Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 106-108). Also available via the Internet.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 15, 2009). Includes bibliographical references.

Thesis (doctoral)--University of Stockholm, 1983. / Includes bibliographical references (p. 39-44 (first group)).

The work presented here describes novel functions for centrosome proteins, specifically for pericentrin and centriolin. The first chapter describes the involvement of pericentrin in ciliogenesis. Cells with reduced pericentrin levels were unable to form primary cilia in response to serum starvation. In addition we showed novel interactions between pericentrin, intraflagellar transport (IFT) proteins and polycystin 2 (PC2). Pericentrin was co-localized with IFT proteins and PC2 to the base of primary cilia and motile cilia. Ciliary function defects have been shown to be involved in many human diseases and IFT proteins and PC2 have been implicated in these diseases. We conclude that pericentrin is required for assembly of primary cilia possibly as an anchor for other proteins involved in primary cilia assembly. The second chapter describes identification of centriolin, a novel centriolar protein that localizes to subdistal appendages and is involved in cytokinesis and cell cycle progression. Depletion of centriolin leads to defects in the final stages of cytokinesis, where cells remain connected by thin intercellular bridges and are unable to complete abscission. The cytokinesis defects seemed to precede the G0/G1 p53 dependant cell cycle arrest. Finally, the third chapter is a continuation of the cytokinesis study and it identifies pericentrin as an interacting partner for centriolin. Like centriolin, pericentrin knockdown induces defects in the final stages of cytokinesis and leads to G0/G1 arrest. Moreover, pericentrin and centriolin interact biochemically and show codependency in their centrosome localization. We conclude that pericentrin and centriolin are members of the same pathway and are necessary for the final stages of cytokinesis.

La glycylation, une modification post-traductionnelle des microtubules, est cruciale dans le maintien des cils primaires. Notre groupe a précédemment identifié un rôle inattendu de la tubuline glycylase TTLL3 dans la régulation de l'homéostasie du colon et de la tumorigénèse. Plus précisément, une diminution du nombre de cils primaires a été observée chez les souris déficientes pour la glycylase TTLL3, qui est la seule glycylase exprimée dans le côlon. Les souris TTLL3 - / - ne présentent pas d'anomalie évidente à l'état stationnaire. Cependant, lorsqu'elles sont exposées à une carcinogenèse du côlon chimiquement induite, les souris TTLL3 - / - sont plus sensibles à la formation de tumeurs. Il est important de noter que les niveaux d'expression de TTLL3 sont significativement réduits dans les carcinomes primaires et métastases colorectales chez l'homme comparativement au tissu de côlon sain, ce qui suggère un lien entre la régulation des cils primaires par TTLL3 et le développement du cancer colorectal.L'objectif de mon projet de thèse était d'explorer l’effet de la modulation des cils primaires sur la carcinogenèse du côlon. J’ai ainsi démontré que le nombre de cils primaires diminue lors de la carcinogenèse du côlon chimiquement induite chez la souris. Notamment, j'ai découvert que les cils primaires du côlon sont principalement exprimés par les cellules mésenchymateuses. Pour mieux caractériser le rôle des cils primaires dans le côlon murin, j'ai étudié les conséquences de leur perte dans les cellules mésenchymateuses intestinaux. Pour cela, j'ai utilisé deux modèles de souris KO conditionnelles, pour la kinesin-3A (Kif3A) et le transport intra-flagellaire 88 (Ift88), deux molécules essentielles pour la formation des cils. Leur délétion spécifique dans les cellules mésenchymateuses intestinaux est obtenue par croisement des souches de souris Kif3Afl/fl et Ift88fl/fl des souris transgéniques collagène VI-cre. Bien que le promoteur colllagène VI ne soit actif que dans un sous-ensemble de cellules mésenchymateuses coliques, j'ai constaté que la diminution du nombre de cils primaires dans ces derniers favorise la colite chimiquement induite et la carcinogenèse. L'analyse par séquençage ARN des cellules mésenchymateuses coliques isolés de souris mutantes suggère un déclenchement de la signalisation Wnt et Notch chez les souris ColVIcre-Kif3Afl/fl. Nous confirmons actuellement ces résultats par qPCR et immunohistochimie. / Glycylation, a posttranslational modification of microtubules, is crucial in the maintenance of PC. Our group previously identified an unexpected role of the tubulin glycylase TTLL3 in the regulation of colon homeostasis and tumorigenesis. Specifically, a decreased number of primary cilia (PC) was observed in mice deficient for the glycylase TTLL3, which is the only glycyclase expressed in the colon. TTLL3-/- mice display no obvious abnormalities in the steady state. However, when exposed to chemically induced colon carcinogenesis, TTLL3-/- mice are more susceptible to tumor formation. Importantly, TTLL3 expression levels were significantly downregulated in human primary colorectal carcinomas and metastases as compared to healthy colon tissue, suggesting a link between TTLL3 regulation of PC and colorectal cancer development.The aim of my thesis project was to explore the relation of PC and colon carcinogenesis. In fact, I could demonstrate that the number of PC decreases during chemically induced colon carcinogenesis in mice. Notably, I discovered that PC in the colon are mostly expressed by fibroblasts. To better characterize the role of PC in murine colon, I studied the consequences of a loss of PC in intestinal fibroblasts. For this, I used two independent ciliary conditional knockout mice, kinesin-3A (Kif3A) and intraflagellar transport 88 (Ift88), both essential for cilia formation. Specific deletion in intestinal fibroblasts is obtained by crossing with colVI-cre transgenic mice. Though the colVI promoter is only active in a subset of colonic mesenchymal cells I found that the decreased number of PC in colonic mesenchymal cells promotes chemically induced colitis and carcinogenesis. RNAseq on isolated colonic mesenchymal cells of mutant mice suggests a triggering of Wnt and Notch signaling in ColVIcre-Kif3aflx/flx mice. We are presently validating these findings by qPCR and immunohistochemistryTaken together, I discovered that PC are expressed by at least a subset of colonic mesenchymal cells, which has not been described before. Decreased numbers of those PC renders mice more susceptible to colitis and colitis associated carcinogenesis.

