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Overgaard, Christian Edmund Yeaman Charles. "Deciliation dramatically alters epithelial function." [Iowa City, Iowa] : University of Iowa, 2009. http://ir.uiowa.edu/etd/416.
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Xu, Qiang, and 徐强. "Modeling the deformation of primary cilium." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47250008.
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In this thesis we developed a new mechanics model of the primary cilium and analyzed its bending behavior. The primary cilium that extends from the cell surface can detect the mechanical signals of the surrounding environment. Moreover, through its deflection and bending angle, the primary cilium can communicate with the cell regarding the extracellular. Scientists have shown that dysfunction of primary cilia can lead to many diseases as cilia are believed to play an important role in transmitting signals in cells.
A good model of primary cilium can aid in the understanding of the mechanism of its bending movement. Furthermore, a good model is important for determining how the primary cilium contributes to convert mechanical signals into biochemical ones. Previous models have ignored the basal body and transition fiber that are located at the base of the primary cilium.
However, it is clear that the elastic basal body and transition fibers should have a significant effect on the deformation of the whole structure. Aiming to address this issue, we established a model with a rotational spring representing the confinement induced by the basal body and transition fibers. Specially, we developed two governing equations for two different conditions, namely uniformly distributed load and spatially varying load. In addition, this model is valid for situations where the deflection is large.
To obtain the results the shooting and Newton-Raphson methods are used to solve the governing equations numerically. Then, we compared the numerical results with experimental data to test the validity of the model.
Comparison between our model predictions and experimental data showed that the governing equation for spatially varying load described the bending behavior of the primary cilium very well under various realistic conditions, including cases where the flow field is not uniform both spatially and temporally fluid flow with variable velocity.published_or_final_version
Mechanical Engineering
Master
Master of Philosophy
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Norton, Michael M. "Modeling problems in mucus viscoelasticity and mucociliary clearance /." Online version of thesis, 2009. http://hdl.handle.net/1850/10822.
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Ghosh, Rajat. "Designing oscillating cilia for regulating particle motion in microfluidic devices." Thesis, Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/33861.
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We design actuated cilia that can maneuver microscopic particles normal to a microfluidic channel wall and transport microscopic particles parallel to the channel wall. For identifying the design specifications, we employ a hybrid LBM/LSM computational model, to simulate hydrodynamic interactions between oscillating elastic cilia and microscopic particles in a microfluidic channel. The oscillating synthetic cilia are elastic filaments tethered to the channel wall and actuated by sinusoidal force acting at their free ends. The cilia are arranged in a square pattern. The microscopic particle is a neutrally buoyant solid sphere, which is sufficiently small compared to the cilium length and inter-cilium distances, so that the particle can move freely inside the ciliated layer.
We study the effect of actuation frequency on the particle motion inside the ciliated layer. We show that depending on the frequency, particles can be either driven away from the ciliated channel wall or drawn towards the wall. We also examine how to use inclined cilia to transport particles along the ciliated layer. We show that the particle transport along the ciliated layer can be regulated by the frequency of cilium oscillation. The results uncover a new route for regulating particle position and transport in microfluidic devices.
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Wan, Yixin. "Modulation and synchronization of eukaryotic flagella." Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708434.
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Pruski, Michal. "ARL13B and IFT172 truncated primary cilia and misplaced cells." Thesis, University of Aberdeen, 2017. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=231675.
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Primary cilia are cellular organelles that protrude into the extracellular space, acting as antennas. They detect a wide range of chemical cues, including SHH and PDGF, as well as fluid flow, and they modulate downstream signalling systems, such as WNT and ERK. Due to this cue-sensing ability and the close association of the primary cilium with the centrosome the organelle is able to influence both cell cycle progression and cell migration. This work investigated the effect of mutations on two genes associated with primary cilia: Arl13b and Ift172. The effects of the HNN genotype of Arl13b and the WIM genotype of Ift172 on cell migration were assessed uniquely within the context of direct current electric fields. Both cell lines showed a decreased migratory response when compared to WT cells, despite no clear involvement of cilia in sensing the direction of the electric field. This corroborated with previous data of in vivo Arl13b cellular migration. Through the use of in utero electroporation the migratory deficits of IFT172 knock down were then confirmed in vivo in the developing mouse neocortex. Further in vitro investigation revealed a slower proliferation rate of HNN and WIM cells, though this was not confirmed in vivo after IFT172 knock down using a standard BrDU protocol. Nevertheless, further in vitro investigations revealed a wide variety of cell cycle and intracellular changes within both cell lines. The commonalities included lower numbers of cells in the S-phase and lower MAPK3 phosphorylation compared to WT, and differences such as GSK3β phosphorylation on Ser9. This work showed for the first time that ciliopathies affect galvanotaxis, and revealed fundamental commonalities in cell migration and proliferation between various ciliary mutations, as well as differences in specific signalling pathways. This will hopefully aid in developing future therapeutic interventions for ciliary diseases.
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Hughes, Rhome. "Immunohistochemical characterization of neuronal cilia in the rat central nervous system." Thesis, University of North Texas, 2002. https://digital.library.unt.edu/ark:/67531/metadc3136/.
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An anti-G"11 antibody was used to label neuronal cilia throughout the rat central nervous system. Immunoreactive cilia were observed in every examined region of the rat CNS, but not in monkey or mouse tissue. Antibodies to G"q and G"q/11 failed to label cilia. Immunoreactive cilia were observed as early as postnatal day 0 in spinal tissue, and postnatal day 3 in hypothalamic tissue. There was a statistically significant negative correlation between a region's mean cilium length and that region's distance to the nearest ventricle; regions nearest ventricles were those with the longest cilia. This correlation suggests neuronal cilia may function as chemosensors, detecting substances as they move out from the cerebrospinal fluid and into the extracellular space of the brain.
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Wilson, Gabrielle. "The role of the parkin co-regulated gene (PACRG) in male fertility /." Connect to thesis, 2009. http://repository.unimelb.edu.au/10187/5806.
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Thesis (Ph.D.)--University of Melbourne, Dept. of Paediatrics, The Bruce Lefroy Centre for Genetic Health Research, The Murdoch Childrens Research Institute, 2009.Typescript. Includes bibliographical references (leaves 183-207)
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Subedi, Ashok. "Roles of Primary Cilia in the Oligodendrocyte Lineage." Thesis, University of North Texas, 2018. https://digital.library.unt.edu/ark:/67531/metadc1404594/.
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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.
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Mahato, Deependra. "Mutation of Polaris, an Intraflagellar Transport Protein, Shortens Neuronal Cilia." Thesis, University of North Texas, 2005. https://digital.library.unt.edu/ark:/67531/metadc4856/.
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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.
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Kwok, Pui-wai. "The effects of gelomyrtol forte on human ciliary beat frequency and intracellular cyclic adenosine monophosphate in vitro /." View the Table of Contents & Abstract, 2007. http://sunzi.lib.hku.hk/hkuto/record/B38296913.
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Kwok, Pui-wai, and 郭佩瑋. "The effects of gelomyrtol forte on human ciliary beat frequency and intracellular cyclic adenosine monophosphate in vitro." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B4501291X.
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Bhattarai, Samip Ram. "Characteristics of Primary Cilia and Centrosomes in Neuronal and Glial Lineages of the Adult Brain." Thesis, University of North Texas, 2015. https://digital.library.unt.edu/ark:/67531/metadc801939/.
