Orientador: Marcelo Fábio Gouveia Nogueira / Co-orientadora: Mariana Fernandes Machado / Banca: Marcelo Marcondes Seneda / Banca: Claudia Lima Verde Leal / Resumo: Em camundongo, estudos recentes demonstraram que o precursor do peptídeo natriurético tipo C (NPPC) e o seu receptor do tipo 2 (NPR2) são essenciais para a manutenção do bloqueio da meiose I no oócito. Além disso, fatores parácrinos secretados pelo oócito, como a proteína morfogenética óssea (BMP15), podem modular esse bloqueio pelo estímulo da expressão do gene NPR2 nas células do cumulus de camundongos. Em recente estudo na espécie bovina, o fluido folicular bovino (FFb), quando adicionado ao meio de maturação, interferiu na maturação nuclear do oócito e, como resultado, melhorou a produção in vitro de embriões. Contudo, não são conhecidos quais os fatores, presentes no FFb, que são responsáveis por esse efeito podendo inclusive haver participação das microvesículas presentes neste. Além de ser um componente do FFb, foi demonstrado que o estradiol pode participar diretamente da estimulação do NPR2. Portanto, os objetivos gerais do presente trabalho envolvem a elucidação do sistema NPPC-NPR2 no ovário bovino e a investigação da influência do NPPC exógeno, do estradiol e das vesículas extracelulares sobre a expressão do gene da PDE3 no oócito bovino e sobre a concentração de nucleotídeos cíclicos nos complexos cumulus-oócito. Foram realizado s quatro experimentos: 1) Ensaio imunoenzimático (ELISA) para investigar a presença e a concentração do NPPC no fluido folicular, no lisado de células da granulosa mural e nas vesículas extracelulares, de acordo com a classe de diâmetro folicular (3 a 6 mm; 6 a 8 mm e >8 mm); 2) Imunolocalização do receptor NPR2 no folículo antral bovino; 3) Expressão do gene da PDE3 no oócito bovino, mediante RT - PCR em tempo real, em CCOs im aturos e maturados in vitro por 6 horas com NPPC exógeno, estradiol e vesículas extracelulares; 4) Mensuração da concentração de AMPc e GMPc, em oócitos e células... / Abstract: In mice, recent studies have demonstrated that the natriuretic peptide precursor C (NPPC) and its type - 2 - receptor (NPR2) are essential for the maintenance of meiosis I arrest in the oocyte. Moreover, paracrine factors secreted by the oocyte such as bone mo rphogenetic protein 15 (BMP15) can modulate this arrest through the expression of the NPR2 gene in cumulus cells of mice. In a recent study in cattle, the bovine follicular fluid (bFF) added to the maturation medium was able to interfere with the oocyte nu clear maturation resulting in higher embryo yield in vitro . However, it is not known what factors present in bFF might be responsible for this effect and one candidate could be the presence of microvesicles in the bFF. Additionally, the estradiol hormone i s component of the bFF and was demonstrated to directly stimulate NPR2 expression. Therefore, the objectives of this work includes the investigation of the NPPC - NPR2 system in the bovine ovary and the assessment of the effects of exogenous NPPC, estradiol and extracellular vesicles on the expression of the PDE3 gene in bovine oocytes and the concentrations of cyclic - nucleotides in cumulus - oocyte complexes. We designed four experiments: 1) enzyme - linked immunosorbent assay (ELISA) for the presence and concen tration of the NPPC in follicular fluid, in the lysate of mural granulosa cells and in the extracellular vesicles, according to follicular diameter (3 to 6 mm, 6 to 8 mm, >8 mm); 2) Immunolocalization of NPR2 receptor in bovine antral follicles; 3) PDE3 ge ne expression in bovine oocyte by RT - PCR in real time, in immature COCs and in COCs in vitro matured for 6 h with exogenous NPPC, estradiol and extracellular vesicles; 4) Measurement of the concentration of cAMP and cGMP in oocytes and cumulus cells, respectively, either immature or matured in vitro for 6 h with exogenous NPPC or extracellular vesicles through specific... / Mestre

The long-term treatment of injured, aging, or pathological skeletal muscle using stem cell therapy requires an abundant source of skeletal muscle progenitors (SMP) that are capable of self-replenishment. While adult SMPs—known as satellite cells and marked by PAX7 expression—can be collected from healthy donors, these satellite cells have limited replication potential once extracted, and may have difficulties providing sufficient numbers for therapy. Therefore, we sought to utilize the near-unlimited replication potential of human embryonic stem cells (hESC) to generate large quantities of SMPs in vitro. We developed a 50-day directed hESC differentiation that produced cultures with up to 90% myogenic identity; roughly 43 ± 4% become PAX7+ SMPs, and 47 ± 3% of cells become skeletal myocytes. We also performed gene expression profiling on our differentiating cultures to better understand in vitro skeletal myogenesis, and to better characterize in vitro hESC-derived SMPs, which remain poorly understood relative to adult satellite cells. 50-day cultures shared gene expression profiles more similar to quiescent rather than activated satellite cells, featuring a number of genes related to FOS/JUN, NOTCH, and TGFB-signaling. Day 50 cultures also expressed surface proteins known to mark adult or embryonic SMPs: CD82, CXCR4, ERBB3, NGFR, and PDGFRA. Transplanting 50-day cultures into cardiotoxin or BaCl2 injured immunodeficient murine muscle showed donor human cells persisted within the host muscle for 1 – 2 months post-injection; however, donor cells were confined to the interstitial space and did not contribute to host myofibers or the satellite cell niche. Together, these studies provide a tool for generating large quantities of embryonic skeletal muscle, and a gene expression resource that can provide insight into signaling factors that might improve or accelerate SMP development, or provide putative new surface receptors that may isolate embryonic SMPs better suited for in vivo transplantation.

