Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified

Cilia motility studies in zebrafish embryos

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

Understanding the Collective Dynamics of Motile Cilia in Human Airways

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

Functional imaging of mucociliary phenomena: High-speed digital reflection contrast microscopy

We present a technique for the investigation of mucociliary phenomena on trachea explants under conditions resembling those in the respiratory tract. Using an enhanced reflection contrast, we detect simultaneously the wave-like modulation of the mucus surface by the underlying ciliary activity and the transport of particles embedded in the mucus layer. Digital recordings taken at a speed of 500 frames per second are analyzed by a set of refined data processing algorithms. The simultaneously extracted data include not only ciliary beat frequency and its surface distribution, but also space-time structure of the mucociliary wave field, wave velocity and mucus transport velocity. Furthermore, we propose the analysis of the space and time evolution of the phase of the mucociliary oscillations to be the most direct way to visualize the coordination of the cilia. In particular, this analysis indicates that the synchronization is restricted to patches with varying directions of wave propagation, but the transport direction is strongly correlated with the mean direction of waves. The capabilities of the technique and of the data-processing algorithms are documented by characteristic data obtained from mammalian and avine trachea

Using cilia mutants to study left-right asymmetry in zebrafish

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

Characterization of an in vitro 3D Human Small Airway Epithelia model for the application of integrated strategies in inhaled drug development

Drug Inhalation is one of the most effective administration routes; in fact, first pass metabolism is bypassed, rapid action onset is enabled and drug doses can be kept relatively low compared to other administration routes. The most recent 3D in vitro models allow to mimic some of the pulmonary tissue functionalities. These models reproduce the air-liquid interface, with beating cilia and mucus production, since different types of cells are present. Therefore, these models could be possibly applied to multi-disciplinary investigations following liquid, dry powder or aerosol treatment. In this thesis, an integrated strategy is proposed, with the aim to increase the rate of success in the drug candidate selection phase. Ideally, safety data should be integrated with early pharmacokinetics (PK) and efficacy indications in order to increase chances to select the candidate with the highest safety margins. With this ambitious objective in mind, a human-based 3D model were evaluated as model for the toxicity assessment and drug permeability evaluation. In particular, a commercially available 3D respiratory model SmallAir\u2122 has been qualified for: a) sensibility and specificity in the evaluation of lung toxicity potential of new compounds; b) permeability to test drug transport through tissues for formulation screening purposes; c) quantitative cytokine secretion on cell supernatant; d) cilia beating and muco-ciliary clearance evaluation by image analysis. These tests performed on a human tissue could provide more reliable results also because all tests were performed in the same model and this could be helpful in data integration. This approach could allow to fill gaps in drug discovery for human-relevant screening of new chemical entities (NCEs), best formulation selection, including physiochemical equivalence evaluation generic drug development. In vitro and in silico data can be helpful in predicting PK and toxicity profiles prior to preclinical and clinical studies. This allows to respect the 3Rs principle of replacement, reduction and refinement of in vivo studies. The proposed integrated testing strategy (ITS) has the potential to reduce the attrition in drug development, to optimize the inhaled formulation, to screen compounds for candidate selection and to reduce in vivo studies.For the toxicity tests, well-known respiratory toxic compound were tested both in the SmallAir\u2122 model and in the A549 cell line model. On the basis of results, the SmallAir\u2122 model seemed to be less sensitive than A549, probably due to the 3D structure physiological features. Cilia beating and mucus production can indeed protect the cells from the toxic effect miming the in vivo response. For the permeability study, well-known inhalation compounds with very different permeability values were evaluated both in SmallAir\u2122 model and in the standard Caco2 cell model. For low and high permeable compounds results obtained were comparable in the two test considered systems. The most evident difference was observed with medium permeable compounds, suggesting that the SmallAir\u2122 model should express different efflux pumps on their surface form the standard Caco2 cell model. The SmallAir\u2122 model was also evaluated as in vitro model for the inflammatory mediators assessment. The treatment with TGF-\u3b2 allowed to confirm the activation of the signalling via Smad2 while, inconclusive results were obtained with regards to cytokines and ROS release following Bleomycin treatment. The SmallAir\u2122 model was finally evaluated as in vitro model for the assessment of the Muco-ciliary Clearance (MCC). Results obtained in this project, showed that the SmallAir\u2122 can be a promising model to assess the MCC in vitro after treatment with compound acting on ATP release and Cystic fibrosis transmembrane conductance Inhibitor-172 (CFTR172inh). More test considering different compound, study design and end points has to be conducted, in order to identify a human relevant in vitro lung model to be applied in many fields of analysis

