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.

<p>Primary cilia are evolutionarily conserved, acetylated microtubule-based organelles that transduce mechanical and chemical signals. Primary cilium assembly is tightly controlled and its deregulation causes a spectrum of human diseases. Formation of primary cilium is a collaborative effort of multiple cellular machineries, including microtubule, actin network and membrane trafficking. How cells coordinate these components to construct the primary cilia remains unclear. In this dissertation research, we utilized a combination of cell biology, biochemistry and light microscopy technologies to tackle the enigma of primary cilia formation, with particular focus on isoform-specific roles of non-muscle myosin II family members. We found that myosin IIB (Myh10) is required for cilium formation. In contrast, myosin IIA (Myh9) suppresses cilium formation. In Myh10 deficient cells, Myh9 inactivation significantly restores cilia formation. Myh10 antagonizes Myh9 and increases actin dynamics, permitting pericentrosomal preciliary complex formation required for cilium assembly. Importantly, Myh10 is upregulated upon serum starvation-induced ciliogenesis and this induction requires Atat1/Mec-17, the microtubule acetyltransferase. Our findings suggest that Atat1/Mec17-mediated microtubule acetylation is coupled to Myh10 induction, whose accumulation overcomes the Myh9-dependent actin cytoskeleton, thereby activating cilium formation. Thus, Atat1/Mec17 and myosin II coordinate microtubules and the actin cytoskeleton to control primary cilium biogenesis.</p> / Dissertation

Cilia are cellular organelles involved in processing components of the hedgehog (hh) signalling pathway and determining left-right (L-R) axis formation in the embryo. An embryonic lethal mouse mutant, called gasping 6 (gpg6), was identified that demonstrated morphological and molecular defects associated with L-R development and hh signalling. gpg6 mutant embryos also demonstrate abnormally short cilia, which was hypothesised to be the primary morphological defect in gpg6 mutants. The underlying genetic lesion in gpg6 is a mutation in the DNA repair gene Atmin. The base pair change results in an amino acid substitution in a critical residue in the third zinc finger of Atmin. The consequence of this change is the failure to activate transcriptional targets of Atmin. This raised the possibility that previously unidentified Atmin target genes are important for ciliogenesis. Consistent with this hypothesis, Dynein light chain-LC8 (Dynll1) is downregulated in gpg6 mutants. LC8 (a homolog of mouse Dynll1) is required, in the single cell eukaryotic organism Chlamydomonas, for retrograde intraflagellar transport (IFT), a process crucial for ciliogenesis. These data led to the following hypothesis: Atmin activates expression of Dynll1, which functions in retrograde IFT to enable normal ciliogenesis. Knockdown of Atmin in a ciliated kidney cell line resulted in abnormally short cilia. Thus, Atmin functions in ciliogenesis. Investigation of gpg6 has therefore identified a novel role for Atmin in ciliogenesis and has added to the growing knowledge of genes that control cilia formation and embryonic development.

Paramecium tetraurelia is a ciliated single cell used as a model organism for the study of ciliopathies. Ciliopathies are mammalian diseases involving the dysfunction of cilia, including cilia maintenance, construction, and signaling. P. tetraurelia and its cilia provides an excellent non-canonical system for the investigation and elucidation of proteins important for the structure, maintenance and function of cilia and ciliary beating. We utilize features of this cell such as its 1000's of cilia and highly organized and patterned cell surface to observe changes in swimming behavior or disruptions in the ordered cell surface which are not feasible in mammalian cells. Here, we present research on three proteins in Paramecium, two of which are homologs to human ciliopathy genes. Using combinations of epitope-tagging, RNA interference (RNAi), immunofluorescence, immunoprecipitations, LC-MS/MS analysis and electrophysiology, we have attempted to elucidate the location, function, and potential interacting partners of these three proteins. The first protein, meckelin (MKS3), is a contributing factor in Meckel-Gruber syndrome, among other ciliopathies. Using epitope tagging, we identified the location of the Mks3 protein above each basal body. Depletion of MKS3 using RNAi leads to global loss of cilia, a severe disruption in the surface organization and a mislocalization of basal bodies out of the anterior-posterior axis of the cell. We show that depletion of Mks3 leads to abnormal backward swimming in ionic stimuli and depleted secretion of trichocysts. Based on our data, we propose two functions for Mks3 in P. tetraurelia. The first function is a transition zone component important for proper regulation of ciliary protein content, consistent with MKS3 function in other organisms. The depletion of MKS3 led to global ciliary loss, but also an imbalance in the ciliary ion channels that was different from the loss of cilia due to interference with intraflagellar transport as observed in cells depleted of IFT88. The second novel role for MKS3 is as a transient connection to the kinetodesmal fiber which is important for basal body guidance when daughter basal bodies migrate away from the mother basal body before cell division. We also examine the contribution of the non-selective cation channel Polycystin-2 (Pkd2) in Paramecium to Mg2+ permeability and Mg2+-induced behavior. When mutated in humans, Pkd2 leads to 15% of the cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD). When PKD2 is depleted using RNAi in Paramecium, cells show short backward swimming in Mg2+ solutions, a resistance to heavy metal paralysis, and depleted membrane permeability to Mg2+. The channel-like protein XntA which is unique to Paramecium and Tetrahymena, is also important for these phenotypes. Therefore, we utilized the Paramecium XntA1 mutant in our studies, which lacks Mg2+-induced behavior. We demonstrate that both Pkd2 and XntA are present in the cell membrane and in the cilia. Co-IP assays show that the IP of XntA-myc co-IPs the Pkd2-FLAG channel, but not vice versa, possibly because of an occluded FLAG epitope due to protein interactions. To tease apart the contributions of Pkd2 in the cilia and the cell membrane, electrophysiology was used to measure membrane potential of ciliated and deciliated cells. Depletion of BBS8 eliminates Pkd2 in the cilia, allowing us to examine Pkd2 activity restricted to the cell membrane of ciliated cells. Depletion of Pkd2 or XntA decreases membrane permeability to Mg2+. When Pkd2 was restricted to the cell membrane via BBS8 depletion, the membrane permeability to Mg2+ increased, much like over-expressing the Pkd2 protein. Depletion of Pkd2, especially in the deciliated XntA1 mutant, leads to a dramatic decrease in Mg2+ membrane permeability. Based on these data, we propose that Pkd2 is the Mg2+ channel in Paramecium and XntA is not a channel, but is perhaps important for stabilizing Pkd2 in membrane microdomains. We have uncovered novel function roles for the proteins mentioned here, leading to a broader understanding of their function. These studies also highlight to usefulness and importance of the model organism Paramecium tetraurelia to the study of human ciliopathy genes.

Les cils et les flagelles sont des organites cellulaires très conservés qui assurent des fonctions essentielles. Chez l'Homme, les défauts d'assemblage des cils et des flagelles conduisent à de multiples pathologies, les ciliopathies. Afin de comprendre comment se forment et fonctionnent les cils, j'ai analysé la fonction de deux nouveaux gènes identifiés comme cible des facteurs de transcription de ciliogenèse RFX. Tout d'abord je me suis focalisée sur le gène CCDC151, évolutivement conservé dans les espèces possédant des cils motiles. J'ai pu montrer que CCDC151 est impliquée dans le transport dépendant de l'IFT des bras de dynéine chez les animaux et qu'elle est nécessaire à la perception sensorielle chez la drosophile. Par ailleurs, j'ai également montré que cette protéine possède des fonctions cellulaires additionnelles puisqu'elle est requise pour l'orientation correcte des plans de division cellulaire et qu'elle est impliquée dans la régulation de la taille du cil primaire chez les mammifères. Je me suis ensuite intéressée au gène LRRC48 également conservée dans les espèces possédant des cils motiles. Cette protéine est nécessaire à la motilité des flagelles de spermatozoïdes et des cils des neurones sensoriels en 9+0 et dans la réponse auditive chez la drosophile. De plus LRRC48 est indispensable au développement des vertébrés puisque son absence chez le poisson zèbre conduit à l'hydrocéphalie, des kystes rénaux et des défauts de motilité des cils. Elle est également essentielle à la biogenèse de l'oreille dans cet organisme.En conclusion, il s'agit de deux nouveaux acteurs de la ciliogenèse potentiellement impliqués dans les pathologies ciliaires chez l'Homme / Cilia are highly conserved structures found from protozoa to mammals where they play essential physiological and developmental functions and cilia dysfunction leads to various syndromes in humans known as ciliopathies. To understand cilia formation and function, I performed functional analysis of two new target genes of the RFX ciliogenic transcription factors. First, I focused on CCDC151 that is evolutionary conserved in motile ciliated species. I showed that CCDC151 is involved in the control of IFT-dependent dynein arm assembly in animals and required for geotaxis behavior of adult flies. In zebrafish, depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts, two phenotypes resulting from impaired ciliary beating. However, I also showed that CCDC151 is also implicated in other cellular functions in vertebrates as it is involved in proper orientation of cell divisions and implicated in the regulation of primary cilium length in mammalian cells. In a second part, I studied LRRC48 that is also conserved in species with motile cilia. I showed that this protein is essential for motility of flagellar spermatozoids and for motility of the 9+0 sensory cilia as well as in the auditory response in drosophila. In zebrafish, morpholinos induced depletion of this protein leads to hydrocephaly, kidney cysts, inner ear abnormalities and cilia motility defects. Moreover this protein is also required for inner ear biogenesis in the model. In conclusion, these two genes are essential for ciliogenesis and they are new candidate genes potentially implicated in human ciliary diseases

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

Thesis advisor: John Wing / Thesis advisor: Stephen Wicks / Molecular biology and genetics, single nucleotide polymorphism genetic mapping, phenotypic assays including behavioral assessment, and fluorescent microscopy of GFP-tagged proteins were used to study ciliary defects in the nematode Caenorhabditis elegans. Mammalian cilia are multifunctional. Some of the physiological roles in which they are involved include sensing developmental signaling molecules and ligands as well as creating flows of mucus and cerebrospinal fluid that function as flow meters and mechanosensors. Due to the multifunctional nature of cilia, it is not surprising that many human diseases can be caused by ciliary defects. Bardet-Biedl Syndrome is a rare genetic ciliopathy characterized by retinal degeneration, polydactyly, obesity, cystic kidneys, mental retardation, and many other ailments. We have identified osm- 12/bbs-7 to be a C. elegans homologue of human BBS7, a gene known to cause Bardet-Biedl Syndrome when mutated. With the help of Michel Leroux’s group, I showed the BBS-7 protein to be localized to the base of cilia and to undergo intraflagellar transport along the ciliary axoneme. Our findings suggest that BBS- 7 plays a role in the assembly and/or functioning of the IFT complex. I also performed a mutagenesis and phenotypic screen for animals defective in the uptake of DiI into a subset of their ciliated neurons in order to identify new components involved in ciliogenesis and IFT. I describe an extended bulked segregant analysis (BSA) mapping methodology, which can save time and resources by filtering out alleles of previously known genes without performing time-consuming interval mapping. In addition, I identified one of the 11 dyefilling defective alleles from the screen to be a novel allele of dyf-3, which encodes a protein required for sensory cilia formation. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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 G&#945;olf, 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 G&#945;olf: 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 G&#945;olf, 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 G&#945;olf 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)

Les forces mécaniques dirigeant la valvulogénèse sont mal connues. Chez le poisson zèbre, le flux induit l’expression du facteur de transcription klf2a de façon endothéliale spécifique afin d’initier ce processus. Nous nous sommes intéressés aux mécanismes moléculaires activés par le flux et aboutissant à la formation des valves cardiaques. En altérant et modifiant le pattern fluidique, nous avons observé le rôle des forces engendrées par le flux pour le contrôle de l’expression de klf2a ainsi que du nombre de cellules endocardiques. Nous avons ensuite regardé les divers mécanosenseurs pouvant intervenir lors de ce processus. Ainsi nous avons mis à jour la présence de cils dans l’endocarde et fait la relation entre les canaux membranaires TRPP2 et TRPV4, la présence de calcium intracellulaire dans les cellules endocardiques et la voie moléculaire PKC-PKD2-HDAC5, composants nécessaires à l’expression du gène klf2a en réponse au flux. Ces données nous permettent de suggérer le rôle de TRPP2, TRPV4 et la voie moléculaire récemment découverte, PKC-PKD2-HDAC5 dans la formation valvulaire dépendante de klf2a. / Mechanical forces that dictate valve formation are not well understood. In zebrafish, blood flow induces expression of the transcription factor klf2a in a specific subset of endocardial cells that will go on to form functional valves. We aimed to identify the molecular mechanisms activating this flow-induced valve formation. By altering and modifying blood flow patterns, we observed that flow-mediated forces are necessary to control early klf2a expression and endocardial cell numbers. We then looked at different mechanosensors operating at early stages of valve development. Using in vivo labelling, we identified primary cilia in the endocardium and showed that the membrane channels TRPP2 and TRPV4 increase intracellular calcium which activates a PKC-PKD2-HDAC5 pathway necessary for klf2a expression in response to flow. Together these data suggest a role for TRPV4, TRPP2 and the recently described PKC-PKD2-HDAC5 signalling pathway in klf2a-mediated valvulogenesis.

Current sperm processing methods used in assisted reproductive technologies can cause damage to the sperm cell. New ways that mimic the natural guidance mechanisms present in the female genital tract may offer ways to sort sperm with better fertility parameters. Sperm that respond to these cues may have better over sperm health. Human sperm exhibit positive rheotaxis by orienting and swimming against the fluid released by the female genital tract. At certain flow rates sperm can actively orient and swim against the flow. Sperm retrieved that exhibit positive rheotaxis have higher motility and better morphology than the original semen sample. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection