As pointed out more and more frequently in the literature, there is a pressing need for research into the health and environmental impact of nanoparticles. This work represents a joint effort between scientists in nanotechnology, chemistry and biology to answer this call and to investigate the environmental effects of carbon nantoubes (CNTs) from a brand new aspect. The results showed clearly the dose-dependent effects of single-walled carbon nanotubes (SWNTs) on the ingestion and digestion of bacteria by Tetrahymena thermophila, a ciliated protozoan, propagated to its prey bacteria, Escherichia coli. Investigated by confocal microscopy Tetrahymena were able to internalize large quantities of SWNTs and then excrete SWNTs and undigested bacteria in aggregates. Inhibition of ciliate bacterivory measured by Ciliate Bacterivory assay was evident at far below lethal concentrations. At high tube concentrations (above 6.8 μg∙ml-1), cell viability was affected. In addition, explored by fluorescence microscopy and scanning electron microscopy, SWNTs stimulated Tetrahymena to abnormally egest viable bacteria inside membrane protected structures, which enhanced bacterial survival during antimicrobial treatments, bacteriostatic or bacteriocidal. This phenomenon may have important implications to public health. In general, research on toxicity of nanoparticles is in a very early stage with most studies on direct fatality (kill or not to kill) of a single organism or certain type of cells. This work is believed to be among the first few investigating extrapolated effects. Hopefully, this wok will stimulate a line of research towards better understanding of the effects of nanomaterials on diverse organisms, and stimulate not only toxicology but also ecotoxicology studies.

As pointed out more and more frequently in the literature, there is a pressing need for research into the health and environmental impact of nanoparticles. This work represents a joint effort between scientists in nanotechnology, chemistry and biology to answer this call and to investigate the environmental effects of carbon nantoubes (CNTs) from a brand new aspect. The results showed clearly the dose-dependent effects of single-walled carbon nanotubes (SWNTs) on the ingestion and digestion of bacteria by Tetrahymena thermophila, a ciliated protozoan, propagated to its prey bacteria, Escherichia coli. Investigated by confocal microscopy Tetrahymena were able to internalize large quantities of SWNTs and then excrete SWNTs and undigested bacteria in aggregates. Inhibition of ciliate bacterivory measured by Ciliate Bacterivory assay was evident at far below lethal concentrations. At high tube concentrations (above 6.8 μg∙ml-1), cell viability was affected. In addition, explored by fluorescence microscopy and scanning electron microscopy, SWNTs stimulated Tetrahymena to abnormally egest viable bacteria inside membrane protected structures, which enhanced bacterial survival during antimicrobial treatments, bacteriostatic or bacteriocidal. This phenomenon may have important implications to public health. In general, research on toxicity of nanoparticles is in a very early stage with most studies on direct fatality (kill or not to kill) of a single organism or certain type of cells. This work is believed to be among the first few investigating extrapolated effects. Hopefully, this wok will stimulate a line of research towards better understanding of the effects of nanomaterials on diverse organisms, and stimulate not only toxicology but also ecotoxicology studies.

Most benthic marine invertebrates have long-lived planktonic ciliated larvae that must feed and grow to reach metamorphosis. Because ciliated cells in animals are unable to divide it is of considerable interest how ciliated larvae are able to grow. To understand how ciliated larvae grow I compared cell proliferation patterns in several species with planktotrophic larvae from five different phyla (Nemertea, Mollusca, Phoronida, Echinodermata, and Annelida). Cell proliferation events were detected using anti-phosphohistone antibody labeling, BrdU assays, and confocal microscopy. Studied larvae included some with monociliated epithelia (pluteus, bipinnaria, actinotroch, and mitraria) and others with multiciliated epithelia (metatrochophore, pilidium, and veliger). Dividing cells were detected in all studied larvae, but the pattern of dividing cells varied among types and correlated with the kind of epithelium (mono- vs. multiciliated) and phylogeny (e.g. protostome vs. deuterostome). Running z-projection movies of the actinotroch, mitraria, veliger and pilidium are included as supplemental files.

