Ces dernières années, de nombreuses études transcriptomiques à grande échelle ont clairement mis en évidence que la grande majorité du génome des eucaryotes est transcrite.Ce réseau complexe de transcrits inclus des petits ARN non codants qui interviennent généralement en tant que régulateurs transcriptionnels, post-transcriptionnels et/ou traductionnels de l’expression de certains ARNm cibles spécifiques. Ils sont classés selon leur origine biologique et leur mode d’action. Une catégorie de petits ARN non codants, les Piwi-interacting RNAs (piRNA), maintient l’intégrité du génome dans les tissus reproducteurs des métazoaires en réprimant les éléments transposables endogènes, des séquences ADN capables de se déplacer et de se dupliquer à l’intérieur du génome. Les piRNA sont produits par deux mécanismes : i) La biogenèse primaire à partir de longs ARN simple brin produits par certains loci spécifiques du génome, les clusters de piRNA, des loci énigmatiques, localisés dans les régions hétérochromatiques et composés de fragments d’éléments transposables actifs, ii) La boucle d’amplification appelée ping-pong. Durant ma thèse, j’ai étudié un cluster de piRNA majeurs dans les cellules somatiques des gonades femelles de Drosophila melanogaster, le locus flamenco. Tout d’abord, j’ai mis en évidence que la transcription de flamenco est initiée à partir d’un promoteur contenant une séquence INR et un élément DPE, reconnu par l’ARN polymerase II, et qu’elle nécessite la présence du facteur de transcription Cubitus Interruptus. Ensuite, j’ai montré que le transcrit de flamenco subit de l’épissage alternatif pour générer divers précurseurs ARN qui seront ensuite maturés en piRNA. De plus, j’ai montré que le promoteur de flamenco serait suffisant pour déclencher l’adressage du transcrit vers la voie de maturation des piRNA. Dans un autre axe, je me suis intéressée à l’organisation tridimensionnelle du locus flamenco au sein du noyau en recherchant ses partenaires d’interaction en utilisant la technique de 4C (capture de la conformation des chromosomes). J’ai pu voir que flamenco semble interagir physiquement avec des régions génomiques fortement transcrites en cis. En trans, le locus flamenco interagit majoritairement avec des régions génomiques péricentromériques et avec d’autres clusters de piRNA. Cette disposition tridimensionnelle particulière pourrait être le reflet d’une organisation fonctionnelle.Dans l’ensemble, ces travaux permettent de mieux comprendre l’expression et le fonctionnement du locus flamenco et ouvrent la voie vers de nouvelles recherches prometteuses. / The past few years it has become clear from many transcriptomic studies that most of the eukaryotic genome is pervasively transcribed. This complex network of transcripts include several types of small RNAs classified as non-coding RNAs. The vast majority of small RNA act as transcriptional, posttranscriptional and/or translational regulators, controlling specific target mRNAs involved in various cellular functions. They are classified based on their biogenesis and mode of action. A subclass of small non-coding RNAs, the Piwi-interacting RNAs (piRNA), ensures genomic stability by silencing endogenous transposable elements, endogenous sequence that are able to move and duplicate into the genome, in both germline and somatic gonadal tissues of metazoan. piRNA are produced through two mechanisms, i) The primary processing pathway from long single-stranded precursors produced by some specific loci in the genome, the piRNA clusters, ii) The secondary pathway by the amplification loop called the ping-pong. piRNA clusters are enigmatic loci localized in heterochromatic region and composed of transposable element fragments.During my PhD, I studied a major piRNA cluster in the somatic cells of Drosophila melanogaster female gonads, the flamenco locus. First, I demonstrated that flamenco transcription is initiated from an RNA Polymerase II promoter containing Inr and DPE elements, and requires the transcription factor, Cubitus interruptus. Then, I showed that the flamenco precursor transcript undergoes differential alternative splicing to generate diverse RNA precursors that are processed into piRNA. Moreover, I showed that the flamenco promoter could be sufficient to target transcripts into the piRNA processing pathway. In an other hand, I was interested to the tridimensional nuclear organization of the flamenco locus using the 4c technology. I saw that the flamenco locus interacts physically with strongly transcribed genomic region in cis. In trans, the flamenco interacts with other peri-centromeric genomic regions and with two other piRNA cluster. This particularly three-dimensional positioning could be the reflect of a functional organization.In the main, this work allows to better understand the expression and the mode ofaction of the flamenco locus and paves the way for new promising research.