RIC-8B é uma proteína que apresenta, in vitro, atividade de fator de troca de nucleotídeos guanina (GEF). No entanto, seu papel in vivo não é conhecido. Dados anteriores do nosso laboratório demonstraram que essa proteína interage especificamente com Gαolf, que é uma proteína G exclusiva do sistema olfatório, presente nos cílios dos neurônios olfatórios, onde ocorre a transdução de sinal ativada pelos odorantes. No camundongo adulto verificou-se, por meio de ensaios de hibridização in situ, que RIC-8B está presente somente em regiões de expressão de Gαolf: no epitélio olfatório maduro e no núcleo estriado do sistema nervoso central. Para avaliar a função fisiológica de RIC-8B in vivo, resolvemos gerar uma linhagem de camundongo knockout para Ric-8B. Verificamos que a linhagem é inviável devido à letalidade dos embriões já em fases precoces do desenvolvimento (por volta de E8,5 e E9,0). A coloração de embriões com X-gal mostra que RIC-8B é especificamente expressa em regiões que darão origem ao sistema nervoso, como na região ventral do tubo neural, e em regiões cefálicas. Interessantemente, mostramos que RIC-8B é expressa na placa do assoalho do tubo neural, de uma maneira muito semelhante ao padrão de expressão de Sonic Hedgehog (SHH), que apresenta um papel fundamental para a organização do sistema nervoso, entre outras funções. Nossos resultados indicam, portanto, que RIC-8B desempenha um papel crucial durante a embriogênese, e que este papel pode estar relacionado com o papel exercido por SHH. Além disso, como a via de sinalização de SHH ocorre em cílios primários nas células alvo, nossos dados levantam a interessante possibilidade de que RIC-8B apresenta funções relacionadas a cílios, tanto no camundongo adulto (neste caso nos cílios dos neurônios olfatórios) como no embrião (neste caso nos cílios primários). / RIC-8B is a protein that, in vitro, acts as a guanine nucleotide exchange factor (GEF). However, its role in vivo remains unknown. Previous data from our laboratory demonstrated that this protein is able to interact specifically with Gαolf, a G protein found only in the olfactory system. This G protein is located in the cilia from olfactory neurons, where odorant signaling occurs. In situ hybridization experiments showed that RIC-8B, in adult mice, is expressed only in regions where Gαolf is expressed, such as the olfactory epithelium and the nucleus striatum in the central nervous system. In order to determine the function of RIC-8B in vivo, we decided to generate a knockout mouse strain for Ric-8B. We found that this strain is not viable due to the lethality of embryos in the early stages of development (around days E8.5 and E9.0). X-gal staining of embryos shows that RIC-8B is specifically expressed in regions that originate the nervous system, such as the ventral neural tube and also cephalic regions. Interestingly, we show that RIC-8B is restrictedly expressed in the floor plate of the neural tube, in a pattern that is very similar to the one shown by Sonic Hedgehog (SHH). The SHH protein plays a fundamental role in the organization of the nervous system, among other functions. Therefore, our results indicate that RIC-8B plays an essential role during the embryogenesis, and that this role can be related to the role played by SHH. Furthermore, because the SHH signaling pathway occurs in primary cilia in the target cells, our data raise the interesting possibility that the role played by RIC-8B is related to ciliary functions, both in adult mice (in this case, in olfactory cilia), and in the embryo (in this case, in primary cilia)

