INTRODUÇÃO: A discinesia ciliar primária é uma doença genética que se caracteriza pela alteração da ultraestrutura e função do cílio móvel, com consequentes alterações do transporte mucociliar, causando infecções das vias aéreas superiores, inferiores e infertilidade. O diagnóstico, realizado por avaliação da ultraestrutura ou pesquisa de mutação genética, é feito mediante critérios de seleção de pacientes e testes de screening. Esta pesquisa avalia a ultraestrutura e frequência de batimento ciliar, propõe um modelo de investigação de discinesia ciliar primária, e caracteriza os pacientes diagnosticados. MÉTODO: Foi realizado um estudo transversal controlado entre janeiro de 2007 e julho de 2009, no Ambulatório de Pneumologia Pediátrica do Instituto da Criança. Foram selecionadas 28 crianças e adolescentes (6 meses a 19 anos, de ambos os sexos), de uma população de 75 crianças com pneumopatias crônicas e de repetição sem diagnóstico definido, que apresentavam ao menos um dos seguintes achados: bronquiectasia de causa desconhecida, doença de vias aéreas superiores com sintomatologia crônica, infecções pulmonares de repetição, dextrocardia e/ou situs inversus acompanhados de sintomas em vias aéreas superiores e/ou inferiores, e asma de difícil controle com sintomas em vias aéreas superiores e/ou inferiores. A presença de pneumopatias crônicas com diagnóstico definido foi utilizada como critério de exclusão por meio dos seguintes exames: dois testes do suor (exclusão de fibrose cística), tomografia computadorizada do tórax (suspeita de bronquiolite obliterante), dosagem de alfa-1 antitripsina (investigação de déficit de alfa-1 antitripsina), e exames de investigação das imunodeficiências mais frequentes (hemograma, dosagens de imunoglobulinas, contagem de linfócitos T e B, sorologias para avaliação da produção ativa de anticorpos, PPD e HIV). Foi desenvolvido um sistema medição da frequência de batimento ciliar, baseado em análise espectral. Dez adultos voluntários saudáveis (maiores ou iguais a 17 anos, de ambos os sexos), sem doença infecciosa respiratória aguda no último mês e não fumantes, formaram o grupo controle para a frequência do batimento ciliar. Foi realizada a coleta ciliar por escovado nasal. A amostra foi dividida para a avaliação da ultraestrutura e verificação da frequência do batimento ciliar. Os pacientes diagnosticados foram avaliados com tomografia de tórax e seios da face (esta última nos maiores de cinco anos), provas de função pulmonar e avaliação otorrinolaringológica. RESULTADOS: Os 28 pacientes selecionados foram submetidos ao escovado nasal. Para 24 dos 28 pacientes foi possível produzir filmes passíveis de avaliação. O grupo com ultraestrutura alterada (diagnóstico de discinesia ciliar primária) mostrou diferença das médias da frequência de batimento ciliar em relação ao grupo com ultraestrutura normal (p<0,001) e em relação ao grupo controle (p<0,001). O grupo com ultraestrutura normal e o grupo controle, quando comparados entre si, também apresentaram diferenças (p<0,005). Foram diagnosticados 12 pacientes com discinesia ciliar primária: dois com ausência de braços externos de dineína, um com encurtamento dos braços externos de dineína, cinco com defeitos nas espículas radiadas e braços internos de dineína, três com defeitos de ausência do par central com transposição, e um com ultraestrutura normal (Kartagener). Os pacientes, sete homens e cinco mulheres, com predomíno da etnia branca (11 pacientes - 91,6%), eram na maior parte fruto de pais consanguíneos (8 pacientes - 66,6%). Sete dos 12 pacientes (58,3%) apresentaram situs inversus. Sete dos dez pacientes que realizaram as provas de função pulmonar (70%) apresentaram distúrbio ventilatório obstrutivo. Achados radiológicos: Dez pacientes (83,3%) apresentaram algum grau de colapso ou consolidação, 11 (91,6%) apresentaram bronquiectasias, e todos (100%) algum grau de espessamento brônquico. A avaliação otorrinolaringológica apontou alterações em seis pacientes (50%): pólipos em três pacientes (25%), otite secretora em dois (16,6%), desvio de septo em dois (16,6%), e esclerose do tímpano em um (8,3%). CONCLUSÃO: Foi padronizada a técnica do escovado nasal para a coleta de material ciliar. Foram estabelecidos critérios de uniformidade quanto à análise dos defeitos ultraestruturais ciliares, baseados em experiência internacional. Foi desenvolvido um novo método de medição da frequência de batimento ciliar baseado em metodologia de análise espectral e vídeos de alta velocidade. Os pacientes diagnosticados com discinesia ciliar primária foram caracterizados, sendo a maioria: da etnia branca, de pais consanguíneos, com predomínio dos defeitos de espículas radiadas e braços internos de dineína, situs inversus, e distúrbio ventilatório obstrutivo. Problemas otorrinolaringológicos foram encontrados em metade dos pacientes. / INTRODUCTION: Primary ciliary dyskinesia (PCD) is a genetic disorder of the ultrastructure and function of mobile cilia, with consequent impairment of mucociliary clearance, leading to upper and lower airways respiratory infection and infertility. The diagnosis, based on ultrastructure evaluation or genetic scan, is performed according to patient selection and screening tests. This research evaluates cilia ultrastructure and beat frequency, proposes a model for investigating primary ciliary dyskinesia, and characterizes the patients diagnosed.METHOD: A controlled and observational study was carried out at the Pediatric Pulmonology Ambulatory of the Instituto da Criança between January 2007 and July 2009. Twenty eight children and teenagers (ages between 6 months and 19 years) were selected, from a population of 75 patients with chronic and repetition pneumopathies without a defined diagnosis, which met at least one of the following inclusion criteria: bronchiectasis of unknown cause, upper respiratory disease with chronic symptoms, repetition pulmonary infections, dextrocardia and/or situs inversus