New methods for the study of Primary Ciliary Dyskinesia

Os cilios e flagelos são projeções celulares encontradas nas células eucariotas, são altamente conservados entre espécies e envolvidos na locomoção e movimentação de fluídos. A Discinésia Ciliar Primária (DCP) é uma doenca genética autossómica recessiva dos cílios móveis, que tem como consequência várias manifestações clínicas. Estima-se que a DCP afete ~1 em cada 10.000 pessoas, mas é mais prevalente em grupos com marcada consanguinidade. A DCP está associada até à data a mais de 40 genes causadores de doença. O diagnóstico da DCP envolve a combinação de vários testes, entre eles a microscopia electrónica (ME), teste determinante na classificação de anomalias ciliares. Neste trabalho foquei-me nos cílios móveis e em como se classificam as derivações à estrutura considerada normal. Este estudo levou ao desenvolvimento de feramentas e diretrizes que tornam o diagnóstico de DCP por EM mais estandardizado, informativo e fidedigno. A DCP necessita de ser modelada em organismos vertebrados como o ratinho, a rã e o peixe-zebra (PZ) para melhor conhecimento dos seus mecanismos moleculares. O PZ é um bom modelo de DCP porque apresenta diversos órgãos ciliados durante os estados larvares (cílios moveis e imoveis) e tem, até agora, homólogos de todos os genes causadores da doença humana. Desta forma a utilização de peixes mutantes tem sido um bom contributo para compreender esta doença humana. Neste trabalho investiguei por ME dois tipos de cílios móveis do PZ concluindo que estes apresentam semelhanças estruturais conservadas com os cílios móveis das vias aéreas do ser humano saudável e com DCP.Cilia and flagella are cellular protrusions found in eucaryotic cells, highly conserved between species and found in almost every cell type. Motile cilia are known for their motility properties and are involved in propelling and moving fluids. Primary ciliary dyskinesia (PCD) is an inherited autosomal-recessive disorder of motile cilia that results in several clinical manifestations. The estimated prevalence of PCD is ∼1 per 10,000 births, but it is more prevalent in populations where consanguinity is common, it is currently associated with mutations in more than 40 genes. To diagnose PCD it involves a combination of tests, in particular, electron microscopy (EM) that is essential for determining the type of ciliary ultrastructural defect. In this work I have focused on motile cilia ultrastructure and how the differences in cilia can be identified and classified, through the development of tools and guidelines to make the quantification and analysis of cilia more reliable and informative. The differential diagnosis of PCD is complex but crucial, and the development of new potential targeted treatments is essential. For better investigating the molecular mechanisms underlying PCD, it has been modelled in several organisms like mice, frogs and Zebrafish (ZF). ZF is a teleost vertebrate used in many areas of research, and a well-known animal model. ZF embryos develop quickly and allow unique advantages for research studies owing to their transparency during larval stages. ZF has many ciliated organs and presents primary cilia as well as motile cilia together with homologs for all the disease causing genes. The use of mutant zebrafish has been contributing to the better understanding of PCD molecular aetiology. Here, I investigated whether zebrafish cilia are ultrastructurally suitable for the study of PCD and concluded that the motile cilia of zebrafish resemble the cilia in the human airway in healthy conditions and in PCD

Moraxella catarrhalis and rhinovirus infection and co-infection of healthy and chronic obstructive pulmonary disease ciliated respiratory epithelium