Made available in DSpace on 2014-06-11T19:26:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-15Bitstream added on 2014-06-13T20:54:01Z : No. of bitstreams: 1 pereirajunior_ej_me_sjrp.pdf: 1176824 bytes, checksum: ea26888e0c90e98ae7bc9367c03e1901 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Caracteres embriológicos possuem valor sistemático e sua utilidade foi demonstrada por diversos autores para elucidar o posicionamento filogenético de certas famílias de angiospermas. Este estudo visa analisar a ontogenia das estruturas reprodutivas de Cybistax antisyphilitica, com o propósito de acrescentar dados embriológicos relevantes ao delineamento filogenético da família, ou de categorias taxonômicas infrafamiliares. As características embriológicas observadas demonstraram similaridade com espécies pertencentes à ―Tabebuia alliance‖ cuja embriologia já foi investigada. Observou-se durante a ontogenia do óvulo de C. antisyphilitica que apenas o ginósporo calazal se desenvolve e, durante sua diferenciação em célula-mãe do saco embrionário, a face micropilar de sua parede celular assume uma conformação côncava, na qual há grande deposição de calose. No estádio octonuclear, há acúmulo de uma substância fibrogranular entre o endotélio e a parede do ginófito fazendo com que o saco embrionário apresente um característico afunilamento mediano. Em relação a ontogenia da antera da espécie estudada, verificou-se que as camadas tapetais são dimórficas, embora ao final do estádio pré-meiótico se tornem similares; embora, após a meiose, o dimorfismo se acentua novamente nas camadas tapetais. Os amiloplastos das células-mãe dos andrósporos são herdados pelos andrósporos e grãos de pólen deles resultantes. Os amiloplastos dos grãos de pólen gradualmente aumentam em número e tamanho em um único ciclo de amilogênese/amilólise. Com base nos dados obtidos conclui-se que a configuração da parede distal do ginósporo calazal e o acúmulo de secreção na porção mediana do saco embrionário são características não relatadas para outras espécies da família e podem... / Embryological characters have systematic value and its usefulness has been demonstrated by several authors to elucidate the phylogenetic position of certain angiosperms families. This study aims to analyze the ontogeny of reproductive structures of Cybistax antisyphilitica, with the purpose of adding relevant embryological data to the phylogenetic design of the family or, infra-familiar taxonomic categories. The embryological features observed was similar to species belonging to ―Tabebuia alliance‖ whose embryology has been investigated. During the ontogey of C. antisyphilitica ovule, only the chalazal megaspore develops, and in the course of its differentiation in the embryo sac mother cell, the micropylar side of its cell wall assumes a concave conformation in which there is an expressive deposition of callose. In the octonuclear stage, there is an accumulation of a fibrogranular substance between the endothelium and megagametophyte wall causing a bottleneck in the middle portion of embryo sac. For anther ontogeny of the species studied, the tapetal layers are dimorphic, although becoming similar at the late pre-meiotic stage. After meiosis, the dimorphism is accentuated between the two tapetal layers. The microspore mother cell amyloplasts are inherited by the microspores and the resulting pollen grains. The pollen grain amyloplasts gradually increasing in number and size in a single amylogenesis/amylolyse cycle. Based on the obtained data we concluded that the configuration of the distal wall of the chalazal megaspore and the accumulation of secretion in the median portion of embryo sac are characters not reported for other species of the family, and may possibly be autapomorphic characters. Regarding the ontogeny of the anther, Cybistax antisyphilitic showed a multistratified fibrous endothecium... (Complete abstract click electronic access below)

