Canola (Brassica napus) contributes $19.3 billion dollars to the Canadian economy each year as a result of its oil- and protein-rich seeds. These economically important seed products are produced in highest concentration in the embryo. Embryo development is supported nutritionally and structurally by the maternal subregions, which include the inner (ISC) and outer distal seed coat (OSC), the chalazal seed coat (CZSC), and the chalazal proliferating tissue (CPT). Research on the maternal seed subregions is limited to the SC as a result of its accessibility; the embedded CZSC and CPT subregions have yet to be characterized in canola. Using light and transmission electron microscopy, I found the CZSC and CPT to be anatomically distinct and experience profound changes throughout seed development. To understand these changes at the RNA level, laser microdissection and RNA sequencing were used to profile these subregions spatially and temporally from the ovule to mature green stage of seed development. Employing vigorous bioinformatics analyses, I found that the maternal subregions are transcriptomically distinct and possess unique RNA populations. From here I began to elucidate the biological processes operating within the maternal subregions. As a whole, the maternal subregions appear to have a critical role in transporting nutrients to the filial subregions as well as in coping with oxidative stress produced during these energy-rich processes. Additionally, using CanEnrich, I was able to generate predictive transcriptional circuits regulating the biological processes occurring within the maternal seed. This research has produced the most comprehensive dataset on the canola seed to date and will provide a valuable resource for research on seed development as well as seed improvement. / October 2016

Introdução: O carcinoma espinocelular (CEC) representa 7% de todos os novos casos de câncer no mundo, sendo o carcinoma espinocelular o tipo mais frequente. Tanto o comportamento biológico quanto o crescimento dos tumores devem ser melhores entendidos, uma vez que a sobrevida dos pacientes apresentou discreta melhora nas últimas décadas. Os modelos PDX foram desenvolvidos para estudar a biologia tumoral e principalmente os mecanismos de crescimento e proliferação através da manutenção da arquitetura e microambiente tumoral do tumor original. Os retinóides possuem a capacidade de restaurar o crescimento e a diferenciação de células normais através da ação dos receptores retinóides nucleares (RARs e RXRs) que são os principais mediadores destas ações que ao sofrerem alterações na sua expressão podem levar ao desenvolvimento e manutenção de tumores. No estudo da carcinogênese o modelo PDX é uma importante ferramenta pois mantém a arquitetura e microambiente do tumor original melhorando a compreensão de algumas vias, entre estas o processo de EMT/MET, na diferenciação das células tronco tumorais e quais receptores nucleares podem estar influenciando nestas vias. Objetivos: Analisar os padrões de comportamento biológico - tempo de formação e expansão do tumor e a manutenção dos padrões histológicos e de arquitetura do tumor original - em F0 e F1 no modelo PDX (xenoenxerto derivado de paciente) das amostras de centro de tumor e epitélio adjacente em camundongos Balb C/nude e avaliar a expressão gênica dos receptores retinóides, ALDH1 e marcadores do processo de EMT/MET por RT-PCR em PDX de carcinoma espinocelular oral em comparação com a amostra dos pacientes doadores nas passagens F(0) e F(1). Método: 24 camundongos Balb C/Nude, divididos em 2 grupos TG(I) – tumor graft paciente (I) e TG(II) – tumor graft paciente (II), subdivididos em 4 grupos de 3 animais: (A) – receberam PDX do centro do tumor; (B) – receberam PDX de epitélio adjacente ao tumor (margem de segurança cirúrgica); (C) receberam PDX de um animal do grupo (A); (D) receberam PDX de um animal do grupo (B). E Após estas fases, as amostras coletadas serão avaliadas por RT-PCR para comparação das expressões gênicas entre a amostra original (CT e EA) com os PDX´s nas passagens F(0) e F(1). Resultados: formação de tumores em todos os grupos – tanto do PDX de fragmento de centro do tumor quanto do PDX do epitélio adjacente. E A expressão gênica dos parâmetros observados não diferem no tumor original e passagem F(0) significativamente diferentes em F(1) (p<0,05). Conclusões: A técnida do PDX para o CEC é possível de ser realizada em menor tempo com a implantação de apenas um fragmento do tumor. Os tumores resultantes do PDX apresentaram tamanho suficiente para novas passagens, bem como para seu 6 uso em estudos de comportamento biológico das células neoplásicas. Quanto ao epitélio adjacente ao tumor (margem de segurança cirúrgica) constatou-se a presença de células tumorais com potencial de promover o crescimento de tumores devendo portanto ser melhor observada nas ressecções. O PDX de primeira passagem F(0) é o que mais se assemelha com o tumor original sendo o melhor para testes terapêuticos e estudos da carcinogênese do CEC oral. Keywords: CECP, modelo PDX, xenoenxerto, margem de segurança cirúrgica, , receptores retinóides, microdissecção a laser. / Introduction: Squamous cell carcinoma (SCC) represents 7% of all new cases of cancer in the world, with squamous cell carcinoma being the most frequent type. Both the biological behavior and the growth of the patients should be better understood, since the patients' survival show unobtrusive improvement in the last decades. PDX models were developed to study a tumor biology and especially the mechanisms of growth and proliferation through maintenance of the architecture and tumor microenvironment of the original tumor. Retinoids have a capacity to restore normal cell growth and differentiation through the action of nuclear retinoid receptors (RARs and RXRs) that are the main mediators and maintenance actions of tumors. In the study of carcinogenesis, the PDX model is an important tool because it maintains an architecture and microenvironment of the original tumor, improving an understanding of some pathways, among them in the EMT / MET process, the difference in tumor stem cells and which nuclear receptors may be influencing these routes. Objectives: To analyze changes in methodology and patterns of biological behavior - time of tumor formation and expansion and maintenance of histological and architectural patterns of the original tumor - in F0 and F1 without PDX model (patient derived xenograft) tumor and adjacent epithelium in Balb C / nude mice and to evaluate the gene expression of retinoid receptors, ALDH1 and EMT / MET process markers by RT-PCR in PDX of oral squamous cell carcinoma compared to a sample of donor patients in F ( 0) and F (1). Method: 24 Balb C / Nude mice, divided into 2 groups TG (I) - patient tumor graft (I) and TG (II) - patient tumor graft (II) subdivided into 4 groups of 3 animals: (A) - received PDX from the center of the tumor; (B) - received epithelial PDX adjacent to the tumor (surgical margin of safety); (C) received PDX from one animal of group (A); (D) received PDX from one animal of group (B). E After these phases, as samples collected for RT-PCR evaluation for comparison of gene expressions between an original sample (CT and EA) with F passages of PDX F (0) and F (1). Results: tumor formation in all groups - both the PDX of the tumor center fragment and the PDX of the adjacent epithelium. E The gene expression of the observed parameters did not differ without original tumor and F (0) differential passage in F (1) (p <0.05). Conclusions: The PDX technique for CPB is possible to be performed in a shorter time with a tumor fragment implantation. Tumors resulting from PDX presented the solution for new passages, as well as for their use in studies of the biological behavior of neoplastic cells. As for the epithelium adjacent to the tumor (surgical margin of safety), a presence of tumor cells with the potential to promote the growth of tumors has been observed and should therefore be better observed in the resections. The first pass PDX F (0) is the one that most closely resembles the 8 original tumor being the best for therapeutic tests and studies of oral SCC carcinogenesis.

O gênero Passiflora é o maior gênero da família Passifloraceae, que compreende mais de 500 espécies, a maioria originária de regiões neotropicais, sendo centenas distribuídas pela América Latina. Algumas dessas espécies apresentam importância econômica na produção de fruta in natura, suco concentrado, uso ornamental e medicinal com propriedades sedativas. Estudos do desenvolvimento reprodutivo e do fruto de espécies de Passiflora são fundamentais para melhor compreensão de aspectos do desenvolvimento que possam contribuir para a produção agronômica e compreensão da evolução de estruturas florais presentes em espécies desta família. A importância das sementes para a sua propagação, para estudos taxonômicos e a presença do arilo, estrutura de interesse fundamental para a comercialização de frutos e produção de suco em espécies desse gênero, estimulou a elaboração de um estudo comparativo entre duas espécies de interesse comercial, P. edulis e P alata, associando o estudo de características morfoanatômicas e moleculares. O presente projeto teve como objetivo caracterizar o desenvolvimento do embrião zigótico e arilo de Passiflora edulis Sims e Passiflora alata Curtis. Flores foram manualmente polinizadas e amostras coletadas periodicamente após a polinização, visando à obtenção de sementes em diferentes fases do desenvolvimento embrionário e do arilo. Primórdios do arilo são observados em pré-antese, quando o saco embrionário é organizado. Células epidérmicas na base do funículo sofrem divisões periclinais formando uma borda em torno da rafe. O desenvolvimento do primórdio do arilo é interrompido, observando-se a reativação de divisões celulares e o arilo recomeça o desenvolvimento em uma estrutura multicelular ao redor da semente em desenvolvimento. Aos 14 dias após a polinização o arilo já cobre dois terços da semente, crescendo rapidamente até a semente ser recoberta totalmente, desde o funículo, até o polo calazal. O endosperma é nuclear e seu desenvolvimento se inicia logo após a fertilização, através de divisões sucessivas, formando um sincício ao redor do proembrião, simultaneamente à diminuição tamanho