Plants have developed intricate mechanisms to control growth in response to a variety of environmental cues, to compensate its immobility and to survive in both normal and adverse conditions. The TCP proteins are a family of plant-specific, basic helix-loop-helix (bHLH) transcription factors that involve in different aspects in plant growth and developmental control. The Arabidopsis TCP20 has been shown to involve in coordinating cell growth and proliferation, and in growth arrest in response to DNA double-stranded breaks (DSB). In this thesis, the main interest is to examine the function of Arabidopsis TCP6, which shares the highest homology with TCP20, and like TCP20, contains a putative ATM phosphorylation motif that suggests potential involvement in the ATM/ATR-mediated DSB responses. Expressional analysis including transcript measurement and reporter gene tagging demonstrated that TCP6 is expressed in flowers, in particular in the first mitotic event of pollen and ovule/embryo sac development, indicating that TCP6 potentially involves in regulating the mitotic cell cycle during gametophyte development. Yet no gametophytic or fertility-affecting mutant phenotype was observed in the tcp6 single and tcp6/tcp20 double mutants, which may be due to high functional redundancy. The tcp6/tcp20 double mutant seedlings exhibited significantly higher growth performances in true leaf growth compared to wild type when treated with gamma radiation, implying that both functional TCP6 and TCP20 are involved in response to gamma radiation-generated DSBs. The work of this thesis provides the first expressional and functional characterizations of TCP6, with the results suggesting that TCP6 and other class I TCPs play a role in regulating growth under both normal and stress conditions.

Sexual conflict has been proposed to be important for evolution, and is often implicated in population divergence and speciation through sexually antagonistic co-evolution (SAC). However, empirical tests of these ideas on field populations are few. How sexual conflict, and SAC, operates in the wild, remains an important unanswered question if we are to fully understand the role of sexual conflict in evolution in nature. Here, I studied sexual conflict over mating in the seed feeding bugs Lygaeus equestris and Lygaeus simulans. Firstly, I show that laboratory adapted populations of L. equestris that differ in the magnitude of sexual conflict also differ in aspects of their reproductive development and mating propensity, with the population displaying greater conflict load also mating more readily. Study of female receptivity to mating as an evolvable trait, that could be involved in conflict over mating, revealed moderate to low heritability at two age groups. To better understand variation in the expression of sexual conflict in the wild, field caught populations of L. equestris, ranging across its distribution, and also of its sister species, L. simulans, were assayed for the magnitude of sexual conflict over mating in common garden laboratory experiments. High female mating costs were apparent across the populations, but the magnitude of these costs did not vary. No consistent patterns of mating costs and life history variation were found however, arguing against close links between mating costs and life-history. Finally, I investigated whether populations displaying sexual conflict over mating have begun to diverge, and evolve reproductive isolation. I found no evidence of reproductive isolation, or variation in mating propensity, between populations of L. equestris when crossed in reciprocal no-choice mating trials. However, L. equestris and L. simulans did show pre-zygotic reproductive isolation albeit with asymmetries between the reciprocal crosses (L. simulans males were able to mate L. equestris females, but male L. equestris were largely unable to mate L. simulans females). As expected for close taxa that perhaps have not been diverged for long, pre-zygotic isolation was perhaps stronger than post-zygotic isolation, as F2 offspring were generated by some of the inter-specific crosses: gene flow can therefore occur between these species contrary to previous studies. My data suggest that sexual conflict over mating may reduce the likelihood of speciation through the evolution of male persistence, as well as promote it through population divergence.