O embrião equino apresenta um desenvolvimento dinâmico por um longo período entre a fertilização, sua entrada no útero, fixação e posterior invasão trofoblástica após o 36º dia da gestação. Durante todo este período o concepto é sustentado pelo histotrofo endometrial. Estas características dos equídeos favorecem o seu uso com modelo experimental in vivo para estudos nos desenvolvimentos e interações fetal e maternal. Os estudos em endométrio equino tiveram foco em eventos fisiológicos nas diferentes fases do ciclo estral, enquanto os estudos aos primeiros momentos da gestação são escassos. O entendimento do processo de remodelação e morfofisiologia do endométrio após a entrada do embrião no útero não é completamente entendido. O objetivo deste trabalho foi estudar a morfofisiologia endometrial da gestação na égua envolvendo os períodos pós-fixação e peri-implantação, por histologia e microscopia eletrônica de varredura. A característica mais marcante da transformação ou adaptação endometrial à gestação foi o quase total desaparecimento das células ciliadas na superfície epitelial em ambos cornos uterinos. A capacidade de secreção das células microvilosas também passou por mudanças com o avanço gestacional. Muitas células secretórias ingurgitadas e protusas formam a maioria da população do epitélio, onde o histotrofo se acumula, e apresentaram erosões em sua superfície, provavelmente pela secreção apócrina de vesículas. A superfície epitelial apresentou pleomorfismo celular e pseudoestratificação, promovida por intensa hiperplasia celular, acompanhada de adensamento das glândulas endometriais, desde o 21º dia da gestação diminuído após o 36º dia. Linfócitos, provavelmente uNK, foram encontrados no epitélio luminal do endométrio já aos 21 dias de gestação em ambos cornos, gravídico e não gravídico. Foi evidenciado o septamento no epitélio luminal, com sulcos formados aos 35 e 36 dias, tornando-se mais profundos aos 42 dias de gestação. Toda esta evolução e adaptação contínua aconteceram principalmente no corno gravídico acompanhados em menor intensidade pelo corno não gravídico. / The equine embryo plays a dynamic development for a long period between fertilization, its entry into the uterus, fixes and subsequent trophoblastic invasion after day 36 of gestation. Throughout this period, the conceptus is supported by endometrial histotrophe. These equids characteristics favor their use as an in vivo experimental model for studying the changes and interactions in fetal and maternal development. Studies in equine endometrium were focused on physiological events in the different phases of the estrous cycle, while studies in early moments of pregnancy are scant. The process of endometrial remodeling and morphophysiology after the maternal recognition of pregnancy is not completely understood. The objective of this work was to study the endometrial morphophysiology in the mare comprising post-fixation and peri-implantation periods, by histology and scanning electron microscopy. The most striking feature of endometrial transformation or adaptation to pregnancy was the almost total ciliated cells disappearance of the epithelial surface in both uterine horns. In addition, the secretory capacity of microvillus cells underwent changes with gestational age. Many engorged and protruded secretory cells were the majority epithelium population where histotroph accumulates, and showed erosions on its surface, probably by apocrine vesicle secretion. The epithelial surface also showed cellular pleomorphic and pseudostratified epithelium as a result of intense cell hyperplasia. It was accompanied by thickening of the endometrial glands from day 21 of gestation, then decreasing after the 36th day. Lymphocytes, probably uNK, were found in the luminal epithelium of the endometrium since 21st day of gestation in both pregnant and not pregnant horns. Septation was evidenced in the luminal epithelium, with sulci formed at 35 days, becoming deeper at 42 days of pregnancy. All this continued evolution and adaptation occurred mainly in the gravid horn accompanied in less intensity by non-gravid horn.

