texture, but only after DNA demethylation. Incontrast, the HDAC class I inhibitor, MS-275, which display additive effect with 5-azadC, isable to induce, alone, MMP-1 gene expression through chromatin remodeling and p300recruitment to its promoter. These data suggest that epigenetic mechanisms play a crucialrole in MMP-1 expression control in HT1080 cells thus influencing the invasive potential ofthese cells.

Os impactos negativos que as espécies invasoras causam aos ecossistemas naturais ou seminaturais são noticiados há pelo menos seis décadas em diferentes partes do mundo. Diversos estudos têm sido realizados visando diagnosticar o processo de invasão, conhecer a ecologia das espécies invasoras e gerar subsídios para o manejo, controle ou erradicação daquelas que causam problemas. No Brasil, a invasão de ecossistemas naturais começou a ser estudada recentemente e muito conhecimento ainda precisa ser gerado para basear a tomada de decisões sobre o problema. Este estudo teve por objetivo descrever o processo de invasão do Cerrado por Pinus elliottii e buscar técnicas de manejo viáveis ecológica e economicamente para o controle da invasora. O estudo foi realizado na Estação Ecológica de Santa Bárbara, no munícipio de Águas de Santa Bárbara, São Paulo, em duas frentes de pesquisa: uma para estudar a ecologia da espécie invasora, e a outra para verificar qual seria a técnica de controle mais vantajosa ecológica e economicamente. O estudo de ecologia baseou-se na amostragem de cinco blocos de 10 parcelas, cada uma com área de 10 x 10 m, para o estudo de plantas nativas com altura a partir de 50 cm e, dentro de cada parcela, cinco subparcelas de 1 x 1 m para o estudo de plantas com altura inferior a 50 cm. Em cada bloco as parcelas foram instaladas de forma que todo o gradiente de invasão por P. elliottii (desde 0 até 100% invadido) fosse contemplado. A densidade e a riqueza de espécies vegetais, agrupadas quanto à forma de vida, foram analisadas como variáveis resposta ao processo de invasão. Nessas parcelas foram caracterizadas, como variáveis explanatórias, a área basal da espécie invasora, a profundidade da camada de acículas e a abertura de dossel, que poderiam explicar as perdas de diversidade. O experimento de manejo foi realizado em uma área de 70 x 50 m, subdividida em 35 parcelas de 10 x 10 m. Sete tratamentos de erradicação (com cinco réplicas) foram testados, correspondendo a diferentes técnicas de manejo, que foram aplicadas isoladas ou combinadas. As técnicas de controle incluíram o corte raso com motosserra, a injeção de herbicida nos troncos e a aplicação de queimada prescrita. Após a aplicação dos tratamentos, a vegetação espontânea de cerrado foi monitorada por dois anos e comparados os tratamentos, utilizando-se a densidade e a riqueza de plantas nativas em regeneração como indicadores ecológicos de sucesso das técnicas. A viabilidade econômica foi analisada com base em todos os custos envolvidos em cada técnica, discutidos mediante a estimativa do custo do controle precoce da invasão. A pesquisa como um todo possibilitou a descrição, em detalhes, do processo de invasão, assim como a quantificação das perdas de diversidade e identificação dos filtros ecológicos envolvidos no processo. O estudo ecológico mostrou que a espessa camada de acículas depositada pela árvores invasoras quando a invasão se adensa é o principal fator responsável pelo desaparecimento de gramíneas, ciperáceas e indivíduos herbáceos, ou seja, quanto mais acículas depositadas no solo, maior a perda dessas formas de vida. Já o fechamento do dossel provocado pela entrada das árvores invasoras no ambiente savânico prejudicou especialmente os arbustos, fazendo com que desaparecessem em ambientes mais sombreados. Não foi notada nenhuma influência das variáveis estudadas sobre espécies arbóreas em regeneração na comunidade invadida. No experimento de manejo, diversos tratamentos foram economicamente viáveis, especialmente quando se prevê a comercialização da lenha das árvores invasoras. Isto porque a receita gerada com a venda da madeira pode cobrir os custos de manejo. Além da viabilidade econômica, ao longo dos dois anos de acompanhamento, o tratamento que combinou corte raso com queima das acículas foi o mais indicado, pois cobriu os custos de erradicação e, dentre os tratamentos testados, foi o que apresentou a melhor regeneração natural da vegetação nativa de cerrado. No entanto, a restauração passiva (regeneração natural) do ecossistema densamente invadido após a erradicação é um processo extremamente lento, de modo que os dois anos de estudo levam a crer que intervenções de restauração por meio de plantio serão necessárias para acelerar o processo. Com esta pesquisa, portanto, foi possível compreender os fatores e processos que levam à perda de biodiversidade decorrente da invasão por Pinus