In nematode Caenorhabditis elegans, the chronic and multi-generational toxicological effects of commercially relevant engineered nanoparticles (ENPs), such as quantum dots (QDs) and silver (AgNP) caused significant changes in a number of physiological endpoints. The increased water-solubility of ENPs in commercial products, for example, makes them increasingly bioavailable to terrestrial organisms exposed to pollution and waste in the soil. Since 2008, attention to the toxicology of nanomaterials in C. elegans continues to grow. Quantitative data on multiple physiological endpoints paired with metal analysis show the uptake of QDs and AgNPs, and their effects on nematode fitness. First, C. elegans were exposed for four generations through feeding to amphiphilic polymer coated CdSe/ZnS (core-shell QDs), CdSe (core QDs), and different sizes of AgNPs. These ENPs were readily ingested. QDs were qualitatively imaged in the digestive tract using a fluorescence microscopy and their and AgNP uptake quantitatively measured using ICP-MS. Each generation was analyzed for changes in lifespan, reproduction, growth and motility using an automated computer vision system. Core-shell QDs had little impact on C. elegans due to its metal shell coating. In contrast, core QDs lacked a metal shell coating, which caused significant changes to nematode physiology. In the same way, at high concentrations of 100 ppm, AgNP caused the most adverse effect to lifespan and reproduction related to particle size, but its adverse effect to motility had no correlation to particle size. Using C. elegans as an animal model allowed for a better understanding of the negative impacts of ENPs than with cytotoxicity tests. Lastly, to test the toxicity of water-dispersed fullerene (nanoC60) using human dermal fibroblast cells, this thesis investigated a suite of assays and methods in order to establish a standard set of cytotoxicity tests. Ten assays and methods assessed nanoC60 samples of different purities to show differences in cytotoxic effects. Washed samples of fullerenes, with negligible traces of THF and other impurities, rendered the solution nontoxic. Even when exposed to UV-irradiation, washed nanoC60 were not photosensitized and did not cause cellular death. This work characterizes ENPs and investigates their impact in C. elegans and cells to assess toxicity risks to the environment and to human health.

ALENCAR, Luciana Magalhães Rebêlo. Estudo da viscoelasticidade de células de câncer renal por microscopia de força atômica. 2010. 155 f. Tese (Doutorado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2010. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2015-05-22T18:40:21Z No. of bitstreams: 1 2010_tese_lmralencar.pdf: 15264548 bytes, checksum: e3df53eb49036e78f623d089ab7e0995 (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2015-05-22T18:42:17Z (GMT) No. of bitstreams: 1 2010_tese_lmralencar.pdf: 15264548 bytes, checksum: e3df53eb49036e78f623d089ab7e0995 (MD5) / Made available in DSpace on 2015-05-22T18:42:17Z (GMT). No. of bitstreams: 1 2010_tese_lmralencar.pdf: 15264548 bytes, checksum: e3df53eb49036e78f623d089ab7e0995 (MD5) Previous issue date: 2010 / The mechanical properties of living cells have a crucial role in the accomplishment of their physiological functions. However, our knowledge on this subject is still limited. Is not fully understood how a cell responds structurally and mechanically to an external pressure or as the elasticity of cells is altered in diseased organisms compared to healthy ones. Recently, the biomechanics of cancer cells, in particular the elasticity or stiffness, has been identified as an important factor that is related to function, adhesion, motility, invasion and transformation of the neoplastic cells. Studies in vivo show that cancerous transformations introduce significant changes in the structure and behavior of cells. These differences can cause changes in mechanical properties, often leading to greater cell deformability. Quantifying the change of elasticity using mechanical tests in conjunction with a microscopic examination, can become a powerful method for the diagnosis of cancer, and open new routes for treatments. In this context, Atomic Force Microscopy (AFM) is presented as an ideal tool for cell research due to its high resolution capability for surface nano-manipulation, ability to work in fluids and for being a noninvasive and nondestructive technique. This study investigates the mechanical response of cancer cells (lines A-498 and ACHN), compared to normal cells (RC-124). Using an AFM and its components as a morphological tool of high resolution characterization and characterization of the cells mechanical properties using the AFM probe as a nano-indenter, and from the strength data obtained by the microscope, and appropriate theoretical models to interpret these data to