Os roedores murídeos são melhor conhecidos em relação à placentação, porém descrições para outros grupos como os cricetídeos incluindo os \"camundongos do Novo Mundo\" ainda são escassos. Recentemente à placenta e as membranas fetais tem sido considerados fontes promissoras para a obtenção de células-tronco. Em particular, o saco vitelino é estruturalmente diverso e desempenha várias e importantes funções. Por essa razão, o objetivo deste trabalho foi descrever a placentação corioalantóidea e vitelina em Necromys lasiurus. Além de avaliar o potencial do saco vitelino como uma fonte de células-tronco mesenquimais. Para tanto, um total de 10 placentas variando de início ao final de gestação foram analisadas através de técnicas de histologia, imunohistoquímica e microscopia eletrônica, bem como através do cultivo e diferenciação celular, citometria de fluxo e imunocitoquímica. A placenta corioalantóidea discoidal era organizada em uma zona labiríntica, zona juncional e decidua. O labirinto era a região mais importante para as trocas materno-fetal. Próximo ao final da gestação ele apresentou uma barreira hemotricorial com espessura média de 2,41 µm. A zona juncional era composta por sincício e citotrofoblasto. Células trofoblásticas gigantes localizavamse entre a zona juncional e a decidua, assim como nas margens laterais da placenta. A morfologia do saco vitelino visceral invertido variou de acordo com a sua localização e relação com a placenta e o útero. Quando cultivadas, as células aderentes do saco vitelino formaram colônias fibroblastóide (92,13%) e expressaram marcadores de células-tronco mesenquimais e alguns para células precursoras de células-hematopoiéticas. As células apresentaram sucesso quanto às diferenciações osteogênica, adipogênica e condrogênica e não desenvolveram formação tumoral quando injetadas em camundongo nude. Com isso, essas células-tronco despontam como uma fonte terapêutica promissora para a terapia celular. / Murine rodents are well investigated in regard to placentation, but data for other groups such as cricetids including New World mice or Sigmodontinae are sparse. Recently the placenta and fetal membranes are regarded to be promising sources for obtaining stem cells. In particular, the yolk sac is structurally diverse and shows important functions throughout gestation. For this reason, this study aims to describe the chorioallantoic and yolk sac placentation in Necromys lasiurus. In addition, the potential of the yolk sac as a source for mesenchymal stem cells that are not well studied so far will be evaluated. In total, 10 individuals from early gestation to near term were investigated by means of histology, immunohistochemistry and electron microscopy as well as cell culture and differentiation, flow citrometry and immunocytochemistry. The discoidal chorioallantoic placenta was organized in a labyrinth zone, junctional zone, and decidua. The labyrinth was most import for maternal-fetal exchange processes. It possessed a hemotrichorial barrier of about 2.41 µm thickness near term. The junctional zone included syncytial areas and cytotrophoblasts. Trophoblast giant cells were located between the junctional zone and decidua as well as in the lateral margins of the placenta. The morphology of the inverted visceral yolk sac varies according to its location and relationship with the placenta and uterus. When cultured, the adherent cells of the yolk sac formed fibroblastoid colonies (92.13%) and expressed mesenchymal stem cells markers, and some hematopoietic precursor cells markers. They showed successful osteogenic, adipogenic, and chondrogenic differentiation and did not develop tumors when transferred to nude mice. Thus, these cells resulted as stem cells with promising therapeutic values for cell therapy.