do nucelo. A celularização do endosperma, com a deposição de paredes celulares é observada aproximadamente 20 dias após a polinização. A embriogênese se inicia com a primeira divisão do zigoto, observada aos 7 dias após a polinização. Essa primeira divisão é transversal dividindo o zigoto em duas células, assimetricamente. A célula apical sofre sucessivas divisões que levam a estádios subsequentes de desenvolvimento do embrião, tais como, 4-8 células, globular, coração e torpedo. Aproximadamente 30 dias após a polinização o embrião atinge o estádio cotiledonar e a partir de então apenas aumenta em tamanho consumindo o endosperma e ocupando seu espaço na semente. Essas observações permitiram que fossem definidos dois estádios específicos do desenvolvimento do arilo para futura captura por microdissecção a laser. A caracterização anatômica do desenvolvimento do embrião e do arilo em ambas as espécies subsidia o estabelecimento de estádios específicos do desenvolvimento que podem servir como alvos para estudos moleculares nessas espécies de Passiflora / Passiflora is the largest genus in Passifloraceae and most of the commercially used species develop an aril around the seed, which is commercially important for fresh fruit consumption, and concentrate juice. Reproductive developmental studies associating morphoanatomical and molecular characteristics are essential for a better understanding of particularities of this genus. The present project aimed to characterize the development of Passiflora edulis Sims and Passiflora alata Curtis zygotic embryo and aril. Pollination of flowers were done manually, fruits and ovaries were collected at regular intervals after pollination and processed for scanning and light microscopy, for analysis of embryos and aril in different stages of development. Aril primordium is observed in pre-anthesis when the embryo sac is organized. Epidermic cells at the base of the funiculus undergo periclinal divisions forming a rim surrounding the raphe. Aril development is arrested until after fertilization when cell divisions are reactivated and the aril resume development into a multicellular structure surrounding the developing seed from the funicle towards the chalazal end. At approximately 14 days after pollination the aril already covers two thirds of the seed, and grows rapidly until the whole seed is covered. The endosperm is nuclear and starts developing soon after fertilization through successive divisions forming a syncytium mostly at the chalazal region, and around the developing embryo, replacing the nucellus. Cell walls are formed and the endosperm begins cellularization approximately 20 days after pollination. Embryogenesis initiates with the first division of the zygote, approximately 7 days after pollination. This first cell division is transversal and asymmetrical; the apical cell undergoes successive divisions leading to the subsequent stages of embryo development such as 4- and 8-celled, globular, heart-shaped, torpedo. Approximately 30 days after pollination, the embryo reaches the cotyledonary stage and thereafter grows only in size, consuming the endosperm and occupying its space in the seed. The first division of the zygote was observed around seven days after pollination (DAP), with the mature embryo formed approximately 30 DAP. Initial development of the aril primordium is observed at the ovule basal region, before anthesis/pollination. Embryo and aril development occurs simultaneously. These observations allowed for the definition of two specific stages of aril development for laser-capture-microdissection and further molecular analysis aiming at the evaluation of the molecular basis of aril differentiation in Passiflora. The morphoanatomical characterization of embryo and aril development in these species will serve as a source of information for the definition of specific developmental stages, which can be targets for molecular studies in Passiflora

Introdução: O carcinoma espinocelular (CEC) representa 7% de todos os novos casos de câncer no mundo, sendo o carcinoma espinocelular o tipo mais frequente. Tanto o comportamento biológico quanto o crescimento dos tumores devem ser melhores entendidos, uma vez que a sobrevida dos pacientes apresentou discreta melhora nas últimas décadas. Os modelos PDX foram desenvolvidos para estudar a biologia tumoral e principalmente os mecanismos de crescimento e proliferação através da manutenção da arquitetura e microambiente tumoral do tumor original. Os retinóides possuem a capacidade de restaurar o crescimento e a diferenciação de células normais através da ação dos receptores retinóides nucleares (RARs e RXRs) que são os principais mediadores destas ações que ao sofrerem alterações na sua expressão podem levar ao desenvolvimento e manutenção de tumores. No estudo da carcinogênese o modelo PDX é uma importante ferramenta pois mantém a arquitetura e microambiente do tumor original melhorando a compreensão de algumas vias, entre estas o processo de EMT/MET, na diferenciação das células tronco tumorais e quais receptores nucleares podem estar influenciando nestas vias. Objetivos: Analisar os padrões de comportamento