Orientadores: Angela Maria Moraes, Paulo de Tarso Vieira e Rosa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-14T08:50:13Z (GMT). No. of bitstreams: 1 Egydio_JulianaAndrade_M.pdf: 10367421 bytes, checksum: 38511c53be9bd466f1d1a050abd0a1e7 (MD5) Previous issue date: 2009 / Resumo: Nos últimos anos o cultivo de células animais tornou-se uma importante tecnologia de produção de vacinas e proteínas terapêuticas recombinantes. Durante o crescimento in vitro de células animais, alterações nas condições ambientais ótimas do cultivo podem resultar em perda de viabilidade. Uma forma de controlar a apoptose, que é um processo de morte celular programado, é a inclusão de agentes antioxidantes no meio de cultura. O licopeno, cuja principal fonte é o tomate, tem despertado crescente interesse devido às suas propriedades antioxidantes e por apresentar atividade de proteção contra o câncer e doenças do coração. A caracterização da atividade biológica do licopeno in vitro é dificultada pelo fato de que solventes potencialmente tóxicos são empregados para sua obtenção. Assim, a extração de licopeno de tomate empregando CO2 supercrítico, consiste em uma opção atraente. Neste trabalho desenvolveu-se um processo de obtenção de licopeno de suco de tomate a partir de extração com CO2 supercrítico, em diferentes condições de temperatura e pressão, sem a etapa prévia de secagem do material, mas pela substituição de parte da água por etanol. Após a caracterização do extrato quanto à atividade antioxidante, a aplicabilidade do extrato como agente antiapoptótico foi avaliada no cultivo de células animais, especificamente da linhagem Drosophila melanogaster S2. Foram obtidos rendimentos na faixa de 7,7% a 76,9%, com efeito estatisticamente significativo apenas para a temperatura. Através da análise por cromatografia líquida de alta eficiência (CLAE), observou-se que o licopeno é o principal composto presente nos extratos obtidos, com picos de 82,7% em média. O estudo da atividade antioxidante indicou que o extrato obtido à 40 ºC e 350 bar apresenta maior capacidade antioxidante, de 49,7% pelo método do DPPH_ e 38,2% pelo método do rubreno. Nos ensaios de citotoxicidade, o mesmo extrato não apresentou efeito citotóxico às células S2 em concentrações inferiores a 0,182 mg/mL quando disperso no solvente DMSO, entretanto quando disperso em THF, mostrou-se citotóxico às células. O extrato dissolvido em DMSO apresentou elevado potencial para ser utilizado como agente antiapoptótico no cultivo de células S2, resultando em proteção superior a 92% contra a apoptose induzida por H2O2. / Abstract: In recent years the cultivation of animal cells has become an important technology for the production of vaccines and therapeutic recombinant proteins. During in vitro culture of animal cells, changes in optimum environmental conditions may result in loss of viability. One way to control apoptosis, a process of programmed cell death is the inclusion of antioxidants in the culture medium. Lycopene, whose main source is tomatoes, has attracted increasing interest due to its antioxidant activity and because it can provide protection against cancer and heart diseases. The characterization of the biological activity of lycopene in vitro is complicated by the fact that potentially toxic solvents are used for their production. Thus, the extraction of lycopene from tomatoes using supercritical CO2 is an attractive option. In this work, a process was developed for obtaining lycopene from tomato juice by extraction with supercritical CO2 at different conditions of temperature and pressure, without the prior step of drying the material, but replacing water by ethanol. After characterizing the extract regarding its antioxidant activity, the applicability of the extract as an antiapoptotic agent was evaluated for the cultivation of animal cells, specifically the Drosophila melanogaster S2 cell line. Yields in the range of 7.7% to 76.9% were observed, with statistically significant effect only for the temperature. The analysis by high performance liquid chromatography (HPLC) indicated that lycopene was the main compound present in the extracts obtained, with peaks of 82.7% in average. The extract obtained at 40 ºC and 350 bar showed the highest antioxidant capacity, equal to 49.7% by the DPPH_ method and 38.2% by the rubrene method. The same extract showed no cytotoxic effect on S2 cells concentrations up to 0.182 mg/mL when dispersed in the solvent DMSO, however, when dispersed in THF, it proved to be cytotoxic to cells. The extract dissolved in DMSO showed strong potential to be used as an antiapoptotic agent in the culture of S2 