Estudos anteriores (Zayas, 1990) tem sugerido a existência de uma melhor transportabilidade por cílio do muco respiratório de pacientes fumantes que não apresentam câncer de pulmão em comparação com pacientes fumantes com câncer de pulmão e semelhante carga tabágica. Nosso principal objetivo foi verificar esta hipótese. Nós estudamos 16 tabagistas com câncer de pulmão (média carga tabágica = 58,78), 16 tabagistas com câncer extra-pulmonar (média carga tabágica = 53,87) e 11 não Tabagistas com Metástase Pulmonar com indicação de broncoscopia diagnóstica. O muco respiratório foi coletado durante a broncoscopia, usando um pequeno cateter através do canal de aspiração do broncoscopio. A transportabilidade por cílio no palato de rã, ângulo de contato (wetabilidade), transportabilidade pela tosse e viscosidade (cone-plate) e análise morfológica do epitélio respiratório foi realizado. Não foi encontrada diferença estatística entre os pacientes Tabagistas (Câncer de pulmão e Câncer extra-Pulmonar) para os parâmetros de muco estudos. Da mesma forma não foi encontrada diferença estatística nas análises do muco coletado de um lado do tumor comparado com o lado contralateral. Entretanto, encontramos diferença estatística entre os grupos não tabagistas com Metástase Pulmonar e Tabagistas com câncer Pulmonar e Extra-Pulmonar para os parâmetros de Transportabilidade pela Tosse (p = 0,018), Viscosidade 10 rpm (p= 0,021), FEV 1 (L) (p= 0,028) e FEV 1 (%) (p= 0,042) e diferença estatística nos Tabagistas para Correlação entre carga tabágica e idade (p=0,038) e Viscosidade (p= 0,029). Na análise histologica observamos 10 Tabagistas (60% alteração, sendo, 30% metaplasia escamosa; 20% hyperplasia e 10% epitélio com ausência de cílios) e 15 não Tabagistas (40% com alteração histológica sendo 20% destes com metaplasia escamosa e em pacientes com Câncer pulmonar ou Câncer extra-pulmonar). Não teve diferença na composição das mucinas entre os tabagistas. Concluímos que não há diferença entre as propriedades físicas do muco respiratório de Tabagistas com Câncer de Pulmão e Câncer Extra-Pulmonar com similar carga tabágica e que essas alterações das propriedades físicas do muco respiratório e alterações morfológicas, devem-se mais à exposição dose-tempo da fumaça do cigarro ao epitélio respiratório / Previous study (Zayas 1990) has suggested the existence of a better transportability by cilia in respiratory mucus of smoking patients who did not present lung cancer in comparison to lung cancer patients smoking similar packages/year. Our aim was to verify this hypothesis. We studied 16 smoking patients with lung cancer (mean packages/year = 58,78), 16 smoking patients with extra pulmonary cancer (esophagus and head and neck), (mean packages/year = 53,87), and 11 non-smoking patients (metastasis) that underwent diagnostic bronchoscopy. Respiratory mucus was collected during bronchoscopy, using a small catheter passed through the aspiration channel of the bronchoscope. Mucus transportability in frog palate, contact angle (wettability), transportability by cough and viscosity (cone-plate) as well as morphological analysis the respiratory epithelium were performed. No statistical differences were found between smoking patients (lung and extra pulmonary cancer) in the mucus parameters studied. In the same way, no difference was found in the analysis of mucus samples collected from the tumor side compared to contra lateral samples. Nevertheless, statistical difference between Smoking (Lung Cancer and Extra-Pulmonary Cancer) and non Smoking (metastasis Pulmononary) for valous Clearance by cough (p = 0,018), viscosity 10rpm (p=0,021) FEV 1 (L) (p= 0,028) and FEV1 (%) (p=0,042) and statistical difference for correlation between smoking history and age (p=0,038) and viscosity 10 rpm (p= 0,029). The analysis histological of the 10 smoking, observed 60% of cases with alteration histological (30% with squamous metaplasia; 20% with hyperplasia and 10% with epithelium with cilia absence) and 15 non smoking presented 40% of cases with alteration histological (205 with squamous metaplasia in patients with lung cancer or exra-pulmonary cancer. We conclude that there is no difference between the physical properties of the respiratory mucus of smokers with lung cancer and extra-pulmonary cancer with similar packages/year and that changes in physical properties of respiratory mucus and morphological changes, due to more exposure to the dose-time of cigarette smoke in the respiratory epithelium

