Nossa proposta de trabalho visava estudar as propriedades do movimento ciliar, aplicando técnicas de microscopia eletrônica de varredura.. Escolheu-se como material de estudo a espécie Macrostomum tuba (turbelário), bastante comum em ambientes naturais de água doce, e em aquários. Sua superfície é inteiramente revestida por cilios, que o animal usa como meio locomoção suave e rápida. Cílios são estruturas em forma de projeções delgadas de células, com uma notável organização interna, constante em todas as espécies animais, e são dotados de movimento oscilatório rítmico e autônomo. O batimento ciliar coordenado origina ondas na superfície das células, e por conseguinte, na superfície do organismo, conhecidas como ondas metacrônicas. No caso em estudo, estas ondas são sufícientes para promover o deslocamento do organismo no meIO. Estudou-se a estrutura [ma destes cílios por microscopia eletrônica de transmissão, e a sua forma durante o batimento, por microscopia eletrônica de varredura. A frequência do batimento ciliar foi determinada por microscopia de luz com fonte estroboscópica, e o movimento do organismo em meio de diferentes viscosidades, foi registrado com câmara de vídeo. Os métodos utilizados neste trabalho permitiram obter-se as seguintes informações sobre o movimento ciliar de M. tuba: 1. Os cílios têm cerca de 5f..lm de comprimento, e estruruta interna típica (\"9+2\"). 2. Os cílios em meio aquoso, batem com frequência de 15 Hz, sendo esta reduzida de modo exponencial para os meios de viscosidade maior. 3. Os cilios trabalham de modo coordenado, produzindo ondas \"metacrônicas\" que apresentam um estágio de batimento efetivo e outro de recôbro, bem distintos. O comprimento de onda é da ordem de 4 a 5 f..lm e pode ser medido diretamente nas imagens de varredura. 4. Os cílios se distribuem ao longo da face ventral em campos, onde as ondas se orientam de modo conspícuo. 5. A velocidade de propagação da onda é da ordem de 78-80 ,.!In/s. e a velocidade média de deslocamento do animal em meio aquoso é cerca de 4 mmls, caindo a menos da metade para meios de viscosidades altas. 6. Os cílios podem ser removidos por diferentes métodos experimentais, tornando acessivel a superfície da célula: isto facilitou a observação de perfis de onda com mutio boa resolução, bem assim, pennitiu contagens de densidade ciliar (cerca de 200/célula ). / The aim of this study was to investigate the properties of ciliary motion in the flatwonn, Macrostomum tuba, using electron microscopy. This is a quite common species inhabiting freshwater ponds and fish aquaria. Its suface is entirely covered with cilia, that povide a smooth and fast gliding motion for the anima!. The fme structure of the cilia has been studied by use of transmission electron microscopy. The profiles assumed by the organelle during its undulatory motion have been described by use of special scanning microscopy techniques. Frequency of the ciliary beating has been detennined with a stroboscope system, and the gliding motion ofthe animal was recorded with a vide o camera. The several approaches used in the present study provide the following conclusions: 1. The cilia are 5 11m long; ultrastructurally they confonn to the \"9+2\" mode!. 2. The beating frequency in water is 15 Hz. This value is exponentially reduced for higher viscosity media. 3. The coordinated beating of a field of cilia gives rise to \"metachronal waves\" of about 4 to 5 11m in wavelength. A distinctive effective and another recovery stroke were characterized in the scanning images. 4. Along the ventral surface of the animal, oriented metachronal waves point to the directions ofwater flow. 5. Calculated metachronal wave velocity is 78-80 l1m/s, and the animal speed in water reaches some 4 mm/s; it slows down rapidly for higher viscosity fluids. 6. Experiments with deciliation have allowed a clear cut view of the wavefronts, as well as, the counting of ciliary density (about 200/cell).