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Primary cilia are sensory organelles that are important for initiating cell division in the brain, especially through sonic hedgehog (Shh) signaling. Several lines of evidence suggest that the mitogenic effect of Shh requires primary cilia. Proliferation initiated by Shh signaling plays key roles in brain development, in neurogenesis in the adult hippocampus, and in the generation of glial cells in response to cortical injury. In spite of the likely involvement of cilia in these events, little is known about their characteristics. Centrosomes, which are associated with primary cilia, also have multiple influences on the cell cycle, and they are important in assembling microtubules for the maintenance of the cell’s cytoskeleton and cilia. The cilia of terminally differentiated neurons have been previously examined with respect to length, incidence, and receptors present. However, almost nothing is known about primary cilia in stem cells, progenitors, or differentiated glial cells. Moreover, it is not known how the properties of cilia and centrosomes may vary with cell cycle or proliferative potential, in brain or other tissues. This dissertation focuses first on neurogenesis in the hippocampal subgranular zone (SGZ). The SGZ is one of the few brain regions in mammals that gives rise to a substantial number of new neurons throughout adulthood. The neuron lineage contains a progression of identifiable precursor cell types with different proliferation rates. This present study found that primary cilia were present in every cell type in the neuronal lineage in SGZ. Cilium length and incidence were positively correlated among these cell types. Ciliary levels of adenylyl cyclase type III (ACIII) levels relative to ADP-ribosylation factor-like protein 13b (Arl13b) was higher in neurons than in precursor cells and glia, and also changed with the cell cycle. G-protein coupled receptors, SstR3, MCHR1, and Gpr161 receptors were only found in neuronal cilia. The levels and distribution of three centrosomal proteins, γ-tubulin, pericentrin and cenexin in neurons was different from the distributions in precursors and glia. The second focus of study is glial responses to injury in the neocortex, which has been widely studied as an injury model. This study found that in the normal adult somatosensory cortex, primary cilia were present in astrocytes and polydendrocytes but not in microglia. Following injury, the incidence of primary cilia decreased in astrocytes. Also, a new cell type expressing GFAP, NG2 and Olig2 was seen 3 days following injury, but was not present in normal mice. The characteristics of primary cilia and centrosome described here suggest that in stem cells and progenitors their characteristics may be well suited for proliferation, whereas in neurons, the cilia and centrosomes are important for other sensory functions.
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Feriani, Luigi. "Understanding the collective dynamics of motile cilia in human airways." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/288418.
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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.
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Coronel, Marco V. "Effects of Brain Injury on Primary Cilia of Glial Cells and Pericytes." Thesis, University of North Texas, 2016. https://digital.library.unt.edu/ark:/67531/metadc955100/.
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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.
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Duncan, Robert Keith. "Finite-element analysis of inner ear hair bundles : a parameter study of bundle mechanics /." Thesis, This resource online, 1993. http://scholar.lib.vt.edu/theses/available/etd-09292009-020226/.
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Aragão, Pedro Henrique Arruda. "Estudo do Movimento Ciliar de Macrostomum Tuba Utilizando Métodos de Microscopia Eletrônica\"." Universidade de São Paulo, 1996. http://www.teses.usp.br/teses/disponiveis/43/43133/tde-02072013-103757/.
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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).
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Blanchon, Sylvain. "Etude de la diversité phénotypique et génotypique des dyskinésies ciliaires primitives : vers une prise en charge personnalisée." Thesis, Paris Est, 2016. http://www.theses.fr/2016PESC0073.
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Jory, Myriam. "Approche biophysique de la fonction muco-ciliaire de l'épithélium bronchique : propriétés d'écoulement du mucus et coordination du battement ciliaire." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTS078.
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Comprendre et soigner les pathologies respiratoires chroniques constituent des enjeux médicaux croissants du fait des modifications de nos modes de vie et de notre environnement. L’épithélium des voies respiratoires assure le rôle de première barrière contre les agressions extérieures grâce à la fonction muco-ciliaire. Les deux éléments fonctionnels de la fonction muco-ciliaire sont la couche de mucus sécrétée par l’épithélium et le battement des cils des cellules ciliées qui contribue à la mise en mouvement de cette couche et à son évacuation des bronches. Le mucus est un fluide complexe et hétérogène et ses propriétés mécaniques couplées aux mécanismes de coordination des cils restent encore mal compris. Mes travaux de thèse ont porté sur une approche biophysique de ces mécanismes. Premièrement, l’étude de la rhéologie du mucus a été menée à deux échelles : en macro-rhéologie de cisaillement et par micro-rhéologie active grâce aux pincettes optiques. Les pincettes optiques permettent également des mesures in situ sur le tissu et des mesures d’adhésion. L’utilisation de la rhéologie comme marqueur des pathologies respiratoires chroniques a été explorée. Deuxièmement, la quantification et la compréhension de la coordination spatiotemporelle de l’activité ciliaire et du transport du mucus engendré ont été conduites, en développant un nouvel outil de traitement d’images de vidéo-microscopie et d’analyse de donnéesUnderstanding and treating chronic respiratory diseases are growing medical challenges due to changes in our lifestyles and environment. The epithelium of the respiratory tract is the first barrier against external aggression, in particular thanks to the mucociliary function. Functional elements are the mucus layer lining the tissue and the beating of cilia from the ciliated cells of the epithelium. Mucus is a complex and heterogeneous fluid that acts as a protective barrier by trapping particles and pathogens present in the inhaled air, while the coordination of the cilia beating allows the directed transport of the mucus layer and its evacuation from the bronchi. The mechanical properties of mucus coupled with the coordination mechanisms of beating cilia are still poorly understood. My thesis work focused on two biophysical aspects of these mechanisms: i) the study of mucus rheology at two scales, in macro-rheology and by active micro-rheology using optical tweezers directly on the tissue; ii) the understanding and quantification of the spatiotemporal coordination of ciliary activity and on the associated mucus transport, by developing a new tool for processing video-microscopy images and data analysis
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Ferreira, Rita Joana Rodrigues da Silva Rua. "Cilia motility studies in zebrafish embryos." Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/7984.
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A thesis submitted in fulfilment of the requirements for the degree of Masters in Molecular Genetics and BiomedicineMotile ciliary dysfunctions cause specific Ciliopathies that affect mainly the respiratory tract, fertilization and left-right body establishment. The embryonic organ where left-right decisions are first taken is called the organizer, a ciliated organ where a leftward cilia driven fluid-flow is generated. The organizer is named node in the mouse and Kupffer’s vesicle (KV) in zebrafish. The correct left-right axis formation is highly dependent on signaling pathways downstream of such directional fluid-flow. Motile cilia need to be coordinated and Ciliary Beat Frequency (CBF) is characteristic of different types of cilia depending on their function. Using zebrafish as a model, our group has been studying cilia length regulation and motility in wild-type and deltaD-/- mutant embryos. Recently, we showed that Notch signalling was directly involved in the control of cilia length in the KV cells given that the deltaD-/- mutant present shorter KV cilia. The goal of this project was to characterize the CBF of deltaD-/- KV cilia vs. wild-type cilia and reveal how potential differences in CBF impact on KV fluid flow, using spectral analysis associated with highspeed videomicroscopy. By decomposing and comparing the obtained CBF with Fast Fourier Transform, we identified two major populations of motile cilia in wild-type as well as in deltaD-/- mutant embryos. However, we found the CBF populations had differential relative contributions and different distributions between wild-type and mutant embryos. Furthermore, by measuring the velocity of native particles we studied the KV fluid-flow and concluded that the dispersion of the flow velocity was much wider in the deltaD-/- mutants. On the other hand, based on a gene expression study of motility genes downstream of DeltaD, we concluded that motility related genes (dnah7, rsph3 and foxj1a) were deregulated in the mutants. During this project we generated data that led to new hypotheses that will allow us to test the causality between the described correlations.
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Shah, Alok Shirish. "Structural maintenance and chemosensory function of human airway motile cilia." Diss., University of Iowa, 2009. https://ir.uiowa.edu/etd/2983.