cells, resulting in protection above 92% against apoptosis induced by H2O2. / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Orientador: Louis Bernard Klaczko / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T02:56:34Z (GMT). No. of bitstreams: 1 Silva_LauraHelenaHafnerda_M.pdf: 2141945 bytes, checksum: 17c00d0d744de62b04cc274305caedb5 (MD5) Previous issue date: 2006 / Resumo: O presente trabalho tem como objetivo caracterizar a variação do tamanho e forma das asas de populações de Drosophila melanogaster em três pontos ao longo de uma grande amplitude latitudinal na costa brasileira. O trabalho foi feito a partir de coletas realizadas no Recife, Rio de Janeiro e Porto Alegre, e os seguintes aspectos foram abordados: 1) variação geográfica; 2) variação temporal; 3) herdabilidade; e 4) a influência de inversões cromossômicas. Para este fim, o método da elipse foi aplicado a imagens digitalizadas das asas, e foram analisados: o tamanho das asas, sua forma e as posições dos pontos de junção e das extremidades das veias (caracterizadas por suas coordenadas angulares é radiais, essas padronizadas pelo tamanho da asa). Os resultados obtidos mostraram que a variação de tamanho em D. melanogaster no Brasil segue a tendência mundial de formação de clines latitudinais, com indiv.íduos maiores sendo encontrados a latitudes também maiores. No entanto, a herdabilidade e a variação temporal entre múltiplas coletas realizadas no Recife e no Rio de Janeiro não apresentou um padrão regular nítido. O único efeito consistente e significativo de inversões cromossômicas que pudemos observar foi o de In(3R)Payne sobre o tamanho corporal, sendo também consistente com achados prévios descritos na literatura. Entretanto, não detectamos efeito significativo de interação genótipo-ambiente, quer entre coletas, quer entre localidades / Abstract: The present work aims to characterize the variation of wing size and shape in Drosophila melanogaster populations from three localities distributed along a wide latitudinal range of the Brazilian coast. The work was performed based on collections made in the cities of Recife, Rio de Janeiro and Porto Alegre. The aspects studied were: 1) geographic variation; 2) temporal variation; 3) heritability; and 4) the influence of chromosomal inversions. To this end, the ellipse method was applied to digitized images of the wings. We analyzed wing size, wing shape and the position of vein junctions and extremities (characterized by their angular and radial coordinates, the latter being standardized by wing size). The results obtained showed that size variatiorn in Brazilian D. melanogaster follows the worldwide tendency toward the formation of latitudinal clines, with larger individuaJs being found at higher latitudes. However, the heritability and temporal variation among multiple collections performed in Recife and Rio de Janeiro did not show a clear regular pattern. The only consistent and significant effect of chromosomal inversions that we could observe was that of In(3R)Payne on body size, which is also consistent with previous findings reported in the literature. However, we did not detect a significant effect of genotype-environment interactions, neither among collections, nor among localities / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Orientadores: Angela Maria Moraes, Carlos Augusto Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-09T04:29:58Z (GMT). No. of bitstreams: 1 Galesi_AdrianaLagesLima_D.pdf: 1381119 bytes, checksum: 756e8fc5c7c30b40f7e2ebe60c293b63 (MD5) Previous issue date: 2007 / Resumo: Células de inseto têm sido freqüentemente empregadas na obtenção de proteínas recombinantes devido a algumas vantagens em relação às células de mamíferos, e a linhagem Drosophila melanogaster Schneider 2 é um dos sistemas de expressão utilizados para este propósito. Entretanto, a literatura é escassa em relação ao metabolismo e ao cultivo destas células, o que motivou o desenvolvimento deste trabalho. O objetivo deste estudo foi determinar, através da técnica estatística de planejamento fatorial de experimentos, um meio de cultura adequado ao crescimento da linhagem Drosophila melanogaster Schneider 2 transfectada para expressão da glicoproteína do vírus da raiva (GPV) e avaliar seu comportamento nos diferentes meios formulados. Para tal, no meio basal TC100, foram estudados os efeitos dos suplementos glicose, glutamina, concentrado protéico de soro de leite, yeastolate (extrato de levedura), hidrolisado de soja, lactoalbumina hidrolisada, emulsão lipídica e Pluronic F68 sobre o crescimento e a viabilidade celular, na tentativa de reduzir o percentual