with symptoms in upper and/or lower respiratory airways, and asthma of difficult control with symptoms in upper and/or lower respiratory airways. The presence of cronic pneumopathies with a defined diagnosis was used as exclusion criterion, based on the following exams: two sweat tests (cystic fibrosis exclusion), lung CT scan (bronchiolitis obliterans exclusion), seric levels of alpha-1 anti-trypsin (alpha-1 anti-trypsin deficit evaluation), and evaluation of more frequent immunodeficiency disorders (white blood cells, T and B lymphocytes levels, and sorology tests for humoral immunity, PPD and HIV). A cilia beat frequency measurement system was developed, based on spectral analysis. Ten healthy adult volunteers (ages greater than or equal to 17 years old, of both sexes), without an acute respiratory disease in the last month, and non-smoking, formed the control group for the cilia beat frequency measurements. Cilia samples were collected employing nasal brushing. The sample was divided for cilia ultrastructure evaluation and for cilia beat frequency measurement. Diagnosed patients were sent to lung and sinuses CT scan (> 5 years), pulmonary function tests and ear, nose and throat evaluation. RESULTS: For the 28 patients selected a nasal brushing was performed. For 24 of the 28 patients it was possible to make films suitable for evaluation. The average cilia beat frequency of the defective ultrastructure group (primary ciliary dyskinesia group) was different from the average frequency of the normal ultrastructure group (p<0.001) and from the control group (p<0.001). The average cilia beat frequency of the normal ultrastructure group was different from the average frequency of the control group (p<0.005). Twelve patients were diagnosed with primary ciliary dyskinesia: two with absence of outer dynein arm, one with a shortened outer dynein arm, five with radial spoke and inner dynein arm, three with absence of central par and transposition, and one with a normal ultrastructure (Kartagener). The patients, seven men and five women, were mostly white (11 patients 91.6%) and had parents who were relatives (eight patients 66.6%). Seven of the twelve patients (58.3%) had Situs inversus. Seven of the ten patients (70%) for whom pulmonary function tests were performed presented a ventilatory obstructive pattern. Radiological findings: Ten patients (83.3%) presented signs of consolidation or collapse, eleven patients (91.6%) had bronchiectasis, and 12 (100%) presented some degree of bronchial wall thickening. Otorhinolaryngologycal evaluation indicated impairments in 6 patients (50%): polips in three patients (25%), effusion otitis in two (16.6%), sept problems in two (16.6%) and timpanus sclerosis in one (8,3%). CONCLUSION: The nasal brushing technique was standardized for the collection of cilia. Uniform criteria for the analysis of ultrastructural cilia defects were established, based on international experience. A new method of cilia beat frequency measurement was developed, based on spectral analysis and high speed video images. Patients diagnosed with primary ciliary dyskinesia were characterized. The majority was white, had parents who were also relatives, had a prevalence of radial spoke and inner dynein arm defects, situs inversus, and ventilatory obstructive pattern. Otorhinolaryngologycal problems were found in 50% of the patients.

The maintenance of the airway/respiratory epithelium during adult homeostasis and repair and its construction during embryonic development require tightly regulated cell fate decisions. This regulation takes the form of complex transcription factor and signalling cascades, much of which are unknown, particularly in human lung development. Multiciliogenesis describes the process of specification/differentiation of airway epithelial progenitors/stem cells into mature multiciliated cells (MCCs). Here, I have identified 2 novel transcription factors, Fank1 and Jazf1 which form part of the transcription factor cascade regulating multiciliogenesis in adult and embryonic mouse tracheas. Mouse tracheal epithelium is representative of epithelium lining the entire human airway and it is possible that we will also be able to extrapolate these findings to the human airway. It is not until we fully understand the regulation of multiciliogenesis that it will be possible to look at ways of pushing basal cells towards a MCC fate for purposes of cell replacement therapy, for example in patients with mucociliary disease. As well as exploring cell fate decisions in the mouse upper airway epithelium using embryonic tracheal explants and mouse tracheal epithelial cell (MTEC) cultures, I have also explored the regulation of cell fate decisions in distal human lung epithelium at the pseudoglandular stage of development. At this stage SOX9+ distal tip cells are self-renewing and multipotent and give rise to SOX2+ stalk descendents, which differentiate into airway epithelium. The regulation of SOX9+ lung tip cell multipotency and migration of SOX2+ stalk descendents during human lung development is poorly understood. I have compared human tip (SOX9+) versus stalk (SOX2+) transcriptomes using gene ontology (GO), which has highlighted some key signalling pathways enriched in tip cells which could be important in maintaining distal tip cell multipotency. These pathways have been utilised in optimising conditions for propagating self-renewing tip-derived organoids. These organoids have the potential to be differentiated into bronchiolar and alveolar fates and as such are an invaluable research tool for studying human lung epithelial development, whilst minimising the use of human embryos and its associated ethical implications. I have also performed human tip versus mouse tip transcriptome GO analysis which highlights that although there are many similarities, there are also differences between human and mouse lung epithelium development, emphasising the need for research on human tissue.