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Cilia are finger-like projections that extend from the surface of most cells. These microtubule-based structures serve important mechanical or sensory functions. Motile cilia have been implicated in fluid movement whereas the non-motile primary cilia have been shown to play a role in sensory signal transduction. There exists a dichotomy in the field that primary cilia have only sensory function and motile cilia only have mechanical function. The central question of this thesis project is "what are the structural and functional components of airway motile cilia and are these cilia sensory?" In Chapter 2, the role of Bardet-Biedl Syndrome (BBS) proteins in maintaining the structure and function of airway motile cilia is examined. We found that BBS proteins localize to the cilium and to ciliary-related structures in human airway epithelia. Using mutant mice we found that BBS proteins play an essential role in motile cilia structure and the loss of BBS proteins results in reduced ciliary beat. These proteins have previously been shown to play a role in primary cilia structure and function, and our studies indicate a novel function for BBS proteins. Chapter 3 examines the sensory role of motile cilia. Our data show that bitter taste receptors and components of the bitter taste signal transduction pathway localize to the motile cilia or to the ciliated cells. Ciliated cells also show an increase in intracellular calcium in response to bitter compounds, accompanied by a corresponding increase in cilia beat. The increase in intracellular calcium originates at the ciliated cells and is propagated to adjacent cells. Chapter 4 delves into the possibility that every motile ciliated cell also contains a single, primary cilium. Using immunostaining and Smoothened as a marker for primary cilia, we found that every group of motile cilia contains a single Smoothened-positive cilium. Furthermore, downstream components of the Sonic Hedgehog pathway are also present in ciliated cells. Chapter 6 is a summary chapter including possible explanations for observed outcomes and plans for future experiments. Our results indicate that the divide between primary and motile cilia may not be as great as has been previously thought.
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Mao, Suifang. "Motile cilia of human airway epithelia mediate noncanonical hedgehog signaling." Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6195.
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During embryogenesis, airway epithelial cells possess primary cilia, and HH signaling guides lung development. As epithelial cells mature, they produce hundreds of motile cilia and continue to produce the sonic hedgehog (SHH) ligand, which is found apically in the thin layer of liquid covering airways. However, whether ciliated airway cells express apical HH signaling components and what their function might be have remained unknown. Here we show that motile cilia are enriched for HH signaling proteins, including patched 1 and smoothened. These cilia are also enriched for proteins affecting cAMP-dependent signaling, including Gαi and adenylyl cyclase 5/6. Surprisingly, SHH in differentiated airway epithelia did not elicit the canonical SHH signaling pathway that regulates transcription during development. But instead, activating HH signaling decreases intracellular levels of cAMP, which reduces ciliary beat frequency and airway surface liquid pH, similar to changes that have been observed in the airway of people with chronic obstructive pulmonary disease (COPD). Furthermore, we observed that significant increase of SHH ligand expression in differentiated airway epithelia with COPD, suggesting a potential role of SHH signaling in the pathogenesis of airway disease. Collectively, our study indicates that airway cilia detect apical SHH to mediate airway physiology through noncanonical HH signaling. SHH may dampen defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses. This may suggest a potential role of SHH signaling in the pathogenesis of airway disease, such as COPD.
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Sampaio, Pedro Rafael Martins de Almeida. "Using cilia mutants to study left-right asymmetry in zebrafish." Master's thesis, Faculdade de Ciências e Tecnologia, 2014. http://hdl.handle.net/10362/13155.
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A thesis submitted in fulfillment of the requirements for the degree of the Masters in
Molecular Genetics and BiomedicineIn vertebrates, internal organs are positioned asymmetrically across the left-right (L-R) body axis. Events determining L-R asymmetry occur during embryogenesis, and are regulated by the coordinated action of genetic mechanisms. Embryonic motile cilia are essential in this process by generating a directional fluid flow inside the zebrafish organ of asymmetry, called Kupffer’s vesicle ﴾KV). A correct L-R formation is highly dependent on signaling pathways downstream of such flow, however detailed characterization of how its dynamics modulates these mechanisms is still lacking. In this project, fluid flow measurements were achieved by a non-invasive method, in four genetic backgrounds: Wild-type (WT), deltaD-/- mutants, Dnah7 morphants (MO) and control-MO embryos. Knockdown of Dnah7, a heavy chain inner axonemal dynein, renders cilia completely immotile and depletes the KV directional fluid flow, which we characterize here for the first time. By following the development of each embryo, we show that flow dynamics in the KV is already asymmetric and provides a very good prediction of organ laterality. Through novel experiments, we characterized a new population of motile cilia, an immotile population, a range of cilia beat frequencies and lengths, KV volumes and cilia numbers in live embryos. These data were crucial to perform fluid dynamics simulations, which suggested that the flow in embryos with 30 or more cilia reliably produces left situs; with fewer cilia, left situs is sometimes compromised through disruption of the dorsal anterior clustering of motile cilia. A rough estimate based upon the 30 cilium threshold and statistics of cilium number predicts 90% and 60% left situs in WT and deltaD-/- respectively, as observed experimentally. Cilia number and clustering are therefore critical to normal situs via robust asymmetric flow. Thus, our results support a model in which asymmetric flow forces registered in the KV pattern organ laterality in each embryo.
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McIntosh, Kate. "The extra ciliary roles of Meckel-Gruber syndrome proteins." Thesis, University of Exeter, 2015. http://hdl.handle.net/10871/20774.
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Meckel-Gruber syndrome (MKS) is a recessive genetic disease that is uniformly lethal in affected children due to resultant developmental defects in the kidney and brain. 13 MKS genes have been identified, and further candidate genes have been linked to this disease, all encoding unrelated proteins. Their role is believed to be in generation and compartmentalisation of the primary cilium, a microtubule-based organelle that functions in signal transduction of developmentally-crucial pathways. However, recent evidence indicates that these proteins are also likely involved in regulation of the actin cytoskeleton. Furthermore, research is beginning to uncover roles of other ciliopathy proteins in regulation of additional subcellular structures, such as the microtubule cytoskeleton, focal adhesions and the Golgi. To begin to understand the roles of the MKS proteins beyond the cilium, I examined a number of cellular features of patient fibroblasts carrying mutations in TMEM216 (MKS2) and TMEM67 (MKS3). In this thesis, I describe the temporal appearance and nature of prominent actin bundles observed in these cells, and analyse the dependency of these on the Rho/ROCK signalling pathway. Furthermore, I identify novel alterations to the microtubule cytoskeleton and organisation of the Golgi complex in MKS patient cells, and subsequently establish a temporal order of these phenotypes, demonstrating microtubule defects as the first to occur in these cells. Finally, I connect these phenotypic defects to Rho/ROCK signalling. In contrast to the prevailing view in the ciliopathy field, I believe that a diffusion barrier at the transition zone is not the primary role of MKS proteins. Instead I propose, supported by these data, that MKS protein complexes play a dual role as effectors of Rho signalling in addition to performing a structural role with particular importance in tethering the cytoskeleton to membranes. I therefore conclude that these, and other ciliopathy protein complexes, may act as important signal transduction and structural components at multiple locations throughout the cell.
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Subramanian, Aswati. "p28 DYNEIN LIGHT CHAINS AND CILIARY MOTILITY IN Tetrahymena thermophila." Miami University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=miami1389719903.
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Mali, Girish Ram. "Multisystem functional characterisation of motile ciliopathy genes HEATR2 and ZMYND10." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/21683.
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Cilia are polarized extensions of the cells microtubule-based cytoskeleton dedicated to sensory, signaling and motility-related functions. In mammals, there are two main types of cilia, immotile and motile, where motile cilia generate/modulate fluid flow at the embryonic node, in respiratory airways, cerebral ventricles and the oviduct in addition to sperm propulsion via the flagellum. Defects in cilia motility cause a rare genetic disorder called Primary Ciliary Dyskinesia (PCD). In this thesis, I present functional and molecular characterisation of two PCD causing genes HEATR2 and ZMYND10. Core cilia genes are transcriptionally activated by members of the winged-helix transcription factors of the RFX family. The forkhead transcription factor FOXJ1, additionally activates motility genes such as the ones encoding components of axonemal dynein motors which transfer the chemical energy released from ATP hydrolysis to kinetic motion necessary for ciliary motility. I present data in this thesis which show that Heatr2 and Zmynd10 are both targets of the RFX3-FOXJ1 transcriptional module which co-operatively switches on genes required to make motile cilia Mutations in both HEATR2 and ZMYND10 cause the same subtype of PCD (loss of inner and outer arm dyneins in cilia). I characterise a human PCD causing mutation in HEATR2 in this thesis. Additionally, using genetic null mouse models generated using the CRISPR technology, I describe the phenotypic effects of complete loss of Zmynd10 in mice. Zmynd10 mutant mice display characteristic PCD-like features. Adding to my functional studies, I present proteomic data to propose mechanisms by which HEATR2 and ZMYND10 proteins control cilia motility. Mass spectrometry and protein interaction studies support distinct roles for HEATR2 and ZMYND10 in intracellular transport and pre-assembly of axonemal dynein motors. The multisystem approaches described in this thesis to characterise the roles of HEATR2 and ZMYND10 highlight the molecular complexity underlying the assembly and delivery of axonemal dyneins to motile cilia and provide novel functional and molecular insights into the pathophysiology of PCD.