Nossa proposta de trabalho visava estudar as propriedades do movimento ciliar, aplicando técnicas de microscopia eletrônica de varredura.. Escolheu-se como material de estudo a espécie Macrostomum tuba (turbelário), bastante comum em ambientes naturais de água doce, e em aquários. Sua superfície é inteiramente revestida por cilios, que o animal usa como meio locomoção suave e rápida. Cílios são estruturas em forma de projeções delgadas de células, com uma notável organização interna, constante em todas as espécies animais, e são dotados de movimento oscilatório rítmico e autônomo. O batimento ciliar coordenado origina ondas na superfície das células, e por conseguinte, na superfície do organismo, conhecidas como ondas metacrônicas. No caso em estudo, estas ondas são sufícientes para promover o deslocamento do organismo no meIO. Estudou-se a estrutura [ma destes cílios por microscopia eletrônica de transmissão, e a sua forma durante o batimento, por microscopia eletrônica de varredura. A frequência do batimento ciliar foi determinada por microscopia de luz com fonte estroboscópica, e o movimento do organismo em meio de diferentes viscosidades, foi registrado com câmara de vídeo. Os métodos utilizados neste trabalho permitiram obter-se as seguintes informações sobre o movimento ciliar de M. tuba: 1. Os cílios têm cerca de 5f..lm de comprimento, e estruruta interna típica (\"9+2\"). 2. Os cílios em meio aquoso, batem com frequência de 15 Hz, sendo esta reduzida de modo exponencial para os meios de viscosidade maior. 3. Os cilios trabalham de modo coordenado, produzindo ondas \"metacrônicas\" que apresentam um estágio de batimento efetivo e outro de recôbro, bem distintos. O comprimento de onda é da ordem de 4 a 5 f..lm e pode ser medido diretamente nas imagens de varredura. 4. Os cílios se distribuem ao longo da face ventral em campos, onde as ondas se orientam de modo conspícuo. 5. A velocidade de propagação da onda é da ordem de 78-80 ,.!In/s. e a velocidade média de deslocamento do animal em meio aquoso é cerca de 4 mmls, caindo a menos da metade para meios de viscosidades altas. 6. Os cílios podem ser removidos por diferentes métodos experimentais, tornando acessivel a superfície da célula: isto facilitou a observação de perfis de onda com mutio boa resolução, bem assim, pennitiu contagens de densidade ciliar (cerca de 200/célula ). / The aim of this study was to investigate the properties of ciliary motion in the flatwonn, Macrostomum tuba, using electron microscopy. This is a quite common species inhabiting freshwater ponds and fish aquaria. Its suface is entirely covered with cilia, that povide a smooth and fast gliding motion for the anima!. The fme structure of the cilia has been studied by use of transmission electron microscopy. The profiles assumed by the organelle during its undulatory motion have been described by use of special scanning microscopy techniques. Frequency of the ciliary beating has been detennined with a stroboscope system, and the gliding motion ofthe animal was recorded with a vide o camera. The several approaches used in the present study provide the following conclusions: 1. The cilia are 5 11m long; ultrastructurally they confonn to the \"9+2\" mode!. 2. The beating frequency in water is 15 Hz. This value is exponentially reduced for higher viscosity media. 3. The coordinated beating of a field of cilia gives rise to \"metachronal waves\" of about 4 to 5 11m in wavelength. A distinctive effective and another recovery stroke were characterized in the scanning images. 4. Along the ventral surface of the animal, oriented metachronal waves point to the directions ofwater flow. 5. Calculated metachronal wave velocity is 78-80 l1m/s, and the animal speed in water reaches some 4 mm/s; it slows down rapidly for higher viscosity fluids. 6. Experiments with deciliation have allowed a clear cut view of the wavefronts, as well as, the counting of ciliary density (about 200/cell).

Primary cilia are non-motile organelles having 9+0 microtubules that project from the basal body of the cell. While the main purpose of motile cilia in mammalian cells is to move fluid or mucus over the cell surface, the purpose of primary cilia has remained elusive for the most part. Primary cilia are shortened in the kidney tubules of Tg737orpk mice, which have polycystic kidney disease due to ciliary defects. The product of the Tg737 gene is polaris, which is directly involved in a microtubule-dependent transport process called intraflagellar transport (IFT). In order to determine the importance of polaris in the development of neuronal cilia, cilium length and numerical density of cilia were quantitatively assessed in six different brain regions on postnatal days 14 and 31 in Tg737orpk mutant and wildtype mice. Our results indicate that the polaris mutation leads to shortening of cilia as well as decreased percentage of ciliated neurons in all brain regions that were quantitatively assessed. Maintainance of cilia was especially affected in the ventromedial nucleus of the hypothalamus. Furthermore, the polaris mutation curtailed cilium length more severely on postnatal day 31 than postnatal day 14. These data suggests that even after ciliogenesis, intraflagellar transport is necessary in order to maintain neuronal cilia. Regional heterogeneity in the effect of this gene mutation on neuronal cilia suggests that the functions of some brain regions might be more compromised than others.