Made available in DSpace on 2015-09-17T15:25:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-03-06. Added 1 bitstream(s) on 2015-09-17T15:48:36Z : No. of bitstreams: 1 000849664.pdf: 1189084 bytes, checksum: cbc1fe6d442a04c2f695f228e768b010 (MD5) / Em camundongo, estudos recentes demonstraram que o precursor do peptídeo natriurético tipo C (NPPC) e o seu receptor do tipo 2 (NPR2) são essenciais para a manutenção do bloqueio da meiose I no oócito. Além disso, fatores parácrinos secretados pelo oócito, como a proteína morfogenética óssea (BMP15), podem modular esse bloqueio pelo estímulo da expressão do gene NPR2 nas células do cumulus de camundongos. Em recente estudo na espécie bovina, o fluido folicular bovino (FFb), quando adicionado ao meio de maturação, interferiu na maturação nuclear do oócito e, como resultado, melhorou a produção in vitro de embriões. Contudo, não são conhecidos quais os fatores, presentes no FFb, que são responsáveis por esse efeito podendo inclusive haver participação das microvesículas presentes neste. Além de ser um componente do FFb, foi demonstrado que o estradiol pode participar diretamente da estimulação do NPR2. Portanto, os objetivos gerais do presente trabalho envolvem a elucidação do sistema NPPC-NPR2 no ovário bovino e a investigação da influência do NPPC exógeno, do estradiol e das vesículas extracelulares sobre a expressão do gene da PDE3 no oócito bovino e sobre a concentração de nucleotídeos cíclicos nos complexos cumulus-oócito. Foram realizado s quatro experimentos: 1) Ensaio imunoenzimático (ELISA) para investigar a presença e a concentração do NPPC no fluido folicular, no lisado de células da granulosa mural e nas vesículas extracelulares, de acordo com a classe de diâmetro folicular (3 a 6 mm; 6 a 8 mm e >8 mm); 2) Imunolocalização do receptor NPR2 no folículo antral bovino; 3) Expressão do gene da PDE3 no oócito bovino, mediante RT - PCR em tempo real, em CCOs im aturos e maturados in vitro por 6 horas com NPPC exógeno, estradiol e vesículas extracelulares; 4) Mensuração da concentração de AMPc e GMPc, em oócitos e células... / In mice, recent studies have demonstrated that the natriuretic peptide precursor C (NPPC) and its type - 2 - receptor (NPR2) are essential for the maintenance of meiosis I arrest in the oocyte. Moreover, paracrine factors secreted by the oocyte such as bone mo rphogenetic protein 15 (BMP15) can modulate this arrest through the expression of the NPR2 gene in cumulus cells of mice. In a recent study in cattle, the bovine follicular fluid (bFF) added to the maturation medium was able to interfere with the oocyte nu clear maturation resulting in higher embryo yield in vitro . However, it is not known what factors present in bFF might be responsible for this effect and one candidate could be the presence of microvesicles in the bFF. Additionally, the estradiol hormone i s component of the bFF and was demonstrated to directly stimulate NPR2 expression. Therefore, the objectives of this work includes the investigation of the NPPC - NPR2 system in the bovine ovary and the assessment of the effects of exogenous NPPC, estradiol and extracellular vesicles on the expression of the PDE3 gene in bovine oocytes and the concentrations of cyclic - nucleotides in cumulus - oocyte complexes. We designed four experiments: 1) enzyme - linked immunosorbent assay (ELISA) for the presence and concen tration of the NPPC in follicular fluid, in the lysate of mural granulosa cells and in the extracellular vesicles, according to follicular diameter (3 to 6 mm, 6 to 8 mm, >8 mm); 2) Immunolocalization of NPR2 receptor in bovine antral follicles; 3) PDE3 ge ne expression in bovine oocyte by RT - PCR in real time, in immature COCs and in COCs in vitro matured for 6 h with exogenous NPPC, estradiol and extracellular vesicles; 4) Measurement of the concentration of cAMP and cGMP in oocytes and cumulus cells, respectively, either immature or matured in vitro for 6 h with exogenous NPPC or extracellular vesicles through specific...

Orientador: Marcelo Fábio Gouveia Nogueira / Co-orientador: José Celso Rocha / Banca: Fernando Frei / Banca: Andrea Cristina Basso / Resumo: A classificação morfológica embrionária possui grande importância para inúmeras técnicas laboratoriais (desde pesquisas básicas às aplicadas na reprodução assistida). Entretanto, o método utilizado para realizar a classificação dos embriões em diferentes graus de qualidade sempre foi baseado na subjetividade do avaliador e, por mais que sejam estabelecidos padrões de graus de qualidade e descrições das características morfológicas que categorizam um embrião em cada grau, não há atualmente um método preciso que possa gerar resultados consistentes e confiáveis. Assim, nosso trabalho resultou no desenvolvimento de um software capaz de realizar a classificação da qualidade morfológica de blastocistos bovinos. Utilizamos como base de funcionamento técnicas de inteligência artificial (mais especificamente de Redes Neurais Artificiais e Algoritmos Genéticos). Resultados indicam uma taxa de acerto global de 79,2% na classificação de blastocistos bovinos em 3 graus de qualidade, sendo que para os blastocistos classificados como Excelentes ou Bons (Classe 1) a taxa de acerto é de 82,6%, para os blastocistos classificados como Regulares (Classe 2) é de 16,7% e para os blastocistos classificados como Pobres (Classe 3) a taxa de acerto é de 91,7% / Abstract: Embryonic morphological classification has great importance for numerous laboratory techniques (from basic to applied research in assisted reproduction). However, the method used to perform the classification of embryos in varying degrees of quality has always been based on the subjectivity of the evaluator. Although quality standards and descriptions of morphological characteristics that categorize an embryo in each grade are established, currently there is not an accurate method that can generate consistent and reliable results. Thus, our work resulted in the development of a software able to perform the classification of morphological quality of bovine blastocysts. Artificial Intelligence techniques (such as Artificial Neural Networks and Genetic Algorithms) were used in the development. Results indicate an overall accuracy of 79.2% in the classification of bovine blastocysts in 3 degrees of quality. For blastocysts classified as Excellent or Good (Class 1) the hit rate is 82.6%, for blastocysts classified as Regular (Class 2) is 16.7% and for blastocysts classified as poor (Class 3) the hit rate is 91.7% / Mestre

O Tubarão azul (Prionace glauca), popularmente conhecido como cação-azul dentre todas as espécies de tubarão é a mais abundante no ambiente marinho, podendo ser encontrado em todos os mares. Com a diversidade das espécies, a descrição de qualquer fígado especifico, dificilmente poderá ser utilizada como um modelo. Juntamente com essa variabilidade, algumas características fisiológicas dos peixes contribuem para ampliar seu polimorfismo hepático, entretanto pode ser considerado o ponto inicial para os estudos comparativos e filogenéticos entre os vertebrados. O fígado dos peixes aparece como em todos os outros vertebrados, como um órgão chave que vai controlar muitas funções vitais e realizar um papel proeminente na fisiologia dos peixes, tanto no anabolismo (proteínas, lipídios e carboidratos) e no catabolismo (nitrogênio, glicogenólises e desintoxicação). Por outro lado, deve ser considerado como um órgão alvo para muitos parâmetros biológicos e ambientais que podem alterar a estrutura e o metabolismo do fígado, como por exemplo, a alimentação, toxinas, parasitas, microrganismos e metais pesados acumulados. Nos peixes o fígado é localizado ventralmente na cavidade celomática, ajustando-se ao espaço disponível na cavidade do corpo. Foi realizada através da microscopia de luz e eletrônica de varredura a morfologia estrutural do desenvolvimento do fígado do tubarão azul nos 33 espécimes, divididos em diferentes tamanhos de embriões e fetos de 4 cm até 45 cm, comparados com um indivíduo adulto fêmea de 2 metros. A contagem de hepatócitos e vacúolos de gordura foi pela morfometria, através da técnica de pontos em fotomicrografias aleatórias. Nos resultados obtidos, pode-se notar que o fígado ocupava 20% do tamanho do animal. Microscopicamente, observou a presença de diferentes tamanhos de vacúolos de armazenamento de lipídio nos hepatócitos, diferença nas proporções de hepatócitos, linfócitos e vasos sanguíneos que diminui à medida que aumenta a estocagem de lipídios, consequentemente diminuindo a visibilidade da estrutura do fígado. Maior visualização de vacúolos translúcidos intracitoplasmáticos microgoticular aumentando gradativamente para macrogoticulares. Sugerindo assim que a presença de lipidios seja para manutenção dos filhotes, flutuabilidade e reserva energéticado animal, indicando que ele armazena gordura em seu fígado desde o início da embriogênese. / The blue shark (Prionace glauca), popularly known as blue dogfish among all shark species is the most abundant in the marine environment, and can be found in all seas. With the diversity of species, the description of any specific liver can hardly be used as a model. Together with this variability, some physiological characteristics of the fish contribute to increase its hepatic polymorphism, however, it can be considered the starting point for comparative and phylogenetic studies among vertebrates. Fish liver appears as in all other vertebrates as a key organ that will control many vital functions and play a prominent role in fish physiology, both in anabolism (proteins, lipids and carbohydrates) and in catabolism (nitrogen, glycogenolysis and detoxification). On the other hand, it should be considered as a target organ for many biological and environmental parameters that can alter the structure and metabolism of the liver, such as food, toxins, parasites, microorganisms and accumulated heavy metals. In fish, the liver is located ventrally in the coelomic cavity, adjusting to the available space in the body cavity. The structural morphology of blue shark liver development in 33 specimens divided into different sizes of embryos and fetuses from 4 cm to 45 cm was compared to a female adult of 2 meters, using light microscopy and scanning electron microscopy. The counts of hepatocytes and fat vacuoles were by morphometry, using the technique of points in random photomicrographs. In the results obtained, it can be noted that the liver occupied 20% of the size of the animal. Microscopically, it observed the presence of different sizes of lipid storage vacuoles in hepatocytes, a difference in the proportions of hepatocytes, lymphocytes and blood vessels that decreases as lipid storage increases, consequently decreasing the visibility of the liver structure. Greater visualization of microcyticular intracytoplasmic translucent vacuoles gradually increasing for macrogoticulares. Thus suggesting that the presence of lipids is for the maintenance of the puppies, buoyancy and energy reserve of the animal, indicating that it stores fat in its liver from the beginning of embryogenesis.