INTRODUÇÃO: A poluição atmosférica é reconhecida como fonte de possíveis agravos à saúde. Estudos tem demonstrado uma clara associação entre aumento da concentração dos poluentes atmosféricos, especialmente o material particulado proveniente de resíduos de exaustão de motores movidos a diesel, com morbidade e mortalidade respiratória e cardíaca na população geral. Até o presente momento, sabe-se que o material particulado possui ações deletérias sobre as vias aéreas superiores e inferiores; contudo, ainda não são completamente conhecidas as ações tóxicas isoladas das diferentes frações do material particulado do diesel. OBJETIVO: O presente estudo teve por finalidade avaliar o grau de toxicidade das frações orgânicas de baixa, média e alta polaridade e da fração inorgânica do material particulado proveniente da emissão de motores movidos a óleo diesel sobre o epitélio mucociliar. MÉTODOS: Para tanto, utilizou-se como modelo experimental a preparação do palato da rã, que possui um epitélio similar ao das vias aéreas de mamíferos. O estudo foi dividido em duas etapas: Fase I - Quarenta palatos foram utilizados com intuito de titular a concentração de material particulado bruto capaz de promover um aumento significante no tempo relativo de transporte mucociliar. Fase II - Uma vez definida a concentração efetiva, cinqüenta palatos foram expostos aos seguintes tratamentos: material particulado bruto do diesel (sem nenhum tipo de tratamento), material particulado do diesel tratado com hexano (solvente que reduz a quantidade dos compostos orgânicos de baixa polaridade), material particulado do diesel tratado metanol (solvente que reduz a quantidade de compostos orgânicos de polaridade intermediária, gerando aumento relativo da concentração de orgânicos de baixa e alta polaridade) e material particulado do diesel tratado ácido nítrico (solvente que reduz a concentração de compostos inorgânicos, gerando aumento relativo da fração orgânica como um todo). Para fins de controle utilizou-se um grupo com ringer-rã. As variáveis analisadas foram: tempo relativo de transporte mucociliar, freqüência de batimento ciliar e análise histológica, na qual foram avaliados o volume proporcional de muco ácido, muco neutro, muco misto, de cílios, vacúolos, dos núcleos celulares e interstício e espessura epitelial. RESULTADOS: Fase I: A concentração de material particulado bruto capaz de promover um aumento significativo no tempo relativo de transporte mucociliar, e também do volume proporcional de muco ácido (p<0,05), correspondeu a 12mg/L. Fase II: Observou-se: a) aumento significativo no tempo relativo de transporte mucociliar e redução significativa do volume proporcional de muco neutro no grupo ácido nítrico; b) maior volume proporcional de muco ácido no grupo metanol (p<0,05); c) ausência de diferenças entre o grupo controle e o grupo hexano, tanto na análise histológica quanto no tempo relativo de transporte mucociliar. CONCLUSÃO: Os dados obtidos sugerem que os compostos orgânicos de baixa polaridade do material particulado proveniente da emissão de motores movidos a óleo diesel desempenham um importante papel na toxicidade aguda ao epitélio ciliado / INTRODUCTION: Air pollution is recognized as a source of potential health problems. Studies have shown a clear association between increasing concentration of atmospheric pollutants, especially particulate matter from waste exhaust of diesel engines, and respiratory and cardiac morbidity and mortality in the general population. To date, it is well known that particulate matter from diesel exhaust has deleterious actions on upper and lower airways; however, the isolated toxic actions of different fractions of the particulate matter are not yet fully understood. OBJECTIVE: This study aimed at evaluating the degree of toxicity of the organic fractions of low, intermediate and high polarity and of the inorganic fraction of particulate matter from diesel engines on the ciliated epithelium. METHODS: The experimental model used was the frog palate preparation, which has a similar epithelium to that found in mammalian airways. The study was divided into two phases: Phase I - Forty palates were used in order to titrate the concentration of intact diesel particulate matter able to elicit a significant increase in the relative time of mucociliary transport. Phase II Once defined the optimal concentration, fifty palates were exposed to dilutions with the following treatments: intact diesel particulate material (without any treatment), particulate matter from diesel exhaust treated with hexane (solvent which reduces the amount of organic compounds of low polarity), particulate matter from diesel exhaust treated with methanol (solvent which reduces the amount of organic compounds with intermediate polarity, generating a relative increase in concentration of organic compounds with low and high polarity) and particulate matter from diesel exhaust treated with nitric acid (solvent which removes inorganic compounds, eliciting a relative increase of the organic fraction as a whole). For control purposes, a group of frog-ringer was used. The following variables were analyzed: relative time of mucociliary transport, ciliary beating frequency and histological analysis, which evaluated proportional volume of acid mucus, neutral and mixed mucus, cilia, vacuoles, cell nuclei and interstice, and epithelial thickness. RESULTS: Phase I: The effective concentration of intact diesel particulate matter in eliciting a significant increase in the relative time of mucociliary transport, and a proportional increase of acid mucus volume (p<0.05), corresponded to 12mg/L. Phase II: a) The nitric acid treatment caused a significant increase in the relative time of mucociliary transport, and decrease in the proportional volume of neutral mucus. b) A higher proportional volume of acid mucus was found in the methanol group (p<0.05). c) There were no differences between control and hexane groups regarding histological findings and relative time of mucociliary transport. CONCLUSION: The results suggest that organic compounds of low polarity from diesel engines exhaust particles play an important role in the acute toxicity on the ciliated epithelium