O embrião equino apresenta um desenvolvimento dinâmico por um longo período entre a fertilização, sua entrada no útero, fixação e posterior invasão trofoblástica após o 36º dia da gestação. Durante todo este período o concepto é sustentado pelo histotrofo endometrial. Estas características dos equídeos favorecem o seu uso com modelo experimental in vivo para estudos nos desenvolvimentos e interações fetal e maternal. Os estudos em endométrio equino tiveram foco em eventos fisiológicos nas diferentes fases do ciclo estral, enquanto os estudos aos primeiros momentos da gestação são escassos. O entendimento do processo de remodelação e morfofisiologia do endométrio após a entrada do embrião no útero não é completamente entendido. O objetivo deste trabalho foi estudar a morfofisiologia endometrial da gestação na égua envolvendo os períodos pós-fixação e peri-implantação, por histologia e microscopia eletrônica de varredura. A característica mais marcante da transformação ou adaptação endometrial à gestação foi o quase total desaparecimento das células ciliadas na superfície epitelial em ambos cornos uterinos. A capacidade de secreção das células microvilosas também passou por mudanças com o avanço gestacional. Muitas células secretórias ingurgitadas e protusas formam a maioria da população do epitélio, onde o histotrofo se acumula, e apresentaram erosões em sua superfície, provavelmente pela secreção apócrina de vesículas. A superfície epitelial apresentou pleomorfismo celular e pseudoestratificação, promovida por intensa hiperplasia celular, acompanhada de adensamento das glândulas endometriais, desde o 21º dia da gestação diminuído após o 36º dia. Linfócitos, provavelmente uNK, foram encontrados no epitélio luminal do endométrio já aos 21 dias de gestação em ambos cornos, gravídico e não gravídico. Foi evidenciado o septamento no epitélio luminal, com sulcos formados aos 35 e 36 dias, tornando-se mais profundos aos 42 dias de gestação. Toda esta evolução e adaptação contínua aconteceram principalmente no corno gravídico acompanhados em menor intensidade pelo corno não gravídico. / The equine embryo plays a dynamic development for a long period between fertilization, its entry into the uterus, fixes and subsequent trophoblastic invasion after day 36 of gestation. Throughout this period, the conceptus is supported by endometrial histotrophe. These equids characteristics favor their use as an in vivo experimental model for studying the changes and interactions in fetal and maternal development. Studies in equine endometrium were focused on physiological events in the different phases of the estrous cycle, while studies in early moments of pregnancy are scant. The process of endometrial remodeling and morphophysiology after the maternal recognition of pregnancy is not completely understood. The objective of this work was to study the endometrial morphophysiology in the mare comprising post-fixation and peri-implantation periods, by histology and scanning electron microscopy. The most striking feature of endometrial transformation or adaptation to pregnancy was the almost total ciliated cells disappearance of the epithelial surface in both uterine horns. In addition, the secretory capacity of microvillus cells underwent changes with gestational age. Many engorged and protruded secretory cells were the majority epithelium population where histotroph accumulates, and showed erosions on its surface, probably by apocrine vesicle secretion. The epithelial surface also showed cellular pleomorphic and pseudostratified epithelium as a result of intense cell hyperplasia. It was accompanied by thickening of the endometrial glands from day 21 of gestation, then decreasing after the 36th day. Lymphocytes, probably uNK, were found in the luminal epithelium of the endometrium since 21st day of gestation in both pregnant and not pregnant horns. Septation was evidenced in the luminal epithelium, with sulci formed at 35 days, becoming deeper at 42 days of pregnancy. All this continued evolution and adaptation occurred mainly in the gravid horn accompanied in less intensity by non-gravid horn.

Mucociliary epithelia are found in the human airways and act as the first line of defence against inhaled foreign agents. Mucus traps potentially damaging particles and the cilia transport the mucus away from the airways to remove the threat. Modelling mucociliary epithelia for research purposes is challenging. This is because the airways are enclosed and are thus difficult to study directly. Instead, tissue is extracted or in vitro techniques are employed. Whilst these systems are useful, there is a need for accessible in vivo models to complement them. In this thesis I assess a new model system for studying mucociliary epithelia. This system is the larval epidermis of the amphibian, Xenopus tropicalis. Its epidermis comprises multi-ciliated cells that beat in a polarised direction reminiscent of those found in the human airways. It is also proposed to have a number of other cell types including mucus-secreting cells, but very little is known about them. The epidermis is open and accessible to manipulation meaning that it has great potential to be used in the study of mucociliary epithelia in live, native conditions. Such a system would be a valuable addition to the current models employed. However, the epidermis has not been thoroughly characterized before so its utility as a model system remains speculative.To develop and evaluate this new model, I fully characterize the epidermis, showing that it has five distinguishable cell types. This includes a population of cells called ionocytes that are shown to be essential for the health and function of the epidermis. I also test for the presence of mucins, the structural component of mucus, secreted from the epidermis in order to evaluate the proposal that mucus-secreting cells are present in the epidermis. A mucin-like protein called otogelin is identified. After characterizing the epidermal cell types, I compare them to the human mucociliary epithelium and consider potential applications and future perspectives for this model.

O embrião equino apresenta um desenvolvimento dinâmico por um longo período entre a fertilização, sua entrada no útero, fixação e posterior invasão trofoblástica após o 36º dia da gestação. Durante todo este período o concepto é sustentado pelo histotrofo endometrial. Estas características dos equídeos favorecem o seu uso com modelo experimental in vivo para estudos nos desenvolvimentos e interações fetal e maternal. Os estudos em endométrio equino tiveram foco em eventos fisiológicos nas diferentes fases do ciclo estral, enquanto os estudos aos primeiros momentos da gestação são escassos. O entendimento do processo de remodelação e morfofisiologia do endométrio após a entrada do embrião no útero não é completamente entendido. O objetivo deste trabalho foi estudar a morfofisiologia endometrial da gestação na égua envolvendo os períodos pós-fixação e peri-implantação, por histologia e microscopia eletrônica de varredura. A característica mais marcante da transformação ou adaptação endometrial à gestação foi o quase total desaparecimento das células ciliadas na superfície epitelial em ambos cornos uterinos. A capacidade de secreção das células microvilosas também passou por mudanças com o avanço gestacional. Muitas células secretórias ingurgitadas e protusas formam a maioria da população do epitélio, onde o histotrofo se acumula, e apresentaram erosões em sua superfície, provavelmente pela secreção apócrina de vesículas. A superfície epitelial apresentou pleomorfismo celular e pseudoestratificação, promovida por intensa hiperplasia celular, acompanhada de adensamento das glândulas endometriais, desde o 21º dia da gestação diminuído após o 36º dia. Linfócitos, provavelmente uNK, foram encontrados no epitélio luminal do endométrio já aos 21 dias de gestação em ambos cornos, gravídico e não gravídico. Foi evidenciado o septamento no epitélio luminal, com sulcos formados aos 35 e 36 dias, tornando-se mais profundos aos 42 dias de gestação. Toda esta evolução e adaptação contínua aconteceram principalmente no corno gravídico acompanhados em menor intensidade pelo corno não gravídico. / The equine embryo plays a dynamic development for a long period between fertilization, its entry into the uterus, fixes and subsequent trophoblastic invasion after day 36 of gestation. Throughout this period, the conceptus is supported by endometrial histotrophe. These equids characteristics favor their use as an in vivo experimental model for studying the changes and interactions in fetal and maternal development. Studies in equine endometrium were focused on physiological events in the different phases of the estrous cycle, while studies in early moments of pregnancy are scant. The process of endometrial remodeling and morphophysiology after the maternal recognition of pregnancy is not completely understood. The objective of this work was to study the endometrial morphophysiology in the mare comprising post-fixation and peri-implantation periods, by histology and scanning electron microscopy. The most striking feature of endometrial transformation or adaptation to pregnancy was the almost total ciliated cells disappearance of the epithelial surface in both uterine horns. In addition, the secretory capacity of microvillus cells underwent changes with gestational age. Many engorged and protruded secretory cells were the majority epithelium population where histotroph accumulates, and showed erosions on its surface, probably by apocrine vesicle secretion. The epithelial surface also showed cellular pleomorphic and pseudostratified epithelium as a result of intense cell hyperplasia. It was accompanied by thickening of the endometrial glands from day 21 of gestation, then decreasing after the 36th day. Lymphocytes, probably uNK, were found in the luminal epithelium of the endometrium since 21st day of gestation in both pregnant and not pregnant horns. Septation was evidenced in the luminal epithelium, with sulci formed at 35 days, becoming deeper at 42 days of pregnancy. All this continued evolution and adaptation occurred mainly in the gravid horn accompanied in less intensity by non-gravid horn.

This doctoral research project aims the study of finite element methods discretized in dynamic meshes in order to simulate fluid-solid interaction and multiphase flow phenomena, particularly flows involving phenomena that are most significant in microfluidic and biofluidic applications. The equations that model multiphase flow will be treated in an arbitrary Lagrangian-Eulerian framework, when required, with several types of boundary conditions at the interfaces, depending on the nature of the phases. The numerical challenges found in this application range from the correct representation of the interface between fluids, passing through geometric challenges in the maintainability of the computer mesh, to the challenges posed by microscales. Special attention is given to squimer models, by means of a general formulation of the swimming problem as well as the steps to transform a standard fluid-solid model to a squirming model. / Este projeto de pesquisa de doutorado visa o estudo de métodos de elementos finitos discretizados em malhas dinâmicas, com o objetivo de simular fenómenos de interação fluídoestrutura e escoamentos multifásicos, em especial escoamentos que envolvem aplicações em microfluídica e biofluídica. As equações que modelam escoamentos multifásicos serão tratadas em um referencial lagrangeano-euleriano arbitrário, quando requerido, com differentes tipos de condições de contorno nas interfaces, dependendo da natureza das fases. Os desafios numéricos impostos por esta aplicação vão desde a correta representação das interfaces, passando por desafios geométricos na manutenibilidade da malha computacional, até os desafios impostos pelas escalas microscópicas. Atenção especial é dada aos modelos de squimer, por meio de uma formulação geral do problema de natação, bem como os passos para transformar um modelo fluido-sólido padrão em um modelo de squirming.

A gestação inicial da égua é um período fascinante que abrange numerosas e intensas mudanças em seu desenvolvimento, muitas das quais são únicas para a espécie equina. Esse desenvolvimento depende da manutenção da função lútea, do estabelecimento de um ambiente uterino e de uma interação precisa e orquestrada entre o concepto e o ambiente uterino. O objetivo deste estudo foi verificar as alterações histológicas do endométrio e a produção histotrófica em éguas cíclicas e prenhes nos dias 7, 10 e 13 pós-ovulação. No primeiro ciclo, biópsias endometriais de 30 éguas foram coletadas no dia 7 (n = 10), 10 (n = 10) e 13 (n = 10) constituindo o grupo éguas cíclicas. No segundo ciclo, as mesmas éguas foram cobertas por um garanhão fértil, acompanhadas diariamente até detectar a ovulação, considerada o dia 0. Foram coletadas biópsias endometriais nos dias7 (n 10), 10 (n 10) e 13 (n 10). Imediatamente após a coleta, o útero foi lavado e as éguas em que foi obtido embrião, foram inseridas no grupo de éguas prenhes. Um maior calibre dos vasos sanguíneos foi observado em prenhez comparados às éguas cíclicas do dia 7 aos 13. No sétimo dia pós-ovulação, uma grande perda de células ciliadas foi evidente no grupo de éguas prenhes, comparadas ao grupo de éguas cíclicas, as células do epitélio endometrial estavam mais protusas e uma pequena quantidade de secreção histotrófica entre as dobras endometriais foi observada. No décimo dia de prenhez, secreção histotrófica glandular e do epitélio luminal estavam mais presentes comparadas às éguas do grupo cíclico. No dia 13 de prenhes, foi observado um grande conteúdo de histotrofo nas aberturas glandulares que estavam cercadas por células ciliares. Ocorreram alterações no ambiente uterino logo após a entrada do embrião no útero. No estroma e no lúmen, essas modificações parecem visar fornecer a nutrição necessária para o desenvolvimento inicial do embrião e estas mudanças nas estruturas celulares irão interagir na sinalização embrionária, futura fixação, implantação e placentação. / The early pregnancy of mare is a fascinating period that encompasses numerous and intense changes in its development, many of which are unique to the equine species. This development depends on the maintenance of the luteal function, the establishment of a favorable uterine environment and a precise and orchestrated interaction between the concept and the uterine environment. The aim of this study was to evaluate histological changes in the endometrium in days 7, 10 and 13 post-ovulation in pregnant and cyclic mares. In the first cycle, endometrial biopsies from 30 cyclic mares (Cyclic group) were collected on days 7, 10 and 13 post-ovulation. In the second cycle, the same mares were bred by a fertile stallion. At days 7, 10 and 13 post-ovulation intrauterine biopsies were collected. Immediately after sample collection, the mare‟s uteri were flushed, and those mares with embryo recovery were assigned to the Pregnant group. A larger blood vessel caliber was observed in pregnant mares than in cyclic from day 7 to 13. On the 7th day a large loss of ciliated cells was evident in the group of pregnant mares in comparison with the Cyclic group and the superficial cells of the endometrium were more protruded, and a small amount of histotrophic material between the folds was observed. On the 10th day of pregnancy, the glandular histotrophic secretion and the secretion of luminal epithelium became more intense than the secretion of cyclic mares. On the 13th day of pregnancy, a very large amount of histotroph was observed within large glandular openings surrounded by ciliated cells. Changes occurred in the uterine environment thereupon the entry of the embryo into the uterus. In the stroma and in the lumen, these modifications seem aim to provide the necessary nutrition for the initial development of the embryo and to promote changes at cellular structures that will interact in the embryonic signaling and future fixation, implantation and placentation.