Orientador: Luis Antonio Violin Dias Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T06:06:09Z (GMT). No. of bitstreams: 1 Justino_MariliaLopes_M.pdf: 4973879 bytes, checksum: 876805a44e4e574031c6f8adea9f285d (MD5) Previous issue date: 2013 / Resumo: O termo e-learning abrange a utilização de ferramentas eletrônicas para mediar o processo de ensino e afeta, portanto, a comunicação entre alunos e professores. Para o cenário da educação presencial a incorporação do e-learning deu origem ao conceito de educação presencial aprimorada, prática que tem sido incitada para educação de nível superior. A percepção desse contexto instigou a possibilidade de aplicação de novas estratégias complementares ao modelo de aula expositiva presencial no ensino de embriologia humana. Aos estudantes da área da saúde essa ciência é particularmente importante por permitir a compreensão de questões de saúde reprodutiva e as bases dos defeitos congênitos, no entanto, como ocorre em geral para as disciplinas da etapa pré-clínica, a retenção de seu conteúdo é bastante pobre. Esse estudo se baseou na utilização do software Embriologia Clínica Humana como recurso didático em disciplinas de graduação da UNICAMP para os cursos de Fonoaudiologia, Enfermagem e Medicina, para os anos de 2010, 2011 e 2012. Foram estabelecidas três estratégias de utilização do software que se diferenciam pelo momento e forma de interferência na dinâmica de aula: (I) com o professor como o único usuário ativo do software em aula expositiva; (II) inicialmente com professor utilizando o software em aula expositiva e retomada desse material pelo aluno para revisão de conteúdo; (III) inicialmente com o aluno utilizando o software para introdução de conteúdo e retomada deste material pelo professor através de exposição dialogada. Para o curso de Medicina empregou-se ainda a utilização do software em estratégias de aprendizagem individual (II/III) e colaborativa (II'/III'). A avaliação do desempenho cognitivo imediato baseou-se no cálculo de valores de ganho normalizado (g) a partir de questões do pré e pós-testes realizados a cada aula. Os desempenhos cognitivos em curto e médio prazo foram dados pela análise de variância (ANOVA) de avaliações somativas das disciplinas e de retenção em médio prazo, respectivamente. Para a cognição imediata, a estratégia I mostrou-se superior as demais. Em curto prazo houve equivalência entre as estratégias e em médio prazo, as estratégias que favorecem comportamentos de aprendizagem ativa (II/II' e III/III') destacaram-se positivamente. A investigação da percepção de estudantes baseou-se em instrumentos de percepção com asserções que compunham uma escala de Likert, itens tipo Likert, e entrevistas semi-estruturadas. A percepção do professor responsável pelas disciplinas de graduação envolvidas no estudo foi coletada através de entrevista. Com relação ao material utilizado e a coerência das atividades, as percepções apresentadas foram positivas. Na percepção dos estudantes diante das estratégias de aula propostas foi notada resistência às estratégias que desconstruíam o modelo de aula expositiva. A estratégia III/III' foi alvo de maior oposição, embora, na percepção do professor responsável, essa tenha sido a estratégia mais motivadora, por valorizar a importância do professor frente aos alunos. A experiência relatada e os resultados obtidos destacam ainda que a introdução de novos métodos de ensino exige o envolvimento íntimo de membros do corpo docente, um aspecto fundamental da qualidade da educação e para o sucesso de qualquer estudante / Abstract: The term e-learning covers the use of electronic tools to mediate the learning process, affecting communication among students and teachers. For the face to face educational scenario, the incorporation of e-learning gives rise to the enhanced learning, a practice that has been encouraged on higher education. The perception of this context instigated the possibility of implementing new strategies to complement the lecture style often adopted in human embryology classes. Students of health science should be particularly interest in this topic, since it allows the understanding of reproductive health issues and the basis of birth defects, however, as occurs in general for the disciplines of pre-clinical stage, the retention of its content is quite poor. This study were based on the use of the software Clinical Human Embryology as a teaching resource in graduate courses at UNICAMP for courses in speech therapy, nursing and medicine, for the years 2010, 2011 and 2012. It was established three strategies using software that differ by time and form of interference in the class' dynamics: (I) with the teacher as the only active user of the software during lecture presentation; (II) initially using the software with teacher during lecture presentation and then by the student to review content; (III) initially with the student using the software to introduce the content and then by the teacher during exposure dialogue. To the medical course it was also possible to use the software trough individual (II / III) and collaborative (II '/ III') learning strategies. The immediate assessment of cognitive performance was based on the calculation of values of normalized gain (g) based on the score of pretests and posttests performed for each class. Cognitive performance in the short and medium term was given by analysis of variance (ANOVA) of summative evaluations and retention assessments, respectively. For immediate cognition, the strategy I figured out with the higher socres. In the short term there was equivalence between strategies and at medium-term the strategies that promote active learning behaviors (II / II 'and III / III') positively stood out. The investigation of the students' perception was based on instruments of perception with assertions that comprised a Likert scale, Likert type items, and semi-structured interviews. The perception of the teacher responsible for undergraduate disciplines involved in the study was collected through an interview. With regard to the material used and the coherence of the activities, perceptions presented were positive. In the perception of the students for the proposed strategies there was noted a resistance to the ones that deconstruct the lecture model. The strategy III / III' was subject to greater opposition, though, in the perception of the teacher in charge, this was the most motivating strategy for valuing the importance of the teacher front students. The reported experience and the results highlight that the introduction of new teaching methods requires the intimate involvement of faculty members, a key aspect of quality of education and to the success of any student / Mestrado / Histologia / Mestra em Biologia Celular e Estrutural

In vitro fertilization treatments are responsible for 1-5% births in industrialized countries. The safest way to generate a pregnancy is to transfer a single embryo to the mother, reducing the likelihood of multiple gestations. Hence, in order to maximize the chance of success, it is extremely important that the embryo prioritised for transfer is the most capable within the cohort of embryos generated by the patient. Along with cytogenetic components, it has been suggested that embryo protein expression patterns may correlate with its ability to implant. However, embryo proteomics strategies have not been easy to harness mainly due to the complexity of the media the embryos are cultured in, and the low concentration of the proteins that are secreted. In this study, the use of the blastocentesis procedure, which allows the safe retrieval of embryo inner fluid (blastosol), was described. The use of the blasocoel fluid as a source of embryonic DNA for preimplantation genetic assessment was also investigated. From this highly purified embryonic sample, a comprehensive catalogue of proteins present in the human blastosol was generated using standard and custom- made mass spectrometry strategies. The embryonic origin of these proteins was validated by gene expression microarray and RNASeq analysis. These experiments also allowed the identification of differentially expressed genes in the first two cell lineages, the Inner Cell Mass and the Trophectoderm. Finally, a targeted proteomics strategy able to measure part of the previously described protein targets in single blastosol samples was employed. The correlation between the presence and abundance of proteins of interest in single blastosols and several biological characteristics of the embryo, including its chromosomal status, was assessed. These data are of major interest for the understanding of human embryo development. The validated embryo-derived protein catalogue and blastocyst gene expression profiles generated in this study, provides access to a thorough document for consultation in human embryology proteomics-based experiment design, paving the way to next-generation proteomic-based embryo assessment.

It is well known that tight coordination of cell division timing is essential for proper cell fate specification and tissue growth during metazoan development. However, how cell divisions are coordinated in vivo is largely unknown. In this thesis, a high- content screening was conducted to identify genes responsible for temporal coordination of cell division during Caenorhabditis elegans embryogenesis. A total of 822 genes were depleted using RNA interference (RNAi). The genes were prioritized based on their degree of conservation in human, as well as their roles in development. In addition to RNAi, an experimental pipeline was established, including 3D time-lapse imaging of an RNAi perturbed C. elegans embryos followed by automated lineaging, which allows systematic quantification of division timing of each individual cells up to approximately 350-cell stage. To identify genes with a significant reduction in the asynchrony of division between sister cells (ADS) upon perturbation, average division timings of each cell between at least two replicate perturbed embryos was compared against the average division timings of 92 wild type embryos. It was found that cell fate determinants were not only important for maintaining fate asymmetries, but are also imperative for establishing ADS regardless of cellular context. Hence, the results demonstrate that fate and temporal asymmetries share a common genetic architecture. The temporal coordination appears to facilitate cell migration during fate specification or tissue growth. Given the observation that perturbations of signalling pathways, especially Wnt and Notch pathways, are frequently associated with the ADS, it would be essential to map the exact signalling event that takes place at cellular level which is responsible for a given ADS or fate asymmetry. To this end, a miniMos transposon-meditated transgenic technique was adopted to insert a fusion between GFP and a promoter derived from individual components of the two signalling pathways into the C. elegans genome. The resulting insertion was crossed into a strain expressing lineaging markers, which allows automated lineaging and gene expression profiling to map the expression of each component with single cell resolution. A combination of cellular expression and cell-cell contact data would lead to a comprehensive map of each signalling event during C. elegans embryogenesis. Our preliminary results on cell-cell contacts and cellular expression validated the existing signalling events and identified potential novel ones that are associated with either ADS and/or fate asymmetry.