de soro fetal bovino do meio de cultura. A combinação de diferentes suplementos permitiu que o soro fetal bovino fosse eliminado do meio de cultura, principalmente em decorrência da adição de yeastolate, e, em várias das formulações desenvolvidas, a densidade de células foi bastante superior àquela obtida empregando o meio basal TC100 suplementado com 10% de soro fetal bovino. O meio de cultura que resultou no melhor desempenho celular foi formulado com o meio TC100 suplementado com 10 g/L de glicose e 3,5 g/L de glutamina (concentrações finais), 3 g/L de yeastolate, 1% (v/v) de emulsão lipídica e 0,1 % (m/v) de Pluronic F68. Inicialmente, os ensaios realizados em biorreator não reproduziram o crescimento celular obtido em menor escala. Porém, com o aumento da concentração de Pluronic F68 para 0,6%, tal limitação foi contornada. Glicose não foi o substrato limitante nos meios formulados e lactato foi produzido em pequenas quantidades. Apesar de produzido em elevadas concentrações, o amônio não influenciou o crescimento celular. A dosagem da glicoproteína mostrou que as células não perderam sua capacidade de expressão após a adaptação em diferentes meios de cultura e que a produção de GPV no meio formulado é superior à verificada com os meios comerciais SF900 II e TC100 suplementado com 10% de soro fetal bovino / Abstract: Insect cells have been intensively employed to obtain recombinant proteins due to some advantages over mammalian cells, and the Drosophila melanogaster Schneider 2 cell line is one of the expression systems used for this purpose. Nevertheless, literature is scarce with regard to metabolism and culture of these cells, what motivated the development of this work. The aim of this study was to establish an adequate culture medium to cultivate Drosophila melanogaster Schneider 2 cells transfected for the expression of the rabies virus glycoprotein (GPV), and to evaluate their behaviour in different formulated media. For this, the factorial design strategy was employed. The effects of glucose, glutamine, whey protein concentrate, yeastolate, soy hydrolysate, lactoalbumin hydrolysate, lipid emulsion and Pluronic F68 were studied over cell growth and viability, aiming to reduce the fetal bovine serum percentage from the culture medium. Adjusting the concentration of these distinct compounds, serum was eliminated, mainly due to the addition of yeastolate, and cell concentration was higher in several of the developed formulations than that achieved with basal TC100 medium supplemented with 10% of serum. The formulated medium which resulted in best cell performance was composed by TC100 containing 10 g/L of glucose, 3.5 g/L of glutamine, 3 g/L of yeastolate, 1% (v/v) of lipid emulsion and 0.1% (w/v) of Pluronic F68. When in bioreactor, initially the cells did not reproduce the growth behavior observed in smaller scale. However, increasing Pluronic F68 percentage to 0.6%, this limitation was circunvended. Glucose was not the limiting substrate in formulated culture media and lactate was produced in low quantities. Despite ammonium was produced in high concentrations, this compound did not influence cell growth. Glycoprotein quantification shows that cells did not loose their expression capacity after adaptation in the different media, and that glycoprotein production in the formulated medium was higher than that obtained in SF900 II medium and TC100 medium containing 10% of fetal bovine serum / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-06T19:20:01Z No. of bitstreams: 2 Dissertação - Igor Gomes de Oliveira - 2011.pdf: 1076733 bytes, checksum: 1faa803ec01cef7512bec500010c985b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-06T19:26:27Z (GMT) No. of bitstreams: 2 Dissertação - Igor Gomes de Oliveira - 2011.pdf: 1076733 bytes, checksum: 1faa803ec01cef7512bec500010c985b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-06T19:26:27Z (GMT). No. of bitstreams: 2 Dissertação - Igor Gomes de Oliveira - 2011.pdf: 1076733 bytes, checksum: 1faa803ec01cef7512bec500010c985b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2011-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Clinical studies have shown that the combinations of chemotherapeutic drugs gemcitabine (GEM) plus cisplatin (CIS) and gemcitabine (GEM) plus doxorubicin (DXR) exert important cytotoxic activity against several types of cancer in advanced stages as well as metastatic cancer. CIS, DXR and GEM have different mechanisms of action. GEM is a pro-drug that must be phosphorylated by deoxycytidine kinase to evolve into its active form. Both gemcitabine diphosphate and gemcitabine triphosphate