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.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23571号 / 医博第4785号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 大森 孝一, 教授 大鶴 繁 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

Les cellules épithéliales nasales humaines (CENH) sont des acteurs importants de la physiopathologie de la polypose naso-sinusienne (PNS), le plus souvent la PNS est primitive (étiologie inconnue), plus rarement, elle est secondaire à la mucoviscidose (PNS CF) ou la dyskinésie ciliaire primitive (PNS DCP). L'objectif de cette thèse est d'identifier des protéines différentiellement exprimées dans les PNS primitives et secondaires par approche protéomique puis de les caractériser par des techniques classiques. Dans cette étude, nous avons utilisé des cultures primaires (interface air/liquide) de CENH, dérivant de déchet per-opératoire (PNS) ou de brossages (muqueuse saine). Le marquage iTRAQ suivie d'une analyse par nano-LC-MALDI-TOF-TOF à en particulier montré : 1) une augmentation de six marqueurs du stress du réticulum endoplasmique (RE) dans la PNS primitive et la PNS CF, 2) une altération du métabolisme du glucose dans la PNS CF (sous-expression de 6 protéines dont la pyruvate kinase, enzyme clef de la glycolyse) et 3) une sous expression de 12 protéines des filaments acto-myosines dans la PNS DCP (dont 3 tropomyosines). Nous avons caractérisé le stress du RE dans la PNS primitive, montrant qu'il est induit par le biais d'une sensibilité des CENH à un stress oxydatif probablement d'origine mitochondrial et qu'il participe activement à l'inflammation (sécrétion d'IL-8 et de LTB4). Ces observations nous permettent de proposer que, dans la PNS primitive, il existe un cercle vicieux entre stress du RE, stress oxydatif et inflammation et que la sensibilité au stress oxydatif observée apparaît comme une nouvelle cible thérapeutique dans le traitement de la PNS. / Human nasal epithelial cells (HNEC) play an important role in nasal polyposis(NP). NP have generally an unknown etiology, less frequently, NP is secondary to cystic fibrosis (CF NP) or primary ciliary dyskinesia (PCD NP). The aim of this thesis is to identify differentially expressed proteins in NP, CF NP and PCD NP using proteomic approach then to characterize them by classical methodes. In this study, we used primary culture (air/liquid)of HNEC derived from NP patients (surgery waste) or healthy patients (brushing). The iTRAQ labeling folowing by nano-LC-MALDI-TOF-TOF analysis shown in particular :1)an increased expression of six endoplasmic reticulum (ER) stress markers in NP and CF NP, 2)an alteration of glucose metabolism (decreased expression of six proteins with in particular the pyruvate kinase, glycolysis key enzyme) and 3)an decreased expression of 12 acto-myosin filament proteins (in particular three tropomyosins). We caracterized the ER stress in NP showing that ER stress is activated by an oxydative stress susceptibility of NP HNEC probably of mitochondrial origin and that it directly participate to inflammation (IL-8 and LTB4 secretions). Altogether, our results underline a vicious circle that exists between oxidative stress, ER stress and inflammation.The oxydative stress susceptibility observed in NP may represent new therapeutic target in such a pathology.

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.

<p>Ultrastructural studies of airway epithelium in airway disease are important for diagnosis and understanding the underlying pathology which helps clinicians to improve the patients' treatment.</p><p>Airway biopsies from a 5-month old boy with respiratory problems and gastro-oesophageal reflux were studied by transmission electron microscopy (TEM). The tracheal columnar cells showed accumulation of lamellar bodies, indicative of lysosomal storage disease. The patient was diagnosed with Gaucher disease type 2.</p><p>Shedding of airway epithelial cells is commonly found in asthma. The attachment of these cells to the basal lamina was investigated by TEM of biopsies from patients with asthma and healthy controls. The contact area between columnar cells and basal lamina in asthmatics was significantly less than in controls. Attachment of columnar cells to the basal lamina occurs mainly indirectly, via desmosomal attachment to basal cells. </p><p>Primary ciliary dyskinesia (PCD) is a congenital disease. It is important to differentiate PCD from acquired (secondary) ciliary dyskinesia (SCD). The number of dynein arms determined by TEM was 1.5 and 1.4 for outer and inner dynein arms, respectively in PCD, versus 7.9 and 5.2 for controls and 8.1 and 5.9 in SCD. Compared to PCD patients, SCD patients have more structurally abnormal cilia. A significant difference was found in orientation of the central microtubule pair between PCD and SCD, but also overlap. </p><p>Leukotriene receptor antagonists are a new treatment for asthma. Both corticosteroids and montelukast caused apoptosis and necrosis of airway epithelial cells, and reduced the expression of intercellular adhesion molecule-1. Treatment of cells with tumor necrosis factor-α or interferon-γ reduced the fraction of the lateral cell membrane occupied by desmosomes and this effect was counteracted by corticosteroids. </p>

Ultrastructural studies of airway epithelium in airway disease are important for diagnosis and understanding the underlying pathology which helps clinicians to improve the patients' treatment. Airway biopsies from a 5-month old boy with respiratory problems and gastro-oesophageal reflux were studied by transmission electron microscopy (TEM). The tracheal columnar cells showed accumulation of lamellar bodies, indicative of lysosomal storage disease. The patient was diagnosed with Gaucher disease type 2. Shedding of airway epithelial cells is commonly found in asthma. The attachment of these cells to the basal lamina was investigated by TEM of biopsies from patients with asthma and healthy controls. The contact area between columnar cells and basal lamina in asthmatics was significantly less than in controls. Attachment of columnar cells to the basal lamina occurs mainly indirectly, via desmosomal attachment to basal cells. Primary ciliary dyskinesia (PCD) is a congenital disease. It is important to differentiate PCD from acquired (secondary) ciliary dyskinesia (SCD). The number of dynein arms determined by TEM was 1.5 and 1.4 for outer and inner dynein arms, respectively in PCD, versus 7.9 and 5.2 for controls and 8.1 and 5.9 in SCD. Compared to PCD patients, SCD patients have more structurally abnormal cilia. A significant difference was found in orientation of the central microtubule pair between PCD and SCD, but also overlap. Leukotriene receptor antagonists are a new treatment for asthma. Both corticosteroids and montelukast caused apoptosis and necrosis of airway epithelial cells, and reduced the expression of intercellular adhesion molecule-1. Treatment of cells with tumor necrosis factor-α or interferon-γ reduced the fraction of the lateral cell membrane occupied by desmosomes and this effect was counteracted by corticosteroids.

Primary ciliary dyskinesia is a rare hereditary disorder with impairment of cilia characterized by chronic cough with sputum, bronchiectasis or pneumonia. Regular exercise training should affect pulmonary function, promote mucociliary clearance and improve quality of life. The aim of this thesis is to compare the aerobic fitness of patients with PCD with a control group.