biológico - tempo de formação e expansão do tumor e a manutenção dos padrões histológicos e de arquitetura do tumor original - em F0 e F1 no modelo PDX (xenoenxerto derivado de paciente) das amostras de centro de tumor e epitélio adjacente em camundongos Balb C/nude e avaliar a expressão gênica dos receptores retinóides, ALDH1 e marcadores do processo de EMT/MET por RT-PCR em PDX de carcinoma espinocelular oral em comparação com a amostra dos pacientes doadores nas passagens F(0) e F(1). Método: 24 camundongos Balb C/Nude, divididos em 2 grupos TG(I) – tumor graft paciente (I) e TG(II) – tumor graft paciente (II), subdivididos em 4 grupos de 3 animais: (A) – receberam PDX do centro do tumor; (B) – receberam PDX de epitélio adjacente ao tumor (margem de segurança cirúrgica); (C) receberam PDX de um animal do grupo (A); (D) receberam PDX de um animal do grupo (B). E Após estas fases, as amostras coletadas serão avaliadas por RT-PCR para comparação das expressões gênicas entre a amostra original (CT e EA) com os PDX´s nas passagens F(0) e F(1). Resultados: formação de tumores em todos os grupos – tanto do PDX de fragmento de centro do tumor quanto do PDX do epitélio adjacente. E A expressão gênica dos parâmetros observados não diferem no tumor original e passagem F(0) significativamente diferentes em F(1) (p<0,05). Conclusões: A técnida do PDX para o CEC é possível de ser realizada em menor tempo com a implantação de apenas um fragmento do tumor. Os tumores resultantes do PDX apresentaram tamanho suficiente para novas passagens, bem como para seu 6 uso em estudos de comportamento biológico das células neoplásicas. Quanto ao epitélio adjacente ao tumor (margem de segurança cirúrgica) constatou-se a presença de células tumorais com potencial de promover o crescimento de tumores devendo portanto ser melhor observada nas ressecções. O PDX de primeira passagem F(0) é o que mais se assemelha com o tumor original sendo o melhor para testes terapêuticos e estudos da carcinogênese do CEC oral. Keywords: CECP, modelo PDX, xenoenxerto, margem de segurança cirúrgica, , receptores retinóides, microdissecção a laser. / Introduction: Squamous cell carcinoma (SCC) represents 7% of all new cases of cancer in the world, with squamous cell carcinoma being the most frequent type. Both the biological behavior and the growth of the patients should be better understood, since the patients' survival show unobtrusive improvement in the last decades. PDX models were developed to study a tumor biology and especially the mechanisms of growth and proliferation through maintenance of the architecture and tumor microenvironment of the original tumor. Retinoids have a capacity to restore normal cell growth and differentiation through the action of nuclear retinoid receptors (RARs and RXRs) that are the main mediators and maintenance actions of tumors. In the study of carcinogenesis, the PDX model is an important tool because it maintains an architecture and microenvironment of the original tumor, improving an understanding of some pathways, among them in the EMT / MET process, the difference in tumor stem cells and which nuclear receptors may be influencing these routes. Objectives: To analyze changes in methodology and patterns of biological behavior - time of tumor formation and expansion and maintenance of histological and architectural patterns of the original tumor - in F0 and F1 without PDX model (patient derived xenograft) tumor and adjacent epithelium in Balb C / nude mice and to evaluate the gene expression of retinoid receptors, ALDH1 and EMT / MET process markers by RT-PCR in PDX of oral squamous cell carcinoma compared to a sample of donor patients in F ( 0) and F (1). Method: 24 Balb C / Nude mice, divided into 2 groups TG (I) - patient tumor graft (I) and TG (II) - patient tumor graft (II) subdivided into 4 groups of 3 animals: (A) - received PDX from the center of the tumor; (B) - received epithelial PDX adjacent to the tumor (surgical margin of safety); (C) received PDX from one animal of group (A); (D) received PDX from one animal of group (B). E After these phases, as samples collected for RT-PCR evaluation for comparison of gene expressions between an original sample (CT and EA) with F passages of PDX F (0) and F (1). Results: tumor formation in all groups - both the PDX of the tumor center fragment and the PDX of the adjacent epithelium. E The gene expression of the observed parameters did not differ without original tumor and F (0) differential passage in F (1) (p <0.05). Conclusions: The PDX technique for CPB is possible to be performed in a shorter time with a tumor fragment implantation. Tumors resulting from PDX presented the solution for new passages, as well as for their use in studies of the biological behavior of neoplastic cells. As for the epithelium adjacent to the tumor (surgical margin of safety), a presence of tumor cells with the potential to promote the growth of tumors has been observed and should therefore be better observed in the resections. The first pass PDX F (0) is the one that most closely resembles the 8 original tumor being the best for therapeutic tests and studies of oral SCC carcinogenesis.