This research project investigated the effects of rhinovirus and Moraxella catarrhalis (M. catarrhalis) infection of the ciliated respiratory epithelium given the clinical infections caused by both pathogens in healthy individuals. However, as both rhinovirus and M. catarrhalis infection are known to exacerbate disease in a number of chronic respiratory diseases we were keen to determine if the underlying cellular response was different in such conditions. We chose to investigate chronic obstructive pulmonary disease (COPD) to allow comparison with results from healthy individuals. COPD is a progressive pulmonary disease characterised by chronic airway inflammation, emphysema, airway remodelling and chronic bronchitis. Exacerbations in COPD are primarily caused by bacterial and viral infections. As most studies looking at viral and bacterial infection of cells have focused on cell lines or basal cells we wanted to determine if differentiation to a ciliated phenotype, mimicking the host more closely affected response to infection. Primary healthy and COPD nasal tissue, differentiated at air-liquid interface allowed us to assess the initial interaction of M. catarrhalis and rhinovirus with the ciliated epithelium in vitro. Host-pathogen interactions were explored utilising high speed video, confocal and electron microscopy, western blotting, flow cytometry and immune-based assays. It was found that M. catarrhalis binds rapidly to the cilia of beating ciliated cells, altering ciliary function and invading epithelial cells, particularly COPD epithelial cells, initiating a cascade of signalling events involving the epidermal growth factor receptor and phosphoinositide-3 kinase pathway, leading to induction of pro-inflammatory cytokines. It was also demonstrated that rhinovirus targets ciliated cells in primary cultures, causing their shedding from the epithelial layer via apoptosis. This resulted in depletion of ciliated cells and a severely disrupted airway epithelium. Investigation of the mechanisms associated with bacterial-viral co-infections showed that rhinovirus pre-infection followed by a subsequent M. catarrhalis infection further decreased ciliary function of epithelial cultures and increased the release of pro-inflammatory mediators. Additionally, it was found that initial viral infection can potentially cause later dissemination of pathogens in the respiratory tract through triggered detachment of ciliated cells which appeared to have bound M. catarrhalis. Detached cells and reduction in ciliary function are likely to exacerbate airways obstruction

Testing the role of extracellular vesicles in early left right patterning

Abstract Bilaterian animals, such as humans, are characterized by an external roughly mirror symmetry along the left – right axis that covers a pronounced internal asymmetric arrangement of the thoracic and abdominal organs. While external symmetry has been associated with health and beauty standards, the internal asymmetry may rely more on efficiency and functionality of the different physiological systems. The left – right asymmetry of visceral organs is established early on during embryonic development within a transient and specialized structure, commonly referred to as the left – right organizer (LRO). The LROs appear in many shapes and sizes, depending on the species, but a common feature in some vertebrates is the requirement of motile cilia. The movement of these tiny hair-like protrusions generate a directional fluid flow, that scales with the cube of cilia length, in order to become capable of triggering a differentiated response on the left side of the LRO. Such flow-dependent response involves Pkd2 channel activation and calcium signaling that subsequently drive the left sided expression of the Nodal signaling cascade. Nodal is a secreted protein that translates the asymmetries established at the LRO to the rest of the embryo, through the lateral plate mesoderm. As embryonic development evolves, at specific time points and locations along the anterior – posterior axis, Nodal induces the expression of genes involved in the formation of the heart, brain, gut and its derivatives, modulating the lateralization of these organs. With this work, we dedicated our efforts to understanding a few molecular and cellular steps missing in the establishment of the left – right axis within the LRO. In the Chapter 2, we explored how the fluid flow is sensed by the LRO cells. Between the two hypotheses in the field, one based on mechanosensing and other on chemosensing properties of the flow, we found that the number of extracellular vesicles is too low and variable to transport sufficient and efficiently a sidedness molecular signal towards the left sided LRO cells. Moreover, pharmacological impairment of distinct endocytic pathways did not impact on heart laterality arrangement. We also found out an upstream regulator of Notch signaling, syntenin-a, involved in the cell fate decision between motile and immotile cilia. We showed that syntenin-a loss-of-function severely affected the left – right axis development. By downregulating the levels of syntenin a, Notch signaling is activated increasing the expression of her12 and resulting in a higher number of immotile cilia, in concordance with our previous published data. We next described a potential molecular switch, downstream of Notch signaling, composed by the Rabconnectin complex. As this complex is known to promote V-ATPase assembly and consequently its activity, we inhibited the V-ATPase activity and we observed an increase in the number of motile cilia. Thus, suggesting that the link between Notch signaling and motile – immotile cilia ratio is through the modulation of pH. Lastly, in Chapter 3, we focused on the impact of ciliary dysfunction in the epithelial respiratory cells. We characterized the distribution pattern of several ciliary proteins in two siblings harboring a primary ciliary dyskinesia causing mutation on Zmynd10 gene. Recent studies showed that ZMYND10 is one of the cytoplasmatic factors responsible for stabilizing and driving axonemal dynein arm assembly. We showed here that outer and inner axonemal dyneins, that become mostly absent from the ciliary axoneme in Zmynd10 mutant respiratory ciliated cells, can sometimes enter the proximal part of the cilium. These results suggest that to a low extent the dynein arms can still assemble and be transported into the cilium in the absence of ZMYND10, thus opening an opportunity for small-molecule therapies that promote protein stability in primary ciliary dyskinesia disease management.Resumo Os animais bilaterais, como os humanos, são caracterizados por uma simetria externa ao longo do eixo esquerda – direita que cobre um arranjo interno pronunciadamente assimétrico dos órgãos torácicos e abdominais. Enquanto a simetria externa tem sido associada a padrões de saúde e beleza, a assimetria interna pode depender maioritariamente da eficiência e funcionalidade da montagem dos diferentes sistemas fisiológicos. Esta assimetria esquerda – direita dos órgãos viscerais é estabelecida durante o desenvolvimento embrionário dentro de uma estrutura transiente e especializada, normalmente conhecida por organizador esquerda – direita. Os organizadores esquerda – direita aparecem em várias formas e tamanhos, dependendo da espécie, mas uma característica comum em alguns dos vertebrados é a existência de cílios. Os cílios são organelos compostos por microtúbulos que são projetados da superfície da célula. E estes podem ser móveis ou imóveis dependendo da presença ou ausência de proteínas motoras, as dineínas do axonema, que geram energia suficiente para mover o cílio. No caso do organizador esquerda – direita, os dois tipos de cílios estão presentes e desempenham funções distintas: os cílios móveis promovem um fluxo direcional do fluido existente no lúmen dos organizadores, cuja velocidade é proporcional ao cubo do comprimento ciliar, e os cílios imóveis são potencialmente responsáveis por detetar esse mesmo fluxo. Por conseguinte, a deteção do fluxo desencadeia uma resposta assimétrica nas células do lado esquerdo do organizador esquerda – direita, que é dependente do canal de cálcio Pkd2 localizado nos cílios. Assim, os iões de cálcio entram pelo cílio e ativam a libertação de mais iões dos organelos internos, o que resulta numa onda de cálcio propagada pela célula que, por sua vez, é necessária para iniciar uma cascada molecular de sinalização composta por Nodal e os seus inibidores. Nodal é um factor secretado da família TGF-β inicialmente expresso em redor do organizador esquerda-direita de forma simétrica. Um dos seus antagonistas expresso no organizador, Dand5, impede a propagação precoce e simétrica de Nodal para a placa lateral da mesoderme. Contudo, a onda de cálcio que se forma nas células do organizador promove a degradação de dand5, tornando-se assim o primeiro gene assimetricamente expresso e libertando Nodal da sua repressão especificamente no lado esquerdo do organizador. Consequentemente, Nodal é capaz de ativar a sua própria expressão na placa lateral da mesoderme do lado esquerdo e a expressão de um segundo inibidor, lefty1, na linha mediana, de forma a impedir que Nodal ative a sua expressão no lado direito. À medida que o desenvolvimento embrionário evolui, Nodal propaga-se pela mesoderme ao longo do eixo anterior - posterior, que em estadios e regiões específicas, leva à expressão de genes envolvidos na formação do coração, cérebro, fígado, pâncreas, entre outros, modulando a lateralização destes órgãos. Este campo da biologia do desenvolvimento tem evoluído bastante ao longo dos últimos anos, contudo algumas questões continuam em aberto. A forma como o fluxo é detetado pelas células do organizador esquerda – direita é uma delas. Historicamente, o campo está dividido em torno de duas hipóteses principais – o modelo quimiossensor e o modelo mecanossensor. Por um lado, o modelo quimiossensor propõe que o fluxo serve para transportar vesículas e moléculas sinalizadoras para o lado esquerdo, onde serão internalizadas pelas células do organizador. Por outro lado, o modelo mecanossensor baseia-se na força hidrodinâmica que o fluxo exerce sobre os cílios imóveis. Com este projeto de doutoramento pretendemos fornecer novos dados do mecanismo biofísico impulsionado pelo fluxo usando o organizador esquerda – direita do peixe-zebra como modelo animal. Inicialmente, dedicámo-nos a inspecionar as características moleculares das células do organizador e o conteúdo de fluído para inferir sobre as possíveis contribuições do modelo quimiosensor na deteção do fluxo de fluidos pelo canal Pkd2. Para tal, gerámos uma linha transgénica para quantificar e permitir o rastreamento de vesículas extracelulares dentro do lúmen do organizador e usámos uma nova configuração de micromanipulação para modificar o conteúdo do fluido do organizador. Os nossos resultados mostram que o número de vesículas extracelulares detetadas é muito baixo e variável para transportar um sinal molecular de lateralidade de forma eficiente para as células do organizador do lado esquerdo. Adicionalmente, a inibição farmacológica de vias endocíticas distintas não teve impacto na lateralidade do coração. De seguida, analisámos a regulação do número de cílios móveis e imóveis no organizador esquerda – direita. No peixe zebra, todos os cílios têm a ultra estrutura necessária para se moverem, contudo, apenas alguns cílios se tornam móveis. O nosso grupo tinha anteriormente descoberto que a decisão entre móvel e imóvel é feita pela via de sinalização de Notch. Com este trabalho, nós identificámos novos moduladores a montante e efetores a jusante da sinalização de Notch envolvidos neste processo. Mostrámos que a perda de função da syntenin-a afeta severamente o desenvolvimento do eixo esquerda – direita, uma vez que ativa a sinalização de Notch e a expressão do seu gene alvo, her12, o que resulta num número maior de cílios imóveis e por conseguinte num fluxo do fluído menor. Também descrevemos um potencial botão molecular, a jusante da sinalização de Notch, composto pelo complexo Rabconnectin. Uma vez que este complexo é conhecido por promover a montagem da V-ATPase e consequentemente sua atividade, inibimos a atividade da V ATPase e observámos um aumento do número de cílios móveis. Assim, sugerimos que a ligação entre a sinalização de Notch e a proporção de cílios móveis – imóveis se dá através da modulação do pH. Por fim, no Capítulo 3, focámo-nos no impacto da disfunção ciliar nas células epiteliais respiratórias. Caracterizámos o padrão de distribuição de várias proteínas ciliares em dois irmãos portadores de discinésia ciliar primária causada por uma mutação no gene Zmynd10. Estudos recentes mostram que ZMYND10 é um dos fatores citoplasmáticos responsáveis por estabilizar e conduzir a montagem do braço de dineína que constituí o axonema do cílio móvel. Mostrámos aqui que as dineínas externas e internas do axonema, que se tornam principalmente ausentes do cílio em células respiratórias mutadas no gene Zmynd10, podem, no entanto, entrar na parte proximal do cílio. Estes resultados sugerem que uma pequena porção dos braços de dineína conseguem ser montados e transportados para o cílio na ausência de ZMYND10, abrindo assim uma oportunidade para terapias com pequenas moléculas que promovam a estabilidade de proteínas na gestão do tratamento da doença de discinésia ciliar primária

The role of hydrodynamic forces in synchronisation and alignment of mammalian motile cilia

Fluid flow generated by a ciliated epithelium is a fascinating evidence of collective behaviour in nature. In many organs and eukaryotic organisms, thousands of microscale whip-like structures called `motile cilia' beat aligned at the same frequency and in a coordinated fashion. This dynamics, known as `metachronal wave', has fundamental physiological roles in microorganisms and many organs of vertebrates. In the airways, the coordinated beatings of motile cilia generate a fluid flow that pushes mucus to the pharynx, and so protects the lungs from inhaled contaminants. The failure of this collective dynamics can precipitate or exacerbate severe infections and chronic inflammatory conditions such as cystic fibrosis (CF), primary ciliary dyskinesia (PCD) or asthma. In the brain, the multiciliated ependymal cells cover all the ventricles. Their cilia beat in a coordinated fashion to ensure the cerebrospinal fluid circulation necessary for brain homoeostasis, toxin washout and orientation of the migration of newborn neurons. Despite the fundamental role in nature, the mechanism underpinning such collective behaviour is still unknown. A recent hypothesis, supported by simulations, experiments with microorganisms and with cilia models, proposed that hydrodynamic interactions between cilia could provide a physical mechanism for their coordination. In contrast, others have proposed a role of the cytoskeletal elastic coupling between cilia. While previous works mainly focused on algae and protists, investigating the conditions that are required for the emergence of the metachronal wave in mammalian tissues can provide important progress in the diagnosis and treatment of human medical diseases. Specifically, I tackled this broad topic by studying the hydrodynamic forces necessary for the synchronisation and alignment of motile cilia from brain and airways. This question was addressed experimentally by measuring cilia motility during treatment with oscillatory and constant external fluid flows. We found that synchronisation and alignment of mammalian cilia in the brain is achieved with flows of similar magnitude of the ones generated by cilia themselves. Our results suggest that hydrodynamic forces between cilia are sufficient for the emergence of their collective behaviour. The first chapter provides basic knowledge on motile cilia structure and functions in microorganisms and humans. Additionally, I introduce the reader to the open questions related to the coordination of a pair and a carpet of cilia, with specific attention on previous works on mammals. This first chapter is followed by a description of a novel microfluidic device that I developed to grow $\textit{in vitro}$ airway and brain cells and apply controlled viscous forces. In Chapter 3, I describe how we have investigated cilia synchronisation of mammalian cilia. Applying external oscillatory flow on brain cells, we studied the susceptibility of cilia motility to hydrodynamic forces similar to the ones generated by cilia themselves. We found that cells with few cilia (up to five) can be entrained at flows comparable to the cilia-driven flows reported in vivo. We suggest that hydrodynamic forces between mammalian cilia are sufficiently strong to be the mechanism underpinning frequency synchronisation. In the second part of my thesis, I looked into the hydrodynamic shear forces needed to align permanently the cilia direction of beating. We tackled this problem by using $\textit{in vitro}$ cultures of mouse brain and human airway cells grown in custom flow channels. We found that cilia from mouse brain do not lock their beating direction after \emph{ciliogenesis}, but can respond and align to physiological shear stress found \emph{in vivo} at any time, in contrast with was previously believed. Moreover, we suggest that cilia alignment depends on the density of cilia, in agreement with a hydrodynamic screening effect of the external flow by the nearby cilia that we aim to investigate in the future. These results are described in Chapter 4. Successively in Chapter 5, I report our approach to study whether physiological shear stress can induce cilia alignment in airway cell cultures. The current hypothesis is that these cilia may also be able to align with external hydrodynamic forces - however, experimental evidence is still needed. There is a lack of experiments on this topic mainly because airway cells are cultured in an air-liquid interface, and so shear stress has to be applied with airflows. We developed novel setups for applying long term shear stress with air and fluid flow on this system, leaving further experiments for the future.EU Horizon 2020 research and innovation program under Marie Sklodowska-Curie 641639 ITN BioPol and ERC CoG HydroSyn