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

This thesis describes the work that I have carried out in the Laboratory of Neurophysiolgy at the Université Libre de Bruxelles, under the supervision of Prof. Serge Schiffmann, in collaboration with Prof. Stéphane Schurmans of Université of Liège.The work is divided in two distinct but related projects and the results section is thus divided into two main chapters. The results described are presented in the form of two manuscripts, the first chapter is named “Ciliary phosphoinositides regulation by INPP5E controls Shh signaling by allowing trafficking of Gpr161 in neural stem cells primary cilium”.The second is named “Regulation of phosphoinositides ciliary levels controls trafficking and ciliogenesis in ependymal cells”.Since both manuscripts are comprehensive regarding the results, and methods, these are inserted as such into the thesis.An expanded introduction to the field, placing the results into context, precedes these two chapters. An extended discussion section follows each chapter; it presents some elements of discussion not included in the manuscripts, the implications of the results and the scope for further research. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Les cils et les flagelles sont des organites présents à la surface cellulaire. Ils sont conservés chez les eucaryotes chez lesquels ils jouent un rôle essentiel dans la régulation de nombreux processus physiologiques. La zone de transition (ZT) est une structure complexe, localisée à la base des cils, qui assure une fonction importante dans l'assemblage et la régulation du trafic des constituants ciliaires. Trois complexes protéiques ont été identifiés à la ZT : MKS-JBTS, NPHP1-4-8 et NPHP5-CEP290. D'autres protéines sont également situées à la ZT telles que CBY et AZI1 mais leur interaction avec ces trois modules reste encore peu connue. Chez l'Homme, des mutations de gènes codant des protéines de la ZT sont associées à des maladies génétiques rares, les ciliopathies. Deux modes d'assemblage des cils ont été décrits : la ciliogenèse compartimentée et la ciliogenèse cytosolique. Alors que la fonction de la ZT au cours de la ciliogenèse compartimentée a été bien étudiée, son rôle dans la ciliogenèse cytosolique reste peu connu. La Drosophile possède deux sortes de cellules ciliées, les neurones sensoriels et les flagelles de spermatozoides dont les cils s'assemblent selon ces deux modes d'assemblage. Au cours de ma thèse, j'ai utilisé ce modèle pour analyser la fonction des protéines de la ZT dans ces deux types cellulaires. Mes résultats montrent que les protéines MKS ne jouent pas un rôle essentiel dans l'assemblage de la ZT dans ces deux types cellulaires. J'ai aussi révélé que CBY et AZI1, coopèrent pour assembler la ZT et qu'elle est nécessaire à l'ancrage du corps basal à la membrane plasmique. De plus, mes travaux ont démontré que KLP59D, une kinésine dépolymérisante des microtubules, est indispensable à la régulation de l'élongation de l'axonème au cours de la ciliogenèse cytosolique. En conclusion, ce travail apporte de nouvelles connaissances sur la dynamique d'assemblage de la ZT des cils et sur les mécanismes qui contrôlent l'élongation de l'axonème / Cilia and flagella are cellular organelles that protrude at the cell surface. They are composed of a microtubular cytoskeleton and they are highly conserved across eukaryotic species from plantae to Human. In mammals, they play essential functions during development and regulate numerous physiological processes in adults. At the ciliary base a complex structure called transition zone (TZ) is necessary for cilia assembly and regulation of ciliary components trafficking inside the cilia. Three protein complexes have been identified at the TZ : MKS-JBTS, NPHP1-4-8 and NPHP5-CEP290. Other TZ proteins such as CBY and AZI1 have been studied but their interaction with these 3 modules is not yet elucidated. In Human, mutations of genes encoding TZ proteins are associated with several genetic diseases called ciliopathies. Two different modes of cilia assembly have been identified: compartimentalized and cytosolic ciliogenesis. While TZ function in compartimentalized ciliogenesis is well studied, its role in cytosolic ciliogenesis remains poorly understood. In Drosophila, there are only two types of ciliated cells, sensory neurons and sperm flagella, representative of these two ciliogenesis pathways. During my PhD, I used Drosophila to study the function of TZ proteins during cilia assembly in these two ciliated cell types. My data show that proteins of the MKS complex do not play an essential role in TZ assembly in the cilia of sensory neurons and in spermatozoon flagella. I also demonstrated that CBY and AZI1 cooperate to assemble the TZ components and that the TZ is necessary to dock the basal bodies to the plasma membrane, one of the first important step in cilia assembly. Finally, I showed that KLP59D, a microtubule-depolymerising kinesin, is required to control axoneme elongation during the cytosolic ciliogenesis. In conclusion, this work brings new insights into the understanding of the dynamic assembly of TZ proteins and the mechanisms that regulate flagella elongation