elliottii e, também, assegurar que é possível a erradicação da espécie invasora. Mas a restauração das áreas densamente invadidas ainda é um obstáculo a ser vencido. Os custos ecológicos e econômicos envolvidos na solução do problema são elevados, mas podem ser minimizados caso seja realizado o controle precoce da invasão. / Damages caused by invasive species in natural or semi-natural environments have been noticed for at least six decades in different parts of the world. Meanwhile around the world several studies aim to diagnose the invasion process, the ecology of invasive species and to generate knowledge about management, control, or eradication of these species. In Brazil, the studies about invasive species started recently and a lot of research is still needed to support the decision-making, and consequently strengthen the connections between scientists and decision makers. This work aims to describe the invasion process of slash pine in the Cerrado and seek for management solutions to deal with the invasion problem. The study took place at the Santa Bárbara Ecological Station, a reserve located at Águas de Santa Barbara municipality, São Paulo state. Two distinct types of research were performed: the first one focused on the study of the invasion ecology of slash pine, and the second aiming to seek for the ecologically and economically most viable management technique to remove the invasive species from this ecosystem. The ecological study was based on sampling of native plants taller than 50 cm, taken from five blocks of 10 plots (plot area = 10 x 10 m). To study the plants smaller than 50 cm height, samples were taken from five 1 x 1 m subplots placed inside each plot. In each block, the plots were placed with the aim of contemplating the whole range of invasion gradient (0% to 100% of invaded area) by P. elliottii. Native plant species were grouped according to their life form, and their density and richness were considered as response variables to the invasion process. In this plots, the basal area of the invasive species, pine needles depth and canopy openness were considered as the explanatory variables, as the oscillations of these variables could justify biodiversity losses. The management experiment was performed in a 70 x 50 m area, divided in 35 plots (10 x 10 m). Seven eradication treatments (with five replicates) were established according to different management techniques applied together or isolated. The control techniques included clear-cut with chainsaw, herbicide injection inside the trunks and prescribed burning. After the application of the treatments, the spontaneous recovery of the grassland vegetation was monitored, and the density and richness of native species were used as indicators of the ecological success of the management techniques. The economical viability was analyzed based on all the costs related to each one of the techniques and compared to an initial invasion control. In general, this work described in details the invasion process at the Cerrado vegetation, quantified biodiversity losses and identified the ecological filters in the invasion process. The increase of the pine needle layer thickness is the main driver of the disappearance of grasses, sedges and forbs. The canopy closure caused by the invasive trees in the open savanna environment was responsible for the decrease of shrubs. None of the explanatory variables influenced the native tree species under regeneration. The management experiment pointed several treatments as economically viable, especially when the timber of the invasive species can be commercialized and, thus, the profits obtained from the sold timber can cover the costs regarding the eradication procedure. In addition to the economical viability, over two years of monitoring the native vegetation, the best treatment was the one that combined clearcut and prescribed fire. Under this combination of treatments, the spontaneous regeneration of native vegetation had the best performance, and the eradication costs could be covered.. Meanwhile, the passive restoration (natural regeneration) from a densely invaded ecosystem is an extremely slow process. Therefore, after two years of research, it seems reasonable to assume that interfering in the restoration process through plantation is necessary in order to speed up the vegetation recovery. In this work, thus, it was possible to understand the factors and process that cause biodiversity losses promoted by slash pine (Pinus elliottii) invasion in the Cerrado, and also to ensure that the eradication of this invasive species is possible in this type of ecosystem. Nevertheless, the restoration of heavily invaded areas is still an issue. The ecological and economic costs related to this problem are high but can be reduced when the early control of the invasion is performed.

Bien que reconnu comme une étape importante vers une meilleur compréhension de l’évolution des tumeurs, de la morphogénèse des tissus et des tests hauts débits de médicaments, l’utilisation de tests cellulaires in vitro en trois dimensions est toujours limitée et ce surtout par la difficulté d’établir un protocole simple et robuste pour leur formation. Dans ce travail, nous présentons d'abord une nouvelle méthode microfluidique pour la formation des sphéroïdes multicellulaires. Cette technologie des Capsules cellulaire est basée sur l'encapsulation et la croissance des cellules à l'intérieur de micro- sphères creuses, perméable, élastiques. Deuxièmement, nous montrons que ces microcapsules servent de capteurs mécaniques pour mesurer la pression exercée par les sphéroïdes expansion. En imagerie en temps réel multi- photons, on observe en outre que le confinement induit une organisation cellulaire stratifiée, avec un noyau nécrotique, solide et dense, entouré d'un rebord de cellules périphériques hyper-mobiles, qui présentent des propriétés invasives. Troisièmement, nous avons adapté la technologie des capsules cellulaires pour former des tubes creux. Cette géométrie cylindrique nous permet d'étudier l'impact de la libération partielle de confinement (le long de l'axe du tube principal) sur la cinétique de croissance d’agrégats cellulaires pseudo-unidimensionnel (nommé cylindroids). Nos données de microscopie et l’analyse d'images suggèrent un mécanisme de croissance par pointe et la prouvent la génération d’une contrainte radiale. La combinaison des configurations sphériques et cylindriques tend vers l'image globale du confinement qui déclenche la motilité cellulaire et l'invasion par la périphérie de l'agrégat cellulaire tandis que la prolifération des cellules est inhibée dans le noyau lorsque la pression augmente. Quatrièmement, nous utilisons l’alginate comme moule pour concevoir des coquilles et tubes multicouches perméables. En particulier, une légère adaptation du protocole nous permet d'ancrer une fine couche de Matrigel (utilisé comme une membrane basale artificielle) sur la paroi interne de l'alginate. Par l'utilisation de ces capsules sphériques décorés de Matrigel, nous montrons que les monocouches sphériques fermés de cellules épithéliales, ou des kystes, peuvent être facilement conçus avec des tailles qui sont imposées par la taille des capsules. De même, les capsules tubulaires décorées de Matrigel sont utilisées pour la formation des organoïds cultivés à partir de cellules extraites des cryptes du côlon de la souris. Enfin, notre technologie offre une nouvelle voie pour produire dans les tests cellulaires in vitro utiles pour développer de nouvelles thérapies anticancéreuses ou des approches d'ingénierie tissulaire et d'étudier l'interaction entre la mécanique et de la croissance dans les agrégats cellulaires in vitro. / Although recognized as an important step towards better understanding of tumor progression, tissue morphogenesis and high throughput screening of drugs, the use of three dimensional in vitro cellular assays is still limited, especially due to the difficulty in establishing simple and robust protocols for their formation. In this work, we first present a novel microfluidics-assisted method for multicellular spheroids formation. This Cellular Capsules technology is based on the encapsulation and growth of cells inside permeable, elastic, hollow micro-spheres. Second, we show that these microcapsules serve as unique mechanical sensors to measure the pressure exerted by the expanding spheroids. By multiphoton live imaging, we additionally observe that confinement induces a layered cellular organization, with a dense, solid, necrotic core surrounded by a rim of hyper-motile peripheral cells, which exhibit enhanced invasive properties. Third, we adapt the Cellular Capsules technology to form hollow tubes. This cylindrical geometry allows us to investigate the impact of partial confinement release (along the main tube axis) on the growth kinetics of pseudo-one dimensional cellular aggregates (named cylindroids). Our microscopy data and image analyses suggest a tip-growing mechanism and evidence radial stress generation. The combination of the spherical and cylindrical configurations leads to the overall picture that confinement triggers cell motility and invasion at the periphery of the cellular aggregate while cell proliferation is inhibited in the core as pressure builds up. Fourth, we use alginate as a template to design multilayered permeable shells and tubes. In particular, slight adaptation of the protocol allows us to anchor a thin layer of Matrigel (used as an artificial basement membrane) to the alginate inner wall. Using these Matrigel-decorated spherical capsules, we show that closed spherical monolayers of epithelial cells, or cysts, can be readily engineered with sizes that are imposed by the size of the capsules. Similarly, Matrigel-decorated tubular capsules are shown to be convenient for the formation of organoids grown from cells extracted from the cypts of mouse colon. Finally, our technology offers a new avenue to produce in vitro cell-based assays useful for developing new anti-cancer therapies or tissue engineering approaches and to investigate the interplay between mechanics and growth of in vitro cellular assemblies.

Résumé : Nous avons d’abord souhaité, dans ce travail de thèse, préciser les mécanismes moléculaires conduisant à l'expression ectopique du récepteur du GIP (glucose-dependent insulinotropic polypeptide receptor, GIPR) dans des adénomes somatotropes provenant de patients présentant une acromégalie avec une réponse paradoxale (stimulation) de l’hormone de croissance au glucose par voie orale. Nous avons montré que l’expression ectopique de GIPR se produit par une activation transcriptionnelle hypomorphe du gène GIPR associée à des anomalies de méthylation dans le corps du gène. L’activation de la voie AMP cyclique par le GIP postprandial dans les adénomes exprimant le GIPR peut représenter un mécanisme alternatif de la tumorigenèse somatotrope en l’absence de mutations de l’oncogène GNAS.Nous rapportons d’autre part une analyse cytogénétique approfondie des adénomes somatotropes, qui nous a permis de définir deux groupes d'adénomes, un groupe à faible altération du nombre de copies et un groupe à forte altération du nombre de copies. Deux tumeurs présentaient des réarrangements chromosomiques complexes avec une signature typique de chromothripsis, et une architecture sous-clonale incluant jusqu’à six populations cellulaires différentes, témoignant d’une hétérogénéité intratumorale importante.Dans une collection d'adénomes hypophysaires invasifs comportant la portion intrasellaire et la portion envahissante le sinus caverneux, nous avons montré par RNA-seq des profils d'expression génique divergents, apportant des arguments supplémentaires en faveur de l'hétérogénéité intratumorale dans ces tumeurs bénignes. Les échantillons tumoraux provenant de portions invasives ont montré une surexpression de la voie de transition épithélio-mésenchymateuse et des marqueurs de cellules souches cancéreuses soulignant leur rôle potentiel dans l’acquisition du phénotype invasif des cellules adénomateuses hypophysaires. / AbstractIn this work, we explored the molecular mechanisms of ectopic glucose-dependent insulinotropic polypeptide receptor (GIPR) expression in somatotroph adenomas from patients with acromegaly displaying a paradoxical GH increase to oral glucose. We showed that ectopic GIPR expression occurs through hypomorphic transcriptional activation of GIPR gene likely driven by DNA methylation changes. Activation of the cAMP pathway by postprandial GIP may represent an alternative tumorigenic mechanism in GIPR expressing somatotroph adenomas without driver mutations in GNAS oncogene. Cytogenetic profiling defined two groups of adenomas, a low-copy-number alteration (CNA) group and a high-CNA group.Two tumor samples displayed complex chromosomal rearrangements compatible with chromothripsis and showed subclonal architecture with up to six distinct cell population in each tumor, demonstrating an important intratumor heterogeneity.In a collection of invasive pituitary adenomas including the non-invasive intrasellar portions and the portions invading the cavernous sinuses, we showed by RNA-seq different gene expression profiles, providing supplemental evidence for the intratumoral heterogeneity in these benign tumors. Tumor samples from invasive portions showed up-regulation of the epithelial-mesenchymal transition pathway and increased expression of cancer stem-cell markers highlighting their potential role in pituitary tumor cell invasive behavior.

Plasmodium falciparum is a protozoan parasite responsible for causing the most severe form of malaria in humans. This species is responsible for over 90% of malaria mortalities which occur predominantly in Africa. An increase in drug resistant parasites in recent years is threatening the progress made against malaria and thus new antimalarial drugs and vaccines are needed to combat this disease. During the intraerythrocytic phase, merozoites egress from mature schizonts to invade new uninfected erythrocytes. Glycophosphatidylinositol (GPI) -anchored proteins cover most of the exterior surface of the merozoite prior to invasion, while other GPI-anchored proteins are released onto the merozoite surface through apical organelle secretions. These proteins are involved in interactions with erythrocytes and are thought to be vital to erythrocyte invasion. GPI-anchored proteins have also been implicated as a cause of pathogenic symptoms and activation of immune components. These proteins are then released or cleaved to enable merozoite entry into the erythrocyte. Several enzymes are thought to be involved in their cleavage including the serine proteases subtilisin-like proteases (SUB) 1 and 2, and phosphatidylinositol-phospholipase C (PIPLC); GPI-anchored proteins are also generally sensitive to phospholipase A2 (PLA2). Cleaved proteins are released into the host blood system, while uncleaved proteins are carried into the erythrocyte during invasion. Merozoites have a limited period in which they retain invasive capacity. A previous lack of available techniques that are specifically adapted to merozoite analysis has resulted in an incomplete understanding of invasion and GPI-anchored protein involvement in invasion. This study aimed to determine how GPI-anchored proteins on the merozoite surface are altered in the invasive phase, and explore the possibility of using merozoite GPI-anchored proteins as potential drug targets to block erythrocyte invasion. Optimised methods of in vitro parasite culturing which produce highly synchronised merozoites was essential to this study. Parasite culturing techniques were optimised by utilising low haematocrit cultures with frequent culture splitting and optimised synchronisation. The “Malarwheel” is a tool that was developed for this research to provide a means for scheduling sorbitol treatments and MACs isolations. This tool and optimised culturing methods enabled large volumes of highly synchronised invasive merozoites to be harvested. Four compounds (vanadate, edelfosine, dioctyl sodium sulfosuccinate (DSS), and gentamicin) suspected to interfere with GPIanchored cleavage or processes were screened on intraerythrocytic stages and merozoites. Antimalarial and anti-invasive properties of these compounds were screened by modified malaria SYBR Green I-based fluorescence (MSF) assay and merozoite invasion assays (MIA) respectively. DSS and gentamicin showed limited potential as antimalarials or as anti-invasive agents. Vanadate and edelfosine both showed antimalarial and anti-invasive activity, while edelfosine was the most potent anti-invasive agent at physiological concentrations. The merozoite GPI-anchored proteome was analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by complete gel lane analyses conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on soluble and pelleted merozoite proteins in samples from either invasive or non-invasive merozoites. Thirteen known or predicted GPI-anchored proteins were identified in samples. Several changes were identified in merozoite GPI-anchored proteins between the invasive phase and after its completion, and minor differences were observed following treatment with edelfosine. Edelfosine showed partial inhibition of erythrocyte invasion, however, the primary cause of inhibition cannot be directly related to interferences with GPI-anchored proteins. These results suggest that GPIanchored proteins are controlled by various complex processes, and are cleaved or processed by diverse mechanisms during the invasive phase. These mechanisms may be controlled by multiple signals which effect proteins or groups of proteins in specific ways. These signals may be influenced by “checkpoints” during invasion processes including the time period after egress from schizonts, and possibly the recognition of erythrocyte targets. These methods and results provide a foundation for future research to enable culturing of P. falciparum parasites specifically for merozoite research, and to identify merozoite proteins active during the invasive phase. These results confirm and challenge previous ideas reported in literature on the GPI-anchored processes of merozoites and further characterise less studied GPIanchored proteins. The results suggest that the processes controlling GPI-anchored proteins may be more complex than previously thought. These results form a basis to further identify and characterise GPI-anchored proteins in the aim to develop antimalarial medications and vaccines that target merozoites and their GPI-anchored processes. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

Le placenta humain est un organe indispensable au bon déroulement de la grossesse. La villosité choriale est l’unité structurale et fonctionnelle du placenta. Elle est constituée essentiellement de cellules trophoblastiques. Les cytotrophoblastes extra-villeux (CTEV) présentent des propriétés invasives et assurent l’ancrage du placenta dans l’endomètre maternel. De plus, une apoptose physiologique assure le renouvellement des cytotrophoblastes tout au long de la grossesse.Le Facteur Préimplantatoire (PIF) est un peptide de 15 acides aminés, sécrété par des embryons viables. Le PIF exerce un effet autocrine positif sur le développement embryonnaire. Le PIF est également impliqué dans le contrôle de l’immunité et de l’inflammation dans divers types cellulaires.Au cours de ce travail de thèse, nous nous sommes intéressés aux rôles du PIF dans le développement placentaire humain. Dans un premier temps, nous avons caractérisé l’expression protéique du PIF dans des placentas humains de 1er et 3ème trimestre de grossesse.Nous avons montré que i) l’expression du PIF diminue au cours de la grossesse et ii) le PIF est majoritairement exprimé dans les CTEV.Dans un second temps, nous avons mis en évidence que le PIF i) favorise l’invasion trophoblaste et ii) inhibe l’apoptose des CTEV en régulant la voie de signalisation de p53.Par ailleurs, des altérations de l’invasion et de l’apoptose trophoblastiques sont associées à des pathologies de la grossesse telles que la pré-éclampsie et le retard de croissance intra-utérin. Ainsi, dans un dernier temps nous avons montré que l’expression du PIF est diminuée dans des placentas humains de 3ème trimestre issus de grossesses pathologiques par comparaison avec des grossesses normales.L’ensemble de ces résultats démontrent que le PIF est un nouvel acteur de la placentation humaine. De plus, le PIF pourrait être considéré comme un nouveau biomarqueur des pathologies de la grossesse. / Human placentation is a critical step in the establishment of a successful pregnancy. The chorionic villus constitutes the structural and functional unit of the placenta. The extravillous trophoblast (EVT) is a placental cell type that differentiates from the highly proliferative cytotrophoblast located at the base of the anchoring villous. EVT have invasive properties, essential for placenta anchoring in the endometrium and uterine artery remodeling. Moreover, programmed cell death is an active process required for normal trophoblastic cell turnover during pregnancy.PreImplantation Factor (PIF) is a 15-amino-acid peptide secreted by developing embryos. PIF exerts autotrophic and protective effects on the embryo. PIF is also implicated in the control of immune and inflammatory processes in various cell types.In this work, we aimed to determine the direct effects of PIF on human placental development.In a first part, we characterized PIF protein expression in first and third trimester human placentas. We showed that PIF protein expression i) decreased over the course of the pregnancy and ii) was higher in EVT compared to villous trophoblast.In a second part, we showed that PIF i) enhanced pro-invasive capacities and ii) prevented cell death by regulating p53 signaling pathway in human EVT.Dysregulation of trophoblastic invasion and apoptosis have been associated with pregnancy pathologies. Thereby, in a last part, we showed that PIF protein expression was lower in placentas from preeclampsia and intra-uterine growth restriction as compared with non-pathological placentas. Altogether, we highlighted for the first time, that PIF is a new positive regulator of placental functions. PIF could be considered as a novel biomarker of a favorable outcome of pregnancy.

Sparvugglan (Glaucidium passerinum) är en invasionsfågel som uppvisar årliga fluktuationer i sina invasioner vid Hammarö Fågelstation. Invasioner av denna art utlöses vanligtvis av brist på föda tidigt under hösten, vilket kan orsaka att hundratals fåglar migrerar på ett oförutsägbart sätt. Syftet med denna studie var att undersöka vilka ekologiska faktorer som ligger bakom detta beteende, och om invasioner vid Hammarö Fågelstation sker samtidigt som invasioner i övriga Sverige. Hypoteserna var att 1) sparvugglefångst uppvisar ingen årlig trend vid Hammarö Fågelstation, 2) sparvugglans populationstrender sammanfaller inte med andra ugglors trender, 3) det finns en korrelation mellan sparvugglans populationstrender vid Hammarö Fågelstation och övriga Sverige, och 4) det finns ett samband mellan sparvugglors populationstrender vid Hammarö Fågelstation och årliga fluktueringar i sorkpopulationer samt andra ekologiska faktorer såsom årlig temperatur och snötäcke. Data samlades in från Hammarö Fågelstations årsrapporter över fångst och ringmärkning av migratoriska fågelarter, och data som uppskattar populationstrender över hela Sverige inhämtades från Svensk Fågeltaxering. Korrelationstester utfördes på dessa parametrar mot sorkpopulationer och andra ekologiska faktorer. Studien fann en positiv korrelation mellan sparvugglefångst på Hammarö och populationstrender från hela Sverige under vintermånaderna. Det fanns även en positiv korrelation mellan årliga fluktuationer i sorkpopulationer vid Vindeln och fångst av sparvuggla i Hammarö. Inga korrelationer hittades när sparvugglefångst mättes mot miljömässiga faktorer. Dessa resultat stämmer överens med tidigare litteratur över området, och öppnar för möjligheter till ny forskning gällande miljö- och beteendemässiga aspekter av invasionsmigratoriska mönster, som ännu är en dåligt förstådd aspekt av fågelbeteende. / The Eurasian pygmy owl (Glaucidium passerinum) is an irruptive migrant that shows yearly fluctuations in its invasions at Hammarö Bird Station, Värmland, Sweden. Invasions in this species are usually triggered by a shortage in food supply during early autumn, which can cause hundreds of birds to migrate in an unpredictable manner. The purpose of this study was to explore what ecological factors cause this behaviour, and if invasions at Hammarö Bird Station occur during the same years as invasions in the rest of Sweden. The hypotheses were that: 1) Eurasian pygmy owl captures at Hammarö Bird Station show no inter-annual trend, 2) Eurasian pygmy owl invasions are not correlated with captures of other owl species, 3) there is a correlation between population trends of the Eurasian pygmy owl at Hammarö Bird Station and trends in the rest of Sweden, and 4) there is a correlation between Eurasian pygmy owl captures and annual fluctuations in vole population as well as other environmental factors such as yearly temperature and snow cover. Data was collected from Hammarö Bird Stations annual review of capture and ringing of migratory bird species, and data approximating population trends nationwide were collected from “Svensk Fågeltaxering”. Correlation tests were conducted on these parameters against vole populations and other ecological factors. The study found a positive correlation between owl captures at Hammarö and population trends from all of Sweden during the winter. A positive correlation was also found between annual vole population fluctuations in Vindeln, Västerbotten and owl captures at Hammarö. No correlations were found when measuring owl captures against environmental factors. These results coincide with previous literature on the subject, and open up possibilities for future research regarding the environmental and behavioral aspects of irruptive migration patterns, which is still a poorly-understood aspect of bird behaviour.