O saco vitelino é o único anexo embrionário presente em todas as espécies animais cuja função é manter o desenvolvimento primário do feto até que a placenta assuma esta função. Além disso, ele desempenha funções importantes como: nutrição do embrião, síntese proteica, atividade fagocitária, transferência de materiais e hematopoiese. A utilização de células-tronco embrionárias gera muitas discussões devido às questões éticas associadas à obtenção destas células, além de questões religiosas e também as relações de baixa plasticidade das células-tronco adultas motivam os pesquisadores ao crescente interesse em outras fontes de célulastronco. Com isso, células provenientes de tecidos como as dos anexos embrionários chamam cada vez mais atenção devido à sua facilidade de acesso, crescimento rápido e boa plasticidade. Nosso grupo tem demonstrado que as células do saco vitelino têm boa capacidade de proliferação e são multipotentes. Vários pesquisadores vêm demonstrando que o período no qual as células são obtidas pode ser de fundamental importância para a diferenciação. Sendo assim, neste projeto tivemos como objetivo estabelecer e caracterizar células-tronco de saco vitelino de cães em dois estágios gestacionais, visando analisar de forma comparativa o perfil de expressão de marcadores mesenquimais, hematopoiéticos e pluripotência de forma a entender o comportamento das mesmas. Neste trabalho observamos que as células de saco vitelino obtidas de gestações com 40 e 50 dias aderiram ao plástico, tiveram morfologia fibroblastóide, boa proliferação celular, se diferenciaram em adipócitos, condrócitos e ostéocitos e expressaram alguns marcadores mesenquimais, sugerindo assim ser uma célula-tronco mesenquimal. Além disso, não levaram à formação tumoral quando injetadas em camundongos imunossuprimidos, nudes. / The yolk sac is an embryonic attachment present in all animal species, which function is to keep the primary development of the fetus until the placenta assumes this function. Moreover, it plays important roles as: nutrition of the embryo, protein synthesis, phagocytic activity, transfer of materials and hematopoiesis. The use of embryonic stem cells generates many discussions because of the ethical and religious issues associated with the obtaintion of these cells. Also the relationship of low plasticity of adult stem cells motivates researchers to explore other sources of stem cells. Thus, cells from tissues such as the embryonic anexes call more attention, due to its ease of access, rapid growth and good plasticity. Our group has shown that cells of the yolk sac has good ability to proliferate and are multipotent. Several researchers have shown that the period in which the cells are obtained can be critical for differentiation. Therefore, in this project we aimed to establish and characterize stem cells from dog yolk sac in two stages of pregnancy, in order to analyze comparatively the expression profile of mesenchymal, hematopoietic and plurypotency markers, and study their behavior and plasticity potencial in vitro. In this study we observed that yolk sac cells obtained from pregnancies at 40 and 50 days adhered to plastic, had fibroblast like-morphology, good cell proliferation and differentiated into adipocytes and osteocytes and expressed some mesenchymal markers, therefore suggesting to be a mesenchymal stem cell. Also, they did not lead to tumor formation when injected into immunosuppressed mice, nude.

Calbindin-D28K is a cytosolic calcium binding protein found in a variety of cells that transport calcium. The chorioallantoic membrane and yolk sac of oviparous squamate reptiles (lizards and snakes) transport calcium from the eggshell and yolk to the developing embryo. I used immunohistochemistry to localize calbindin-D28K expression in the chorioallantoic membrane and yolk sac of two species of oviparous scincid lizards, Plestiodon fasciatus and Saproscincus mustelinus. Calbindin-D28K was detected in the chorioallantoic membrane and yolk sac of both lizard species by a polyclonal anti-snake calbindin antibody and a monoclonal anti-cow calbindin antibody. Calbindin-D28K was localized in the chorionic epithelium and allantoic epithelium of the chorioallantoic membrane and in endodermal cells scattered throughout the yolk mass of both species. This is the first demonstration of calbindin-D28K in allantoic epithelium and in endodermal cells of the yolk sac of lizards.

Le problème du sac-à-dos à contraintes disjonctives (DCKP) est une variante du sac-à-dos normal. C'est un problème dans lequel certains objets peuvent être incompatibles avec d'autres. Le DCKP apparait, souvent, comme sous problème d'autres problèmes d'optimisation combinatoire plus complexes.