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Moore, Daniel John. "Identification and characterisation of conserved ciliary genes expressed in Drosophila sensory neurons." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/17918.
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Drosophila provide an excellent model organism in which to study cilia as there are only two ciliated cell types; the sensory neurons and sperm cells. The chordotonal neuron is one such ciliated cell and is required for hearing, proprioception and gravitaxis. Mechanical manipulation of the cilium that extends from the neuronal dendrite is required for signal transduction. Chordotonal neuronal differentiation is regulated by a transcription factor cascade. Atonal begins the cascade, which is then continued by RFX and Fd3F for ciliary genes (Cachero et al 2011, Newton et al 2012). Genes expressed in developing chordotonal neurons are downstream of these transcription factors and their characterisation can further elucidate how neuronal differentiation is regulated. Ciliary genes are highly enriched in developing chordotonal cells; uncharacterised genes enriched in these cells can therefore be considered candidate ciliary genes (Cachero et al 2011). A behavioural assay was conducted to identify further genes that could have a role in ciliary formation and function. Candidate genes were identified by combining enrichment data with previous genomic, proteomic and transcriptomic studies of cilia. A climbing assay of RNAi mediated knock down of these genes identified a number of candidates for future work. One gene found to be highly enriched in developing chordotonal neurons is CG11253. CG11253EY10866 P element insertion mutant flies show a mild uncoordinated phenotype in a climbing assay consistent with reduced chordotonal organ function. Male flies are also infertile due to a lack of motile sperm. CG11253 is expressed in motile ciliated cells and is conserved in organisms with motile cilia. CG11253 expression is also regulated by RFX and Fd3F, suggesting that it is involved in cilium motility. This was confirmed by electron microscopy, which showed disruption of axonemal dynein arm localisation in chordotonal cilia and sperm flagella. A CG11253::mVenus fusion protein was found to localise mainly to the cytoplasm and to a lesser extent the cilia of chordotonal neurons. Patients with symptoms consistent with Primary Ciliary Dyskinesia (PCD), a condition caused by cilium immotility, have subsequently been found to have point mutations in ZMYND10, the human homologue of CG11253. The identification of PCD patients with ZMYND10 mutations showed that investigating cilium motility in Drosophila chordotonal neurons could identify novel PCD genes. It was thought that investigating previously uncharacterised targets of Fd3F could identify novel genes involved in cilium motility and thus candidate PCD genes. CG31320 is a gene regulated by RFX and Fd3F and conserved in organisms with motile cilia. RNAi mediated knock down of CG31320 resulted in both a mild uncoordinated phenotype and male infertility due to a lack of motile sperm. Electron microscopy showed a complete loss of axonemal dynein arms in chordotonal neuron cilia. An mVenus fusion protein of CG6971, an inner dynein arm component, was also mislocalised from the cilia in CG3132027 deletion mutant larvae. This shows that CG31320 is required for the appropriate localisation of the axonemal dynein arms and thus cilium motility. This further showed that uncharacterised genes enriched in chordotonal neurons and regulated by Fd3F could be novel ciliary genes required for cilium motility. Our collaborators and Horani et al (2012) showed that the human homologue of CG31320 (HEATR2) is mutated in patients with PCD, further confirming that this method can be used to identify PCD genes. I have identified two factors required for cilium motility. Disruption of the axonemal dynein arms in both cases results in reduced coordination, and lack of fertility due to sperm immotility. Mutations in the human homologues of these genes have been found to result in PCD. This indicates that further PCD genes could be identified from genes enriched in Drosophila chordotonal neurons that are regulated by Fd3F.
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Nozaki, Shohei. "Architecture of the BBSome and its role in ciliary protein trafficking." Kyoto University, 2019. http://hdl.handle.net/2433/242664.
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Deckman, Cassandra M. "DEPHOSPHORYLATION OF INNER ARM 1 IS REQUIRED FOR CILIARY REVERSALS IN TETRAHYMENA THERMOPHILA." Miami University / OhioLINK, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=miami1054064051.
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Liu, Siming. "TESTING THE MULTI-DYNEIN HYPOTHESIS BY MUTATING INNER ARM DYNEIN HEAVY CHAINS IN TETRAHYMENA THERMOPHILA." Miami University / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=miami1077152822.
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Failler, Marion. "Mise en évidence et caractérisation de nouveaux gènes impliqués dans les ciliopathies rénales." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05T031/document.
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Mokhtarpour, Vanaki Shayan. "Numerical investigation of muco-ciliary transport." Thesis, Queensland University of Technology, 2020. https://eprints.qut.edu.au/203744/1/Shayan_Mokhtarpour%20Vanaki_Thesis.pdf.
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The human airways are protected from inhaled external substances by an extremely thin layer called airway surface liquid. This film of liquid captures most of the inhaled toxic particles and is constantly propelled back out of the airway by a dense mat of beating hair-like structures, thus cleansing the airways of inhaled pathogens. It is vital to better understand this clearance process under diseased conditions and to predict the fate of therapeutic drug particles after deposition. An advanced numerical model is developed to investigate these objectives, given that the complex nature of lung clearance limits the ability to conduct experiments.
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KABI, AMRITA. "Role of Inner Arm Dyneins and Hydin in Ciliary Motility in Tetrahymena thermophila." Miami University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=miami1271977227.
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Inganni, Johan. "Nitric Oxide in Primary Ciliary Dyskinesia : Missing in action?" Thesis, Södertörn University College, School of Life Sciences, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:sh:diva-1608.
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Bright, Alison R. "A Role for Intraflagellar Transport Proteins in Mitosis: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/682.
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Disruption of cilia proteins results in a range of disorders called ciliopathies. However, the mechanism by which cilia dysfunction contributes to disease is not well understood. Intraflagellar transport (IFT) proteins are required for ciliogenesis. They carry ciliary cargo along the microtubule axoneme while riding microtubule motors. Interestingly, IFT proteins localize to spindle poles in non-ciliated, mitotic cells, suggesting a mitotic function for IFT proteins. Based on their role in cilia, we hypothesized that IFT proteins regulate microtubule-based transport during mitotic spindle assembly. Biochemical investigation revealed that in mitotic cells IFT88, IFT57, IFT52, and IFT20 interact with dynein1, a microtubule motor required for spindle pole maturation. Furthermore, IFT88 co-localizes with dynein1 and its mitotic cargo during spindle assembly, suggesting a role for IFT88 in regulating dynein-mediated transport to spindle poles. Based on these results we analyzed spindle poles after IFT protein depletion and found that IFT88 depletion disrupted EB1, γ-tubulin, and astral microtubule arrays at spindle poles. Unlike IFT88, depletion of IFT57, IFT52, or IFT20 did not disrupt spindle poles. Strikingly, the simultaneous depletion of IFT88 and IFT20 rescued the spindle pole disruption caused by IFT88 depletion alone, suggesting a model in which IFT88 negatively regulates IFT20, and IFT20 negatively regulates microtubulebased transport during mitosis. Our work demonstrates for the first time that IFT proteins function with dynein1 in mitosis, and it also raises the important possibility that mitotic defects caused by IFT protein disruption could contribute to the phenotypes associated with ciliopathies.
