Recent studies have demonstrated that phages can be co-transported with motile non-host bacteria, thereby enabling their invasion of biofilms and control of biofilm composition. Here, we developed a novel approach to isolate non-host bacteria able to co-transport phages from soil. It is based on the capability of phage-carrying non-host bacteria to move along mycelia out of soil and form colonies in plaques of their co-transported phages. The approach was tested using two model phages of differing surface hydrophobicity, i.e., hydrophobic Escherichia virus T4 (T4) and hydrophilic Pseudoalteromonas phage HS2 (HS2). The phages were mixed into soil and allowed to be transported by soil bacteria along the mycelia of Pythium ultimum. Five phage-carrying bacterial species were isolated (Viridibacillus sp., Enterobacter sp., Serratia sp., Bacillus sp., Janthinobacterium sp.). These bacteria exhibited phage adsorption efficiencies of ≈90–95% for hydrophobic T4 and 30–95% for hydrophilic HS2. The phage adsorption efficiency of Viridibacillus sp. was ≈95% for both phages and twofold higher than T4-or HS2-adsorption to their respective hosts, qualifying Viridibacillus sp. as a potential super carrier for phages. Our approach offers an effective and target-specific way to identify and isolate phage-carrying bacteria in natural and man-made environments.

Cataract surgery is currently the most common surgical procedure done in the world. However, within 5 years, approximately half of these patients will develop posterior capsule opacification (PCO). In cataract surgery, the biological lens is replaced with an intraocular lens (IOL). PCO is caused by migration and transformation of residual lens epithelial cells (LEC) that remain in the capsule following the surgery. LECs which have migrated to the posterior capsule within the first month of surgery are thought to be the major contributors to PCO since after this time, the capsule completely seals. A mathematical model has been developed in order to better understand the process of LEC migration during PCO. The model addresses the impact of substrate and substrate modification as well as the presence and absence of the growth factors transforming growth factor beta (TGF(beta)) and fibroblast growth factor (FGF2). It was developed from a first order rate of decay model taken from process control. If the cell speed is divided by the distance travelled by the cell up to the point of posterior capsule breach, the time for the LECs to breach the capsule posterior can be calculated. The model was tested with literature data and was able to predict the effects of cell speed on the presence of various extracellular matrix components and growth factors. It was determined that potentially modification with fibronectin may be useful for the prevention of PCO Preliminary experimental validation of the model was performed by modifying silicone substrates with various extracellular matrix derived peptides. Results demonstrate that peptide modified surfaces may be more resistant to EMT by increasing cell adhesion and decreasing cell migration. Therefore, this LEC migrational model will be a useful tool in the development of superior IOLs and materials. / Thesis / Master of Applied Science (MASc)

Chez les vertébrés, le battement coordonné des cils motiles présents par centaines à la surface apicale des cellules multiciliées (MCC) est requis pour propulser directionnellement les fluides biologiques à l’intérieur de certains organes (voies respiratoires, ventricules cérébraux, trompes utérines ou certaines structures embryonnaires). De nombreuses pathologies humaines sont associées à des défauts ciliaires ou à une perte des MCC (dyskinésies ciliaires, mucoviscidose, asthme,...). Dans ce contexte, mon travail de thèse a consisté à élucider les mécanismes complexes contrôlant la différenciation des MCC et donc la formation des cils motiles (multiciliogenèse). Par des approches de génomiques fonctionnelles à partir de deux modèles d’épithéliums multiciliés évolutivement éloignés (épithélium respiratoire humain et épiderme d’embryon de Xénope) nous avons identifié la famille des microARN (petits ARN non-codants régulateurs de l’expression génique) miR-449 comme majoritairement exprimée dans les MCC. Nous avons montré que miR-449 contrôle la multiciliogenèse i) en bloquant le cycle cellulaire, ii) en réprimant directement la voie de signalisation Notch et iii) en inhibant l’expression de la petite GTPase R-Ras. Enfin, nos travaux montrent que l’ensemble de ces mécanismes est conservé chez les vertébrés. En conclusion, miR-449 est un nouveau régulateur clé de la multiciliogenèse conservé au cours de l’évolution. Nos résultats pourraient ouvrir la voie à de nouvelles stratégies thérapeutiques utilisant des petits ARN régulateurs dans le traitement de certaines pathologies associées à des défauts ciliaires. / In vertebrates, the coordinated beating of hundreds of motile cilia present at the apical surface of multiciliated cells (MCC) is required for propel directionally flow of biological fluids inside some organs (airways, cerebral ventricles, fallopian tubes or some embryonic structures). Many human diseases are associated with ciliary defects or loss of MCC (ciliary dyskinesia, cystic fibrosis, asthma ...). In this context, my thesis has sought to elucidate the complex mechanisms that control the differentiation of MCC and thus the formation of motile cilia (multiciliogenesis). By functional genomic approaches from two evolutionarily distant models of multiciliated epithelia (human respiratory epithelium and epidermis of Xenopus embryo) we identified the miR-449 family of microRNAs (small non-coding RNAs regulating gene expression) as mainly expressed in MCC. Then, we showed that miR-449 controlled multiciliogenesis by i) blocking the cell cycle ii) directly suppressing the Notch pathway and iii) by inhibiting the expression of the small GTPase R-Ras. Finally, we have demonstrated that all these mechanisms were conserved in vertebrates. In conclusion, miR-449 is a new key and conserved regulator of multiciliogenesis. Our findings could pave the way for new therapeutic strategies using small regulatory RNAs in the treatment of several diseases associated with ciliary defects.

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.

Chez les vertébrés, le battement coordonné des cils motiles présents par centaines à la surface apicale des cellules multiciliées (MCC) est requis pour propulser directionnellement les fluides biologiques à l'intérieur de certains organes (voies respiratoires, ventricules cérébraux, trompes utérines ou certaines structures embryonnaires). De nombreuses pathologies humaines sont associées à des défauts ciliaires ou à une perte des MCC (dyskinésies ciliaires, mucoviscidose, asthme,...). Dans ce contexte, mon travail de thèse a consisté à élucider les mécanismes complexes contrôlant la différenciation des MCC et donc la formation des cils motiles (multiciliogenèse). Par des approches de génomiques fonctionnelles à partir de deux modèles d'épithéliums multiciliés évolutivement éloignés (épithélium respiratoire humain et épiderme d'embryon de Xénope) nous avons identifié la famille des microARN (petits ARN non-codants régulateurs de l'expression génique) miR-449 comme majoritairement exprimée dans les MCC. Nous avons montré que miR-449 contrôle la multiciliogenèse i) en bloquant le cycle cellulaire, ii) en réprimant directement la voie de signalisation Notch et iii) en inhibant l'expression de la petite GTPase R-Ras. Enfin, nos travaux montrent que l'ensemble de ces mécanismes est conservé chez les vertébrés. En conclusion, miR-449 est un nouveau régulateur clé de la multiciliogenèse conservé au cours de l'évolution. Nos résultats pourraient ouvrir la voie à de nouvelles stratégies thérapeutiques utilisant des petits ARN régulateurs dans le traitement de certaines pathologies associées à des défauts ciliaires.

Macroinvertebrates constitute the backbone of megafaunal communities in benthic ecosystems around the globe. Many macroinvertebrates have vital roles in benthic ecosystems, ranging from enhancing habitat complexity to providing staple food sources for other organisms. Regardless of how familiar macroinvertebrates are to the general public, very few studies have attempted to describe benthic macroinvertebrate assemblages across large spatial scale in the continental shelf waters of the Pacific Northwest. This study describes different subtidal macroinvertebrate assemblages off Washington and Oregon based on species-substrata associations and the key species that distinguish one assemblage from another. Two data sets were used for this study: underwater footage collected by the submersible Delta during 1993-1995 geological surveys, and footage collected by the remotely operated vehicle (ROV) Hammerhead during macroinvertebrate surveys in late summer 2011. Footages from these surveys were used to document species-substrata associations and distinguish different assemblages based on species composition similarities and dissimilarities. In addition, I determined if a specific group of invertebrates, Asteroids (Echinodermata), were useful in explaining different assemblage patterns, after all other environmental parameters were taken into account. Findings of this study can be used not only to shed light on the structure of macroinvertebrate communities in the Pacific Northwest, but also as baseline data for future research on the direct and indirect effects of potential offshore installations on macroinvertebrate communities across the continental shelf waters. / Graduation date: 2012

Motile cilia play critical roles in the human body, including expelling mucus from the lungs and facilitating the transport of oocytes and sperm through the fallopian tubes. Understanding the complex structure and motility of cilia, as well as the diseases associated with them, is of big importance. This study investigates the proteins expressed in ciliated cells from both respiratory and reproductive tissues using multiplex immunofluorescence. We determined the subcellular localization of 134 proteins in the fallopian tube, endometrium, cervix, nasopharynx, and bronchus, focusing on five subcellular regions: the cilia tip, transition zone, basal body, cytoplasm, and nucleus. This analysis was conducted using an automated image analysis method developed specifically for this project. Our findings revealed a high correlation in protein expression across all tissues, although several proteins exhibited distinct expression patterns between different tissues. Notably, the fallopian tube showed a higher correlation with the nasopharynx and bronchus than with the endometrium and cervix. Within these proteins, six gene clusters were identified, with the two largest clusters being strongly associated with ciliary structure. This study enhances our understanding of motile ciliary structures and ciliated cells, identifying key proteins for further research into cilia motion, function, and related diseases.