Chez la souris, peu de choses sont connues sur les marqueurs de surface qui caractérisent les cellules souches hématopoïétiques (CSHs) embryonnaires. Durant ma thèse, j'ai étudié au niveau du placenta (Pl) et du sac vitellin (SV) les populations CD34+c-kithi, CD144+CD45+ et Sca-1+AA4.1+, déjà décrites comme enrichies en CSHs dans d'autres contextes. Le projet dans son ensemble a permis de montrer que l'enrichissement en CSHs n'implique pas les mêmes marqueurs selon le tissu considéré : dans les sites d'émergence (AGM) et d'émergence/amplification des CSHs (SV), la population la plus enrichie en CSHs a pour phénotype CD144+CD45+ ; concernant le Pl, organe d'amplification des CSHs, elle a pour phénotype CD34+c-kithiSca-1+ alors que dans le foie fœtal (FF), organe d'amplification/différenciation des CSHs, elle a pour phénotype CD34+c-kithiSca-1+AA4.1+. L'analyse moléculaire de ces populations permettra de révéler des molécules régulatrices spécifiques de l'émergence et de l'amplification des CSHs. Par ailleurs, nous avons utilisé les souris transgéniques VECR pour étudier l'origine des CSHs CD34+c-kithi du Pl et il semblerait qu'à E11.5, toutes ne proviennent pas de l'endothélium. Les résultats préliminaires réalisés sur les souris Mpl-/-, qui présentent un défaut de contenu en CSHs dans le Pl et le FF, indiquent qu'à E11.5, le potentiel hématopoïétique de la population CD34+c-kithi du Pl Mpl-/- est inférieur à celui de la population CD34+ckithi du Pl C57Bl6 ; cette différence n'est plus visible à E12.5. Le Pl constitue donc une niche transitoire d'amplification/maturation des CSHs mais il est possible qu'il puisse également produire des CSHs

Les composés organiques volatils (COV) sont une catégorie de polluants de l'air dont les techniques d'analyse et méthodes d'échantillonnage peuvent être affectées par l'humidité. Deux voies ont été explorées pour éliminer l'humidité des échantillons de COV avant leur analyse. D'une part, l'élimination de l'humidité par diffusion à travers les parois des sacs d'échantillonnage en film polymère, méthode appelée Sample Water Removal. En plaçant des sacs en Tedlar et Nalophan dans un caisson balayé d'air sec, des échantillons humides de 10 L ont été séchés en respectivement 7 h et 3,5 h. Aucune perte significative de COV jusqu'à 500 ppbv n'a été constatée avec les sacs en Tedlar et 10 µg/m3 avec le sac en Nalophan. D'autre part, l'utilisation de nouveaux adsorbants hydrophobes à base de nanostructures de carbone élaborés au laboratoire ont été testés. Durant les essais d'échantillonnage d'un mélange de COV à 500 ppbv en air humide, ces adsorbants n'ont pas donné les résultats espérés.

The extraembryonic endoderm (XEN) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recently, the committed XENprecursor was identified in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related, anatomically indistinguishable epiblast (EPI)- precursor that gives rise to the fetus. In vitro, the EPI-precursor is represented by the well-known embryonic stem (ES) cell lines, but cell lines representing the XENprecursor are not known. Furthermore, since the XEN-precursor cells were discovered only very recently, the unexpected fact that they express the key pluripotency marker Oct4 has not been explored. Recently, however, our laboratory has isolated rat XEN cell lines that express Oct4, leading to the following two questions: (i) Do these new XEN cell lines represent XEN-precursor cells? (ii) Is their Oct4 expression regulated similarly as previously known from ES cells? These two questions are addressed here by lineage marker and reporter gene analyses. Whole culture analyses showed that rat XEN cell lines expressed markers of all XEN stages including XEN-precursor, primitive endoderm (PrE) and/or visceral endoderm (VE), and parietal endoderm (PE) but trophoectoderm and EPI-precursor markers were missing. In line with this, immunocytochemistry demonstrated heterogeneity and directly visualized the XEN-precursor, PrE/VE, and PE subpopulations. Low-density plating and time-dependent immunocytochemistry on resulting colonies strongly suggested that XEN-precursor cells generate the other XEN stages. Moreover, by analyzing single-cell derived clones, it was shown that culture heterogeneity results from the self-renewal and differentiation of a single cell. Reporter gene analyses using the 5’ regulatory region of the mouse Oct4 gene revealed that a DNA fragment containing the previously described distal enhancer drove reporter gene expression only in ES cells whereas inclusion of an upstream fragment led to high expression in both mouse ES and rat XEN cells. In conclusion, our rat XEN cell lines contain XEN-precursor cells that differentiate extensively, providing for the first time an in vitro model that mimics the natural process of early XEN differentiation. In addition, they regulate Oct4 gene transcription differently than ES cells suggesting heterogeneous Oct4 regulation within the mammalian ICM.

No description available.