Hair cells are transducers found found in the inner ear of vertebrates. They convert a mechanical signal, detected by the deflection of a bundle of cilia extending from their top surface, into an electrochemical signal. This dissertation studies the mechanical influence of the structure and materials on the function of the cells. I introduce two methods to conduct the mechanical analysis. The first uses strength of materials formulae to solve the simplified hair cell bundle models. The second is a finite element analysis, used to better account for the observed complexity of the structure. I then use these two techniques to build a fundamental understanding of the hair cell bundle structure. By first studying simplified models, then adding complexity, the effects of geometric and material variation can be deduced. I then study three actual bundles. These are all taken from vestibular organs of turtles, two from the posterior semicircular canal and one from the utricle. I present estimations of stiffness, tip link tensions, and nonlinear response. Finally, I investigate a single cilium forced by a fluid flow. The problem is solved by finite difference technique. Three different initial conditions are solved. / Ph. D.

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).

<p>α₁- and α₂-Adrenoceptors are involved in various physiological events in the eye: blood flow regulation, aqueous humor dynamics and pupil regulation. The α₁- and α₂-adrenoceptors can be further subdivided into six subtypes (α_1A, α_1B, α_1D, α_2A , α_2B, and α_2C ). Currently available α1- and α₂-adrenergic drugs are not selective for the different subtypes and some ocular adrenergics have undesirable side-effects, both local and systemic. A better understanding of the subtype distribution in the eye would be useful when designing new drugs with greater efficacy and fewer adverse effects; this applies especially to the treatment of glaucoma. The purpose of the thesis was therefore to identify and localize the different subtypes of α₁- and α₂-adrenoceptors in the eye. </p><p>The identities of the α₁-adrenoceptor subtypes were studied in various parts of pig and albino rabbit eyes by radioligand binding. In the pig retina and in the albino rabbit iris, ciliary body and retina, mixed populations of α_1A- and α_1B-adrenoceptors were localized. In the rabbit choroid only the α_1A-adrenoceptor subtype was detected. </p><p>The α₂-adrenoceptor subtypes were also characterized by radioligand binding, in different parts of the pig eye. In the iris, ciliary body and choroid, only α_2A-adrenoceptors were localized, while in the retina, mostly α_2A-adrenoceptors and a minor population of α_2C-adrenoceptors were identified. High densities of α_2A-adrenoceptors were found in the ciliary body and choroid.</p><p>The effect of α₂-adrenoceptor agonists on the porcine ciliary artery was studied on a small-vessel myograph. α₂-Adrenoceptor agonists proved to be potent vasoconstrictors in the porcine ciliary artery and it was found that the vasoconstriction induced by brimonidine was mediated by the α_A-adrenoceptor.</p>

α1- and α2-Adrenoceptors are involved in various physiological events in the eye: blood flow regulation, aqueous humor dynamics and pupil regulation. The α1- and α2-adrenoceptors can be further subdivided into six subtypes (α1A, α1B, α1D, α2A , α2B, and α2C ). Currently available α1- and α2-adrenergic drugs are not selective for the different subtypes and some ocular adrenergics have undesirable side-effects, both local and systemic. A better understanding of the subtype distribution in the eye would be useful when designing new drugs with greater efficacy and fewer adverse effects; this applies especially to the treatment of glaucoma. The purpose of the thesis was therefore to identify and localize the different subtypes of α1- and α2-adrenoceptors in the eye. The identities of the α1-adrenoceptor subtypes were studied in various parts of pig and albino rabbit eyes by radioligand binding. In the pig retina and in the albino rabbit iris, ciliary body and retina, mixed populations of α1A- and α1B-adrenoceptors were localized. In the rabbit choroid only the α1A-adrenoceptor subtype was detected. The α2-adrenoceptor subtypes were also characterized by radioligand binding, in different parts of the pig eye. In the iris, ciliary body and choroid, only α2A-adrenoceptors were localized, while in the retina, mostly α2A-adrenoceptors and a minor population of α2C-adrenoceptors were identified. High densities of α2A-adrenoceptors were found in the ciliary body and choroid. The effect of α2-adrenoceptor agonists on the porcine ciliary artery was studied on a small-vessel myograph. α2-Adrenoceptor agonists proved to be potent vasoconstrictors in the porcine ciliary artery and it was found that the vasoconstriction induced by brimonidine was mediated by the αA-adrenoceptor.

No description available.

Eukaryotic organisms rely on the coordinated beating of motile cilia for a multitude of fundamental reasons. In smaller organisms, such as Paramecium and the single cell alga Chlamydomonas reinhardtii, it is a matter of propulsion, to swim towards a higher concentration of nutrients or away from damaging environments. Larger organisms use instead the coordinated motion of cilia to push fluid along an epithelium: examples common to mammals are the circulation of cerebrospinal fluid in the brain, the transport of ovules in the fallopian tubes, and breaking the left/right symmetry in the embryo. Another notable example, and one that is central to this thesis, is mucociliary clearance in human airways: A carpet of motile cilia helps keeping the cell surface free from pathogens and foreign particles by constantly evacuating from lungs, bronchi, and trachea a barrier of mucus. The question of how motile cilia interact with one another to beat in a coordinated fashion is an open and pressing one, with immediate implications for the medical community. In order for the fluid propulsion to be effective, the motion of cilia needs to be phase-locked across significant distances, in the form of travelling waves (``metachronal waves''). It is still not known how this long-range coordination emerges from local rules, as there is no central node regulating the coordination among cilia. In the first part of this thesis I will focus on studying the coordination in carpets of cilia with a top-down approach, by proposing, implementing, and applying a new method of analysing microscope videos of ciliated epithelia. Chapter 1 provides the reader with an introduction on motile cilia and flagella, treating their structure and motion and reporting the different open questions currently tackled by the scientific community, with particular interest in the coordination mechanisms of cilia and the mucociliary clearance apparatus. Chapter 2 introduces Differential Dynamic Microscopy (DDM), a powerful and versatile image analysis tool that bridges the gap between spectroscopy and microscopy by allowing to perform scattering experiments on a microscope. The most interesting aspects of DDM for this work are that it can be applied to microscope videos where it is not possible to resolve individual objects in the field of view, and it requires no user input. These two characteristics make DDM a perfect candidate for analysing several hundred microscope videos of weakly scattering filaments such as cilia. In Chapter 3 I will present how it is possible to employ DDM to extract a wealth of often-overlooked information from videos of ciliated epithelia: DDM can successfully probe the ciliary beat frequency (CBF) in a sample, measure the direction of beating of the cilia, and detect metachronal waves and read their direction and wavelength. In vitro ciliated epithelia however often do not show perfect coordination or alignment among cilia. For the analysis of these samples, where the metachronal coordination might not be evident, we developed a new approach, called multiscale DDM (multiDDM), to measure a coordination length scale, a characteristic length of the system over which the coordination between cilia is lost. The new technique of multiDDM is employed in Chapter 4 to study how the coordination among cilia changes as a response to changes in the rheology of the mucous layer. In particular, we show that cilia beating under a thick, gel-like mucus layer show a larger coordination length scale, as if the mucus acted as an elastic raft effectively coupling cilia over long distances. This is corroborated by the coordination length scale being larger in samples from patients affected by Cystic Fibrosis than in healthy samples, and much shorter when the mucus layer is washed and cilia therefore beat in a near-Newtonian fluid. We then show how it is possible to employ multiDDM to measure the effectiveness of drugs in recovering, in CF samples, a coordination length scale typical of a healthy phenotype. In the second part I will focus instead on the single cilium scale, showing how we can attempt to link the beating pattern of cilia to numerical simulations studying synchronisation in a model system. In particular in Chapter 5 I will describe our approach to quantitatively describe the beating pattern of single cilia obtained from human airway cells of either healthy individuals or patients affected by Primary Ciliary Dyskinesia. Our description of the beating pattern, and the selection of a few meaningful, summary parameters, are then shown to be accurate enough to discriminate between different mutations within Primary Ciliary Dyskinesia. In Chapter 6 instead I report the results obtained by coarse-graining the ciliary beat pattern into a model system consisting of two ``rotors''. The rotors are simulated colloidal particles driven along closed trajectories while leaving their phase free. In my study, the trajectories followed by the rotors are analytical fits of experimental trajectories of the centre of drag of real cilia. The rotors, that are coupled only via hydrodynamics interactions, are seen to phase-lock, and the shape of the trajectory they are driven along is seen to influence the steady state of the system.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 86-111).

L'épuration mucociliaire est la première ligne de défense de l'appareil respiratoire. L'altération de l'épuration mucociliaire se traduit par une stagnation et/ou une accumulation de mucus, conduisant potentiellement à des obstructions plus ou moins partielles et à des infections chroniques des voies aériennes. On compte deux grands types d'altération de l'épuration mucociliaire. Le premier est lié aux caractéristiques du mucus, comme dans le cas de la mucoviscidose. Le deuxième est lié à des anomalies de l'ultrastructure du cil et /ou de son battement regroupées sous le terme de "ciliopathies"qui peuvent être soit innées soit acquises.Ce travail, qui s'inscrit dans le cadre clinique de la problématique des ciliopathies respiratoires et de leur prise en charge, a pour objectif de mieux caractériser, à partir des outils de la Biomécanique, les mécanismes du battement ciliaire (incluant une mesure de l'efficacité globale du battement des cils) et à transférer ces connaissances au profit de la clinique.Pour cela une méthodologie de caractérisation systématique de la mécanique ciliaire à partir de prélèvements biologiques issus de patients observés par vidéo-microscopie à haute vitesse a été développée parallèlement au développement d'un modèle numérique intégrant le couplage entre battement ciliaire et transport de fluide. Cette association entre expériences et modèle numérique a permis de proposer un nouvel index utilisable par les cliniciens pour caractériser l'efficacité du battement ciliaire. Cet index présente l'avantage de ne pas exiger de modification de la pratique clinique concernant la collecte de données biologiques / Mucociliary clearance is the first line of defense of the respiratory tract. Alteration of this clearance leads to a stagnation and/or accumulation of mucus, potentially resulting in more or less partially obstructions and in chronic infections of the airways. There are two main types of mucociliary clearance alterations. The first one is linked to the mucus characteristics, as in cystic fibrosis. The second one is connected to cilium ultrastructure abnormalities and/or ciliary beating defects encompassed by the term of "ciliopathies" which may be primary or acquired.This work, which takes place in the clinical issue of respiratory ciliopathies and their care, aims to better characterize, through biomechanics tools, the mechanisms of ciliary beating (including a measurement of the global efficiency of cilia) and to transfer these knowledges in favor of the clinical management.A methodology of systematic characterization of the ciliary mechanics, from biological samples derived from patients observed through high-speed video-microscopy analysis, has been developed as the same time as the development of a numerical model incorporating the coupling between ciliary beating and fluid motion. This association between experiments and numerical model allowed to propose a new index usable by the clinicians to characterize the ciliary beating efficiency. This index has the advantage of not requiring a modification of the clinical practice of biological data collection

Over the past several decades, the rate of pulmonary nontuberculous my- cobacterial (PNTM) disease has been increasing. PNTM patients gener- ally consist of lean and tall women presenting with symptoms in the sixth decade of life. They have a de nitive morphophenotype, but no consistent immunological abnormalities despite extensive investigation. I hypothesized that respiratory epithelial dysfunction might play a critical role in PNTM disease predisposition because diseases with defects of mucociliary transport have high rates of PNTM disease that increase with age, suggesting a direct connection between airway epithelial function and PNTM disease. I found that PNTM patients have a distinct respiratory epithelial phenotype ex vivo and decreased nasal nitric oxide levels in vivo. The PNTM ex vivo phenotype consists of an abnormally low resting ciliary beat frequency (CBF) and abnormal CBF response to toll-like receptor (TLR) agonists. The depressed baseline CBF response in PNTM patient cells can be normalized ex vivo by augmenting the nitric oxide-cyclic guanosine monophosphate pathway without appreciable e ect on CBF in healthy controls. In healthy controls, bacterial TLR agonists increase CBF and viral TLR agonists decrease CBF. In PNTM patients these responses are impaired and are not normalized with the normalization of the resting CBF rate. Inhibitor-induced disruption of signalling pathways associated with CBF regulation demonstrated that the majority of the CBF response to TLR agonists involves the PI-3K pathway and PKC. Inhibition of the PI-3K pathway (PI-3K , Akt1, and PDK1) closely mimicked the ex vivo phenotype seen in PNTM patient respiratory epithelia. These data identify a novel aspect of PNTM disease with in vivo and ex vivo correlates that suggest that PNTM infection is associated with abnormal function at both the CBF and TLR response levels. This phenotype is novel, reproducible, and provide a foundation with which to determine the genetic basis of PNTM infection.