The skin epidermis ensures critical vital functions: it represents the first line of defense against external aggressions.<p>The Epidermal Proliferative Unit (EPU) and the stochastic model are two opposite theories that have been proposed to explain the homeostasis of the interfollicular epidermis (IFE). During my thesis, we addressed this question. We used two inducible CREER transgenic mouse models (K14CREER and InvCREER) to labeled basal cells of the IFE using the RosaYFP reporter system, allowing us to follow their fate. Using confocal microscopy to analyze the basal YFP positive clones induced, we demonstrated the existence of two distinct basal progenitors. The mathematical modeling of our data set confirmed this observation and clearly demonstrated the existence of a quiescent stem cell (SC) population (targeted by the K14 promoter), which eventually gives rise to a more rapidly proliferating cells, the committed progenitors (CPs, targeted by the Inv promoter) that participate actively to replace the IFE cells lost during the normal turnover of the tissue. By using an independent genetic mouse model (K5tTA tetO-H2BGFP) and by studying the transcriptional profiling of the two basal populations, we confirmed our hypothesis, and thus reconciling the two seemingly opposite EPU and stochastic models. Finally, we challenged the function of the K14 SCs and Inv CPs during IFE wound healing. Conversely to what happens during homeostasis, we observed that SCs are massively proliferating and participating to the repair and maintenance of the damaged area, while CPs are poorly recruited to the wound.<p>The skin is also a highly sensitive organ, which contains many different receptors specialized in a precise sensation, and richly innervated by somatosensory neurons.<p>The Merkel Cells (MCs) are mechanoreceptors present in the basal layer of the IFE that have features of both neurons and keratinocytes, and might be implicated in the development of a rare but very aggressive skin tumor. Since their discovery in 1875, many studies claimed the neural crest cells (NCCs) or the epidermal progenitors are at the origin of MC, but no clear evidence has been proposed. <p>During my thesis, we were also interested in the embryonic origin of the MC. We used different transgenic mouse models to assess the NCC (Wnt1CRE and Pax3CRE RosaYFP) and the epidermal (K14CRE RosaYFP) hypotheses and we finally demonstrated that MC arise from embryonic epidermal progenitors. Using induction of adult epidermis transgenic mouse (K14CREER RosaYFP, K15CREPR RosaYFP), we showed that the epidermis also participates to the renewing of adult MC. Finally, we deleted the Math1 gene in all the MC progenitors (K14CRE Mathfl/fl) of mouse embryos, resulting in the absence of MCs in adult mice, demonstrating the importance of this transcription factor for MCs specification during embryogenesis.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Adult Stem Cells (SCs) have been found in almost every organ. They are responsible for<p>homeostasis and tissue repair after injury. SCs reside and self-renew in the adult body<p>throughout the life of the organism. In rapid self-renewing organs, such as the skin, the<p>intestine and the blood, SCs divide many times during the life of the animal in order to sustain<p>the homeostatic needs of the tissue.<p>All cells of the body, including SCs, are constantly subjected to DNA assaults arising from<p>endogenous sources, such as reactive oxygen species (ROS) generated by cellular<p>metabolism, or exogenous assaults arising from the environment. The DNA damage response<p>(DDR) and DNA repair mechanisms protect cells from accumulating DNA damage by<p>inducing transient cell cycle arrest allowing DNA repair, triggering senescence or apoptosis.<p>DNA damages trigger the activation of the effectors of the DDR inducing a transient cell<p>cycle arrest, allowing DNA repair, or triggering a permanent arrest of the cell cycle or<p>apoptosis if damages are too extensive.<p>As skin is the outermost barrier of the body, epidermal cells, including SCs, are<p>continuously subjected to genotoxic stress, such as UV rays, ionizing radiation (IR) and<p>chemicals. The skin epidermis is composed of hair follicles (HFs), its associated sebaceous<p>gland (SG) and the surrounding inter-follicular epidermis (IFE). Different types of SCs<p>maintain the homeostasis of the skin; multipotent adult bulge SCs ensure the cyclic<p>regeneration of the HF and the repair of the epidermis after injury, while individual unipotent<p>SCs ensure homeostasis of the SG and the IFE.<p>In tissues with high cellular turnover, such as the epidermis, the numerous divisions that a<p>SC undergoes could result in the accumulation of replication-associated DNA damage. It has<p>been suggested that adult SCs may undergo asymmetric divisions in which the daughter SC<p>retains the older (thus “immortal”) DNA strand, while the daughter cell committed to<p>differentiation inherits the newly synthesized strand that may have incorporated replicationderived<p>mutations. The in vivo relevance of this mechanism is still a matter of intense debate.<p>We used multiple in vivo experimental approaches to investigate precisely how bulge SCssegregate their chromosomes during HF morphogenesis, SC activation and skin homeostasis.<p>Using pulse-chase experiments with two different uridine analogs together with DNAindependent<p>chromatin labelling, we showed that multipotent HF SCs segregate their<p>chromosomes randomly, and that the label-retention observed in the skin epidermis derives<p>solely from relative quiescence of skin SCs 1.<p>We investigated the in vivo response of multipotent adult HF bulge SCs to DNA damage<p>induced by IR. We showed that bulge SCs are profoundly resistant to DNA damage-induced<p>cell death compared to their more mature counterparts. Interestingly, we demonstrated that<p>resistance of bulge SCs to IR-induced apoptosis does not rely on their relative quiescence.<p>Moreover, we showed that DDR in SCs does not lead to premature senescence. We found that<p>two intrinsic cellular mechanisms participate in the resistance of bulge SCs to DNA damageinduced<p>cell death. Bulge SCs express higher level of the anti-apoptotic Bcl-2 and present<p>more transient activation of p53 due to a faster DNA repair activity mediated by a nonhomologous<p>end joining (NHEJ) mechanism. Since NHEJ is not error free, this property<p>might be a double-edged sword, supporting short-term survival of bulge SCs but impairing<p>long-term genomic integrity 2.<p>While we unveiled the relevance of DSBs repair by NHEJ in the skin epidermis, little is<p>known about the role of homologous recombination (HR) during the morphogenesis of the<p>skin epidermis. Brca1 is an essential protein for HR. Conditional deletion of Brca1 in the<p>developing epidermis leads to congenital alopecia accompanied by a decreased density of hair<p>placodes. The remaining HFs never produce mature hair and progressively degenerate due to<p>high levels of apoptosis. Multipotent adult HF bulge SCs cannot be detected in adult HF in<p>the Brca1 cKO epidermis. Brca1 deletion in the epidermis triggers p53 activation throughout<p>the epidermis, which activates apoptosis. Interestingly, IFE and the isthmus region of the HF<p>do not present any pathological phenotype by constitutive deletion of Brca1. Our results<p>demonstrated the critical role of Brca1 during HF morphogenesis. Future studies will be<p>required to understand the molecular mechanisms controlling this phenotype / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

A taxonomia vegetal atualmente exige um grande esforço dos botânicos, desde o processo de aquisição do espécime até a morosa comparação com as amostras já catalogadas em um herbário. Nesse contexto, o projeto TreeVis surge como uma ferramenta para a identificação de vegetais por meio da análise de atributos foliares. Este trabalho é uma ramificação do projeto TreeVis e tem o objetivo de identificar vegetais por meio da análise do corte transversal de uma folha ampliado por um microscópio. Para tanto, foram extraídas assinaturas da cutícula, epiderme superior, parênquima paliçádico e parênquima lacunoso. Cada assinatura foi avaliada isoladamente por uma rede neural pelo método leave-one-out para verificar a sua capacidade de discriminar as amostras. Uma vez selecionados os vetores de características mais importantes, os mesmos foram combinados de duas maneiras. A primeira abordagem foi a simples concatenação dos vetores selecionados; a segunda, mais elaborada, reduziu a dimensionalidade (três atributos apenas) de algumas das assinaturas componentes antes de fazer a concatenação. Os vetores finais obtidos pelas duas abordagens foram testados com rede neural via leave-one-out para medir a taxa de acertos alcançada pelo sinergismo das assinaturas das diferentes partes da folha. Os experimentos consitiram na identificação de oito espécies diferentes e na identificação da espécie Gochnatia polymorpha nos ambientes Cerrado e Mata Ciliar, nas estações Chuvosa e Seca, e sob condições de Sol e Sombra / Currently, taxonomy demands a great effort from the botanists, ranging from the process of acquisition of the sample to the comparison with the species already classified in the herbarium. For this reason, the TreeVis is a project created to identify vegetal species using leaf attributes. This work is a part of the TreeVis project and aims at identifying vegetal species by analysing cross-sections of leaves amplified by a microscope. Signatures were extract from cuticle, adaxial epiderm, palisade parenchyma and sponge parenchyma. Each signature was analysed by a neural network with the leave-one-out method to verify its ability to identify species. Once the most important feature vectors were selected, two different approachs were adopted. The first was a simple concatenation of the selected feature vectors. The second, and more elaborated approach, consisted of reducing the dimensionality (three attributes only) of some component signatures before the feature vector concatenation. The final vectors obtained by these two approaches were tested by a neural network with leave-one-out to measure the correctness rate reached by the synergism of the signatures of different leaf regions. The experiments resulted in the identification of eight different species and the identification of the Gochnatia polymorpha species in Cerradão and Gallery Forest environments, Wet and Dry seasons, and under Sun and Shadow constraints

A taxonomia vegetal atualmente exige um grande esforço dos botânicos, desde o processo de aquisição do espécime até a morosa comparação com as amostras já catalogadas em um herbário. Nesse contexto, o projeto TreeVis surge como uma ferramenta para a identificação de vegetais por meio da análise de atributos foliares. Este trabalho é uma ramificação do projeto TreeVis e tem o objetivo de identificar vegetais por meio da análise do corte transversal de uma folha ampliado por um microscópio. Para tanto, foram extraídas assinaturas da cutícula, epiderme superior, parênquima paliçádico e parênquima lacunoso. Cada assinatura foi avaliada isoladamente por uma rede neural pelo método leave-one-out para verificar a sua capacidade de discriminar as amostras. Uma vez selecionados os vetores de características mais importantes, os mesmos foram combinados de duas maneiras. A primeira abordagem foi a simples concatenação dos vetores selecionados; a segunda, mais elaborada, reduziu a dimensionalidade (três atributos apenas) de algumas das assinaturas componentes antes de fazer a concatenação. Os vetores finais obtidos pelas duas abordagens foram testados com rede neural via leave-one-out para medir a taxa de acertos alcançada pelo sinergismo das assinaturas das diferentes partes da folha. Os experimentos consitiram na identificação de oito espécies diferentes e na identificação da espécie Gochnatia polymorpha nos ambientes Cerrado e Mata Ciliar, nas estações Chuvosa e Seca, e sob condições de Sol e Sombra / Currently, taxonomy demands a great effort from the botanists, ranging from the process of acquisition of the sample to the comparison with the species already classified in the herbarium. For this reason, the TreeVis is a project created to identify vegetal species using leaf attributes. This work is a part of the TreeVis project and aims at identifying vegetal species by analysing cross-sections of leaves amplified by a microscope. Signatures were extract from cuticle, adaxial epiderm, palisade parenchyma and sponge parenchyma. Each signature was analysed by a neural network with the leave-one-out method to verify its ability to identify species. Once the most important feature vectors were selected, two different approachs were adopted. The first was a simple concatenation of the selected feature vectors. The second, and more elaborated approach, consisted of reducing the dimensionality (three attributes only) of some component signatures before the feature vector concatenation. The final vectors obtained by these two approaches were tested by a neural network with leave-one-out to measure the correctness rate reached by the synergism of the signatures of different leaf regions. The experiments resulted in the identification of eight different species and the identification of the Gochnatia polymorpha species in Cerradão and Gallery Forest environments, Wet and Dry seasons, and under Sun and Shadow constraints

Orientador: Nelson Sabino Bittencourt Júnior / Resumo: Compreender a diversidade das Nymphaeales, considerado como o segundo ramo da filogenia das angiospermas, é de considerável interesse como modelo da evolução inicial das angiospermas. Este trabalho objetivou investigar a ontogenia da antera, dos óvulos e sementes em espécies de Nymphaea. Ovários e sementes de N. amazonum Mart. & Zucc. subsp. amazonum, N. caerulea Savigny e N. lotus L. e, anteras de N. amazonum Mart. & Zucc. subsp. amazonum e N. caerulea Savigny, em diversos estádios de desenvolvimento, foram fixados, infiltrados em historesina e as secções obtidas com 1-4 µm foram coradas com azul-de-toluidina O ou submetidas a testes histoquímicos. O desenvolvimento da parede do androsporângio é do tipo básico. Após a meiose, a citocinese simultânea dá origem a tétrades tetraédricas de andrósporos nas quais já são detectáveis a intina e a primexina, há um atraso na liberação dos andrósporos das tétrades em N. caerulea. A partir do estádio de andrósporos livres observa-se uma abertura anelar equatorial e, no estádio de andrósporos vacuolados observa-se o ‘pollenkitt’ na superfície da esporoderme. A mitose do andrósporo ocorre perpendicularmente à esporoderme e, a intina se espessa, principalmente abaixo da abertura, onde há a contribuição da endexina para a formação do oncus. O tapete de N. amazonum e N. caerulea é secretor e não apresenta ciclos invasivo como relatado para N. colorata. O tipo básico de desenvolvimento do androsporângio também foi relatado para Amborella tric... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Understanding the diversification of Nymphaeales, the second branch in the angiosperm phylogenetic tree, has a considerable interest to propose models of early evolution of the angiosperms. This work aims to analyze the anther and ovule ontogeny in species of Nymphaea. Ovaries and seeds of N. amazonum Mart. & Zucc. subsp. amazonum, N. caerulea Savigny and N. lotus L. and, anthers of N. amazonum Mart. & Zucc. subsp. amazonum and N. caerulea Savigny, in several development stages, were fixed infiltrated in historesin and the sections with 1-4 µm were stained with toluidine blue O or submitted to histochemical tests. The androsporangium wall follows the basic type of development. After meiosis, a simultaneous cytokinesis give rise a tetrahedral tetrad of androspores, the intine and primexine is detectable in this stage and, in N. caerulea occurs a delay in the release of androspores from the tetrads. We observe an equatorial ring-like aperture in the free androspores stage and, in vacuolated androspores the ‘pollenkitt’ is visualized in the sporoderm surface. The mitosis of the androspore occurs perpendicularly to sporoderm and, the intine begins to thicken beneath the aperture and, together with the endexine originate the oncus. The tapetum of Nymphaea amazonum and N. caerulea are secretor and do not show invasive cycles as reported to N. colorata. The basic type of androsporangium wall development also are reported to Amborella trichopoda, Nuphar pumila and for the species of ... (Complete abstract click electronic access below) / Doutor