Introdução: O carcinoma espinocelular (CEC) representa 7% de todos os novos casos de câncer no mundo, sendo o carcinoma espinocelular o tipo mais frequente. Tanto o comportamento biológico quanto o crescimento dos tumores devem ser melhores entendidos, uma vez que a sobrevida dos pacientes apresentou discreta melhora nas últimas décadas. Os modelos PDX foram desenvolvidos para estudar a biologia tumoral e principalmente os mecanismos de crescimento e proliferação através da manutenção da arquitetura e microambiente tumoral do tumor original. Os retinóides possuem a capacidade de restaurar o crescimento e a diferenciação de células normais através da ação dos receptores retinóides nucleares (RARs e RXRs) que são os principais mediadores destas ações que ao sofrerem alterações na sua expressão podem levar ao desenvolvimento e manutenção de tumores. No estudo da carcinogênese o modelo PDX é uma importante ferramenta pois mantém a arquitetura e microambiente do tumor original melhorando a compreensão de algumas vias, entre estas o processo de EMT/MET, na diferenciação das células tronco tumorais e quais receptores nucleares podem estar influenciando nestas vias. Objetivos: Analisar os padrões de comportamento biológico - tempo de formação e expansão do tumor e a manutenção dos padrões histológicos e de arquitetura do tumor original - em F0 e F1 no modelo PDX (xenoenxerto derivado de paciente) das amostras de centro de tumor e epitélio adjacente em camundongos Balb C/nude e avaliar a expressão gênica dos receptores retinóides, ALDH1 e marcadores do processo de EMT/MET por RT-PCR em PDX de carcinoma espinocelular oral em comparação com a amostra dos pacientes doadores nas passagens F(0) e F(1). Método: 24 camundongos Balb C/Nude, divididos em 2 grupos TG(I) – tumor graft paciente (I) e TG(II) – tumor graft paciente (II), subdivididos em 4 grupos de 3 animais: (A) – receberam PDX do centro do tumor; (B) – receberam PDX de epitélio adjacente ao tumor (margem de segurança cirúrgica); (C) receberam PDX de um animal do grupo (A); (D) receberam PDX de um animal do grupo (B). E Após estas fases, as amostras coletadas serão avaliadas por RT-PCR para comparação das expressões gênicas entre a amostra original (CT e EA) com os PDX´s nas passagens F(0) e F(1). Resultados: formação de tumores em todos os grupos – tanto do PDX de fragmento de centro do tumor quanto do PDX do epitélio adjacente. E A expressão gênica dos parâmetros observados não diferem no tumor original e passagem F(0) significativamente diferentes em F(1) (p<0,05). Conclusões: A técnida do PDX para o CEC é possível de ser realizada em menor tempo com a implantação de apenas um fragmento do tumor. Os tumores resultantes do PDX apresentaram tamanho suficiente para novas passagens, bem como para seu 6 uso em estudos de comportamento biológico das células neoplásicas. Quanto ao epitélio adjacente ao tumor (margem de segurança cirúrgica) constatou-se a presença de células tumorais com potencial de promover o crescimento de tumores devendo portanto ser melhor observada nas ressecções. O PDX de primeira passagem F(0) é o que mais se assemelha com o tumor original sendo o melhor para testes terapêuticos e estudos da carcinogênese do CEC oral. Keywords: CECP, modelo PDX, xenoenxerto, margem de segurança cirúrgica, , receptores retinóides, microdissecção a laser. / Introduction: Squamous cell carcinoma (SCC) represents 7% of all new cases of cancer in the world, with squamous cell carcinoma being the most frequent type. Both the biological behavior and the growth of the patients should be better understood, since the patients' survival show unobtrusive improvement in the last decades. PDX models were developed to study a tumor biology and especially the mechanisms of growth and proliferation through maintenance of the architecture and tumor microenvironment of the original tumor. Retinoids have a capacity to restore normal cell growth and differentiation through the action of nuclear retinoid receptors (RARs and RXRs) that are the main mediators and maintenance actions of tumors. In the study of carcinogenesis, the PDX model is an important tool because it maintains an architecture and microenvironment of the original tumor, improving an understanding of some pathways, among them in the EMT / MET process, the difference in tumor stem cells and which nuclear receptors may be influencing these routes. Objectives: To analyze changes in methodology and patterns of biological behavior - time of tumor formation and expansion and maintenance of histological and architectural patterns of the original tumor - in F0 and F1 without PDX model (patient derived xenograft) tumor and adjacent epithelium in Balb C / nude mice and to evaluate the gene expression of retinoid receptors, ALDH1 and EMT / MET process markers by RT-PCR in PDX of oral squamous cell carcinoma compared to a sample of donor patients in F ( 0) and F (1). Method: 24 Balb C / Nude mice, divided into 2 groups TG (I) - patient tumor graft (I) and TG (II) - patient tumor graft (II) subdivided into 4 groups of 3 animals: (A) - received PDX from the center of the tumor; (B) - received epithelial PDX adjacent to the tumor (surgical margin of safety); (C) received PDX from one animal of group (A); (D) received PDX from one animal of group (B). E After these phases, as samples collected for RT-PCR evaluation for comparison of gene expressions between an original sample (CT and EA) with F passages of PDX F (0) and F (1). Results: tumor formation in all groups - both the PDX of the tumor center fragment and the PDX of the adjacent epithelium. E The gene expression of the observed parameters did not differ without original tumor and F (0) differential passage in F (1) (p <0.05). Conclusions: The PDX technique for CPB is possible to be performed in a shorter time with a tumor fragment implantation. Tumors resulting from PDX presented the solution for new passages, as well as for their use in studies of the biological behavior of neoplastic cells. As for the epithelium adjacent to the tumor (surgical margin of safety), a presence of tumor cells with the potential to promote the growth of tumors has been observed and should therefore be better observed in the resections. The first pass PDX F (0) is the one that most closely resembles the 8 original tumor being the best for therapeutic tests and studies of oral SCC carcinogenesis.