A gestação inicial da égua é um período fascinante que abrange numerosas e intensas mudanças em seu desenvolvimento, muitas das quais são únicas para a espécie equina. Esse desenvolvimento depende da manutenção da função lútea, do estabelecimento de um ambiente uterino e de uma interação precisa e orquestrada entre o concepto e o ambiente uterino. O objetivo deste estudo foi verificar as alterações histológicas do endométrio e a produção histotrófica em éguas cíclicas e prenhes nos dias 7, 10 e 13 pós-ovulação. No primeiro ciclo, biópsias endometriais de 30 éguas foram coletadas no dia 7 (n = 10), 10 (n = 10) e 13 (n = 10) constituindo o grupo éguas cíclicas. No segundo ciclo, as mesmas éguas foram cobertas por um garanhão fértil, acompanhadas diariamente até detectar a ovulação, considerada o dia 0. Foram coletadas biópsias endometriais nos dias7 (n 10), 10 (n 10) e 13 (n 10). Imediatamente após a coleta, o útero foi lavado e as éguas em que foi obtido embrião, foram inseridas no grupo de éguas prenhes. Um maior calibre dos vasos sanguíneos foi observado em prenhez comparados às éguas cíclicas do dia 7 aos 13. No sétimo dia pós-ovulação, uma grande perda de células ciliadas foi evidente no grupo de éguas prenhes, comparadas ao grupo de éguas cíclicas, as células do epitélio endometrial estavam mais protusas e uma pequena quantidade de secreção histotrófica entre as dobras endometriais foi observada. No décimo dia de prenhez, secreção histotrófica glandular e do epitélio luminal estavam mais presentes comparadas às éguas do grupo cíclico. No dia 13 de prenhes, foi observado um grande conteúdo de histotrofo nas aberturas glandulares que estavam cercadas por células ciliares. Ocorreram alterações no ambiente uterino logo após a entrada do embrião no útero. No estroma e no lúmen, essas modificações parecem visar fornecer a nutrição necessária para o desenvolvimento inicial do embrião e estas mudanças nas estruturas celulares irão interagir na sinalização embrionária, futura fixação, implantação e placentação. / The early pregnancy of mare is a fascinating period that encompasses numerous and intense changes in its development, many of which are unique to the equine species. This development depends on the maintenance of the luteal function, the establishment of a favorable uterine environment and a precise and orchestrated interaction between the concept and the uterine environment. The aim of this study was to evaluate histological changes in the endometrium in days 7, 10 and 13 post-ovulation in pregnant and cyclic mares. In the first cycle, endometrial biopsies from 30 cyclic mares (Cyclic group) were collected on days 7, 10 and 13 post-ovulation. In the second cycle, the same mares were bred by a fertile stallion. At days 7, 10 and 13 post-ovulation intrauterine biopsies were collected. Immediately after sample collection, the mare‟s uteri were flushed, and those mares with embryo recovery were assigned to the Pregnant group. A larger blood vessel caliber was observed in pregnant mares than in cyclic from day 7 to 13. On the 7th day a large loss of ciliated cells was evident in the group of pregnant mares in comparison with the Cyclic group and the superficial cells of the endometrium were more protruded, and a small amount of histotrophic material between the folds was observed. On the 10th day of pregnancy, the glandular histotrophic secretion and the secretion of luminal epithelium became more intense than the secretion of cyclic mares. On the 13th day of pregnancy, a very large amount of histotroph was observed within large glandular openings surrounded by ciliated cells. Changes occurred in the uterine environment thereupon the entry of the embryo into the uterus. In the stroma and in the lumen, these modifications seem aim to provide the necessary nutrition for the initial development of the embryo and to promote changes at cellular structures that will interact in the embryonic signaling and future fixation, implantation and placentation.