Les cils et les flagelles sont des organites conservés chez les eucaryotes où ils jouent des rôles essentiels et variés comme la motilité et la signalisation cellulaire. La zone de transition (ZT) est une structure complexe, localisée à la base des cils, indispensable à leur assemblage et pour la sélection des constituants ciliaires. Chez l'Homme, de nombreuses pathologies appelées ciliopathies sont associées à des défauts d'assemblage ou de fonctionnement des cils. Les plus sévères sont liées à des défauts de protéines de la ZT. Cette dernière est composée principalement de trois complexes protéiques nommés MKS, NPHP et CEP290 interagissant étroitement entre eux. D'autres protéines, dont CBY conservée des mammifères à la drosophile, s'ajoutent à ces modules mais leur interconnections ne sont pas connues Deux modes d'assemblage ciliaire ont été décrits : la ciliogenèse compartimentée et cytosolique. La fonction de la ZT au cours de la ciliogenèse compartimentée a fait l'objet de nombreuses études mais son rôle dans la ciliogenèse cytosolique reste peu connu. Au cours de ma thèse j'ai analysé la fonction de nouvelles protéines de la ZT en utilisant le modèle de la drosophile qui présente les 2 types de ciliogenèse. J'ai d'une part réalisé un crible protéomique en cellules murine IMCD3 et caractérisé le module protéique CBY, composé de CBY, FAM92A et DZIP1L. Ce module est conservé chez la drosophile à la ZT. Il est nécessaire à la ciliogenèse notamment pour l'assemblage de la ZT et pour l'ancrage du corps basal à la membrane plasmique. L'absence de ces protéines entraine des défauts ciliaires importants dans l'assemblage des flagelles de spermatozoïde et des cils des neurones sensoriels chez les drosophiles.En conclusion, ce travail apporte de nouvelles connaissances sur l'assemblage de la ZT et sur le rôle de CBY dans les mécanismes qui contrôlent la ciliogenèse / Cilia and flagella are highly conserved organelles among eukaryotes species. They are composed of a microtubular cytoskeleton and play essential functions during development and in numerous physiological processes. As a result, in humans, cilia dysfunction leads to a wide range of pathologies, called ciliopathies.At the ciliary base, the transition zone (TZ), a complex structure, is required for proper cilia assembly and regulates the traffic of ciliary components in and out cilia. Defects in TZ proteins lead to severe ciliopathies. The TZ is composed of 3 protein complexes, MKS, NPHP et CEP290 that closely interact. Additional proteins, like CBY, conserved between mammals and Drosophila, have been described at the TZ but their precise role and relationships with the other TZ complexes are unknown. Two modes of cilia assembly have been described: compartmentalized and cytosolic ciliogenesis. Whereas the function of the TZ in compartmentalized ciliogenesis is well documented, its role in cytosolic ciliogenesis remains poorly characterized. During my PhD, I characterized new TZ proteins conserved in mammals and Drosophila and analyzed their function during cilia assembly in Drosophila. First, I performed a proteomic screen in murine IMCD3 cells and characterized the CBY module composed of CBY, FAM92A1 and DZIP1L. This complex is conserved in Drosophila and locates at the TZ. Moreover, I showed that this module is necessary for TZ assembly and centriolar docking to the plasma membrane and hence required for cilia and flagella assembly. In absence of these proteins, Drosophila show severe ciliogenesis defects both in sperm cells and in sensory neurons.In conclusion, this work brings new insights into the understanding of TZ assembly and of the mechanisms, that control ciliogenesis

INTRODUÇÃO: A discinesia ciliar primária é uma doença genética que se caracteriza pela alteração da ultraestrutura e função do cílio móvel, com consequentes alterações do transporte mucociliar, causando infecções das vias aéreas superiores, inferiores e infertilidade. O diagnóstico, realizado por avaliação da ultraestrutura ou pesquisa de mutação genética, é feito mediante critérios de seleção de pacientes e testes de screening. Esta pesquisa avalia a ultraestrutura e frequência de batimento ciliar, propõe um modelo de investigação de discinesia ciliar primária, e caracteriza os pacientes diagnosticados. MÉTODO: Foi realizado um estudo transversal controlado entre janeiro de 2007 e julho de 2009, no Ambulatório de Pneumologia Pediátrica do Instituto da Criança. Foram selecionadas 28 crianças e adolescentes (6 meses a 19 anos, de ambos os sexos), de uma população de 75 crianças com pneumopatias crônicas e de repetição sem diagnóstico definido, que apresentavam ao menos um dos seguintes achados: bronquiectasia de causa desconhecida, doença de vias aéreas superiores com sintomatologia crônica, infecções pulmonares de repetição, dextrocardia e/ou situs inversus acompanhados de sintomas em vias aéreas superiores e/ou inferiores, e asma de difícil controle com sintomas em vias aéreas superiores e/ou inferiores. A presença de pneumopatias crônicas com diagnóstico definido foi utilizada como critério de exclusão por meio dos seguintes exames: dois testes do suor (exclusão de fibrose cística), tomografia computadorizada do tórax (suspeita de bronquiolite obliterante), dosagem de alfa-1 antitripsina (investigação de déficit de alfa-1 antitripsina), e exames de investigação das imunodeficiências mais frequentes (hemograma, dosagens de imunoglobulinas, contagem de linfócitos T e B, sorologias para avaliação da produção ativa de anticorpos, PPD e HIV). Foi desenvolvido um sistema medição da frequência de batimento ciliar, baseado em análise espectral. Dez adultos voluntários saudáveis (maiores ou iguais a 17 anos, de ambos os sexos), sem doença infecciosa respiratória aguda no último mês e não fumantes, formaram o grupo controle para a frequência do batimento ciliar. Foi realizada a coleta ciliar por escovado nasal. A amostra foi dividida para a avaliação da ultraestrutura e verificação da frequência do batimento ciliar. Os pacientes diagnosticados foram avaliados com tomografia de tórax e seios da face (esta última nos maiores de cinco anos), provas de função pulmonar e avaliação otorrinolaringológica. RESULTADOS: Os 28 pacientes selecionados foram submetidos ao escovado nasal. Para 24 dos 28 pacientes foi possível produzir filmes passíveis de avaliação. O grupo com ultraestrutura alterada (diagnóstico de discinesia ciliar primária) mostrou diferença das médias da frequência de batimento ciliar em relação ao grupo com ultraestrutura normal (p<0,001) e em relação ao grupo controle (p<0,001). O grupo com ultraestrutura normal e o grupo controle, quando comparados entre si, também apresentaram diferenças (p<0,005). Foram diagnosticados 12 pacientes com discinesia ciliar primária: dois com ausência de braços externos de dineína, um com encurtamento dos braços externos de dineína, cinco com defeitos nas espículas radiadas e braços internos de dineína, três com defeitos de ausência do par central com transposição, e um com ultraestrutura normal (Kartagener). Os pacientes, sete homens e cinco mulheres, com predomíno da etnia branca (11 pacientes - 91,6%), eram na maior parte fruto de pais consanguíneos (8 pacientes - 66,6%). Sete dos 12 pacientes (58,3%) apresentaram situs inversus. Sete dos dez pacientes que realizaram as provas de função pulmonar (70%) apresentaram distúrbio ventilatório obstrutivo. Achados radiológicos: Dez pacientes (83,3%) apresentaram algum grau de colapso ou consolidação, 11 (91,6%) apresentaram bronquiectasias, e todos (100%) algum grau de espessamento brônquico. A avaliação otorrinolaringológica apontou alterações em seis pacientes (50%): pólipos em três pacientes (25%), otite secretora em dois (16,6%), desvio de septo em dois (16,6%), e esclerose do tímpano em um (8,3%). CONCLUSÃO: Foi padronizada a técnica do escovado nasal para a coleta de material ciliar. Foram estabelecidos critérios de uniformidade quanto à análise dos defeitos ultraestruturais ciliares, baseados em experiência internacional. Foi desenvolvido um novo método de medição da frequência de batimento ciliar baseado em metodologia de análise espectral e vídeos de alta velocidade. Os pacientes diagnosticados com discinesia ciliar primária foram caracterizados, sendo a maioria: da etnia branca, de pais consanguíneos, com predomínio dos defeitos de espículas radiadas e braços internos de dineína, situs inversus, e distúrbio ventilatório obstrutivo. Problemas otorrinolaringológicos foram encontrados em metade dos pacientes. / INTRODUCTION: Primary ciliary dyskinesia (PCD) is a genetic disorder of the ultrastructure and function of mobile cilia, with consequent impairment of mucociliary clearance, leading to upper and lower airways respiratory infection and infertility. The diagnosis, based on ultrastructure evaluation or genetic scan, is performed according to patient selection and screening tests. This research evaluates cilia ultrastructure and beat frequency, proposes a model for investigating primary ciliary dyskinesia, and characterizes the patients diagnosed.METHOD: A controlled and observational study was carried out at the Pediatric Pulmonology Ambulatory of the Instituto da Criança between January 2007 and July 2009. Twenty eight children and teenagers (ages between 6 months and 19 years) were selected, from a population of 75 patients with chronic and repetition pneumopathies without a defined diagnosis, which met at least one of the following inclusion criteria: bronchiectasis of unknown cause, upper respiratory disease with chronic symptoms, repetition pulmonary infections, dextrocardia and/or situs inversus with symptoms in upper and/or lower respiratory airways, and asthma of difficult control with symptoms in upper and/or lower respiratory airways. The presence of cronic pneumopathies with a defined diagnosis was used as exclusion criterion, based on the following exams: two sweat tests (cystic fibrosis exclusion), lung CT scan (bronchiolitis obliterans exclusion), seric levels of alpha-1 anti-trypsin (alpha-1 anti-trypsin deficit evaluation), and evaluation of more frequent immunodeficiency disorders (white blood cells, T and B lymphocytes levels, and sorology tests for humoral immunity, PPD and HIV). A cilia beat frequency measurement system was developed, based on spectral analysis. Ten healthy adult volunteers (ages greater than or equal to 17 years old, of both sexes), without an acute respiratory disease in the last month, and non-smoking, formed the control group for the cilia beat frequency measurements. Cilia samples were collected employing nasal brushing. The sample was divided for cilia ultrastructure evaluation and for cilia beat frequency measurement. Diagnosed patients were sent to lung and sinuses CT scan (> 5 years), pulmonary function tests and ear, nose and throat evaluation. RESULTS: For the 28 patients selected a nasal brushing was performed. For 24 of the 28 patients it was possible to make films suitable for evaluation. The average cilia beat frequency of the defective ultrastructure group (primary ciliary dyskinesia group) was different from the average frequency of the normal ultrastructure group (p<0.001) and from the control group (p<0.001). The average cilia beat frequency of the normal ultrastructure group was different from the average frequency of the control group (p<0.005). Twelve patients were diagnosed with primary ciliary dyskinesia: two with absence of outer dynein arm, one with a shortened outer dynein arm, five with radial spoke and inner dynein arm, three with absence of central par and transposition, and one with a normal ultrastructure (Kartagener). The patients, seven men and five women, were mostly white (11 patients 91.6%) and had parents who were relatives (eight patients 66.6%). Seven of the twelve patients (58.3%) had Situs inversus. Seven of the ten patients (70%) for whom pulmonary function tests were performed presented a ventilatory obstructive pattern. Radiological findings: Ten patients (83.3%) presented signs of consolidation or collapse, eleven patients (91.6%) had bronchiectasis, and 12 (100%) presented some degree of bronchial wall thickening. Otorhinolaryngologycal evaluation indicated impairments in 6 patients (50%): polips in three patients (25%), effusion otitis in two (16.6%), sept problems in two (16.6%) and timpanus sclerosis in one (8,3%). CONCLUSION: The nasal brushing technique was standardized for the collection of cilia. Uniform criteria for the analysis of ultrastructural cilia defects were established, based on international experience. A new method of cilia beat frequency measurement was developed, based on spectral analysis and high speed video images. Patients diagnosed with primary ciliary dyskinesia were characterized. The majority was white, had parents who were also relatives, had a prevalence of radial spoke and inner dynein arm defects, situs inversus, and ventilatory obstructive pattern. Otorhinolaryngologycal problems were found in 50% of the patients.

Les cils sont des organites très conservés au cours de l’évolution des eucaryotes et présents à la surface de presque tous les types cellulaires. Ils sont constitués d’une structure microtubulaire, l’axonème, entourée d’une membrane en continuité avec la membrane plasmique. Ils sont nucléés par un corps basal, centriole ancré à la surface cellulaire. Grâce aux nombreux récepteurs qu’ils concentrent à leur membrane, tous les cils sont des senseurs de leur environnement. Ils peuvent aussi être motiles et assurer, par leur battement coordonné, le déplacement relatif de la cellule et du fluide environnant. Tandis que cil et structure centriolaire, hérités du premier eucaryote, ont été perdus par certains champignons et par les plantes supérieures, certains gènes codant des protéines ciliaires et centriolaires sont pourtant retrouvés dans le génome de ces espèces. Cette conservation de protéines sans l’organite suggère soit que ces protéines interviennent dans un même processus moléculaire utilisé dans plusieurs organites, soit qu’elles jouent des rôles dans des processus moléculaires distincts via leur interaction avec différents types de partenaires.J’ai choisi d’étudier l’une de ces protéines ciliaires et centriolaires, Bug22p, hautement conservée en séquence protéique entre l'homme et la paramécie, mais également présente chez les plantes supérieures. J’ai mené cette étude principalement sur la paramécie, système modèle pour la biogénèse des corps basaux et des cils, mais aussi sur des cellules de mammifère et de végétaux supérieurs. Si Bug22p est impliquée dans la détermination du battement ciliaire chez la paramécie, elle se localise également dans des cils immotiles de cellules de mammifère suggérant que son activité ciliaire n’est pas réduite à cette seule fonction. Des expériences d’inactivation génique suggèrent par ailleurs un lien entre l’activité de Bug22p et la polyglycylation. Sa surexpression dans les cellules de mammifère en culture entraîne l’apparition d’extensions cellulaires et une augmentation des réseaux de tubulines acétylées probablement associées à une stabilisation des microtubules. L'ensemble de mes résultats suggère donc un rôle de Bug22p dans la régulation de modifications post-traductionnelles. En plus d’être présente dans les structures ciliaires, Bug22p se localise aussi bien dans les noyaux de la paramécie que dans ceux des cellules humaines et des plantes supérieures Arabidopsis et Nicotiana. Ces observations ouvrent un nouveau champ d’études. En effet, si l’on sait que les tubulines ciliaires sont soumises à différentes modifications post-traductionnelles telles que polyglycylation ou acétylation, ce type de modifications touchent également des protéines nucléaires régulant ainsi le trafic de protéines nucléaires ou l’expression génique. Nous pouvons donc avancer l’hypothèse selon laquelle Bug22p agirait sur la régulation de ces modifications dans le cil et dans le noyau. Il serait donc intéressant de caractériser les modifications post-traductionnelles chez les plantes supérieures afin de vérifier une possible implication de Bug22p dans leur régulation et donc comprendre les raisons de sa conservation chez les végétaux. / Cilia, organelles that have been conserved throughout the evolution of eukaryotes, are found at the surface of most cell types. They are composed of a microtubular structure, the axoneme, surrounded by a membrane continuous to the plasma membrane. Cilia are nucleated by basal body, which is a centriole anchored to the cell surface. Cilia are environmental sensors concentrated in the ciliary membrane. Cilia can be motile and ensure the relative movement of the cell with respect to the surrounding fluid by their coordinated beating. While cilia and centriolar structures inherited from the first eukaryote have been lost by certain fungi and higher plants, certain genes encoding ciliary and centriolar proteins are found in the genomes of organisms lacking these structures. The conservation of these proteins without organelle suggests that these proteins are involved in the same molecular process into different organelles or proteins are involved into different processes through some interactions with different partners.I chose to study the ciliary and centriolar protein, Bug22p, highly conserved between human and Paramecium proteins sequences, and also present in higher plants. My work addressed this study primarily on Paramecium, a model system for biogenesis of basal bodies and cilia, and I also pursued investigation of mammalian cells and higher plants. I was able to show that Bug22p is necessary for efficient Paramecium ciliary beating, but I also localized in the immotile cilia of mammalian cells suggesting that Bug22p is not only restricted to the motile ciliary function. By knockdown experiments in Paramecium, I obtained evidence that Bug22p is involved in polyglycylation. Bug22p overexpression in mammalian cells led to the appearance of cell extensions and increased acetylated tubulin networks consistent with microtubule stabilization. My results suggest that Bug22p may regulate post-translational proteins modifications.Bug22p is also localized in the nuclei of Paramecium, human and higher plants such as Arabidopsis and Nicotiana. These observations open a new field of study. The axoneme microtubules are highly modified by post-translational modifications such as acetylation and polyglycylation; we know that in the nucleus, theses modifications are involved in the control of nuclear trafficking of some proteins and the regulation of gene expression. We can therefore speculate that Bug22p acts on the regulation of these changes in the cilium and in the nucleus. Finally, it would be interesting to characterize the post-translational modifications in higher plants to verify the possible involvement of Bug22p in their regulation and thus understand the meaning of its conservation in higher plants lacking cilia.

Bardet-Biedl syndrome (BBS) is a rare genetic disease that causes retinal degradation, obesity, kidney dysfunction, polydactyly, and other cilium-related disorders. To date, more than 20 BBS genes, whose mutants cause BBS phenotypes, have been identified, and eight of those (BBS1-2, 4-5, 7-9, and 18) are known to form the BBSome complex. Recent studies have revealed that the BBSome is closely involved in the trafficking of signaling proteins in the primary cilium. Mutations in BBS genes are highly pathogenic because trafficking in the primary cilium is not fully functional when BBS mutations impair assembly of the BBSome. However, the functional links between onset of BBS and BBSome assembly are not well understood. To address this gap in knowledge, we examined the structure of a BBSome assembly intermediate, the BBSome core complex (BBS2, 7, and 9). We employed a combination of chemical crosslinking coupled with mass spectrometry (XL-MS) and electron microscopy (EM) to determine the structure. We applied this structural information to BBS mutations in the core complex to understand how these mutations might cause the disease. These results provide the first structural model of the BBSome core complex and give insight into the molecular basis of Bardet-Biedl syndrome. We have also investigated the mechanism of assembly of the two mTOR kinase complexes (mTORC1 and 2). mTOR is a master regulator of cell metabolism, growth and proliferation. As such, mTOR is a high-value drug target. We investigated the mechanism of assembly of these mTOR complexes and found that the cytosolic chaperonin CCT contributes to mTOR signaling by assisting in the folding of mLST8 and Raptor, components of mTORC1 and mTORC2. To understand the function of CCT in mTOR complex assembly at the molecular level, we have isolated the mLST8-CCT complex and performed a structural analysis using chemical cross-linking couple with mass spectrometry (XL-MS) and cryogenic EM. We found that mLST8 binds CCT deep in its folding cavity, making specific contacts with the CCTα and γ subunits and forming a near-native β-propeller conformation. This information can be used to develop new therapeutics that regulate mTOR activity by controlling mTOR complex assembly.

Neonatal hydrocephalus is a common neurological disorder leading to expansion of the cerebral ventricles. This disease is associated with significant morbidity and mortality and is often fatal if left untreated. Hydrocephalus was first described over 2500 years ago by Hippocrates, the father of medicine, and remains poorly understood today. Current therapies still rely on invasive procedures developed over 60 years ago that are associated with high failure and complication rates. Thus, the identification of molecular mechanisms and the development of non-invasive medical treatments for neonatal hydrocephalus are high priorities for the medical and scientific communities. The prevailing doctrine in the field is that hydrocephalus is strictly a "plumbing problem" caused by impaired cerebrospinal fluid (CSF) flow. Recently, animal models with impaired cilia have provided insight into the mechanisms involved in communicating (non-obstructive) hydrocephalus. However, as a result of a poor understanding of hydrocephalus, no animal studies to date have identified an effective non-invasive treatment. The goal of this thesis project is to investigate the molecular mechanisms underlying this disease and to identify a non-invasive, highly effective treatment strategy. In Chapter 2, we utilize a novel animal model with idiopathic hydrocephalus, mimicking the human ciliopathy Bardet-Biedl Syndrome (BBS), to examine the role of cilia in hydrocephalus. We find that these mice develop communicating hydrocephalus prior to the development of ependymal "motile" cilia, suggesting that this phenotype develops as a result of dysfunctional "primary" cilia. Primary cilia are non-motile and play a role in cellular signaling. These results challenge the current dogma that dysfunctional motile cilia underlies neonatal hydrocephalus and implicate a novel role for primary cilia and cellular signaling in this disease. Chapter 3 focuses on identifying the link between primary cilia and neonatal hydrocephalus. In this chapter, we report that disrupting the molecular machinery within primary cilia leads to faulty PDGFRα signaling and the loss of a particular class of neural progenitor cells called oligodendrocyte precursor cells (OPCs). We find that the loss of OPCs leads to neonatal hydrocephalus. Importantly, we identify the molecular mechanism underlying both the loss of OPCs and the pathogenesis of neonatal hydrocephalus. Chapter 4 explores the therapeutic potential of targeting the defective cellular signaling pathways to treat neonatal hydrocephalus. By targeting the faulty signaling, we restore normal development of oligodendrocyte precursor cells, and curtail the development of hydrocephalus. This work challenges the predominant view of hydrocephalus being strictly a "plumbing problem" treatable solely by surgical diversion of CSF. Here, we propose that hydrocephalus is a neurodevelopmental disorder that can be ameliorated by non-invasive means. Importantly, we introduce novel molecular targets and a non-invasive treatment strategy for this devastating disorder. To our knowledge, we are the first to successfully treat neonatal hydrocephalus in any model organism by targeting neural progenitor cells.

Cilia are finger-like projections that extend from the surface of most cells. These microtubule-based structures serve important mechanical or sensory functions. Motile cilia have been implicated in fluid movement whereas the non-motile primary cilia have been shown to play a role in sensory signal transduction. There exists a dichotomy in the field that primary cilia have only sensory function and motile cilia only have mechanical function. The central question of this thesis project is "what are the structural and functional components of airway motile cilia and are these cilia sensory?" In Chapter 2, the role of Bardet-Biedl Syndrome (BBS) proteins in maintaining the structure and function of airway motile cilia is examined. We found that BBS proteins localize to the cilium and to ciliary-related structures in human airway epithelia. Using mutant mice we found that BBS proteins play an essential role in motile cilia structure and the loss of BBS proteins results in reduced ciliary beat. These proteins have previously been shown to play a role in primary cilia structure and function, and our studies indicate a novel function for BBS proteins. Chapter 3 examines the sensory role of motile cilia. Our data show that bitter taste receptors and components of the bitter taste signal transduction pathway localize to the motile cilia or to the ciliated cells. Ciliated cells also show an increase in intracellular calcium in response to bitter compounds, accompanied by a corresponding increase in cilia beat. The increase in intracellular calcium originates at the ciliated cells and is propagated to adjacent cells. Chapter 4 delves into the possibility that every motile ciliated cell also contains a single, primary cilium. Using immunostaining and Smoothened as a marker for primary cilia, we found that every group of motile cilia contains a single Smoothened-positive cilium. Furthermore, downstream components of the Sonic Hedgehog pathway are also present in ciliated cells. Chapter 6 is a summary chapter including possible explanations for observed outcomes and plans for future experiments. Our results indicate that the divide between primary and motile cilia may not be as great as has been previously thought.

Mon travail de thèse a porté sur l’étude chez la paramécie d’une protéine à localisation à la fois ciliaire et nucléaire. Les cils sont des organites conservés chez la plupart des eucaryotes qui pointent à la surface cellulaire vers le milieu extérieur. Leur squelette microtubulaire est nucléé par la structure centriolaire sous-jacente, le corps basal, qui transmet sa structure à symétrie 9. Une parenté évolutive lointaine existe entre le compartiment cil, isolé du cytoplasme par un filtre moléculaire appelé zone de transition, et les noyaux dont les contacts avec le cytoplasme sont réduits aux pores nucléaires. Cette homologie fonctionnelle est soutenue par l’existence de mécanismes et de partenaires apparentés dans la communication avec le cytoplasme. Les dysfonctionnements des cils conduisent à des maladies graves appelées ciliopathies. La croissance des cils est réalisée au moyen de complexes protéiques appelés IFT (intraflagellar transport) qui incluent au moins 17 protéines regroupés en deux sous complexes, IFTB, lié au moteur kinésine pour le mouvement antérograde vers la pointe du cil et IFTA lié au moteur dynéine pour le mouvement rétrograde vers la base du cil. L’objet principal de ma thèse, dans le cadre de l’étude de l’IFT chez la paramécie, a porté sur la protéine IFT57, aussi connue sous le nom de HIPPI chez l’homme, où elle assure, en plus de sa fonction ciliaire, une fonction d’activateur transcriptionnel associé à l’apoptose dans la maladie de Huntington. Chez la paramécie, il y a quatre gènes codant IFT57 provenant de duplications globales de génome, IFT57A et B d’une part et IFT57C et D d’autre part, suffisamment proches deux à deux pour que l’inactivation d’un gène puis éteindre également l’autre. Dans un premier temps, j’ai étudié la fonction ciliaire d’IFT57 et j’ai montré qu’il était nécessaire à la croissance ciliaire, mais apparemment pas à sa maintenance, contrairement à d’autres protéines de l’IFT telles qu’IFT46. L’action croisée d’inactivation d’une IFT sur la localisation d’une autre IFT fusionnée à la GFP on permis de suggérer des interactions entre IFT 46 et IFT57 dans le cytoplasme, en amont de leur site habituel d’interaction que représente le corps basal. Je me suis ensuite intéressé à la localisation nucléaire d’IFT57. La protéine IFT57A-GFP entre dans le macronoyau, en plus de sa localisation ciliaire, alors que IFT57C-GFP en est exclue. J’ai essayé de déterminer la différence de séquence qui permet de distinguer ces deux molécules proches pour en envoyer une dans le noyau et pas l’autre. L’aspect le plus marquant de la localisation nucléaire d’IFT57A est le changement de localisation au cours des événements sexuels, où le marquage quitte l’ancien macronoyau pour rejoindre les nouveaux macronoyaux en formation. Cette relocalisation évoque celle observée avec des protéines impliquées dans le métabolisme de petits ARN importants pendant les événements sexuels. Une idée est que IFT57A, protéine associée au transport, puisse gouverner ces mouvements internucléaires et j’ai entrepris d’analyser l’effet de l’inactivation de cette protéine sur les événements sexuels. La difficulté qui est apparue est que l’extinction d’IFT57A est rapidement létale. J’ai réalisé plusieurs approches méthodologiques pour contourner ce problème, dont la mise au point d’une nouvelle méthode d’inactivation par ARN en épingle à cheveux, mais sans pouvoir répondre à la question. En utilisant des constructions chimères entre les deux protéines, puis en comparant les séquences au sein du genre Paramecium, j’ai déterminé une région de 90 acides aminés, L129-N219, avec deux positions critiques pour distinguer fonctionnellement ces deux protéines. / My thesis work consisted in the study of cilia and flagella, organelles highly conserved in many eukaryotes, which protrude at the cell surface as a microtubular backbone, the axoneme, bounded by an extension of the plasma membrane. A major interest in the study of cilia is that they are involved in the regulation of many cell events, such as re-entry in the cell cycle, and that their dysfunction in vertebrates provokes multi-symptomatic diseases called ciliopathies. Ciliary growth is under control of IFT (intraflagellar transport) mechanism, first discovered in Chlamydomonas. The IFT complex includes at least 17 members and is is composed of two subcomplexes, IFTB linked to kinesin for anterograde transport in ciliary building, and IFTA linked to dynein for retrograde transport in recycling. In my thesis on IFT in Paramecium, I focused on IFT57/Hippi, a member of IFTB, which seems to display two different functions in human cells: biogenesis of cilia as member of the IFTB particle and gene regulation in Huntington’s diseases, as part of a complex with Hip1 that binds caspase promoters involved in apoptosis. Some recent work also showed that in the cytoplasm of non-ciliate cells, IFT57, associated with IFT20 and IFT88, regulates T-cell antigen receptor (TCR) recycling in immune synapse. In Paramecium, four genes issued from two successive genome duplications encode IFT57 (IFT57A – D). The isoforms A and B on the one hand and C and D on the other hand are sufficiently close in DNA sequence to get homology dependent RNAi silencing with only one gene of each pair. I first confirmed that IFT57 Paramecium genes have a conserved IFT function in cilia formation, but apparently not in maintenance, in contrast to other IFT proteins such as IFT46. The combination of RNAi and GFP fusion localization allowed us to suggest interactions between IFT46 and IFT57 in the cytoplasm, upstream from the usual site of interaction in the basal body. I also found that IFT57A-GFP, but not IFT57C-GFP, can enter the macronucleus and that the labelling shifts from the old to the new macronucleus during sexual events (autogamy and conjugation). This result must be analysed in light of the mechanism that governs genome rearrangements during nuclear reorganization, which are dependent on transport of RNA as well as of piwi and other RNA-binding molecules from old to new macronucleus. By extension of its ciliary role, one interesting possibility is that IFT57 is a partner involved in this transport. I tried to detect the putative roles of IFT57A in sexual process and worked out a new RNAi method by hairpin RNA expression. I expressed a specific IFT57A hairpin under the control of the NOWA1 promoter (only expressed in autogamy or conjugation) and found surprisingly that this hairpin stops the autogamy process. Since another hairpin contain NSF sequence displayed a similar phenotype, I could not draw any conclusion about the role of IFT57A during autogamy. Using chimeras by exchanging parts of IFT57A and IFT57C, then by comparing this sequence to other in the Paramecium genus, I could determine the shortest region of IFT57A necessary for nuclear localization is the peptide encompassing L129 to N219, with two critical positions to functionally distinguish these two proteins.