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Bright, Alison R. "A Role for Intraflagellar Transport Proteins in Mitosis: A Dissertation." eScholarship@UMMS, 2006. http://escholarship.umassmed.edu/gsbs_diss/682.
Full textAbstract:
Disruption of cilia proteins results in a range of disorders called ciliopathies. However, the mechanism by which cilia dysfunction contributes to disease is not well understood. Intraflagellar transport (IFT) proteins are required for ciliogenesis. They carry ciliary cargo along the microtubule axoneme while riding microtubule motors. Interestingly, IFT proteins localize to spindle poles in non-ciliated, mitotic cells, suggesting a mitotic function for IFT proteins. Based on their role in cilia, we hypothesized that IFT proteins regulate microtubule-based transport during mitotic spindle assembly. Biochemical investigation revealed that in mitotic cells IFT88, IFT57, IFT52, and IFT20 interact with dynein1, a microtubule motor required for spindle pole maturation. Furthermore, IFT88 co-localizes with dynein1 and its mitotic cargo during spindle assembly, suggesting a role for IFT88 in regulating dynein-mediated transport to spindle poles. Based on these results we analyzed spindle poles after IFT protein depletion and found that IFT88 depletion disrupted EB1, γ-tubulin, and astral microtubule arrays at spindle poles. Unlike IFT88, depletion of IFT57, IFT52, or IFT20 did not disrupt spindle poles. Strikingly, the simultaneous depletion of IFT88 and IFT20 rescued the spindle pole disruption caused by IFT88 depletion alone, suggesting a model in which IFT88 negatively regulates IFT20, and IFT20 negatively regulates microtubulebased transport during mitosis. Our work demonstrates for the first time that IFT proteins function with dynein1 in mitosis, and it also raises the important possibility that mitotic defects caused by IFT protein disruption could contribute to the phenotypes associated with ciliopathies.
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Mohieldin, Ashraf M. "Cellular Function and Structure of Primary Cilia." University of Toledo Health Science Campus / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=mco1438784302.
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Rachev, Ev [Verfasser], Achim [Akademischer Betreuer] Gossler, and Dietmar J. [Akademischer Betreuer] Manstein. "Characterization of CFAP43 and its function in motile cilia / Ev Rachev ; Akademische Betreuer: Achim Gossler, Dietmar Manstein ; Institut für Molekularbiologie." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2018. http://nbn-resolving.de/urn:nbn:de:gbv:354-20171115154.
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Rachev, Ev [Verfasser], Achim Akademischer Betreuer] Gossler, and Dietmar J. [Akademischer Betreuer] [Manstein. "Characterization of CFAP43 and its function in motile cilia / Ev Rachev ; Akademische Betreuer: Achim Gossler, Dietmar Manstein ; Institut für Molekularbiologie." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2018. http://d-nb.info/1153482509/34.
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Rachev, Ev Verfasser], Achim [Akademischer Betreuer] Gossler, and Dietmar J. [Akademischer Betreuer] [Manstein. "Characterization of CFAP43 and its function in motile cilia / Ev Rachev ; Akademische Betreuer: Achim Gossler, Dietmar Manstein ; Institut für Molekularbiologie." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2018. http://d-nb.info/1153482509/34.
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Chavez, Garcia Edison. "Phosphoinositides regulation and function in the ciliary compartment of Neural stem cells and Ependymal cells." Doctoral thesis, Universite Libre de Bruxelles, 2014. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/221625.
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This thesis describes the work that I have carried out in the Laboratory of Neurophysiolgy at the Université Libre de Bruxelles, under the supervision of Prof. Serge Schiffmann, in collaboration with Prof. Stéphane Schurmans of Université of Liège.The work is divided in two distinct but related projects and the results section is thus divided into two main chapters. The results described are presented in the form of two manuscripts, the first chapter is named “Ciliary phosphoinositides regulation by INPP5E controls Shh signaling by allowing trafficking of Gpr161 in neural stem cells primary cilium”.The second is named “Regulation of phosphoinositides ciliary levels controls trafficking and ciliogenesis in ependymal cells”.Since both manuscripts are comprehensive regarding the results, and methods, these are inserted as such into the thesis.An expanded introduction to the field, placing the results into context, precedes these two chapters. An extended discussion section follows each chapter; it presents some elements of discussion not included in the manuscripts, the implications of the results and the scope for further research.Doctorat en Sciences biomédicales et pharmaceutiques
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Follit, John A. "Building the Cell's Antenna: Protein Targeting to the Ciliary Membrane: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/594.
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Protruding from the apical surface of nearly every cell in our body lies a specialized sensory organelle—the primary cilium. Eukaryotic cells use these ubiquitous structures to monitor the extracellular environment, defects in which result in an ever-growing list of human maladies termed ciliopathies including obesity, retinal degeneration and polycystic kidney disease. The sensory functions of primary cilia rely on the unique complement of receptors concentrated within the ciliary membrane. Vital to the proper functioning of the cilium is the cell's ability to target specific proteins to the ciliary membrane yet little is known how a cell achieves this highly polarized distribution. IFT20, a subunit of the intraflagellar transport particle is localized to the Golgi complex that is hypothesized to sort proteins to the ciliary membrane. We show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP-210 and mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction and heart defects. Cilia on GMAP210 mutant cells have reduced amounts of the membrane protein polycystin-2 localized to them suggesting IFT20 and GMAP-210 function together in the sorting or transport of proteins to the ciliary membrane. To better understand the mechanism of ciliary protein trafficking, we identify a ciliary targeting sequence (CTS) contained within fibrocystin, the gene mutated in autosomal recessive polycystic kidney disease, and investigate a series of proteins required for the delivery of this sequence to the primary cilium. We demonstrate the small G protein Rab8 interacts with the CTS of fibrocystin and controls the ciliary levels of the CTS. Arf4 is another small G protein deemed a key regulator of ciliary protein trafficking. We show Arf4 binds the CTS of fibrocystin but is not absolutely required for trafficking of the fibrocystin CTS to cilia. Arf4 mutant mice are embryonic lethal and die at mid-gestation likely due to defects in the non-ciliated visceral endoderm, where the lack of Arf4 caused defects in cell structure and apical protein localization. This suggests Arf4 is not only important for the efficient transport of fibrocystin to cilia, but also plays critical roles in non-ciliary processes. Together this work aims to elucidate the mechanisms of protein targeting to the ciliary membrane.
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Fowler, Cedar. "Identification of a ciliary defect associated with pulmonary nontuberculous mycobacterial disease." Thesis, University of Cambridge, 2013. https://www.repository.cam.ac.uk/handle/1810/245062.
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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.
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Puybareau, Elodie. "Motion analysis for Medical and Bio-medical applications." Thesis, Paris Est, 2016. http://www.theses.fr/2016PESC1063/document.
