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 $in\; vitro$ 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 $in\; vitro$ 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 $\backslash emphciliogenesis$, but can respond and align to physiological shear stress found $\backslash emphinvivo$ 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.