obtain qualitative and quantitative values of the elastic response these cells. / As propriedades mecânicas de células vivas possuem um papel crucial no bom desempenho de suas funções fisiológicas. Porém, nosso conhecimento nesse assunto ainda é limitado. Não é totalmente compreendido como uma célula responde, estrutural e mecanicamente, a uma tensão externa ou como a elasticidade das células altera-se em organismos doentes em comparação a organismos sadios. Recentemente, a biomecânica de células do câncer (em particular, a elasticidade ou rigidez) tem sido apontada como um fator importante que está relacionado à função, adesão, motilidade, transformação e invasão da célula neoplásica. Estudos in vivo mostram que transformações cancerosas introduzem alterações significativas na estrutura e comportamento celular. Essas diferenças também podem causar alterações nas propriedades mecânicas, geralmente levando a uma maior deformabilidade da célula. A quantificação da alteração de elasticidade, utilizando ensaios mecânicos em conjunto com um exame microscópico, pode tornar-se um poderoso diagnóstico do câncer e abrir caminhos para novos tratamentos. Neste contexto, a Microscopia de Força Atômica (AFM) se apresenta como uma ferramenta ideal para a investigação de células por sua alta resolução, capacidade de nano-manipulação de superfícies, possibilidade de trabalhar em meios líquidos e por ser uma técnica não destrutiva. Neste trabalho, propõe-se a investigação da resposta mecânica de células cancerígenas (linhagens A-498 e ACHN), comparando-se com células normais (RC-124), utilizando-se um Microscópio de Força Atômica e seus componentes como ferramentas de caracterização morfológica de alta resolução e caracterização das propriedades mecânicas dessas células. Utilizando a sonda de AFM como nano-indentador e a partir dos dados de força obtidos pelo microscópio, analisados por meio de modelos teóricos adequados, temos por objetivo obter valores qualitativos e quantitativos da resposta elástica dessas células.

No description available.

Ever increasing applications of nanomaterials (materials with one or more dimension less than 100 nm) has raised awareness of their potential genotoxicity. They have unique physico-chemical properties and so could have unpredictable effects. Zinc oxide (ZnO) and titanium dioxide (TiO2) are widely used in a number of commercial products. There are published studies indicating that some forms of these compounds may be photo-clastogenic in mammalian cells. What has not been investigated before is the effect of nanoparticles from these compounds in human germ cells. Thus the present study has examined their effects in the presence and absence of UV light in human sperm and compared responses to those obtained with human lymphocytes using the Comet assay to measure DNA damage. The effect of nanoparticles (40-70nm range) was studied in human sperm and lymphocytes in the dark, after pre-irradiation with UV and simultaneous irradiation with UV. The studies do provide some evidence that there are photo-genotoxic events in sperm and lymphocytes in the absence of overt toxicity. The cytotoxic and genotoxic potentials of ZnO and TiO2 as well as their effect on phosphotyrosine expression, were examined in the human epithelial cervical carcinoma cells (Hela cells). This was done to try and determine the underlying molecular events resulting from their exposure to ZnO and TiO2 nanoparticles occurring at the same time as DNA is damaged. Concentration- and time-dependent cytotoxicity, and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations were reported in this study. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Nanotechnology has raced ahead of nanotoxicology and little is known of the effects of nanoparticles in human systems, let alone in diseased individuals. Therefore, the effects of TiO2 nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases (lung cancer, chronic obstructive pulmonary disease (COPD) and asthma) were compared with those in healthy individuals using genotoxic endpoints to determine whether there are any differences in sensitivity to nano-chemical insult between the patient and control groups. The results have shown concentration dependent genotoxic effects of TiO2 in both respiratory patient and control groups in the Comet assay and an increasing pattern of cytogenetic damage measured in the micronucleus assay without being statistically significant except when compared with the untreated controls of healthy individuals. Furthermore, modulation of ras p21 expression was investigated. Regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels that showed modulation as the result of nanoparticle treatment. Results have suggested that both ZnO and TiO2 nanoparticles can be genotoxic over a range of concentrations without either photoa-ctivation or being cytotoxic.