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L’analyse du mouvement, ou l’analyse d’une séquence d’images, est l’extension naturelle de l’analyse d’images à l’analyse de séries temporelles d’images. De nombreuses méthodes d’analyse de mouvement ont été développées dans le contexte de la vision par ordinateur, incluant le suivi de caractéristiques, le flot optique, l’analyse de points-clef, le recalage d’image, etc. Dans ce manuscrit, nous proposons une boite a outils de techniques d’analyse de mouvement adaptées à l’analyse de séquences biomédicales. Nous avons en particulier travaillé sur les cellules ciliées qui sont couvertes de cils qui battent. Elles sont présentes chez l’homme dans les zones nécessitant des mouvements de fluide. Dans les poumons et les voies respiratoires supérieures, les cils sont responsables de l’épuration muco-ciliaire, qui permet d’évacuer des poumons la poussière et autres impuretés inhalées. Les altérations de l’épuration mucociliaire peuvent être liées à des maladies touchant les cils, pouvant être génétiques ou acquises et peuvent être handicapantes. Ces maladies peuvent être caractérisées par l’analyse du mouvement des cils sous un microscope avec une résolution temporelle importante. Nous avons développé plusieurs outils et techniques pour réaliser ces analyses de manière automatiques et avec une haute précision, à la fois sur des biopsies et in-vivo. Nous avons aussi illustré nos techniques dans le contexte d’éco-toxicité en analysant le rythme cardiaque d’embryons de poissonsMotion analysis, or the analysis of image sequences, is a natural extension of image analysis to time series of images. Many methods for motion analysis have been developed in the context of computer vision, including feature tracking, optical flow, keypoint analysis, image registration, and so on. In this work, we propose a toolbox of motion analysis techniques suitable for biomedical image sequence analysis. We particularly study ciliated cells. These cells are covered with beating cilia. They are present in humans in areas where fluid motion is necessary. In the lungs and the upper respiratory tract, Cilia perform the clearance task, which means cleaning the lungs of dust and other airborne contaminants. Ciliated cells are subject to genetic or acquired diseases that can compromise clearance, and in turn cause problems in their hosts. These diseases can be characterized by studying the motion of cilia under a microscope and at high temporal resolution. We propose a number of novel tools and techniques to perform such analyses automatically and with high precision, both ex-vivo on biopsies, and in-vivo. We also illustrate our techniques in the context of eco-toxicity by analysing the beating pattern of the heart of fish embryo
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Shebani, Eyman. "Ultrastructural Studies of the Airway Epithelium in Airway Diseases." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6632.
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Lorenzo, Moldero Ivan. "Localization and regulation of trpv4 channels in CILIATED epithelia." Doctoral thesis, Universitat Pompeu Fabra, 2008. http://hdl.handle.net/10803/7185.
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La neteja del moc i dels patògens dels pulmons, i el transport de gàmets i embrions en els òrgans reproductius de les femelles són funcions clau en els epitelis ciliats, tals com aquells que es troben presents en les vies respiratòries i l'oviducte. La taxa de transport mucociliar és funció de la freqüència de batut ciliar (CBF) i aquesta freqüència és augmentada per increments en la concentració de Ca2+ intracelul·lar. El canal catiònic "transient potential vanilloid 4" (TRPV4) intervé en l'entrada de Ca2+ en resposta a estímuls mecànics i osmòtics. L'expressió del TRPV4 en l'epiteli ciliat de les vies respiratòries i de l'oviducte és confirmada mitjançant la localització per immunofluorescència del canal iònic a la membrana apical de l'epiteli ciliat i polaritzat, allà on la senyalització de Ca2+ és requerida per la regulació de la CBF. Cèl·lules ciliades de la tràquea de ratolins TRPV4-/- no expressen el canal TRPV4, no responen a l'activador específic del TRPV4, el 4α-phorbol 12,13-didecanoate (4α-PDD) i presenten respostes de Ca2+ reduïdes a temperatures mitjanes (~25ºC- 8ºC), un altre estímul dels canals TRPV4. L'activació dels canals TRPV4 per solucions altament viscoses i per hypotonicitat depèn de l'activació de la via de la fosfolipasa A2(PLA2)i la subseqüent producció de àcid epoxieicosatrienoic (EET). En condicions de baixa activació de la PLA2, estímuls mecànics i hipotònics alliberen ATP per a l'activació de la via de la fosfolipasa C (PLC)-inositol trifosfat (IP3) per contribuir a l'activació dels canals TRPV4. Descrivim que el metabòlit IP3 sense ser un agonista per ell mateix, sensibilitza el TRPV4 per a l'activació de EET, essent aquest un mecanisme general. L'acoblament funcional entre els canals TRPV4 de la membrana plasmàtica i els receptors de IP3 (IP3R) és necessari tant per iniciar com mantenir la senyalització oscil·latòria del Ca2+ desencadenada per estímuls viscosos i hipotònics. Un dels principals activadors de la CBF, la adenosina-5'-trifosfat (ATP), desencadena una resposta cel·lular mediada per Ca2+ en la que es desencadena tant l'alliberament de Ca2+ des dels dipòsits intracel·lulars com l'entrada de Ca2+. És destacable la contribució de el TRPV4 en l'augment de la CBF mediada per ATP. És més, el nostre treball implica als canals TRPV4 exclusivament en l'entrada de Ca2+ activada per receptor (ROCE). Tot plegat, aquesta tesi doctoral mostra el paper dels canals TRPV4 en l'acoblament d'estímuls fisiològics tipus mecànic, osmòtic i químic a la regulació de la CBF en l'epiteli ciliat destinat al transport mucociliar.Clearance of mucus and pathogenic agents from lungs and the transport of gametes and embryos in the female reproductive organs are key functions of ciliated epithelia such as those present in the airways and the oviduct. The rate of mucociliary transport is a function of ciliary beat frequency (CBF) and this, in turn, is increased by increases in intracellular calcium. Transient potential vanilloid 4 (TRPV4)cation channel mediates Ca2+ influx in response to mechanical and osmotic stimuli. TRPV4 expression in ciliated epithelia from airways and oviduct is confirmed by immunofluorescence localization of the channel at the apical membrane of the polarized ciliated epithelia, where the Ca2+ signalling is required for CBF regulation. Ciliated tracheal cells from TRPV4-/-mice show no TRPV4 expression, neither increases in intracellular Ca2+ and CBF in response to the TRPV4-specific activator 4α- phorbol 12,13- idecanoate (4α-PDD), and reduced responses to mild temperatures (~25ºC - 38ºC), another TRPV4-activating stimulus. TRPV4 gating by high viscous loads and hypotonicity depends on phospholipase A2 (PLA2) pathway activation and subsequent production of epoxyeicosatrienoic acid (EET). Under conditions of low PLA2 activation, mechanical and hypotonic stimuli use extracellular ATP release-mediated activation of phospholipase C (PLC)-inositol triphosphate(IP3)signalling to support TRPV4 gating. We describe that IP3, without being an agonist itself, sensitizes TRPV4 to EET activation. Besides, the functional coupling between plasma membrane TRPV4 channels and IP3 receptors (IP3R) is required to initiate and maintain the cellular oscillatory Ca2+ signal triggered by high viscous loads and hypotonic stimuli. One of the main CBF activators, adenosine-5'-triphosphate (ATP), triggers both Ca2+ release from intracellular Ca2+ stores and Ca2+ entry. Interestingly, TRPV4 contributes to ATP-induced increase in CBF. Furthermore, our work implicates TRPV4 channel exclusively in receptor-operated Ca2+ entry. Collectively, this PhD thesis shows the role of TRPV4 channels coupling physiologically relevant mechanical, hypotonic and chemical stimuli to CBF regulation in motile ciliary epithelia.
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Laligné, Chloé. "Étude de la fonction de la protéine Bug22p dans différents organismes." Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00746881.
Full textAbstract:
Les cils sont des organites très conservés au cours de l'évolution des eucaryotes et présents à la surface de presque tous les types cellulaires. Ils sont constitués d'une structure microtubulaire, l'axonème, entourée d'une membrane en continuité avec la membrane plasmique. Ils sont nucléés par un corps basal, centriole ancré à la surface cellulaire. Grâce aux nombreux récepteurs qu'ils concentrent à leur membrane, tous les cils sont des senseurs de leur environnement. Ils peuvent aussi être motiles et assurer, par leur battement coordonné, le déplacement relatif de la cellule et du fluide environnant. Tandis que cil et structure centriolaire, hérités du premier eucaryote, ont été perdus par certains champignons et par les plantes supérieures, certains gènes codant des protéines ciliaires et centriolaires sont pourtant retrouvés dans le génome de ces espèces. Cette conservation de protéines sans l'organite suggère soit que ces protéines interviennent dans un même processus moléculaire utilisé dans plusieurs organites, soit qu'elles jouent des rôles dans des processus moléculaires distincts via leur interaction avec différents types de partenaires.J'ai choisi d'étudier l'une de ces protéines ciliaires et centriolaires, Bug22p, hautement conservée en séquence protéique entre l'homme et la paramécie, mais également présente chez les plantes supérieures. J'ai mené cette étude principalement sur la paramécie, système modèle pour la biogénèse des corps basaux et des cils, mais aussi sur des cellules de mammifère et de végétaux supérieurs. Si Bug22p est impliquée dans la détermination du battement ciliaire chez la paramécie, elle se localise également dans des cils immotiles de cellules de mammifère suggérant que son activité ciliaire n'est pas réduite à cette seule fonction. Des expériences d'inactivation génique suggèrent par ailleurs un lien entre l'activité de Bug22p et la polyglycylation. Sa surexpression dans les cellules de mammifère en culture entraîne l'apparition d'extensions cellulaires et une augmentation des réseaux de tubulines acétylées probablement associées à une stabilisation des microtubules. L'ensemble de mes résultats suggère donc un rôle de Bug22p dans la régulation de modifications post-traductionnelles. En plus d'être présente dans les structures ciliaires, Bug22p se localise aussi bien dans les noyaux de la paramécie que dans ceux des cellules humaines et des plantes supérieures Arabidopsis et Nicotiana. Ces observations ouvrent un nouveau champ d'études. En effet, si l'on sait que les tubulines ciliaires sont soumises à différentes modifications post-traductionnelles telles que polyglycylation ou acétylation, ce type de modifications touchent également des protéines nucléaires régulant ainsi le trafic de protéines nucléaires ou l'expression génique. Nous pouvons donc avancer l'hypothèse selon laquelle Bug22p agirait sur la régulation de ces modifications dans le cil et dans le noyau. Il serait donc intéressant de caractériser les modifications post-traductionnelles chez les plantes supérieures afin de vérifier une possible implication de Bug22p dans leur régulation et donc comprendre les raisons de sa conservation chez les végétaux.
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Thomas, Lucie. "Bases moléculaires et cellulaires de l'assemblage de l'axonème des cils mobiles." Electronic Thesis or Diss., Sorbonne université, 2023. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2023SORUS097.pdf.
Full textAbstract:
Les bras de dynéine (BDs), moteurs du mouvement ciliaire et flagellaire, sont ancrés à intervalles réguliers le long de l'axonème des cils mobiles et des flagelles. Les dyskinésies ciliaires primitives (DCP) sont des maladies respiratoires rares, de transmission essentiellement autosomique récessive, liées à un défaut des cils mobiles, souvent associées à une infertilité masculine. Environ 25% des patients DCP présentent une absence des BDs externes et internes. Ces patients portent des mutations dans un des 13 gènes impliqués qui codent, pour la plupart, des constituants des complexes cytoplasmiques d'assemblage des BDs. L'objectif de cette thèse était de préciser le rôle de deux co-chaperonnes appartenant au(x) complexe(s) d'assemblage cytoplasmique des BDs, codées par des gènes nouvellement (TTC12) ou récemment (PIH1D3) impliqués dans les DCP. Nos travaux sur TTC12 ont révélé pour la première fois l'existence de mécanismes d'assemblage des BDs différents entre les cils mobiles et les flagelles chez l'Homme, expliquant le phénotype particulier de ces patients : infertilité masculine prédominante et atteinte respiratoire modérée. Nos données démontrent également l'existence de mécanismes d'assemblage distincts entre les différents sous-types de BDs internes. Ces travaux nous ont conduits à développer un modèle de cellules épithéliales nasales humaines (CENH) différenciées in vitro en interface air-liquide avec invalidation de gène par CRISPR-Cas9. Cet outil ouvre de nouvelles perspectives pour l'étude de la physiopathologie de maladies respiratoires. Notre étude sur PIH1D3, responsable de la seule DCP non syndromique récessive liée à l'X, a montré que les femmes porteuses hétérozygotes d'un défaut de PIH1D3 présentaient des symptômes respiratoires variables (absents à sévères) en relation avec le taux d'inactivation du chromosome X qui porte l'allèle muté. Ce taux d'inactivation a été déterminé par l'évaluation de la méthylation des dimères CG selon deux approches : digestion enzymatique au locus HUMARA et conversion au bisulfite au locus PIH1D3. Ces résultats incitent donc à rechercher une mutation hétérozygote de PIH1D3, en vue d'un conseil génétique, chez (i) des femmes apparentées aux patients masculins avec DCP liée à PIH1D3, et (ii) des femmes avec infections respiratoires chroniques sans étiologie identifiée. Dans les deux cas, le diagnostic de DCP est difficile à porter chez ces femmes : absence de l'infertilité masculine, du situs inversus, signes respiratoires modérés, NO nasal normal. Ce travail indique que dans la perspective d'une thérapie génique ou ARN, une compensation d'environ 30% du niveau de l'expression de PIH1D3 pourrait améliorer significativement le phénotype respiratoire des patientsDynein arms (DAs), the motors of ciliary and flagella beating, are anchored with a regular spacing along cilia and flagella axonemes. Primary ciliary dyskinesia (PCD) is a rare respiratory disease due to defects in motile cilia. This mainly recessive condition combines laterality defects in half of the patients and frequent male and female hypofertility. About 25% of PCD patients show an absence of both outer and inner DAs. Those patients carry mutations in one of the 13 implicated genes that drive, for most of them, the cytoplasmic assembly of DAs. The aim of this thesis was to clarify the role of two co-chaperones, part of the cytoplasmic DA assembly complexes and encoded by PCD genes newly (TTC12) or recently (PIH1D3) identified. Our work on TTC12 revealed for the first time the existence of different mechanisms of DA assembly between respiratory cilia and sperm flagella in humans. This is in keeping with the peculiar phenotype of patients with TTC12 mutations: a predominant male infertility associated with mild respiratory signs. Our data also show the existence of a distinct DA assembly process for the different subspecies of inner DAs. Within the framework of this study, we developed an original cellular model of human airway epithelial cells (HAECs) differentiated in vitro at air-liquid interface, in which genes can be CRISPR-Cas9-invalidated. This tool opens new avenues for the study of the pathophysiology of airway diseases. Our study on PIH1D3, which mutations underlie the unique known X-linked non-syndromic PCD, showed that women who carry a heterozygous PIH1D3 mutation displayed variable airway phenotypes (from asymptomatic to severe respiratory symptoms) in close relation to the X-inactivation rate of the mutated allele. This has been shown by two approaches that assess DNA methylation of CG dimers: enzymatic digestion at the HUMARA locus and bisulfite conversion at the PIH1D3 locus. Those results prompt to search for heterozygous mutations in PIH1D3, in anticipation of a genetic counselling, in (i) females relatives of male PCD patients with a PIH1D3 defect, and (ii) females with mild chronic respiratory symptoms of unknown etiology. In both cases, diagnosis is difficult to estabilsh in those women given the absence of situs inversus, mild respiratory signs, and normal nasal NO. At the prospect of gene or RNA therapy, this work suggests that reaching 30% of PIH1D3 expression could significantly improve the airway phenotype in patients
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Lacouture, Loïc. "Modélisation et simulation du mouvement de structures fines dans un fluide visqueux : application au transport mucociliaire." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS139/document.
Full textAbstract:
Une grande part des muqueuses à l’intérieur du corps humain sont recouvertes de cils qui, par leurs mouvements coordonnés, conduisent à une circulation de la couche de fluide nappant la muqueuse. Dans le cas de la paroi interne des bronches, ce processus permet l’évacuation des impuretés inspirées à l’extérieur de l’appareil respiratoire.Dans cette thèse, nous nous intéressons aux effets du ou des cils sur le fluide, en nous plaçant à l’échelle du cil, et on considère pour cela les équations de Stokes incompressible. Due à la finesse du cil, une simulation directe demanderait un raffinement important du maillage au voisinage du cil, pour un maillage qui évoluerait à chaque pas de temps. Cette approche étant trop onéreuse en terme de coûts de calculs, nous avons considéré l’asymptotique d’un diamètre du cil tendant vers 0 et d’une vitesse qui tend vers l’infini : le cil est modélisé par un Dirac linéique de forces en terme source. Nous avons montré qu’il était possible de remplacer ce Dirac linéique par une somme de Dirac ponctuels distribués le long du cil. Ainsi, nous nous sommes ramenés, par linéarité, à étudier le problème de Stokes avec en terme source une force ponctuelle. Si les calculs sont ainsi simplifiés (et leurs coûts réduits), le problème final est lui plus singulier, ce qui motive une analyse numérique fine et l’élaboration d’une nouvelle méthode de résolution.Nous avons d’abord étudié une version scalaire de ce problème : le problème de Poisson avec une masse de Dirac en second membre. La solution exacte étant singulière, la solution éléments finis est à définir avec précaution. La convergence de la méthode étant dégradée dans ce cas-là, par rapport à celle dans le cas régulier, nous nous sommes intéressés à des estimations locales. Nous avons démontré une convergence quasi-optimale en norme Hs (s ě 1) sur un sous-domaine qui exclut la singularité. Des résultats analogues ont été obtenus dans le cas du problème de Stokes.Pour palier les problèmes liés à une mauvais convergence sur l’ensemble du domaine, nous avons élaboré une méthode pour résoudre des problème elliptiques avec une masse de Dirac ou une force ponctuelle en terme source. Basée sur celle des éléments finis standard, elle s’appuie sur la connaissance explicite de la singularité de la solution exacte. Une fois données la position de chacun des cils et leur paramétrisation, notre méthode rend possible la simulation directe en 3d d’un très grand nombre de cils. Nous l’avons donc appliquée au cas du transport mucociliaire dans les poumons. Cet outil numérique nous donne accès à des informations que l’on ne peut avoir par l’expérience, et permet de simuler des cas pathologiques comme par exemple une distribution éparse des cilsNumerous mucous membranes inside the human body are covered with cilia which, by their coordinated movements, lead to a circulation of the layer of fluid coating the mucous membrane, which allows, for example, in the case of the internal wall of the bronchi, the evacuation of the impurities inspired outside the respiratory system.In this thesis, we integrate the effects of the cilia on the fluid, at the scale of the cilium. For this, we consider the incompressible Stokes equations. Due to the very small thickness of the cilia, the direct computation would request a time-varying mesh grading around the cilia. To avoid too prohibitive computational costs, we consider the asymptotic of a zero diameter cilium with an infinite velocity: the cilium is modelled by a lineic Dirac of force in source term. In order to ease the computations, the lineic Dirac of forces can be approached by a sum of punctual Dirac masses distributed along the cilium. Thus, by linearity, we have switched our initial problem with the Stokes problem with a punctual force in source term. Thus, we simplify the computations, but the final problem is more singular than the initial problem. The loss of regularity involves a deeper numerical analysis and the development of a new method to solve the problem.We have first studied a scalar version of this problem: Poisson problem with a Dirac right-hand side. The exact solution is singular, therefore the finite element solution has to be defined with caution. In this case, the convergence is not as good as in the regular case, and thus we focused on local error estimates. We have proved a quasi-optimal convergence in H1-norm (s ď 1) on a sub-domain which does not contain the singularity. Similar results have been shown for the Stokes problem too.In order to recover an optimal convergence on the whole domain, we have developped a numerical method to solve elliptic problems with a Dirac mass or a punctual force in source term. It is based on the standard finite element method and the explicit knowl- edge of the singularity of the exact solution. Given the positions of the cilia and their parametrisations, this method permits to compute in 3d a very high number of cilia. We have applied this to the study of the mucociliary transport in the lung. This numerical tool gives us information we do not have with the experimentations and pathologies can be computed and studied by this way, like for example a small number of cilia
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Mianné, Joffrey. "Thérapie génique par CRISPR/Cas9 pour corriger des épithéliums bronchiques dérivés de cellules souches pluripotentes induites (iPSCs) de patients atteints de dyskinésie ciliaire primitive (DCP) : une preuve de concept." Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTT045.
Full textAbstract:
La dyskinésie ciliaire primitive (DCP) est une maladie génétique rare et hétérogène affectant la structure et la fonction des cils motiles. Dans l'épithélium des voies respiratoires, l’altération du mouvement ciliaire entraîne des infections chroniques responsables du déclin progressif et définitif des fonctions pulmonaires. Il n'existe actuellement aucun traitement efficace pour la DCP. Par ailleurs, la recherche de nouvelles thérapies reste limitée par le manque de modèles fiables.Dans ce contexte, les deux objectifs de cette thèse sont : 1) de développer un nouveau modèle in vitro de la DCP basé sur la différenciation dirigée de cellules souches pluripotentes induites (CSPi) en épithélium des voies respiratoires multiciliées, et 2) d'utiliser ce modèle pour étudier le potentiel d'une approche de thérapie génique basée sur la technologie CRISPR/Cas9.Dans ce but, nous avons dérivé deux lignées de CSPi, une d'un individu sain et une seconde d'une patiente atteinte de la DCP contenant des mutations hétérozygotes composites dans le gène CCDC40. En utilisant la lignée « saine » et la technologie CRISPR/Cas9, nous avons généré des contrôles isogéniques knock-out pour trois gènes impliqués dans la DCP incluant CCDC40, DNAH5 et MCIDAS. Parallèlement, la lignée de CSPi dérivée de la patiente a été corrigée à l’aide de la technologie CRISPR/Cas9 et de l'approche de réparation dirigée par homologie. En appliquant à ces lignées cellulaires notre protocole de différenciation préalablement optimisé, nous générons efficacement un épithélium multicilié fonctionnel des voies aériennes récapitulant les phénotypes ciliaires en fonction du génotype. En outre, ce nouveau modèle nous a permis d'étudier le potentiel d'une approche de thérapie génique basée sur la technologie CRISPR/Cas9 pour restaurer le cadre de lecture et de rétablir le phénotype ciliaire dans la lignée DCP.En conclusion, le nouveau modèle développé dans ce travail pourrait représenter un outil majeur pour la modélisation de la DCP in vitro. Ce modèle sera particulièrement intéressant pour étudier directement sur le tissu humain concerné la faisabilité et l'efficacité de thérapies innovantes. Notre pipeline pourrait donc permettre d’accélérer la recherche et le développement de nouvelles thérapies pour la DCP ainsi que d'autres pathologies pulmonairesPrimary Ciliary dyskinesia (PCD) is a rare and heterogeneous genetic disorder affecting the structure and function of motile cilia. In the airway epithelium, impaired ciliary motion results in chronic airway infections responsible for progressive and definitive decline of lung functions. There is currently no effective treatment for PCD, and research is limited by the lack of convenient models to study this disease and investigate innovative therapies.In this context, the main goals of this thesis are: 1) to develop a new in vitro PCD model based on the directed differentiation of patient-derived or genetically-engineered induced pluripotent stem cells (iPSC) into multiciliated airway epithelium, and 2) to use this model to investigate the potential of an innovative CRISPR/Cas9 gene therapy approach.To this aim, we have derived two iPSC lines, one from an healthy individual and a second from a PCD patient harbouring compound heterozygous mutations in the CCDC40 gene. Using the “healthy” iPSC line and the CRISPR/Cas9 technology we have generated isogenic knock-out controls for three PCD genes including CCDC40, DNAH5 and MCIDAS. In parallel, using the CRISPR/Cas9 technology and the homology directed repair approach, we have corrected the patient-derived iPSC line. By applying our optimized differentiation protocol to these cell lines, we are efficiently generating functional multiciliated airway epithelium recapitulating the ciliary phenotypes in function of the genotype. Furthermore, this new model has allowed us to investigate the potential of a CRISPR/Cas9-mediated reframing gene therapy approach to rescue ciliary phenotype in the patient line.In conclusion, the new model developed in this work could represent a major tool for in vitro PCD modelling. This model will be of particular interest for investigating the feasibility and efficacy of personalized therapies directly on the relevant human tissue. Our pipeline could therefore accelerate the development and translation of new therapeutics for PCD and other lung diseases