O gênero Passiflora é o maior gênero da família Passifloraceae, que compreende mais de 500 espécies, a maioria originária de regiões neotropicais, sendo centenas distribuídas pela América Latina. Algumas dessas espécies apresentam importância econômica na produção de fruta in natura, suco concentrado, uso ornamental e medicinal com propriedades sedativas. Estudos do desenvolvimento reprodutivo e do fruto de espécies de Passiflora são fundamentais para melhor compreensão de aspectos do desenvolvimento que possam contribuir para a produção agronômica e compreensão da evolução de estruturas florais presentes em espécies desta família. A importância das sementes para a sua propagação, para estudos taxonômicos e a presença do arilo, estrutura de interesse fundamental para a comercialização de frutos e produção de suco em espécies desse gênero, estimulou a elaboração de um estudo comparativo entre duas espécies de interesse comercial, P. edulis e P alata, associando o estudo de características morfoanatômicas e moleculares. O presente projeto teve como objetivo caracterizar o desenvolvimento do embrião zigótico e arilo de Passiflora edulis Sims e Passiflora alata Curtis. Flores foram manualmente polinizadas e amostras coletadas periodicamente após a polinização, visando à obtenção de sementes em diferentes fases do desenvolvimento embrionário e do arilo. Primórdios do arilo são observados em pré-antese, quando o saco embrionário é organizado. Células epidérmicas na base do funículo sofrem divisões periclinais formando uma borda em torno da rafe. O desenvolvimento do primórdio do arilo é interrompido, observando-se a reativação de divisões celulares e o arilo recomeça o desenvolvimento em uma estrutura multicelular ao redor da semente em desenvolvimento. Aos 14 dias após a polinização o arilo já cobre dois terços da semente, crescendo rapidamente até a semente ser recoberta totalmente, desde o funículo, até o polo calazal. O endosperma é nuclear e seu desenvolvimento se inicia logo após a fertilização, através de divisões sucessivas, formando um sincício ao redor do proembrião, simultaneamente à diminuição tamanho do nucelo. A celularização do endosperma, com a deposição de paredes celulares é observada aproximadamente 20 dias após a polinização. A embriogênese se inicia com a primeira divisão do zigoto, observada aos 7 dias após a polinização. Essa primeira divisão é transversal dividindo o zigoto em duas células, assimetricamente. A célula apical sofre sucessivas divisões que levam a estádios subsequentes de desenvolvimento do embrião, tais como, 4-8 células, globular, coração e torpedo. Aproximadamente 30 dias após a polinização o embrião atinge o estádio cotiledonar e a partir de então apenas aumenta em tamanho consumindo o endosperma e ocupando seu espaço na semente. Essas observações permitiram que fossem definidos dois estádios específicos do desenvolvimento do arilo para futura captura por microdissecção a laser. A caracterização anatômica do desenvolvimento do embrião e do arilo em ambas as espécies subsidia o estabelecimento de estádios específicos do desenvolvimento que podem servir como alvos para estudos moleculares nessas espécies de Passiflora / Passiflora is the largest genus in Passifloraceae and most of the commercially used species develop an aril around the seed, which is commercially important for fresh fruit consumption, and concentrate juice. Reproductive developmental studies associating morphoanatomical and molecular characteristics are essential for a better understanding of particularities of this genus. The present project aimed to characterize the development of Passiflora edulis Sims and Passiflora alata Curtis zygotic embryo and aril. Pollination of flowers were done manually, fruits and ovaries were collected at regular intervals after pollination and processed for scanning and light microscopy, for analysis of embryos and aril in different stages of development. Aril primordium is observed in pre-anthesis when the embryo sac is organized. Epidermic cells at the base of the funiculus undergo periclinal divisions forming a rim surrounding the raphe. Aril development is arrested until after fertilization when cell divisions are reactivated and the aril resume development into a multicellular structure surrounding the developing seed from the funicle towards the chalazal end. At approximately 14 days after pollination the aril already covers two thirds of the seed, and grows rapidly until the whole seed is covered. The endosperm is nuclear and starts developing soon after fertilization through successive divisions forming a syncytium mostly at the chalazal region, and around the developing embryo, replacing the nucellus. Cell walls are formed and the endosperm begins cellularization approximately 20 days after pollination. Embryogenesis initiates with the first division of the zygote, approximately 7 days after pollination. This first cell division is transversal and asymmetrical; the apical cell undergoes successive divisions leading to the subsequent stages of embryo development such as 4- and 8-celled, globular, heart-shaped, torpedo. Approximately 30 days after pollination, the embryo reaches the cotyledonary stage and thereafter grows only in size, consuming the endosperm and occupying its space in the seed. The first division of the zygote was observed around seven days after pollination (DAP), with the mature embryo formed approximately 30 DAP. Initial development of the aril primordium is observed at the ovule basal region, before anthesis/pollination. Embryo and aril development occurs simultaneously. These observations allowed for the definition of two specific stages of aril development for laser-capture-microdissection and further molecular analysis aiming at the evaluation of the molecular basis of aril differentiation in Passiflora. The morphoanatomical characterization of embryo and aril development in these species will serve as a source of information for the definition of specific developmental stages, which can be targets for molecular studies in Passiflora

The atrioventricular junction (AVJ) is a complex anatomical structure. It has an important role in maintaining synchronised atrioventricular conduction and protects from ventricular tachycardia, as well as bradycardia. Its embryological development and function is under tight transcription factor control. Heart failure is a chronic systemic condition, affecting one million people in the UK alone. Slowing of atrioventricular conduction in heart failure is associated with increased morbidity and mortality. The molecular and anatomical basis of abnormal atrioventricular conduction was studied in a rabbit model of heart failure due to aortic insufficiency and abdominal aortic constriction. The PR interval was significantly prolonged in heart failure animals. Using laser-assisted microdissection, the tiny tissues of the AVJ were collected for RT-PCR analysis. HCN1, Cav1.3, Cx40 and Cx43 transcripts were significantly downregulated by heart failure, with a compensatory increase in CLCN2, Nav1.1, Navβ1, SUR2A and PAK1. Immunolabelling for Cx43 showed reduction in protein level and longitudinal dissociation not only in the inferior nodal extension but also in the His bundle in heart failure animals. Anatomical studies of the AVJ have previously been limited by its small size and inaccessible location. Contrast-enhanced micro-CT scanning allowed non-destructive imaging of the AVJ anatomy. AVJ length and volume were increased in the rabbit model of heart failure, which is expected to contribute to atrioventricular conduction abnormalities. Micro-CT additionally resolved the anatomy of the canine AVJ and atria, including fibre orientation in the pulmonary vein sleeves and Bachmann’s bundle. The physiological effects of loss of T-box transcription factor 5 (Tbx5) in the AVJ were studied in a transgenic inducible Tbx5 knockout mouse model using optical mapping. Tbx5-deficient mice had a prolonged PR interval in vivo and a higher incidence of atrioventricular block and ventricular conduction abnormalities in Langendorff-perfused hearts.

Le carcinome épidermoïde cutané (CEC) représente un enjeu important par sa fréquence et sa gravité potentielle.L’agressivité de ce cancer est liée à l’invasion péri-nerveuse (IPN), mode d’envahissement tumoral reconnu comme un facteur de mauvais pronostic.L’objectif de ce travail est de s’intéresser aux mécanismes favorisant l’IPN, en comparant 2 groupes appariés de CEC humains, avec et sans IPN.Pour cela nous avons réalisé une étude de facteurs et récepteurs neurotrophiques, de marqueurs de la transition épithélio-mésenchymateuse (TEM), et de la molécule NCAM1, par analyse immunohistochimique à partir de pièces chirurgicales de CEC et par analyse moléculaire en droplet digital PCR sur des cellules tumorales microdisséquées.L’analyse immunohistochimique a trouvé une forte expression de BDNF, TrkB, p75NGFR, Snail 1 et NCMA1 dans les cellules tumorales péri-nerveuses, contrastant avec une faible expression de ces marqueurs dans les cellules tumorales à distance du nerf. L’E-cadhérine était diminuée dans les cellules tumorales péri-nerveuses.L’analyse moléculaire en ddPCR montrait une diminution d’expression de l’E-cadhérine et une surexpression de BDNF, TrkB, p75NGFR, Snail1, Slug, Zeb2, Twist1 et NCAM1 dans les cellules tumorales péri-nerveuses par rapport aux cellules tumorales distantes du nerf.Nous avons démontré dans ce travail que l’invasion péri-nerveuse dans les CEC humains est liée aux neurotrophines, à la TEM et implique NCAM1. / Cutaneous squamous cell carcinoma (SCC) is an important issue because of its frequency and potential severity.The aggressiveness of this cancer is related to perineural invasion (PNI), a mode of tumor dissemination recognized as a poor prognosis factor.The aim of this work is to study the mechanisms of PNI, comparing 2 matched- groups of human SCC with and without PNI.For this, we studied neurotrophins, epithelial-mesenchymal transition (EMT) markers, and the NCAM1 molecule, by immunohistochemistry analysis on surgical pieces of SCC and by molecular analysis with digital-droplet PCR on laser-microdissected tumor cells.Immunohistochemistry analysis found strong expression of BDNF, TrkB, p75NGFR, Snail 1 and NCMA1 in perineural tumor cells, contrasting with weak expression of these markers in tumor cells distant from the nerves. E-cadherin was decreased in perineural tumor cells.Molecular analysis in ddPCR showed decreased expression for E-cadherin and overexpression of BDNF, TrkB, p75NGFR, Snail1, Slug, Zeb2, Twist1 and NCAM1 in perineural tumor cells compared to tumor cells distant from the nerves.We have demonstrated in this work that PNI in human SCC is linked to neurotrophins and EMT, and involves NCAM1.

Many omics-level studies have been undertaken on Aiptasia, however, our understanding of the genes and processes associated with symbiosis regulation and maintenance is still limited. To gain deeper insights into the molecular processes underlying this association, we investigated this relationship using multipronged approaches combining next generation sequencing with metabolomics and immunohistochemistry. We identified 731 high-confident symbiosis-associated genes using meta-analysis. Coupled with metabolomic profiling, we exposed that symbiont-derived carbon enables host recycling of ammonium into nonessential amino acids, which may serve as a regulatory mechanism to control symbiont growth through a carbon-dependent negative feedback of nitrogen availability to the symbiont. We then characterized two symbiosis-associated ammonium transporters (AMTs). Both of the proteins exhibit gastrodermis-specific localization in symbiotic anemones. Their tissuespecific localization consistent with the higher ammonium assimilation rate in gastrodermis of symbiotic Aiptasia as shown by 15N labeling and nanoscale secondary ion mass spectrometry (NanoSIMS). Inspired by the tissue-specific localization of AMTs, we investigated spatial expression of genes in Aiptasia. Our results suggested that symbiosis with Symbiodiniaceae is the main driver for transcriptional changes in Aiptasia. We focused on the phagosome-associated genes and identified several key factors involved in phagocytosis and the formation of symbiosome. Our study provided the first insights into the tissue specific complexity of gene expression in Aiptasia. To investigate symbiosis-induced response in symbiont and to find further evidence for the hypotheses generated from our host-focused analyses, we explored the growth and gene expression changes of Symbiodiniaceae in response to the limitations of three essential nutrients: nitrogen, phosphate, and iron, respectively. Comparisons of the expression patterns of in hospite Symbiodiniaceae to these nutrient limiting conditions showed a strong and significant correlation of gene expression profiles to the nitrogen-limited culture condition. This confirmed the nitrogen-limited growing condition of Symbiodiniaceae in hospite, and further supported our hypothesis that the host limits the availability of nitrogen, possibly to regulate symbiont cell density. In summary, we investigated different molecular aspects of symbiosis from both the host’s and symbiont’s perspective. This dissertation provides novel insights into the function of nitrogen, and the potential underlying molecular mechanisms, in the metabolic interactions between Aiptasia and Symbiodiniaceae.

Le carcinome hépatocellulaire (CHC) est la forme la plus fréquente de cancer du foie et l’une des principales causes de mortalité par cancer dans le monde. Il s’agit d’une maladie de mauvais pronostic, aux ressources thérapeutiques limitées, hétérogène sur le plan immunophénotypique et génomique, qui se développe très souvent sur un foie remanié cirrhotique. Les études moléculaires ont révélé plusieurs sous-classes de CHC caractérisés par des signatures génomiques et protéomiques distinctes. Au cours de mon travail de thèse, nous avons contribué à améliorer notre compréhension de la biologie des CHC et des classifications moléculaires en cartographiant le génome non-codant de tumeurs de CHC induites par des virus hépatotropes (VHB, VHC) et en étudiant la sous-classe moléculaire de CHC la plus agressive KRT19-positif. Nous avons établi la première carte de transcriptome à ARN non codants du CHC et révélé une forte activation intra-tumorale des rétrotransposons à LTR, qui sont principalement inhibés dans les cellules hépatiques normales, dans des CHC induits par les VHB et VHC. Certains des transcrits dérivés de LTR se sont révélés être des régulateurs clés de l’expression génique et donc activer la croissance cellulaire. Dans la deuxième étude, nous identifions une nouvelle voie de régulation des CHC KRT19-positif affectant le métabolisme énergétique de ces tumeurs. Les CHC KRT19-positif sont des tumeurs fortement glycolytiques liée à une activation de la réponse à l’hypoxie. L’excès de production par les CHC KRT19-positif de l’oncométabolite 2-hydroxyglutarate en absence de mutation des gènes IDH1 et IDH2 était associé à un profil aberrant hyperméthylé sur la lysine 9 de l’histone H3 (H3K9me3) suggérant une répression de la transcription notamment des gènes impliqués dans la différenciation cellulaire. / Hepatocellular carcinoma (HCC) is the main primary liver cancer and one of the most leading cause of cancer-related death worldwide. This heterogeneous disease with a worse prognosis has been subjected of numerous studies aimed to establish global phenotypic profiles. During my thesis, I dedicated my work to improve these classifications by identifying signatures on the non-coding genome and working on a very aggressive form of HCC expressing progenitors markers. With help of a Japanese team, we demonstrated that LTR-derived ncRNAs were active in HCC and that correlation correlates with expression of common cancer markers (GPC3) ans TP53 mutations. This signature can also be used to discriminate HCCs at high risk of recurrence. Finally, we have showed that these LTRs are detectable on prenoplastic stages in the Mdr2 KO mouse model. In parallel, I worked on HCC that expresses progenitor markers such as cytokeratin 19. Using proteomic and transcriptionnal approaches and in silico analyses, we propose that the occurrence of this type of cancer id due to an hypoxic event likely related to trans-arterial chemoembolization. These tumors have a highly glycolytic phenotype with production of an oncometabolite (2-hydroxyglutarate) that has been generally foubd in IDH1/2 mutated cholangiocarcinomas. Finally we suggest the use of metformin, type 2 diabetes drug, to reverse metabolic reprogramming and restore sensitivity to chemotherapy