Estudos recentes avaliaram o papel do Magnetic Activated cell Sorting (MACS, separação celular por ativação magnética) para reduzir a percentagem de espermatozoides apoptóticos e melhorar a qualidade seminal. No entanto, a eficiência do uso do MACS isoladamente, antes ou depois do processamento seminal pelos métodos clássicos, como o centrifugação em gradiente de densidade (DGC), ainda não foi estabelecida, de modo que o protocolo de uso do método não foi adequadamente estabelecido. Desta forma, o objetivo do presente estudo foi avaliar se o MACS sozinho, antes (MACS-DGC) ou depois do processamento seminal pelo DGC (DGC-MACS) melhora a seleção espermática quando comparado ao processamento pelo DGC. Realizou-se um estudo prospectivo experimental avaliando amostras de sêmen de 15 homens saudáveis. A mesma amostra foi dividida em 4 alíquotas e processada por: DGC, DGC-MACS, MACS-DGC e MACS. Após o processamento, foram analisadas a integridade do DNA espermático pelo método TUNEL - TdT-mediated dUTP Nick End Labeling (marcação de quebras no DNA por dUTP e deoxinucleotidil terminal transferase) assim como a concentração de espermatozoides, a motilidade progressiva e a morfologia, segundo os últimos critérios adotados pela Organização Mundial da Saúde. A percentagem de dano ao DNA foi significativamente menor no grupo MACSDGC, quando comparado com DGC e MACS, e semelhante a do grupo DGC-MACS. A concentração de espermatozoides recuperados nos grupos de DGC e MACS foi similar e significativamente maior do que MACS-DGC e DGC-MACS, que foram semelhantes entre si. A motilidade progressiva dos espermatozoides recuperados foi semelhante nos grupos MACS-DGC e DGC e significativamente maior do que nos grupos DGC-MACS e MACS. O percentual de espermatozoides com morfologia normal foi significativamente maior no grupo MACS-DGC quando comparado ao DGC-MACS e MACS, e semelhante quando comparado com DGC. Desta forma, evidenciou-se que ambos os métodos combinados promovem a recuperação de espermatozoides com menor percentagem de dano ao DNA espermático. O MACS isoladamente ou aplicado após o DGC promove redução significativa dos espermatozoides progressivos recuperados, assim como da percentagem de espermatozoides com morfologia normal. Todavia, o MACS aplicado antes do DGC promove a recuperação de amostras com elevada percentagem de espermatozoides com motilidade progressiva e morfologia normal, aliada a baixa percentagem de DNA fragmentado, sugerindo ser o melhor protocolo de uso desta metodologia, cuja importância clínica precisa ser avaliada em estudos clínicos bem delineados. / Recent studies evaluated the role of Magnetic Activated Cell Sorting (MACS) in order to reduce the percentage of apoptotic sperm and improve seminal quality. However, the effectiveness of using MACS alone, before or after seminal processing by classical methods, such as the density gradient centrifugation (DGC), has not yet been established. Thus, the role of the present study was evaluate whether: MACS alone, before (MACS-DGC) or after seminal processing by DGC (DGC-MACS) improves sperm selection when compared to DGC. A prospective experimental study was conducted evaluating semen samples from 15 healthy men. The same sample was divided into 4 aliquots and processed by: DGC, DGC-MACS, MACS-DGC and MACS. After processing, the integrity of the sperm DNA was analyzed by TUNEL - TdT - mediated dUTP method Nick End Labeling (marking DNA breaks by dUTP and deoxynucleotidyl terminal transferase), sperm concentration, progressive motility and morphology according to the last criteria adopted by the World Health Organization. The percentage of DNA damage was significantly lower in the MACS-DGC group, when compared to DGC and MACS, and similar to the DGC-MACS group. The concentration of spermatozoa recovered in the DGC and MACS groups was similar and significantly higher than MACS-DGC and DGC-MACS, which were similar to each other. The progressive motility was similar in the MACS-DGC and DGC groups and significantly higher than in the DGC-MACS and MACS groups. The percentage of spermatozoa with normal morphology was significantly higher in the MACS-DGC group when compared to DGC-MACS and MACS, and similar when compared to DGC. Thus, it was evidenced that both methods combined promote the recovery of spermatozoa with a lower percentage of DNA damage. MACS alone or applied after the DGC promotes a significant reduction of the progressive sperm retrieved, as well as the percentage of spermatozoa with normal morphology. However, the MACS applied before the DGC promotes the recovery of samples with a higher percentage of spermatozoa with progressive motility and normal morphology, combined with low percentage of fragmented DNA, suggesting to be the best protocol, whose clinical importance needs to be evaluated in well-delineated clinical studies.

Estudos recentes avaliaram o papel do Magnetic Activated cell Sorting (MACS, separação celular por ativação magnética) para reduzir a percentagem de espermatozoides apoptóticos e melhorar a qualidade seminal. No entanto, a eficiência do uso do MACS isoladamente, antes ou depois do processamento seminal pelos métodos clássicos, como o centrifugação em gradiente de densidade (DGC), ainda não foi estabelecida, de modo que o protocolo de uso do método não foi adequadamente estabelecido. Desta forma, o objetivo do presente estudo foi avaliar se o MACS sozinho, antes (MACS-DGC) ou depois do processamento seminal pelo DGC (DGC-MACS) melhora a seleção espermática quando comparado ao processamento pelo DGC. Realizou-se um estudo prospectivo experimental avaliando amostras de sêmen de 15 homens saudáveis. A mesma amostra foi dividida em 4 alíquotas e processada por: DGC, DGC-MACS, MACS-DGC e MACS. Após o processamento, foram analisadas a integridade do DNA espermático pelo método TUNEL - TdT-mediated dUTP Nick End Labeling (marcação de quebras no DNA por dUTP e deoxinucleotidil terminal transferase) assim como a concentração de espermatozoides, a motilidade progressiva e a morfologia, segundo os últimos critérios adotados pela Organização Mundial da Saúde. A percentagem de dano ao DNA foi significativamente menor no grupo MACSDGC, quando comparado com DGC e MACS, e semelhante a do grupo DGC-MACS. A concentração de espermatozoides recuperados nos grupos de DGC e MACS foi similar e significativamente maior do que MACS-DGC e DGC-MACS, que foram semelhantes entre si. A motilidade progressiva dos espermatozoides recuperados foi semelhante nos grupos MACS-DGC e DGC e significativamente maior do que nos grupos DGC-MACS e MACS. O percentual de espermatozoides com morfologia normal foi significativamente maior no grupo MACS-DGC quando comparado ao DGC-MACS e MACS, e semelhante quando comparado com DGC. Desta forma, evidenciou-se que ambos os métodos combinados promovem a recuperação de espermatozoides com menor percentagem de dano ao DNA espermático. O MACS isoladamente ou aplicado após o DGC promove redução significativa dos espermatozoides progressivos recuperados, assim como da percentagem de espermatozoides com morfologia normal. Todavia, o MACS aplicado antes do DGC promove a recuperação de amostras com elevada percentagem de espermatozoides com motilidade progressiva e morfologia normal, aliada a baixa percentagem de DNA fragmentado, sugerindo ser o melhor protocolo de uso desta metodologia, cuja importância clínica precisa ser avaliada em estudos clínicos bem delineados. / Recent studies evaluated the role of Magnetic Activated Cell Sorting (MACS) in order to reduce the percentage of apoptotic sperm and improve seminal quality. However, the effectiveness of using MACS alone, before or after seminal processing by classical methods, such as the density gradient centrifugation (DGC), has not yet been established. Thus, the role of the present study was evaluate whether: MACS alone, before (MACS-DGC) or after seminal processing by DGC (DGC-MACS) improves sperm selection when compared to DGC. A prospective experimental study was conducted evaluating semen samples from 15 healthy men. The same sample was divided into 4 aliquots and processed by: DGC, DGC-MACS, MACS-DGC and MACS. After processing, the integrity of the sperm DNA was analyzed by TUNEL - TdT - mediated dUTP method Nick End Labeling (marking DNA breaks by dUTP and deoxynucleotidyl terminal transferase), sperm concentration, progressive motility and morphology according to the last criteria adopted by the World Health Organization. The percentage of DNA damage was significantly lower in the MACS-DGC group, when compared to DGC and MACS, and similar to the DGC-MACS group. The concentration of spermatozoa recovered in the DGC and MACS groups was similar and significantly higher than MACS-DGC and DGC-MACS, which were similar to each other. The progressive motility was similar in the MACS-DGC and DGC groups and significantly higher than in the DGC-MACS and MACS groups. The percentage of spermatozoa with normal morphology was significantly higher in the MACS-DGC group when compared to DGC-MACS and MACS, and similar when compared to DGC. Thus, it was evidenced that both methods combined promote the recovery of spermatozoa with a lower percentage of DNA damage. MACS alone or applied after the DGC promotes a significant reduction of the progressive sperm retrieved, as well as the percentage of spermatozoa with normal morphology. However, the MACS applied before the DGC promotes the recovery of samples with a higher percentage of spermatozoa with progressive motility and normal morphology, combined with low percentage of fragmented DNA, suggesting to be the best protocol, whose clinical importance needs to be evaluated in well-delineated clinical studies.

Made available in DSpace on 2016-12-12T20:12:29Z (GMT). No. of bitstreams: 1 PGEF14MA015.pdf: 271974 bytes, checksum: ebd1c9622a65b4e8b500be551ca4bc58 (MD5) Previous issue date: 2014-06-26 / The objective of this work to determine effective treatment for breaking dormancy of seeds of Ocotea puberula, check the behavior of the seed during drying and storage and evaluate the appropriate method for detecting pathogens, asepsis, seeds prior to detection of pathogens and genera of fungi which infest seeds of this species. The seeds were collected in five counties of the State of Santa Catarina (Fraiburgo Joaçaba, Curitibanos, Ponte Alta and Brunópolis), and each collection site was considered a lot. To break dormancy were tested four treatments: 1- control, 2- seeds without tegument; 3- sulfuric acid for 5 minutes and 4- drying the seeds at 25 °C for 12 hours. After performing the tests break dormancy, seeds were subjected to germination test gerboxes substrate with blotting paper in BOD at 30 oC with 4 replicates of 20 seeds per treatment/lot. Regarding the types of drying seeds with an initial moisture content of 38% was dried to 18% with gradient of 2% in oven (35 °C) in a desiccator with silica gel (25 °C). After drying, was determined the water content and viability (tetrazolium and germination tests). In the armazenament study, the seeds were stored with and without fruit, in a dry camera (40% RH and Temp. 10 ± 2 oC) for periods of 0, 3, 6 and 9 months. At each time interval were determined water content and the viability of the seeds by germination, tetrazolium and DNA integrity testing. Determination of seed health was held in PDA culture medium, medium culture V8 and "Blotter Test". In each test were used seeds With and without disinfection (disinfection with sodium hypochlorite and alcohol) totaling 80 seeds for each test. Incubation of seeds was realized in a chamber with controlled temperature of 22 °C ± 3 °C, with a photoperiod of 12 hours for seven days when the assessment and identification of fungi occurred. It was observed that seeds without tegument started germination after 14 days of the start of the test, with stabilizing stand at 36 days, with average scores of 71% germination. Not was observed seed germination for other treatments and control in all lots used. Regarding drying, it was observed that up to 32% humidity no change in seed quality, regardless of the type of drying and verified significant loss of germination after this value. Seeds of Ocotea puberula lost their viability after 3 months of storage, with or without fruit. Nine genera of fungi were observed: Penicillium sp., Phomopsis sp., Epicocum sp., Curvularia sp., Colletotrichum sp., Aspergillus sp., Alternaria sp., Fusarium sp. and Trichoderma sp.. It was concluded that the method of seed coat removal was effective in breaking dormancy of seeds of Ocotea puberula and the type of drying does not affect the quality of the seed; however, reducing the water content below 32% decreased germination. Seeds of Ocotea puberula lose their viability after 3 months of storage in dry chamber and agarized media were more sensitive for the detection of fungi in weed seeds and seed disinfection with sodium hypochlorite and alcohol reduces the incidence of these fungi, is indicated when sanity test is performed with seeds of this species / Objetivou-se com este trabalho determinar tratamento eficiente para superação de dormência de sementes de Ocotea puberula, verificar o comportamento das sementes durante a secagem e o armazenamento e avaliar o método adequado para detecção de patógenos, da assepsia, das sementes antes da detecção dos patógenos e quais gêneros fúngicos infestam sementes desta espécie. As sementes foram colhidas em cinco municípios do Estado de Santa Catarina (Fraiburgo, Joaçaba, Curitibanos, Ponte Alta e Brunópolis), e cada local de coleta foi considerado um lote. Para a superação da dormência, foram testados quatro tratamentos: 1- testemunha, 2- sementes sem o tegumento; 3- ácido sulfúrico por 5 minutos e 4- secagem das sementes a 25 oC por 12 horas. Após a realização dos testes de superação da dormência, as sementes foram submetidas ao teste de germinação em caixas gerbox com substrato de papel mata borrão, em BOD a 30 oC, com 4 repetições de 20 sementes por tratamento/lote. Em relação aos tipos de secagem, sementes com teor de água inicial de 38% foram secas até 18%, com gradientes de 2%, em estufa (35 ºC) e em dessecador com sílica-gel (25 ºC). Após cada secagem, foram determinados o teor de água e a viabilidade (testes de tetrazólio e germinação). No estudo de armazenamento, as sementes foram armazenadas com e sem fruto, em câmara seca (UR 40% e Temp. 10 ± 2 oC), pelos períodos de 0, 3, 6 e 9 meses. A cada intervalo de tempo, foram determinados o teor de água e a viabilidade das sementes, pelos testes de germinação, tetrazólio e a integridade do DNA. A determinação da sanidade das sementes foi realizada em meio de cultura BDA, meio de cultura V8 e Blotter Test . Em cada teste, foram utilizadas sementes com e sem assepsia (desinfestação com hipoclorito de sódio e álcool), totalizando 80 sementes para cada teste. A incubação das sementes foi realizada em câmara com temperatura controlada a 22 °C ±3 oC, com fotoperíodo de 12 horas, durante sete dias, quando ocorreu a avaliação e identificação dos fungos. Foi observado que sementes sem o tegumento iniciaram a germinação após 14 dias do início do teste, com estabilização do estande aos 36 dias, com resultados médios de 71% de germinação. Não foi observada germinação para sementes dos demais tratamentos e testemunha, em todos os lotes utilizados. Em relação à secagem, foi observado que até 32% de umidade não houve alteração na qualidade das sementes, independente do tipo de secagem, sendo verificada significativa perda de germinação após esse valor. Sementes de Ocotea puberula perderam sua viabilidade após 3 meses de armazenamento, com ou sem fruto. Foram observados nove gêneros fungícos: Penicillium sp., Phomopsis sp., Epicocum sp., Curvularia sp., Colletotrichum sp., Aspergillus sp., Alternaria sp., Fusarium sp. e Trichoderma sp. Conclui-se que o método de retirada do tegumento foi eficiente na superação da dormência de sementes de Ocotea puberula e que o tipo de secagem não influencia na qualidade dessas sementes; porém, a redução do teor de água abaixo de 32% prejudica a germinação. Sementes de Ocotea puberula perdem sua viabilidade após 3 meses de armazenamento em câmara seca e os meios agarizados foram mais sensíveis para a detecção de fungos infestantes nas sementes e a assepsia das sementes com hipoclorito de sódio e álcool reduz a incidência desses fungos, sendo indicada quando se realiza teste de sanidade com sementes dessa espécie

L’ADN tumoral circulant (ADNtc) porte des altérations spécifiques de la tumeur des patients, qui sont détectables par un acte minimalement invasif. L’ADNtc représente donc un biomarqueur d’intérêt pour le suivi de l’évolution du cancer. Sa détection requière une technique hautement sensible et quantitative. Dans ce contexte, ce travail de thèse a porté sur la quantification et le suivi de l’ADNtc par PCR digitale en gouttelettes (PCRdg). Cet outil permet la détection d’altérations à l’échelle d’un ADN unique, offrant ainsi une sensibilité allant jusqu’à 0.001%. La détection de cet ADNtc a été réalisée par l’évaluation des biomarqueurs tels qu’une mutation spécifique de la tumeur, la fragmentation de l’ADNtc et l’hyperméthylation de séquences cibles. D’une part, nous avons observé que chez les patients atteints de cancer, l’ADN muté circulant est plus fragmenté que l’ADN non muté, et que cet ADN circulant de patients est globalement plus fragmenté que chez les sujets sains. D’autre part, une corrélation entre les pourcentages d’ADN muté et d’ADN hyperméthylé circulants a été observée au cours du suivi de patients. Ceci suggère la possibilité d’un suivi précis et quantitatif de l’ADNtc par l’évaluation de l’hyperméthylation en alternative à la détermination du statut mutationnel. Nous avons ensuite appliqué nos tests de détection de l’ADNtc dans le cadre de deux études cliniques. L’étude PLACOL, incluant 82 patients atteints de cancer colorectal métastatique, a permis de mettre en évidence deux facteurs pronostiques : un seuil de 0.1 ng/mL et la mesure de la pente de décroissance de la concentration en ADN muté ou hyperméthylé circulant. Dans la seconde étude, portant sur le mélanome métastatique dans le contexte d’une thérapie ciblée (vémurafenib), une corrélation inverse entre les concentrations d’ADNtc et de vémurafenib a été observée. Ces résultats suggèrent le potentiel clinique de l’ADNtc pour l’orientation thérapeutique des patients atteints de cancer avancé. / Circulating tumor DNA (ctDNA) carries tumor-specific alterations that are detectable by minimally invasive sampling. It represents a highly pertinent marker for cancer monitoring during patients’ follow-up. CtDNA detection requires a highly sensitive and quantitative technique. In this context, this project focused on ctDNA quantification and monitoring by picoliter-droplet digital PCR. Thanks to the compartmentalization in millions of picoliter droplets, this tool allowed the detection of single DNA molecule with a sensitivity reaching 0.001%. Testing of ctDNA was performed through the evaluation of different potential biomarkers: specific mutations, ctDNA fragmentation, and hypermethylation of target sequences. On one hand, we observed in cancer patients that ctDNA is more fragmented than wild-type DNA, and, globally more fragmented than circulating DNA in healthy individuals. On the other hand, a strong correlation between percentages of hypermethylated and mutated DNA was observed during the follow-up of patients. Such results suggest the feasibility to precisely and quantitatively monitor ctDNA by the evaluation of hypermethylation as an alternative to the determination of mutational status. We have applied such ctDNA detection strategies in the context of two clinical studies. The PLACOL study, enrolling 82 metastatic colorectal cancer patients, allowed to highlight two prognostic factors: a ctDNA concentration threshold of 0.1 ng / mL, and the evaluation of ctDNA decreasing slope. In the second study, ctDNA was monitored in 11 melanoma patients in the context of a targeted therapy (vemurafenib). An inverse correlation between the concentrations of vemurafenib and ctDNA was demonstrated. These results suggest the clinical relevancy of ctDNA in advanced cancer patients, for the optimization of therapeutic management.

La fitness et a fortiori la survie d'une population dépendent de la stratégie et des performances de reproduction façonnées par son environnement. Par conséquent, les biomarqueurs traduisant une altération de la fonction de reproduction présentent un intérêt particulier. L'atteinte de la fertilité mâle au sein de la faune sauvage a notamment été adressée comme une problématique majeure susceptible de représenter une menace pour le maintien des populations. Toutefois, peu de méthodologies sont aujourd'hui disponibles chez des espèces pertinentes pour aborder cette problématique dans le cadre de programmes de biosurveillance environnementale, notamment chez les crustacés en dépit de leur représentativité au sein du règne animal et de leurs indispensables fonctions au sein des écosystèmes. Dans ce contexte, les présents travaux avaient pour objectif de proposer une ou plusieurs méthodologies basées sur la mesure de marqueurs de fonctionnalité et d'intégrité spermatique chez des crevettes Palaemonidae. Les investigations se sont portées sur deux espèces, une estuarienne, Palaemon longirostris, et une côtière, Palaemon serratus, pour leur complémentarité vis-à-vis du continuum estuaire - littoral. Compte tenu des nombreuses spécificités structurelles et fonctionnelles des spermatozoïdes de crustacés, I nombre de marqueurs transposables vers ces espèces s'est finalement avéré limité. Aussi, après une brève prospection, l'effort de recherche a rapidement été recentré sur la mesure de l'intégrité de l'ADN. Dès lors, la démarche scientifique a été construite de sorte à évaluer, point par point, la pertinence de la méthodologie développée dans une perspective d'application de l'outil dans le cadre de la surveillance environnementale. Une étape préliminaire d'optimisation et de validation méthodologique du test Cornet a démontré que, contrairement à une grande majorité de type spermatique, l'adaptation de ce test sur les spermatozoïdes de Palaemonidae ne nécessite aucune modification particulière du protocole. La dynamique de la réponse biologique en termes d'apparitions, de rémanence et d'effets possibles sur la fitness a été évaluée en conditions contrôlées au laboratoire. Ainsi, des expositions ex vivo et in vivo ont été conduites en utilisant une variété de génotoxiques modèles présentant différents modes d'actions. Les résultats ont attesté de la sensibilité, de la reproductibilité et du caractère intégrateur de la réponse. En revanche, aucun lien entre un ADN spermatique endommagé et une altération du succès de reproduction pré-éclosion n'a pu être établi. Parallèlement, une approche in situ a été conduite en vue de caractériser la valeur basale de la réponse mesurée. Différentes stratégies ont dû être adoptées en fonction des contraintes propres au milieu de vie de chacune des deux espèces. Un référentiel et une valeur seuil, communs aux deux espèces, ont pu être définis, soulignant le potentiel de transférabilité inter-espèces de l'outil. La méthodologie ainsi finalisée, a été éprouvée dans le cadre de plusieurs campagnes de suivi de différentes populations indigènes de l'estuaire et de la baie de Seine en 2015 et 2016. Les résultats se sont révélés très cohérents au regard de la pression de contamination et de la dynamique hydro-sédimentaire de la baie de Seine. En définitive, l'intégrité de l'ADN spermatique chez les Palaemonidae est opérationnelle en l'état pour un déploiement in situ en tant que biomarqueur d'exposition à un stress génotoxique. De futurs études devront néanmoins être conduites (1) pour mieux discerner les implications de ces dommages spermatiques en termes d'impact sur le recrutement des nouvelles cohortes et (2) éprouver la transférabilité de la méthodologie à d'autres espèces de crevettes et sur une plus large échelle géographique. / The environment shapes the reproduction's strategy and performance of a population, influencing its fitness and a fortiori its survival. Therefore, biomarkers that alter reproductive functions represent a great interest in ecotoxicology. The reduction of male fertility in wildlife can represent a threat to the population's survival. Moreover, fcw methodologies are available for species that are relevant to address this issue on envimnmental biomonitoring programs, especially for crustaceans, despite their representativeness in the wildlife and their essential functions within ecosysterns. The present work aimed to propose methodologies based on the measurement of functionality and integrity spermatic biomarkers on Palaemonidae shrimps. We studied two species, an estuarine, Palaemon longirostris, and a coastal species. Palaemon serratus. due to their complementaiity on the continuum estuary-littoral ecosystem. Howevcr, crustaceans' sperm has many structuraI and functional characteristics, the number of transposable markers is limited. Thereafter, the research effort was refocused on the measurement of the DNA integrity and this inethod was evaluated for its adequacy for a biomonitoring study. We optitrtized and validate the Cornet assay for the Palaemonidae species, and the dynamic of the biological response in ternis of appearances, persistence, and possible effects on fitness was evaluated under controlled conditions in the laboratoiy. Furthermore, ex-vivo and in-vivo exposures were conducted using genotoxic models with different modes of action. On the one hand, results attested to the sensibility, the reproducibility and the integrating character of the response, on the other hand, no correlation between damaged sperm DNA and an altération of the pre-hatch stage of development was established. In paralIel, an in-situ approach was conducted to characterize the baseline level of the measured response, taking into consideration the specific constraints of each species' habitat. We were able to define a common baseline level and a threshold value for both species, highlighting the potential of the tool for inter-species transferability. This method was tested with native populations from the estuary and from the Seine Bay in 2015 and 20] 6. And, the results proved to be consistent with the contamination pressure and the hydro-sedimentary dynamics of the Seine Bay. Ultimately, the DNA integrity of sperm in Palaemonidae seems to be functional for in-situ deploytnents as a biomarker of exposure to genotoxic stress. Nevertheless, future studies should be conducted (1) to botter discern the implications of this spermatic damage on the recruitment of new cohorts and, (2) to test the transferability of the methodology to other shrimp species and on a wider geographical scale.

Chez les animaux, la conformation unique du noyau du spermatozoïde dont la chromatine est organisée avec des protéines chromosomiques spécifiques telles que les protamines le rend totalement inactif. Le remodelage de la chromatine paternelle à la fécondation par des activités d'origine maternelle sont donc des processus essentiels à la formation d'un embryon diploïde, dont les mécanismes restent très mal connus. Lors de ma thèse j'ai essayé de mieux comprendre ces processus par l'étude, chez la drosophile, d'un mutant létal embryonnaire à effet maternel : maternal haploid (mh). Ce mutant affecte l'incorporation des chromosomes paternels à la première division zygotique menant à la formation d'embryons haploïdes gynogénétiques. L'identification du gène de mh comme CG9203 m'ont permis de caractériser sa fonction. Dans les œufs mh, les chromosomes paternels se condensent anormalement et ne parviennent pas à se diviser correctement lors de la première mitose de l'embryon. Récemment, des études sur son orthologue humain, appelé Spartan/DVC1, ont montré qu'il était impliqué dans la synthèse translésionnelle (TLS), un mécanisme de tolérance aux dommages d'ADN. J'ai pu démontrer que dans les cellules somatiques, la fonction de Spartan dans le TLS est conservée chez la drosophile. Cependant, la fonction maternelle de MH ne relève pas du TLS canonique, mais permet de maintenir l'intégrité de l'ADN paternel avant la réplication. Ensemble, mes travaux soulignent la singularité du pronoyau mâle et la complexité que présente le maintien de son intégrité à la fécondation / In animals, sexual reproduction requires the union between two distinct parental gametes: the spermatozoon and the oocyte. The unique nuclear conformation of the sperm, in which the chromatin is organized with sperm-specific chromosomal protein like protamines, abolishes its activity. The paternal chromatin remodeling and the maintenance of its integrity at fertilization by maternal activities are therefore essential processes for zygote formation. However, although their mechanisms are crucial, they remain poorly understood. During my thesis, I tried to better understand the processes involved during de novo paternal chromatin assembly in Drosophila through the study of a maternal embryonic lethal mutation: maternal haploid (mh). The mutant affects the incorporation of paternal chromosomes during the first zygotic division, leading to the development of gynogenetic haploid embryos. The identification of the mh gene as CG9203, and the generation of the null allele mh2 allowed me to characterize its function. In eggs led by mh mutant females, paternal chromosomes abnormally condense and fail to divide leading to the formation of chromatin bridges at the first embryonic division. Recently, its human ortholog Spartan/DVC1, has been described to be involved in translesion synthesis (TLS), a DNA damage tolerance pathway that ensures replication fork progression. Combining genetic and cytological approaches, I demonstrated that the Spartan function in TLS is conserved in Drosophila. However, I discovered that the critical function of MH during the first embryonic division, was not consistent with a canonical TLS. Alternatively, it is specifically required to maintain paternal integrity and to allow its proper replication at the first cycle. The mh phenotype characterization, led me to compare it with others phenotypes induced by the knock-down of replication factors and to study parental chromosome condensation in the zygote. Surprisingly, one of the proteins allowing the establishment of the pre-replication complex is dispensable for the proper paternal chromosome segregation contrarily to the maternal counterpart. Altogether, these works highlight the difference that exists between the two parental pronuclei and the complexity of maintaining their integrity at fertilization