inhibit processes required for DNA synthesis. CIS induces a variety of DNA structural changes, mainly intra- and interstrand cross-links between adjacent purine bases. DXR acts as a topoisomerase II inhibitor. This study compares the genetic toxicity effects induced by GEM+CIS and GEM+DXR co-treatments with the single drug treatments. We used the Somatic Mutation And Recombination Test (SMART), which simultaneously detects and quantifies mutagenic and recombinogenic toxicological endpoints through loss of heterozygosity of two genetic markers involved in the metabolic pathways of the Drosophila melanogaster wing hairs formation. Using the standard cross, the third-stage larvae were chronically treated with different concentrations of GEM (0.008, 0.010, 0.012 and 0.014 mM) combined with CIS (0.05 mM) or DXR (0.2 mM). Comparing with GEM single treatment, GEM+CIS and GEM+DXR co-treatments induced a synergistic effect manifested as an increment in the mutant clones frequencies. Homologous recombination was the main genotoxic effect observed. / As combinações dos quimioterápicos gemcitabina (GEM) com cisplatina (CIS) e gemcitabina (GEM) com doxorrubicina (DXR) têm demonstrado uma importante atividade citotóxica contra vários tipos de câncer em estágio avançado e/ou metastático em diversos estudos clínicos. CIS, DXR e GEM possuem diferentes mecanismos de ação, sendo este um fator importante para o sucesso da associação entre quimioterápicos. A GEM é uma pró-droga análoga de desoxicitidina que deve ser fosforilada pela desoxicitidina quinase para se tornar ativa. Ambos, gemcitabina difosfato e gemcitabina trifosfato, atuam inibindo os processos necessários para a síntese de DNA. Já a CIS induz uma variedade de mudanças estruturais, principalmente por meio de ligações cruzadas intra e intercadeias entre bases purínicas adjacentes do DNA. A DXR age como um inibidor da topoisomerase II, formando um complexo entre esta proteína e o DNA, inibindo os processos necessários para a síntese do DNA. Este estudo teve como objetivo comparar os efeitos de toxicidade genética induzidos pelos tratamentos utilizando os combinados de GEM+CIS e GEM+DXR com os tratamentos usando as drogas isoladas. Utilizamos o Teste de Mutação e Recombinação Somática (SMART), que detecta e quantifica, simultaneamente, parâmetros mutagênicos e recombinogênicos através da perda de heterozigose de dois marcadores genéticos envolvidos nas vias metabólicas da formação dos pêlos da asa de Drosophila melanogaster. Usando o cruzamento padrão, as larvas de terceiro estágio foram tratadas cronicamente com diferentes concentrações de GEM (0,008, 0,010, 0,012 e 0,014 mM), combinado com CIS (0,05 mM) ou DXR (0,2 mM). Comparando os combinados GEM+CIS e GEM+DXR com o tratamento isolado com GEM, notou-se que nos combinados houve um efeito sinérgico demonstrado pelo aumento nas frequências de clones mutantes destes em comparação com o tratamento isolado com GEM. A recombinação homóloga foi o principal tipo de efeito genotóxico observado nas combinações.

O metabolismo das células S2 (Schneider 2) de Drosophila melanogaster ainda não é totalmente conhecido. Existem poucos estudos específicos sobre o metabolismo de células S2, sejam elas selvagens ou recombinantes (rS2), como por exemplo aquelas transfectadas para a expressão da glicoproteína do vírus da raiva (GPV). Como o genoma da Drosophila melanogaster já foi mapeado, as principais enzimas que atuam nos processos metabólicos em geral já foram identificadas e estão à disposição no KEGG (Kyoto Encyclopedia of Genes and Genomes). Assim, o KEGG apresenta todas as possíveis vias metabólicas com as enzimas que podem ser codificadas. Diante deste quadro, foi proposto um modelo metabólico baseado em um conjunto de vias de assimilação de glicose e glutamina e foram encontrados os modos elementares característicos do sistema através do programa Metatool. Em seguida, foi definido o modelo matemático mediante o equacionamento desses modos elementares. Esse processo se repetiu até se encontrar um conjunto de vias metabólicas que, através da modelagem matemática, respondesse coerentemente a um conjunto de dez ensaios em diferentes condições de concentrações iniciais de glicose, glutamina e oxigênio dissolvido. Chegou-se então, a um metabolismo básico para a rS2 contendo 33 vias metabólicas englobando a glicólise, a via das pentoses, o ciclo de Krebs e a fosforilação oxidativa. Dados anteriores indicavam elevada flexibilidade metabólica dessa célula, o que foi prevista através de algumas reações propostas como reversíveis nas vias de degradação e síntese de glutamina. Essa proposta de metabolismo resultou em 37 modos elementares. Outra característica interessante da modelagem foi a utilização da produção de purinas e pirimidinas para a estimativa do crescimento celular. Depois de realizada a modelagem, as mesmas condições iniciais dos ensaios foram simuladas através de um programa de simulação do comportamento cinético das células rS2 desenvolvido em MATLAB. Esse simulador foi utilizado também para simulação com diferentes meios e condições iniciais de cultivo. Chegando-se a um ajuste geral entre valores experimentais e simulados com coeficiente de correlação de 0,88. / The metabolism of the S2 cells (Schneider 2) Drosophila melanogaster is not yet fully known. There have been few specific studies on the metabolism of S2 cells, whether recombinant or wild (rS2), such as those transfected for expressing the rabies virus glycoprotein (RVGP). As the genome of Drosophila melanogaster have been mapped, the key enzymes that act on the metabolic processes in general have been identified and are available in the KEGG (Kyoto Encyclopedia of Genes and Genomes). Thus, KEGG presents all possible pathways with the enzymes that can be encoded. Given this context, it was proposed a metabolic model based on a set metabolic glucose and glutamine assimilation pathways and were found characteristic elementary modes of the system through the Metatool program. Then the mathematical model was defined by addressing these elementary modes. This process was repeated until a set of metabolic pathways, by mathematical modelling, consistently responded to a set of ten experiments (in various conditions). We came to a basic metabolism for rS2 containing 33 pathways comprising glycolysis, pentose, Krebs cycle and oxidative phosphorylation. Previous data indicate that rS2 is a cell with high metabolic flexibility, which was confirmed by some reactions in the process proposed as reverse breakdown and synthesis of glutamine. The proposed metabolism resulted in 37 elementary modes. Another interesting model characteristic was the use of the production of purines and pyrimidines for the estimation of cell growth. After the modelling performed, the same initial runs conditions were simulated using a software of Simulation of the Kinetic behaviour of rS2 cells, developed in MATLAB. This simulator was also used for simulation of other experiments with different initial conditions and methods of cultivation. Coming to a general adjustment of experimental and simulated values with correlation coefficient of 0.88.

Une surprolifération cellulaire associée à de l’aneuploïdie est un marqueur couramment retrouvé dans les cancers et une faible instabilité génétique peut-être un élément aggravant (sinon déclencheur) de la tumorigénèse. Récemment, il a été montré sur un modèle de cellules cancéreuses en culture qu’une forte aneuploïdie compromet la prolifération cellulaire en entraînant la mort de ces dernières. Au cours de ma thèse, nous avons souhaité tester si cette hypothèse se vérifiait in vivo en utilisant comme modèle, les tumeurs du système nerveux central de la larve de D. melanogaster. Nous avons fait le choix d’utiliser des mutants pour des gènes impliqués dans la formation du fuseau mitotique et la ségrégation des chromosomes (Sas-4 ou AurA) afin d’induire ces tumeurs. Pour générer l’aneuploïdie, nous avons choisi d’associer les mutations sas-4 ou aurA avec des mutations pour des gènes essentiels du SAC, Mad2 ou BubR1ken. Nous avons ensuite analysé par immunofluorescence et microscopie l’effet de la perte du SAC sur la prolifération des Nb. Pour sas-4, la perte du SAC cause l’apparition d’une forte aneuploïdie et une baisse du nombre de Nb associée à une forte réduction de taille des cerveaux. Cela compromet totalement la capacité des cerveaux mutants à induire des tumeurs lorsqu’on les injecte dans l’abdomen de mouches adultes saines. Dans le cas d’aurA, ni hausse de l’aneuploïdie dans le tissu ni baisse de la prolifération des Nbs n’ont été observés. Par ailleurs, la même forte proportion de mouches injectées avec des cerveaux aurA ou aurA mad2 développant une tumeur a été constaté. Afin de mieux comprendre pourquoi le mutant aurA ne réagit pas comme le mutant sas-4 à la déplétion du SAC, nous avons entrepris une analyse détaillée des mutants aurA et aurA mad2. Nous avons d’abord observé que, malgré la perte du SAC, 1) il existe toujours un délai en mitose dans aurA mad2 et 2) il existe un délai entre la satisfaction du SAC et l’entrée en anaphase dans aurA. Comme l’entrée en anaphase est dépendante de la dégradation de la CycB et de la Sécurine via l’APC/C, nous avons analysé le comportement de la CycB (couplé à une étiquette GFP) par vidéo-microscopie en temps réel et observé un défaut de la régulation de la dégradation de cette dernière dans le mutant aurA ainsi que dans le double mutant aurA mad2. Ces observations nous ont permis de proposer un nouveau rôle pour la kinase AurA dans la régulation de la dégradation de la CycB en fin de mitose. / Cellular overproliferation associated with aneuploidy is a common hallmark of cancers. Low genetic instability may be a contributing factor of tumorigenesis. Recently, it was shown on a cellular cancer model in culture that strong aneuploidy compromises cell proliferation by causing cell death. During my thesis, we have test if this hypothesis was verified in vivo by using as a model, the tumours of the larval central nervous system of D. melanogaster. We decided to use mutants involved in mitotic spindle formation and chromosome segregation (Sas-4 or AurA) to induce these tumours. To generate aneuploidy, we chose to associate these mutations with mutations in genes essential for the SAC, Mad2 or BubR1ken. We then analysed the effect of the SAC depletion on the Nb proliferation. For sas-4, loss of the SAC leads to high aneuploidy and a decrease in Nb number associated with brain size reduction. It completely undermines the ability of mutant brain to induce tumors when injected into the abdomen of healthy adult flies. In the case of aurA, nor increase of aneuploidy in tissue or decrease in nb proliferation have been observed. Moreover, the same proportion of flies injected with aurA or aurA mad2 brains developed tumours. To better understand why the aurA mutant not react as the sas-4 mutant to the SAC depletion, we undertook a detailed analysis of aurA and aurA mad2 mutants. We first observed that despite the SAC depletion, 1) there is always a delay in mitosis in aurA mad2 and 2) there is a delay between SAC satisfaction and anaphase onset in aurA. Since anaphase onset is dependent of the CycB and Securine degradation via the APC / C, we analysed the behaviour of the CycB (coupled with a GFP tag) by real-time videomicroscopy and observed a defect in the regulation of CycB degradation in aurA and in the double aurA mad2 mutant. These observations lead us to propose a new role for AurA kinase in regulating the degradation of CycB at the end of mitosis.

Les rétrovirus endogènes sont des parasites génétiques qui s’insèrent dans l’ADN génomique. Bien que leurs insertions délétères soient éliminées par la sélection naturelle, ils prolifèrent et sont une source de plasticité génomique. L’étude de l’impact de leur mobilité sur le génome hôte est rendue difficile par le faible taux de transposition de ces éléments, réprimés par des petits ARN appelés piARNs. Nous avons développé une approche génétique permettant d’inactiver ce contrôle et de déterminer l’impact sur le génome de la drosophile, d’une transposition réplicative. Nous avons remarqué la mise en place d’autres mécanismes de répression des rétrovirus endogènes lors de la perte des piARNs pouvant ainsi limiter leur propagation. Nous avons aussi identifié de nouveaux sites d’intégrations des rétrovirus endogènes après un cycle de transposition réplicative. Cependant, le taux de transposition reste faible. Ce projet combinant différentes approches (génétique, séquençage à haut débit et bioinformatique) a permis de démontrer que la voie des piARNs n’est pas cruciale pour le maintien de l’intégrité du génome, et que d’autres mécanismes semblent intervenir afin de maintenir sa stabilité. / Endogenous retrovirsuses are genetic parasites which are inserted in the genomic DNA. Although their deleterious insertions are eliminated by natural selection, they proliferate and are a source of genomic plasticity. The study of the impact of their mobility on the host genome is made difficult by the transposition’s low rate of these elements, suppressed by a class of small RNA, called piRNA. We have developed a genetic approach to inactivate this control and determine the impact on Drosophila’s genome, after one replicative transposition. We noticed the establishment of other endogenous retroviruses repression mechanisms that are awakened after the loss of the piRNA and they are able to limit their spread. We identified new integrations sites of endogenous retrovirus after one replicative transposition. But we noticed that the transposition rate still low. This project combines different approaches (genetics, high-throughput sequencing and bioinformatics) and show the piRNA pathway is not essential to maintain genome integrity but other mechanisms involving small RNA can be implicated in the genome stability.

Des perturbations de l’horloge circadienne imposées par nos modes de vie désynchronisés «24/7» peuvent être néfastes à long terme. Ces horloges contrôlent des rythmes biologiques avec une périodicité d’environ 24h et se synchronisent aux cycles journaliers de lumière-obscurité. Les perturbations de l’horloge peuvent influencer le vieillissement normal et celui de patients atteints de la maladie de Parkinson (MP). Ces patients présentent souvent des symptômes non moteurs incluant des troubles du sommeil et/ou des rythmes circadiens, qui se manifestent avant les déficits moteurs ou cognitifs. L’objectif de ce travail de thèse a été d’explorer l’impact des perturbations de l’horloge circadienne sur le déclin fonctionnel lié à l’âge au cours du vieillissement normal et dans des modèles de MP chez la drosophile. Les résultats obtenus confirment l’impact négatif de l’arythmie circadienne sur la longévité, et révèlent que l’inactivation du gène d’horloge Clock (Clk) augmente le niveau de stress oxydatif dans le cerveau et accélère le déclin des capacités locomotrices au cours du vieillissement. Ce dernier effet a pu être relié à une fonction de Clk dans les neurones d’horloge qui contrôlent les rythmes d’activité-repos en obscurité constante, et leurs connections à un groupe spécifique de neurones dopaminergiques. Nous avons aussi observé que des décalages horaires chroniques mènent également à une accélération du déclin locomoteur lié à l’âge, qui peut être sauvée en adaptant les rythmes lumière-obscurité à la période endogène des drosophiles. Enfin, notre travail souligne l’impact négatif de l’arythmie circadienne sur le déclin locomoteur lié à l’âge dans un modèle de MP. / Long-term disruption of circadian clocks, as occurs often in our "24/7 societies", can be detrimental. These clocks control self-sustained biological rhythms with ~24h periodicity and synchronize to the daily light-dark cycle. Clock disruption may impact normal brain aging as well as Parkinson disease (PD) pathogenesis: PD patients frequently exhibit non-motor symptoms including sleep and/or circadian rhythm disruptions that occur before motor or cognitive deficits, and decrease quality of life. The objective of this PhD work was thus to explore the impacts of circadian disruptions on age-related functional decline, both during normal aging and in a fly model of PD-like conditions using well established Drosophila models. Our results confirmed the negative impact of circadian arrhythmia on longevity and revealed that inactivation of a specific circadian gene, Clock (Clk), increases brain oxidative stress levels and accelerates locomotor decline during aging. The latter effect could be associated with Clk function in the clock neurons that drive circadian rest-activity rhythms in constant darkness, and their connections with a specific cluster of dopaminergic neurons. We also observed that chronic jet lag led to an accelerated age-related locomotor decline that could be rescued by adapting the light-dark to the flies’ endogenous period. Finally, our work demonstrated the negative impact of environmentally imposed circadian arrhythmia on age-related locomotor decline in a fly model of PD.

Drosophila are used extensively for genetic, developmental and now molecular biology research. At present, germline transformation of these organisms can only be achieved by microinjection of P-element vectors into the pole cells of young embryos. The technique of microinjection however, requires a delicate touch and is quite laborious. Therefore, the development of a rapid and simple technique was investigated. Electroporation, like microinjection, is a physical means of introducing DNA into a cell and is therefore potentially applicable to all cell types. Electroporation involves the use of an electrical current to create pores in the membrane of a cell. Macromolecules, such as DNA may enter a cell via these pores. Electroporation is a quick, reproducible, and efficient method for transforming cells. Through studies of the survival and permeability of Drosophila melanogaster embryos exposed to electrical currents, it was discovered that although the survival of the embryos decreased steadily as field strength increased, the embryos did not become permeable to a water soluble dye unless a pulse of 10 kV/cm was applied. Few embryos survived this extreme voltage required for dye uptake. Attempts to introduce DNA into dechorionated Drosophila embryos utilizing this technique however, produced no transformants. These results suggested that the remaining protective coatings of the dechorionated embryo were obstructing efficient pore formation, thus preventing DNA penetration. In view of these results, methods to eliminate the wax layer, present between the chorion and vitelline membrane of laid eggs, were examined. Wax removal by detergent solubilization, solvent extraction and melting by heating were investigated, yet did not produce a satisfactory procedure. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate