Indiana University-Purdue University Indianapolis (IUPUI) / Primary cilia are solitary cellular appendages that function as signaling centers for cells in adult energy homeostasis. Here in chapter 1, I introduce cilia and how dysfunction of these conserved organelles results in ciliopathies, such as Bardet-Biedl Syndrome (BBS), which present with childhood obesity. Furthermore, conditional loss of primary cilia from neurons in the hypothalamus leads to hyperphagia and obesity in mouse models of ciliopathies. Classically, cilia coordinate signaling often through specific G-protein coupled receptors (GPCRs) as is the case in both vision and olfaction. In addition, neurons throughout the brain including hypothalamic neurons possess primary cilia whose dysfunction contributes to ciliopathy-associated obesity. How neuronal cilia regulate the signaling of GPCRs remains unclear and many fundamental cell biology questions remain about cilia mediated signaling. For example, how cilia coordinate signaling to influence neuronal activity is unknown. To begin to address some of these cell biology questions around neuronal cilia, chapter 2, describes the development and use of a system for primary neuronal cultures from the hypothalamus. Using this system, we found that activation of the cilia regulated hedgehog pathway, which is critical in development, influenced the ability of neurons to respond to GPCR ligands. This result highlights the role of the developmentally critical hedgehog pathway on terminally differentiated hypothalamic neurons. One challenge facing the cilia field is our ability to assess cilia in large numbers without potential bias. This is especially true in tissues like the brain, where cilia appear to have region-specific characteristics. Work included in Chapter 3 describes the use of a computer-assisted artificial intelligence (Ai) approach to analyze cilia composition and morphology in a less biased and high throughput manner. Cilia length and intensities are important parameters for evaluation of cilia signaling. Evidence suggests that activation of some ciliary GPCRs results in shortening of cilia whereas deviations from normal cilia length in mutant phenotypes affects normal physiological processes such as decreased mucociliary clearance. Therefore, to analyze a large number of cilia, we describe the use of the Ai module from in vitro and in vivo samples in a reproducible manner that minimizes user bias. Using this approach, we identified that Mchr1 expression is significantly stronger in the cilia of paraventricular nucleus than that in the arcuate nucleus of adult mice. Work in Chapter 4 continues to explore the integration between hedgehog pathway and ciliary GPCR signaling in the central nervous system, and its relevance with energy homeostasis. We evaluated the hedgehog ligand in the plasma of mice in acute and long-term metabolic changes and identified that the activity of the ligand changed under altered metabolic conditions. We also developed a genetic mouse model where hedgehog signaling was constitutively active in neuronal cilia. These mice become hyperphagic and obese. These results further emphasize the potential role of the hedgehog signaling pathway in regulation of feeding behavior in adult vertebrates. Overall, results from this work will provide a better understanding of the defects not only underlying ciliopathy-associated obesity but may also reveal more common mechanisms of centrally mediated obesity. In addition, the tools I have developed will help in understanding how neuronal cilia are used for intercellular communications and ultimately how they regulate behaviors like feeding.

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.

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.

Tubulin post-translational modifications (tPTMs) are currently studied as vital, yet obscure, cytoskeletal regulators. Their regulatory function relies on the spatiotemporal control over the activity of multiple tubulin modifying enzymes that functionalize microtubules, enabling their differentiation. The cilium, one of the organelles with the richest tPTMs diversity, has been studied with determination for the last decades, allowing the interrogation of the molecular processes that give rise to its function. The inner structure of this thin organelle, the axoneme, comprises a microtubule scaffold periodically decorated with macromolecular complexes whose characterization has been achieved with pseudoatomic detail. The molecular distribution and mechanism of action of tPTMs in cilia remain elusive. Using a combination of immunolabelling and cryoelectron tomography we interrogated the molecular function two tPTMs in the axonemal context. We showed that tubulin polyglycylation spanned most of the microtubular surface and was required for axonemal dynein activity regulation and male fertility. Additionally, there was an enrichment of polyglutamylation on a single microtubule protofilament, forming a pattern complementary to that of polyglycylation, that was required for proper coupling of microtubule sliding and bending forces during ciliary beating.

Neurons commonly have a primary cilium, which is a non-motile organelle extending from the centrosome into the extracellular space. In most brain regions, neuronal cilia are enriched in either somatostatin receptor type 3 (SstR3) or melanin concentrating hormone receptor type 1 (MCHR1), or both. The present immunohistochemical study provides novel evidence that primary cilia regulate neuronal excitability via G-protein coupled receptors (GPCRs), and that their identity is governed by brain region and by competition, both in adulthood and in postnatal development. The hippocampus, which is particularly vulnerable to seizures, has opposing gradients of SstR3(+) and MCHR1(+) ciliary GPCRs. We hypothesized that there is a competition between these two ciliary GPCRs, which might take place on any level from gene expression to presence in the cilium. We examined whether receptor colocalization occurs transiently in development before ciliary GPCR dominance is established in neurons in the CNS. In postnatal CA1 and CA3, the first GPCR to appear in cilia was the one that will dominate in adults: MCHR1 in CA1 and SstR3 in CA3. Some days later, the second GPCR was expressed along with the first; dual-receptor cilia were the exclusive type until single-receptor cilia emerged again around P14. Single-receptor cilia then increased in numbers through adulthood. By identifying ciliary receptors that modulate seizure activity in mice, the present study lays a foundation for therapeutic approaches to reduce neuronal excitotoxicity underlying cell death in epilepsy, CNS injury, and neurodegenerative diseases.

La scoliose est une déformation de la colonne vertébrale dans les trois plans de l’espace (frontal, sagittal et transverse), et la forme la plus fréquente est la scoliose idiopathique (SI) dont la cause est encore très mal comprise. La scoliose idiopathique de l’adolescent (SIA) est une forme de SI qui se développe au cours de la puberté, plus souvent chez les filles, et présente une importante hétérogénéité phénotypique. Les très nombreuses recherches afin de comprendre les mécanismes qui entrent en jeu dans le développement de cette maladie et afin de déterminer l’étiologie ont permis de mettre en évidence un très grand nombre de théories regroupant des facteurs endocriniens et métaboliques, des facteurs biomécaniques et musculosquelettiques, des facteurs neurologiques et vestibulaires ainsi que des facteurs génétiques et épigénétiques. L’identification du gène candidat POC5 dans la SI par notre laboratoire, un gène crucial dans la formation du cil primaire, a été un tournant majeur apportant la première pièce d’un puzzle plaçant le cil primaire au centre des hypothèses concernant la mise en place de la maladie. Le cil primaire est une organelle non-motile retrouvée à la surface de la totalité des cellules du corps humain à l’exception des cellules sanguines. Grâce à son axonème formé de tubuline capable de subir des modifications post-traductionnelles appelées « code tubuline » et sa membrane ciliaire ultra spécialisée, le cil primaire joue un rôle mécanosenseur important tout au long de la vie, dès le développement embryonnaire. Il prend part à de nombreux mécanismes dont la formation et l’homéostasie osseuse et a déjà été mis en cause dans les maladies osseuses. En effet, lorsque le cil primaire est défectueux, il est responsable de syndromes complexes regroupés sous le nom de ciliopathie dont un des symptômes est la scoliose. L’hypothèse générale de la présente thèse est que des variants rares dans des gènes ciliaires, particulièrement le gène POC5 et le gène TTLL11, agissent sur les mécanismes cellulaires responsables de la mise en place de la SIA faisant d’elle une maladie appartenant à la grande famille des ciliopathies. L’objectif de cette thèse a été de documenter l’implication et la pathogénicité des gènes POC5 et TTLL11 dans la SIA. Nous avons d’abord déterminé la prévalence des variants du gène POC5 dans une cohorte de patients d’origine franco-canadienne ou britannique atteints de SIA (Manuscrit 1). Ensuite, nous avons créé trois modèles de souris Knock-in pour l’équivalent des variants du gène POC5 humain afin d’analyser la pathogénicité et le phénotype éventuel de ciliopathie (Manuscrit 2). Finalement, la pathogénicité du gène TTLL11 muté a été analysée dans des cellules issues de patients atteints de SIA et dans un modèle animal, le poisson zèbre (Manuscrit 3). Les travaux réalisés au cours de cette thèse ont montré que les deux gènes ciliaire POC5 et TTLL11 sont des gènes candidats probablement impliqués dans les mécanismes cellulaires fondamentaux induisant la mise en place de la SIA et ont permis d’identifier un mécanisme, la polyglutamylation du cil primaire, comme étant relié à la SIA. / Scoliosis is a 3D spinal curvature, in the frontal, sagittal and transverse plans, and the most common form is the idiopathic scoliosis (IS) with a cause that remains very poorly understood. Adolescent Idiopathic Scoliosis (AIS) is a subtype of IS that develops during the puberty, most commonly in girls, and has a significant phenotypic heterogeneity. To date, a large amount of research on scoliosis etiology highlighted various hypotheses based on endocrine and metabolic factors, biomechanical and musculoskeletal factors, neurological and vestibular factors, as well as genetic and epigenetic factors. The identification of the IS candidate gene POC5 by our laboratory, a crucial gene in the formation of the primary cilium, was a major turning point bringing the first piece of a puzzle where the primary cilium is a central hypothesis concerning the onset of the disease. The primary cilium is a non-motile organelle found at the cell surface of all the human body cells, except for blood cells. The primary cilium plays an important mechano-sensor role throughout life, from embryonic development through its axoneme composed by tubulin that hosted post-translational modifications called “tubulin code”, and its ultra-specialized ciliary membrane. It is involved in many mechanisms including bone formation and homeostasis and has already been implicated in bone diseases. Indeed, the defective primary cilium is responsible for human ciliopathy syndromes that are associated with scoliosis. The main hypothesis of this thesis is that rare variants in ciliary genes, especially the POC5 gene and the TTLL11 gene, participate on cellular mechanisms responsible for the onset of AIS supporting the idea that scoliosis is a form of ciliopathy. The specific objective of this thesis was to document the involvement and pathogenicity of the POC5 and TTLL11 genes in AIS. We first determined the prevalence of POC5 gene variants in a cohort of French-Canadian and British AIS patients (Manuscript 1). Then, we created three knock-in mouse models carrying the equivalent of the human POC5 gene variants to analyze the pathogenicity and the possible phenotype of ciliopathy (Manuscript 2). Finally, the pathogenicity of the mutated TTLL11 gene was analyzed in cells extracted from AIS patients and in an animal model, the zebrafish (Manuscript 3). The work presented in this thesis showed that the two ciliary genes POC5 and TTLL11 are candidate genes probably involved in the fundamental cellular mechanisms inducing the onset of AIS and allowed us to identify the primary cilium polyglutamylation, as a mechanism being related to AIS.

The primary cilium is an immotile, microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Critically, the transport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, this work employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. Using SPEED microscopy and single molecule tracking, we mapped the movement of membrane and soluble proteins at the base of the primary cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary cilium - passive diffusional and vesicle transport routes - that are adopted by proteins for cytoplasmic-cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins. While the 3D transport pathways in this work are always replicated multiple times with a high degree of consistency, several experimental parameters directly affect the 3D transport routes’ error, such as single molecule localization precision and the number of single molecule localizations. In fact, if these experimental parameters do not meet a minimum threshold, the resultant 3D transport pathways may not have significant resolution to determine any biological details. To estimate the 3D transport routes’ error, this work will explain in detail the component of SPEED microscopy that estimates 3D sub-diffraction-limited structural or dynamic information in rotationally symmetric bio-structures, such as the primary cilium. This component is a post-localization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability distributions based on prior known structural knowledge. This analysis is ideal in cases where the ultrastructure of a cellular structure is known but the sub-structural localization of a particular protein is not. This work will demonstrate how the 2D-to-3D component of SPEED microscopy can be successfully applied to achieve 3D structural and functional sub-diffraction-limited information for 25-300 nm subcellular organelles that meet the rotational symmetry requirement, such as the primary cilium and microtubules. Furthermore, this work will provide comprehensive analyses of this method by using computational simulations which investigate the role of various experimental parameters on the 3D transport pathway error. Lastly, this work will demonstrate that this method can distinguish different types of 3D transport pathway distributions in addition to their locations. / Biology

The objective of this study was to investigate primary cilia and their mechanotransduction role in lung adenocarcinoma tumor progression. The main focus investigated the effect of primary cilia on cell cycle progression, survival, adhesion and migration analysis of these cells and the role of sonic hedgehog signaling pathway in mechanotransduction. Human Non-Small Cell Lung Cancer (NSCLC) adenocarcinoma biopsies contain more primary cilia than non-tumor lung sections. To observe the effects of primary cilia presence in lung cancer cells in-vitro, formation of primary cilia is inhibited using small interfering RNA. A549 cells with intact primary cilia observe less cell cycle progression than cells deficient in primary cilia under static and cyclic stretch conditions. Primary cilia cause higher cell survival and adhesion. Increase in cell adhesion also increases the migration and wound closure rates in control samples compared to samples treated with inhibition of IFT88, thereby increasing the metastasis of these cells. Several downstream regulatory genes in sonic hedgehog signaling pathway observe significantly decreased gene expressions in primary cilia deficient cells, thus indicating inefficient mechanotransduction. Therefore, cancer cells need primary cilia to survive, adhere and migrate and continue tumor progression.

The objective of this study is to investigate the role of microtubule based organelle, the primary cilia in lung adenocarcinoma by i) Quantifying the presence of primary cilia in several Non Small Cell Lung Cancer (NSCLC) cell lines in response to mechanical stimuli, ii) Attempting to determine the role of primary cilia in cell migration, iii) Investigating the effects of Paclitaxel(Taxol) resistance in lung cancer cells, iv) Analyzing the response of lung cancer cells to Smoothened Inhibitors and v) Determining the effects of Transforming Growth Factor Beta-1(TGF-β1) induced Epithelial to Mesenchymal Transition(EMT) in lung cancer cells. To ascertain the effects of primary cilia in the hall marks of tumor progression, several experiments involved prohibition of primary cilia formation by silencing IFT88, the gene responsible using small interfering RNA. Three out of the five cell lines tested, showed increased expression of primary cilia under mechanical stretch. IFT88 inhibition of H460 cells decreased their migration rate to the injury site under stretch conditions. Smoothened (SMO) Inhibitors decreased proliferation and migration rates in human lung adenocarcinoma cell lines (A549luc) similar to the effects observed in IFT88 silenced cells. IFT88 silenced A549luc cells showed a partial reversal of TGF-beta1 induced up-regulation of a mesenchymal marker. These results indicate that primary cilia play a role in the progression and metastasis of lung cancer by aiding the adhesion, proliferation, migration and EMT of lung cancer cells.

A ventilação mecânica (VM) pode ser causa de lesão pulmonar sendo este fato reconhecido na literatura como Lesão Pulmonar Induzida pela Ventilação Mecânica (LPIV), onde alterações tanto fisiológicas quanto morfológicas são evidenciadas no pulmão. O objetivo desse trabalho foi avaliar a repercussão das forças envolvidas na ventilação mecânica através de diferentes mecanismos de LPIV sobre o sistema mucociliar pela análise funcional e histopatológica desse aparelho. Em um estudo controlado e randomizado vinte e sete coelhos machos da raça Nova Zelândia foram separados em quatro grupos. Nos primeiros trinta minutos foram submetidos à VM com volume corrente de 8 ml/kg peso, fluxo de 3 L/minuto, e pressão positiva expiratória final (PEEP) de 5 cm H2O e FIO2 de 0,4, sendo que o grupo Sham (n=6) foi ventilado por apenas 10 minutos. Os grupos Baixo Volume/BV (n=6; Vt 8, Ppico 17, Pmédia 9, PEEP 5, Fluxo 3), Alto Volume/AV (n=7; Vt 16, Ppico 27, Pmédia 12, PEEP 5, Fluxo 5) e Alta Pressão/AP (n=8; Vt 8, Ppico 30, Pmédia 20, PEEP 12, Fluxo 9) foram ventilados por mais 3 horas. A mecânica do sistema respiratório foi registrada pelo sistema Labview®. Foram acompanhados os valores de gasometria e sinais vitais. Amostras de tecido pulmonar foram coradas com H&E para análise de infiltrado inflamatório. Analisou-se a frequência de batimento ciliar (FBC), transporte mucociliar na traqueia (TMCT), transportabilidade em palato de rã (MCT) e ângulo de contato (AC). Amostras de pulmão e traquéia foram coradas pela técnica PAS/AB para avaliação do muco. Observamos diminuição da complacência do sistema respiratório (p<0,05) no grupo AP em comparação com os demais grupos ventilados. Os grupos BV, AV e AP apresentaram um aumento de células polimorfonucleares por área de parênquima pulmonar (p<0,001) em comparação com o grupo Sham. Não foram observadas diferenças quanto ao TMCT e AC nos grupos ventilados. A transportabilidade em palato diminuiu no grupo BV (p=0,007) ao final do protocolo de ventilação (1,42 com variações de 2,11-0,99 para 0,95 com variações de 1,15-0,92). A FBC diminuiu (p=0,047) no grupo AP quando comparamos os valores iniciais (13,51 variação de 14,49-11,62) e finais ao protocolo de VM (11,69 variação de 14,18-10,12). Respostas fisiológicas e microscopia eletrônica (ME) da traquéia corroboram a disfunção da FBC no grupo AP. Na traquéia os grupos ventilados apresentaram uma diminuição da quantidade de muco total (BV 2.018, AV 3.219 e AP 2.883) e de muco ácido (BV 672, AV 240 e AP 480) por m2 de tecido pulmonar por campo estudado (p<0.05) quando comparados com o grupo Sham (4.660 m2 de muco total e 1.873 m2 de muco ácido). Em bronquíolos distais a quantidade de muco total diminuiu nos grupos BV e AP (p<0.05) quando comparados aos grupos Sham e AV. O mesmo fenômeno foi observado com relação ao muco neutro. Concluímos que as forças geradas pela ventilação mecânica têm impacto direto sobre o aparelho mucociliar, alterando suas propriedades funcionais e sua morfologia. Aparentemente a VM acarreta desequilíbrio na produção de muco, sendo essa alteração mais visível quando utilizado um alto volume corrente e uma maior pressão média pulmonar (associada a um fluxo de ar aumentado). O aumento da pressão média com alto fluxo dos gases ventilados também ocasiona maior sofrimento celular do aparelho mucociliar, disfunção da frequência de batimento ciliar e provável perda ciliar. Tais alterações se devem provavelmente ao efeito das forças físicas geradas pela VM associadas a prováveis oscilações na resposta inflamatória e no fluxo sanguíneo local. / Mechanical ventilation (MV) can result in a medical complication named Ventilator-Induced Lung Injury (VILI), where physiologic and morphologic alterations in the lungs have been reported. In this study, we have hypothesized that MV-induced injury can have a major impact on the mucociliary system. In a randomized controlled trial twenty-seven male New Zealand rabbits were separated into four groups. During the first thirty minutes all rabbits were subjected to MV with tidal volume of 8 ml/kg of body weight, 3 L/minute of flow, positive end expiratory pressure (PEEP) of 5 cm H2O and FIO2 of 0.4. After that the study animals were divided into 4 groups: Sham (n=6) was ventilated for ten minutes, Lower Volume/LV (n=6; Vt 8, P peak 17, P mean 9, PEEP 5, Flow 3), High Volume/HV (n=7; Vt 16, P peak 27, P mean 12, PEEP 5, Flow 5) and High Pressure/HP (n=8; Vt 8, P peak 30, P mean 20, PEEP 12, Flow 9) and ventilated for 3 hours more. Respiratory system mechanics was recorded using Labview®. Blood gasometry and vital signals were monitored. Lung tissue sections were stained by H&E to analyze inflammation. We also studied the ciliary beating frequency (CBF), tracheal mucociliary transport (TMCT) in situ and in vitro, mucus contact angle (CA) and respiratory mucus viscosity. To quantify mucosubstances, tracheal samples were stained with PAS/AB. As a result we have that the respiratory system compliance decreased (p<0.05) in the HP group compared to other ventilated groups. All ventilated groups showed an increase in polymorphonuclear cells quantity per area of lung parenchyma (p<0.001) compared to the Sham group. There were no differences in TMCT and CA among ventilated groups, when initial and final measurements were compared. MCT significantly decreased in the LV group (p=0.007) after the protocol (1.42 range of 2.11-0.99 to 0.95 range of 1.15-0.92). The CBF significantly decreased (p=0.047) in the HP group when the initial (13.51 range of 14.49-11.62) and final (11.69 range of 14.18-10.12) measurements were compared. Physiological data and tracheal electronic microscopy confirm the CBF dysfunction observed in the AP group. In the trachea all ventilated groups showed a significant decrease in total mucus (LV 2,018, HV 3,219 and HP 2,883) and acid mucus (LV 672, HV 240 and HP 480) per um2 of lung tissue (p<0.05) compared to the sham group (4,660 um2 of total mucus and 1,873 um2 of acid mucus). In distal bronchioles there was a significant decrease in total mucus in the LV and HP group (p<0.05) compared to Sham and HV groups. The same phenomenon was observed regarding neutral mucus. We concluded that mechanical forces involved in MV affect mucociliary function. Mechanical ventilation probably leads to the dysfunction on the mucus production. This phenomenon is better perceived when higher volume and higher mean pressure (associated with inspired gases high flow) are used. An increase of the mean pressure combined with inspired gases high flow also leads to mucociliary cells suffering, to ciliary beat frequency dysfunction and probably cilia loss. These alterations probably occur due to the effect of the physical forces generated by the mechanical ventilation in association with the oscillation of the inflammatory response and local blood flow.