Glial cells maintain homeostasis that is essential to neuronal function. Injury to the nervous system leads to the activation and proliferation of glial cells and pericytes, which helps to wall off the damaged region and restore homeostatic conditions. Sonic hedgehog is a mitogen which is implicated in injury-induced proliferation of glial cells and pericytes. The mitogenic effects of sonic hedgehog require primary cilia, but the few reports on glial or pericyte primary cilia do not agree about their abundance and did not address effects of injury on these cilia. Primary cilia are microtubule-based organelles that arise from the centrosome and are retracted before cells divide. Depending on cell type, proteins concentrated in cilia can transduce several mitotic, chemosensory, or mechanosensory stimuli. The present study investigated effects of stab wound injury on the incidence and length of glial and pericyte primary cilia in the area adjacent to the injury core. Astrocytes, polydendrocytes and pericytes were classified by immunohistochemistry based on cell-type markers. In normal adult mice, Arl13b immunoreactive primary cilia were present in a majority of each cell type examined: astrocytes, 98±2%; polydendrocytes, 87±6%; and pericytes, 79±13% (mean ± SEM). Three days post-injury, cilium incidence decreased by 24% in astrocytes (p< 0.008) and 41% in polydendrocytes (p< 0.002), but there was no significant effect in pericytes. Polydendrocytes labeled with the cell cycle marker Ki67 were less likely to have cilia compared to resting, Ki67- polydendrocytes. Considering post-injury rates of proliferation for astrocytes and polydendrocytes, it appears that resorption of cilia due to cell cycle entry may account for much of the loss of cilia in polydendrocytes but was not sufficient to account for the loss of cilia in astrocytes. Under normal conditions, astrocytes rarely divide, and they maintain non-overlapping territories. However, three days after injury, there was a 7-fold increase in the number of paired mirror-image astrocytes (p< 0.018), which are most likely daughter cells from astrocytes that recently divided. Cilia incidence tended to decrease in these pairs compared to single astrocytes (p< 0.057) in injured mice. This is the first systematic investigation of cilia of astrocytes, polydendrocytes, and pericytes in the brain. Moreover, the examination of effects of brain injury on cilia adds to the understanding of injury-induced proliferation in these cells.

Primary cilia are nonmotile, hair-shaped organelles that extend from the basal body in the centrosome. The present study is the first investigation of this organelle in the oligodendrocyte lineage in vivo. I used immunohistochemical approaches in normal and cilia-deficient mutant mice to study cilia in relation to oligodendrogenesis and myelination. Primary cilia immunoreactive for Arl13b and ACIII were commonly present in NG2+ oligodendrocyte progenitor cells (OPCs), in which cilia-associated pathways control proliferation, differentiation, and migration. The loss of primary cilia is generally associated with enhanced Wnt/β-catenin signaling, and Wnt/β-catenin signaling has been shown to promote myelin gene expression. I examined whether the lack of cilia in the oligodendrocyte lineage is associated with elevated Wnt/β-catenin activity. I found that absence of a primary cilium was associated with with higher levels of TCF3, and with β-galactosidase in Axin2-lacZ Wnt reporter mice. This evidence supports the proposal that cilia loss in oligodendrocytes leads to enhanced Wnt/β-catenin activity, which promotes myelination. Cilia are dependent on the centrosome, which assembles microtubules for the cilium, the cytoskeleton, and the mitotic spindle. Centrosomes are the organizing center for microtubule assembly in OPCs, but this function is decentralized in oligodendrocytes. I found that the intensity of centrosomal pericentrin was reduced in oligodendrocytes relative to OPCs, and γ-tubulin was evident in centrosomes of OPCs but not in mature oligodendrocytes. These decreases in centrosomal proteins might contribute to functional differences between OPCs and oligodendrocytes. The importance of cilia in the oligodendrocyte lineage was examined in Tg737orpk mice, which have a hypomorphic IFT88 mutation resulting in decreased cilia numbers and lengths. These mice showed marked, differential decreases in numbers of oligodendrocytes and myelin, yet little or no change in OPC populations. It appears that sufficient cells were available for maturation, but lineage progression was stalled. There were no evident effects of the mutation on Wnt/β-catenin. Factors that might contribute to the abnormalities in the oligodendrocyte lineage of Tg737orpk mice include decreased cilia-dependent Shh mitogenic signaling and dysregulation in cilia-associated pathways such as Notch and Wnt/β-catenin.

Inner ear hair cells have been identified as the sites of mechanoelectrical transduction from a mechanical event (e.g. hearing, motion) to an electrical event (e.g. neural response). Deflection of bundles of hair-like stereocilia extending from these cells has been associated with the transduction process. Stereocilia bundle structure and stiffness controls deflection and thus the fundamental sensitivity of the transduction process. The finite-element method was used along with analytical techniques to characterize individual stereocilium and stereocilia bundle stiffnesses. A three ‘stack’ bundle with a Young’s modulus of 3 GPa (F-actin protein) and Poisson’s ratio of 0.4 (nearly incompressible) resulted in a stiffness of K = 2.1 x 10⁻³ N/m. This value is within the range of experimentally determined stiffmesses. Tip-link and subapical band interconnecting structures each contribute significantly to bundle stiffness and each could act as the gating-spring in transduction models, which propose gating structures as a means of regulating ionic activity and therefore neural activity. Stiffness depends most strongly on individual stereocilium geometry and material description, tip-link orientation and material description, and stereocilia bundle width. Stiffness depends least on stereocilia height variations and subapical bands configuration. Linear analysis was reliable up to deflections of 3.5 um, the upper limit of physical response. Preliminary dynamic response indicates a natural frequency of 382 kHz for the vibration mode resembling physical deformation behavior. Future models should include hexagonal bundle arrangements, transversely isotropic stereocilia material descriptions, and viscoelastic tip-link behavior. / Master of Science

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