Hierdie studie het ten doel gehad om die effekte van Se en β-karoteentoediening op vit A, vit E, konsepsietempo en progesteroonvlakke tydens embrio-oorplasings in ontvangerkoeie te ondersoek. Die effekte van die verskillende faktore op mekaar sou ook bepaal word. In Totaal van 142 multipare koeie wat in vier groepe verdeel is, was as ontvangers vir die studie gebruik. Die groepe was as volg behandel: Groep 1 (35 koeie), 50 mg Se en 1500 mg vit E intramuskulêr 6 en 3 weke voor die oorplasings; Groep 2 (36 koeie), 300 mg l1-karoteen per os per bees per dag vanaf 6 weke voor die oorlasings; Groep 3 (36 koeie), vit E/Se en β-karoteen soos in Groepe 1 en 2 respektiewelik; Groep 4 (35 koeie) negatiewe kontrole groep met geen behandeling nie. Elke ontvanger het 6 weke voor oorplasing vit ADE oraal ontvang. Embrios is na 20, 19, 19 en 18 ontvangers van Groepe 1, 2, 3 en 4 onderskeidelik oorgeplaas. Bloedmonsters is van elke dier vir ontledings in die middel van die oorplantingsperiode getrek. Die toediening van Se was hoogs betekenisvol met RBS GSH-Px aktiwiteit gekorreleer R2 = 0,32960, (n = 136, P = 0,0000). Die toediening van β-karoteen was ook hoogs betekenisvol gekorreleer met die RBS GSH-Px aktiwiteit, R2 = 0,18950, (n=136, P< 0,0000). Die RBS GSH-Px aktiwiteit was 7,49,6,61,10,75 en 4,5 EE 11x1010 RBS vir Groepe 1, 2, 3 en 4 onderskeidelik. Groepe 1 en 3,1 en 4, 2 en 3, 2 en 4, en 3 en 4 het betekenisvol van mekaar verskil maar nie Groepe 1 en 2 nie. Daar was geen verband tussen β-karoteen plasmavlakke en RBS GSH¬Px aktiwiteit nie. Die orale toediening van β-karoteen het plasmavlakke duidelik laat styg en was betekenisvol met die β-karoteen plasmavlakke gekorreleer R2 = 0,09296, (n = 134, P=O,0003). Die groepe wat β-karoteen ontvang het, Groepe 2 en 3, se plasmavlakke was merkbaar hoër as die van Groepe 1 en 4 wat niks ontvang het nie. Die groep se waardes was 8,72, 10,13, 9,53 en 8,50 µmol/l vir Groepe 1, 2, 3 en 4 respektiewelik. Groepe 1 en 2, 2 en 4, en 3 en 4 het betekenisvol van mekaar verskil (P=0,0021) maar nie Groepe 1 en 3, en 1 en 4 nie. Daar was 'n positiewe korrelasie tussen die toediening van vit E/Se en die a¬tocoferol plasmavlakke, R2 = 0,06344, (n = 134, P=0,0033). Daar was egter geen aanduiding van 'n verband tussen RBS GSH-Px aktiwiteit en α-tocoferol plasmavlakke nie. Daar was wel In betekenisvolle korrelasie tussen α-tocoferol en β-karoteen plasmavlakke (P=0,0001). Die groepswaardes was 11,90, 10,59, 11,67 en 10,56 vir Groepe 1, 2, 3 en 4 onderskeidelik. Groepe 1 en 2, en 1 en 4 het betekenisvol van mekaar verskil (P=0,0334) maar nie die ander groepe, Groepe 1 en 3, 2 en 3, 2 en 4, en 3 en 4 nie. Die korrelasie tussen die toediening van Se en die retinol plasmavlakke was nie statisties betekenisvol nie (P=0,0671) maar tussen RBS GSH-Px aktiwiteit en retinol plasmavlakke was dit wel betekenisvol R2 = 0,03086, (n = 133, P=0,0431). Daar was ook In waarskynlike korrelasie tussen die toediening van β-karoteen en die retinol plasmavlak (P=0,0835) maar geen korrelasie tussen β-karoteen plasmavlakke en retinol plasmavlakke nie. Die groepwaardes was 1,068, 1,065, 1,154 en 1,026 µmol/l vir Groepe 1, 2, 3 en 4 onderskeidelik. Die dragtigheidstempo na die oorplasing van embrios was 50,0%, 47,4%, 31,5% en 27,8% vir Groepe 1, 2, 3 en 4 onderskeidelik. Die verskille was nie betekenisvol nie. Daar was 'n negatiewe korrelasie tussen die β-karoteen plasmavlakke en progesteroon plasmavlakke vir die eerste 5-9 dae van die estrussiklus. Retinol plasmavlakke en progesteroon plasmavlakke was wel positief gekorreleer (P=O,0317). Die groepswaardes was 5,67, 2,90, 4,28 en 5,60 µmol/l vir Groepe 1, 2, 3 en 4 onderskeidelik. RBS GSH-Px was betekenisvol gekorreleer met corpus luteum grootte maar corpus luteum grootte was nie met progesteroon waardes gekorreleer nie. Daar was geen statistiese verskille tussen die groepe ten opsigte van estruslengte en die tydsduur tussen prostaglandientoediening en estrus nie. Op individuele basis was daar wel In betekenisvolle korrelasie van β-karoteen plasmavlak met beide estruslengte (P=O,0298) en die tydsduur tussen prostaglandientoediening en estrus (P=0,0130). Estruslengte en die tydsduur tussen prostaglandien en estrus was ook betekenisvol met mekaar gekorreleer (P < 0,0000). / This study addressed some of the effects that selenium and β-carotene administration have on vit A, vit E, conception rates, progesterone levels, oestrus length, interval from PG to oestrus and corpus luteum size in embryo recipients. A total of 142 multiparous cows asigned to 4 test groups, were used as recipients in this study. Group 1 (35 cows) received 50 mg Se and 1500 mg vit E intramuscularly 6 and 3 weeks before transfer, Group 2 (36 cows) received 300 mg β-carotene orally per cow per day starting 6 weeks prior to transfer, Group 3 (36 cows) received both vit ElSe and β-carotene as per Group 1 and 2 and Group 4 (35 cows) was a negative control, with no treatment. All recipients received vit ADE orally 6 weeks before transfers. Embryos were transferred to 20, 19, 19, and 18 recipients of the Groups 1, 2, 3 and 4 respectively. Blood for analysis was drawn from all the recipients in the middle of the transferal week, being day 3 of the 5-day period. The administration of selenium was highly significantly correlated with the corresponding RBS GSH-Px activity R2 = 0,3290, (n = 135, P < 0,0000). The β-¬carotene administration was also highly significantly correlated with the RBS GSH¬Px activities R2 = 0,18950, (n = 136, P < 0,0000). However, there were no correlation between the β-carotene plasma levels and that of the RBS GSH-Px activities. The RBS GSH-Px were 7,49, 6,61, 10,75 and 4,5 Enzyme Units 11x1010 RBS for Groups 1, 2, 3 and 4 respectively. Groups 1 and 3, 1 and 4, 2 and 3, 2 and 4, and 3 and 4 differ significantly from each other but not Groups 1 and 2. There were no connection between B-carotene plasma levels and RBS GSH-Px activities. The treatment of β-carotene itself was succesful and was significantly correlated with the resulting β-carotene plasma levels R2 = 0,09296, (n = 134, P = 0,0033). The β-carotene plasma levels of the groups that received β-carotene, Groups 2 and 3, were noticeably higher than those of Group 1 and 4, which did not receive β-carotene. The values were 8,72, 10,13, 9,53 and 8,5 µmol/l for Groups 1, 2, 3 and 4 respectively. Groups 1 and 2, 2 and 4, and 3 and 4 differed significantly from one another (P = 0,0021) but not those of Groups 1 and 3, and 1 and 4. There was a positive correlation between the administration of Se and the retinol plasma levels (P = 0,0671) and a positive significant correlation between the RBS GSH-Px and the retinol plasma levels R2 = 0,03086, (n = 133, P = 0,0431). There was also a positive correlation between the administration of β-carotene and the retinol plasma levels (P = 0,0835) but no correlation between β-carotene plasma levels and retinol plasma levels. The values were 1,068, 1,065, 1,154 and 1,026 µmol/ for Groups 1, 2, 3 and 4 respectively. The pregnancy rate after embryo transferal were 50%, 47,4%, 31,5% and 27,8% for Groups 1, 2, 3 and 4 respectively. The differences were not significant. There was a negative correlation between β-carotene plasma levels and progesterone plasma levels for the first 5 to 9 days of the oestrus cycle. Retinol plasma levels however were significantly correlated with progesterone plasma levels (P = 0,0317). The progesterone levels were 5,67, 2,90, 4,28 and 5,60 µmol/ for Groups 1, 2, 3 and 4 respectively. The RBS GSH-Px activity was significantly correlated with corpus luteum size (P = 0,0445) but corpus size was not correlated with progesterone values. There were no statistical differences amongst the groups, between oestrus length and the interval from prostaglandin to oestrus. However on an individual basis there was a significant correlation between β-carotene plasma levels and both the oestrus length (P = 0,0298) and the interval from prostaglandin to oestrus (P = 0,0130). Oestrus length and the interval from prostaglandin to oestrus were also significantly correlated with each other (P < 0,0000). / Dissertation (MMedVet)--University of Pretoria, 2000. / Production Animal Studies / MMedVet / Unrestricted