La biogenèse des ribosomes eucaryotes est un processus complexe qui implique la production et l'assemblage de 4 ARNr et 80 protéines. La production des deux sous-unités du ribosome, 40S et 60S, débute dans le nucléole par la synthèse d'un long précurseur commun contenant les séquences des ARNr matures et se termine dans le cytoplasme où ont lieu les dernières étapes d'assemblage des protéines ribosomiques et de clivage des ARNr. La production de ribosomes nécessite la participation de plus de 200 co-facteurs, qui catalysent les clivages et modifications des ARNr, coordonnent leur repliement et leur association aux protéines ribosomiques, et assurent des étapes de contrôle-qualité. Ces protéines sont associées aux particules en cours de maturation et absentes des sous-unités matures. Cette voie de synthèse, globalement conservée chez les eucaryotes, a été principalement étudiée chez la levure. Cependant, des études récentes ont montré des différences importantes de ce processus entre levure et mammifères. Un des verrous importants pour comprendre la fonction des co-facteurs, est l'absence de données sur la structure des précurseurs des sous-unités ribosomiques. J'ai donc entrepris une étude structurale de l'assemblage cytoplasmique de la petite sous-unité ribosomique chez l'homme par cryo-microscopie électronique à transmission. Le but de ma thèse était de déterminer la structure 3D des précurseurs de la petite sous-unité ribosomique purifiés à différentes étape de leur maturation. Ce travail a été conduit en collaboration avec l'équipe du Pr. Ulrike Kutay (ETH Zurich) pour la purification des particules pré-40S à partir de cellules humaines. La première structure 3D de particule pré-40S intermédiaire purifiée en étiquetant le co-facteur LTV1 a été déterminée à 19Å de résolution. Dans un deuxième temps, la structure 3D de la particule pré-40S tardive purifiée à via RIO1(KD) a aussi été déterminée à 15Å de résolution. Ces données nous ont permis de proposer un modèle de localisation des co-facteurs sur les précurseurs de la petite sous-unité ribosomique et de montrer une nouvelle différence dans la formation de la petite sous-unité chez l'Homme comparé à la levure, du fait de la présence de la protéine RACK1 sur les particules pré-40S humaines. La comparaison des structures des précurseurs de la petite sous-unité obtenues a permis de mettre en lumière l'existence de remodelages structuraux de la particule pré-40S au cours de sa maturation. Ce travail met en lumière les premières structures 3D de particules pré-40S humaines et pose les fondements méthodologiques d'explorations futures de la dynamique structurale des particules pré-ribosomiques. / Ribosome biogenesis is a complex process that requires the production and the correct assembly of the 4 rRNAs with 80 ribosomal proteins. In Human, the production of the two subunits, 40S and 60S, is initiated by the transcription of a pre-ribosomal rRNA precursor to the mature 18S, 5.8S, and 28S rRNAs by the RNA polymerase I, which is chemically modified and trimmed by endo- and exoribonuclease, in order to form the mature rRNAs. The nascent pre rRNA associated with ribosomal proteins, small ribonucleoprotein particles (snoRNP) and so called co-factors leading to the assembly of an initial 90S particle. This particle is then split into pre-40S and pre-60S pre-ribosomal particles that fallow independent maturation to form the mature subunit into the cytoplasm. Production of eukaryotic ribosomes implies the transient intervention of more than 200 associated proteins and ribonucleoprotein particles, that are absent from the mature subunits. Synthesis of ribosome, globally conserved in eukaryotes, has been principally studied in yeast. However, recent studies reveal that this process is more complex in human compared in yeast. An important bottleneck in this domain is the lack of structural data concerning the formation of intermediate ribosomal subunits to understand the function of assembly factors. Determination of the structural remodeling of pre-ribosomal particles is crucial to understand the molecular mechanism of this complex process. So I have undertaken a structural study on the assembly of the small ribosomal subunit using cryo-electron microscopy and image analysis. The goal of my thesis is to determine the 3D structures of human pre-40S particles at different maturation stages to see the structural remodeling that occurs during the biogenesis of the small ribosomal subunit. We are collaborating with the group of Pr Ulrike Kutay at ETH Zurich, who purify human pre-40S particles. The 3D structures of human pre-40S particles purified at an intermediate and late maturation stages, has been determined with a resolution of 19 and 15Å respectively. Supplementary densities, compared to the mature subunit, indicate the presence of assembly factors and show the unexpected presence of the RACK1 protein in the precursor of the human small ribosomal subunit in the cytoplasm. The comparison of the 3D structures of human pre-40S particle allows showing the structural remodeling that occur during the maturation of the small ribosomal subunit. This work provides the first 3D structure of human pre-40S particles and laid the methodological foundations for future exploration of the structural dynamics of pre-ribosomal particles.

Ever increasing applications of nanomaterials (materials with one or more dimension less than 100 nm) has raised awareness of their potential genotoxicity. They have unique physico¿chemical properties and so could have unpredictable effects. Zinc oxide (ZnO) and titanium dioxide (TiO2) are widely used in a number of commercial products. There are published studies indicating that some forms of these compounds may be photo-clastogenic in mammalian cells. What has not been investigated before is the effect of nanoparticles from these compounds in human germ cells. Thus the present study has examined their effects in the presence and absence of UV light in human sperm and compared responses to those obtained with human lymphocytes using the Comet assay to measure DNA damage. The effect of nanoparticles (40-70nm range) was studied in human sperm and lymphocytes in the dark, after pre-irradiation with UV and simultaneous irradiation with UV. The studies do provide some evidence that there are photo-genotoxic events in sperm and lymphocytes in the absence of overt toxicity. The cytotoxic and genotoxic potentials of ZnO and TiO2 as well as their effect on phosphotyrosine expression, were examined in the human epithelial cervical carcinoma cells (Hela cells). This was done to try and determine the underlying molecular events resulting from their exposure to ZnO and TiO2 nanoparticles occurring at the same time as DNA is damaged. Concentration- and time-dependent cytotoxicity, and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations were reported in this study. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Nanotechnology has raced ahead of nanotoxicology and little is known of the effects of nanoparticles in human systems, let alone in diseased individuals. Therefore, the effects of TiO2 nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases (lung cancer, chronic obstructive pulmonary disease (COPD) and asthma) were compared with those in healthy individuals using genotoxic endpoints to determine whether there are any differences in sensitivity to nano-chemical insult between the patient and control groups. The results have shown concentration dependent genotoxic effects of TiO2 in both respiratory patient and control groups in the Comet assay and an increasing pattern of cytogenetic damage measured in the micronucleus assay without being statistically significant except when compared with the untreated controls of healthy individuals. Furthermore, modulation of ras p21 expression was investigated. Regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels that showed modulation as the result of nanoparticle treatment. Results have suggested that both ZnO and TiO2 nanoparticles can be genotoxic over a range of concentrations without either photoa-ctivation or being cytotoxic.

Les ribosomes sont des macrocomplexes ribonucléoprotéiques sophistiqués, essentiels pour décoder l’information génétique et la traduire en protéines fonctionnelles. Chez les organismes eucaryotes, le ribosome est constitué de deux sous-unités, la petite (40S) et la grande (60S). Leur biogenèse est un processus fondamental, très complexe, qui mène à la synthèse et l’assemblage de 4 ARNr et 80 protéines ribosomiques (79 chez la levure). La biogenèse du ribosome a longtemps été étudiée chez Saccharomyces cerevisiae. Près de 20 ans de recherches ont été nécessaires à la communauté scientifique pour identifier les quelques 200 facteurs de synthèse du ribosome levurien. Alors que le schéma global de cette voie de biosynthèse semble conservé chez les organismes eucaryotes, de nombreux éléments suggèrent qu’elle serait plus élaborée chez l’homme et nécessiterait un plus grand nombre de facteurs que chez la levure. De plus, la caractérisation de nombreuses ribosomopathies, ou maladies du ribosome prédisposant aux cancers, a suscité un intérêt accru pour l’étude de la voie de biosynthèse du ribosome dans le paradigme expérimental le plus approprié, la cellule humaine.<p><p>Au cours de ma thèse de doctorat, j’ai contribué à un projet systématique d’identification de facteurs d’assemblage (FA) du ribosome chez l’homme. Pratiquement, nous avons identifié 286 FA humains, dont beaucoup sont homologues aux facteurs levuriens connus, et 74 sont sans équivalent chez la levure. Par ailleurs, j’ai caractérisé en détail certains facteurs. En particulier, Trm112 pour lequel j’ai montré qu’il agit comme un stabilisateur de la méthyltransférase (MTase) Bud23, spécifique à l’ARNr 18S de la sous-unité levurienne 40S. J’ai également participé à la caractérisation de mutations à l’interface du complexe Bud23-Trm112. Enfin, j’ai contribué à l’étude de trois FA que nous avons identifiés chez l’homme, DIMT1L et WBSCR22-TRMT112. J’ai montré que ces protéines sont les orthologues des MTases levuriennes Dim1 et Bud23-Trm112, qu’elles sont requises pour la synthèse et la modification de l’ARNr mature de la petite sous-unité ribosomique, et qu’elles seraient impliquées dans un mécanisme conservé contrôlant la qualité de la voie de biosynthèse du ribosome.<p><p>La totalité des FA que nous avons identifiés en cellule humaine sont à la disposition de la communauté scientifique dans une base de données en ligne accessible sur la page www.RibosomeSynthesis.com. Nous espérons que cette ressource contribuera à une meilleure compréhension des mécanismes moléculaires sous-jacents au développement des ribosomopathies et à l’élaboration d’agents thérapeutiques efficaces.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Les cardiolipines (CL) sont des phospholipides possédant de nombreux rôles dans la structure et le fonctionnement des mitochondries. Elles sont, par exemple, impliquées dans la stabilisation des complexes des oxydations phosphorylantes, la fusion/fission des membranes mitochondriales, l’import de protéines mitochondriales, la biogénèse des centres fer-soufre (Fe-S), l’apoptose, la protection des mitochondries contre le stress oxydatif…L’ensemble de ces fonctions nécessitent que les chaînes d’acides gras de la CL soient majoritairement insaturées. Le maintien de cette composition en chaînes insaturées requiert une activité acyltransférase portée par la protéine tafazzine, qui est codée par le gène nucléaire TAZ. Des mutations dans ce gène sont la cause du syndrome de Barth (BTHS), qui se caractérise notamment par des myopathies cardiaques et squelettiques, une neutropénie (responsable de nombreuses infections) et des défauts de la chaîne respiratoire. Malgré des progrès considérables dans la compréhension des mécanismes conduisant à la pathogénicité, il n’existe toujours aucune thérapie pour traiter cette maladie. Nous avons donc utilisé la levure Saccharomyces cerevisiae, chez qui la voie de remodelage des CL par la tafazzine est bien conservée, pour modéliser le BTHS et, ainsi non seulement étudier les mécanismes moléculaires sous-jacents de cette maladie, mais aussi identifier différentes voies thérapeutiques potentielles (suppresseurs génétiques et molécules pharmacologiques). Nous avons tout d’abord construit une levure délétée pour le gène orthologue TAZ (TAZ1 chez la levure), la souche Δtaz1. En accord avec des études précédentes, la souche Δtaz1 présente une diminution quantitative de la CL accompagnée d’un changement qualitatif des chaînes d’acides gras1,2 (plus d’acides gras saturés et moins d’insaturés). Nous montrons aussi que cette levure mutante a un défaut de croissance en milieu respiratoire à température élevée (36°C) ainsi que des défauts dans plusieurs composants impliqués dans les oxydations phosphorylantes2. De façon intéressante, alors que le défaut primaire (diminution des CL et changement qualitatif des chaines d’acide gras) est toujours présent, nous montrons que les oxydations phosphorylantes sont restaurées dans la souche Δtaz1 surexprimant Odc1p2, un transporteur mitochondrial d’intermédiaires du cycle de Krebs, ou par plusieurs composés chimiques. Plusieurs de ces drogues sauvant le mutant, dont la cycloheximide, sont des inhibiteurs partiels de la synthèse protéique cytosolique. Cet effet a été confirmé génétiquement par des mutations affectant les ribosomes cytosoliques. L’ensemble des résultats suggère qu’un défaut au niveau des CL provoquerait un stress protéostatique probablement impliqué dans le processus pathologique. / The phospholipid cardiolipin (CL) has many roles in mitochondrial structure and function, ranging from assembly/stability and functioning of the oxidative phosphorylation (OXPHOS) system, fusion and fission of mitochondrial membranes, mitochondrial protein import, iron-sulfur (Fe-S) biogenesis, apoptosis, and protection of mitochondria against oxidative damage. The maintenance of a proper unsaturated acyl chain composition of CL involves the acyltransferase tafazzin in which mutations cause Barth syndrome (BTHS), resulting in cardiac and skeletal myopathy, cyclic neutropenia and respiratory chain defects. Despite considerable progress in the understanding of the underlying pathogenic mechanisms, there are still no effective therapies to treat this disease. We are using the yeast Saccharomyces cerevisiae, in which the tafazzin-based cardiolipin remodeling pathway is conserved, as a model system for the exploration of potential therapeutic pathways against BTHS, by way of genetic suppressors and chemical screening. We first constructed a yeast strain lacking the orthologous taffazin gene (Δtaz1). Consistent with previous studies, our Δtaz1 yeast failed to grow on non-fermentable carbon sources at elevated temperatures (36°C) and exhibited defects in several components of the mitochondrial respiratory system. Interestingly, we found that oxidative phosphorylation was fully restored in Δtaz1 yeast by overexpressing Odc1p [1]-a mitochondrial carrier that transports Krebs cycle intermediates- and by a number of chemical compounds. Some of the rescuing drugs, especially cycloheximide, act by partially inhibiting cytosolic protein synthesis leading to a full recovery of oxidative phosphorylations. Our findings identify potential cellular components and pathways for the pharmacological treatment of BTHS patients.

Telomeres are nucleoprotein structures that cap the physical ends of eukaryotic chromosomes. They consist of repetitive DNA sequences 5’-TTAGGG-3’ assembled with proteins which form the shelterin complex. This complex protects the ends of chromosomes by inhibiting DNA repair pathways at telomeres and avoid their recognition as double-strand breaks. Telomeres have been identified as a transcriptionally silent zone until 2007 when a noncoding RNA called TERRA (TElomeric Repeat containing RNA) transcribed from telomeres was discovered. This RNA gave rise to many questions: How is TERRA regulated? How is TERRA expressed? Does TERRA interact with proteins, DNA or RNA? After several studies, we know that TERRA is frequently expressed in cancer cells and it interacts with a large proteome. Nevertheless, its specific function remains unknown. In this thesis, we studied this RNA in human cancer cells using live-cell microscopy which allowed us to get information on TERRA’s dynamics, localization and its interactome. Moreover, we used single-molecule imaging on TERRA 15q labeled by the MS2-GFP system, which allowed the visualization of TERRA transcripts. This study resulted in the discovery of two types of TERRA population from telomere 15q: one of the population is characterized by the formation of clusters and a second population is constituted of unique molecules more dynamic in the nucleus. Finally, in order to better understand TERRA’s functions, we developed a new approach which consists on immunoprecipitating TERRA using the MS2 stem-loops as a tag to identify TERRA-interacting proteins such as the telomeric factor TRF2 or RNA-binding proteins like hnRNP -A1 or FUS. / Les télomères forment les extrémités des chromosomes chez les eucaryotes. Ces séquences répétées en tandem 5’-TTAGGG-3’ font partie d’un complexe nucléoprotéique appelé shelterin. En effet, cet assemblage de protéines télomériques permet la protection des extrémités des chromosomes, permettant à celles-ci de ne pas être reconnues comme des cassures dans l’ADN et d’activer les voies de réparation de l’ADN. Les télomères ont longtemps été reconnus comme étant des zones de transcription inactives, ce jusqu’en 2007 lorsqu’une équipe de recherche découvrit un ARN non codant appelé TERRA (Telomeric Repeat containing RNA). Ce dernier a suscité de nombreuses questions : quel est le rôle de cet ARN? Comment est-il exprimé et régulé? Interagit-il avec d’autres facteurs cellulaires? Les différentes recherches menées sur cet ARN ont permis de conclure que celui-ci était fréquemment induit dans les cellules de cancer, que ses partenaires d’interactions sont nombreux, mais que ses fonctions restent encore mal définies. Par ailleurs, ces différentes études ont toujours été ou presque réalisées sur des cellules fixées, sur une population totale d’ARN télomérique TERRA. Afin d’apporter de nouvelles réponses et de mieux caractériser cet ARN, nous avons étudié ce transcrit dans des cellules de cancer humain en utilisant la technique de microscopie en temps réel, qui permet de récolter des données sur la dynamique, la localisation de cet ARN et ses éventuels partenaires. De plus, nous nous sommes intéressés à des molécules uniques de TERRA issues du télomère 15q en exploitant la technique de marquage avec des tiges-boucles MS2 (MS2-GFP). Cette étude de microscopie a permis de découvrir deux types de population de l’ARN TERRA 15q : une population caractérisée par des assemblages d’ARN dit clusters (agrégats d’ARN) et une population plus singulière qui semble avoir une diffusion plus importante dans le noyau de la cellule. Par ailleurs, l’expression de l’ARN TERRA semble être différente d’un type cellulaire à un autre et nous avons donc cherché à connaître le niveau d’expression de cet ARN au sein de la lignée étudiée au cours de ce projet de recherche. Enfin, afin de découvrir de nouveaux rôles pour cet ARN, nous avons développé une approche de co-immunoprécipitation de l’ARN TERRA pour identifier des interactions avec des protéines du complexe shelterin comme TRF2, ou des protéines liant l’ARN comme hnRNP-A1 ou encore FUS.

Ma thèse a porté sur des complexes de polyélectrolytes en solution et en films LbL pour la transfection de cellules et le contrôle des interactions cellule-surface. Il est possible de doser un agent de transfection et de l'ADN plasmidique dans des films LbL en ajustant le nombre de couches. Les efficacités de transfection avec différentes lignées cellulaires ont été au moins aussi bonnes que celles rapportées dans la littérature, mais sont restées globalement faibles. Différents nanobags ont également été systématiquement testés menant à un protocole de transfection très efficace avec une faible cytotoxicité pour des fibroblastes humains qui sont difficiles à transfecter. Nous avons pu identifier les architectures LbL qui permettent de contrôler l'adhésion cellulaire même en présence de sérum. Cela nous a permis d'introduire une nouvelle technique pour le suivi in situ de la transfection par QCM-D en suivant la mobilité du cytosquelette qui sera poursuivie dans un futur projet. / My PhD work was focused on polyelectrolyte complexes in bulk and in LbL-films for cell transfection and for controlling cell-surface interactions. It is possible to dose transfection agent and plasmid DNA in LbL-films by adjusting the number of layers. Transfection efficiencies with different cell lines were at least as good as reported in the literature, but remained overall weak. Different nanobags were also tested systematically leading to a highly efficient transfection protocol with low cytotoxicity for human fibroblasts which are difficult to transfect. We were able to identify multilayer architectures that allow to control cell adhesion even in the presence of serum. This allowed us also to introduce a new technique for the in-situ monitoring of transfection by QCM-D through monitoring cytoskeleton mobility which will be further pursued in a future research project.