La diapause embryonnaire se manifeste par un arrêt réversible du développement embryonnaire durant la période de préimplantation et induit un retard de l’implantation. Chez le vison américain, une diapause embryonnaire obligatoire caractérise chaque gestation. Si les mécanismes de contrôle de la diapause embryonnaire obligatoire chez cette espèce sont bien connus, le rôle utérin impliqué dans la réactivation de l’embryon demeure, quant à lui, encore inconnu. Le sujet de ce doctorat a consisté dans un premier temps à explorer l’environnement utérin à la sortie de la diapause embryonnaire afin de caractériser, dans un deuxième temps, les principaux acteurs utérins qui provoquent la réactivation de l’embryon. Nous avons effectué une analyse du transcriptome utérin à l’émergence de la diapause embryonnaire ce qui a permis de construire une librairie de 123 séquences d’ADNc utérines différentiellement exprimées à la réactivation de l’embryon et homologues à des séquences de gènes connues chez d’autres espèces. Ces gènes sont impliqués dans la régulation du métabolisme (25 %), de l’expression génique (21 %), de la transduction de signal (15 %), du cycle cellulaire (15 %), du transport (10 %) et de la structure cellulaire (9 %), reflétant ainsi d’importantes modifications utérines à la réactivation embryonnaire. Nous avons validé l’expression différentielle de dix gènes ainsi identifiés : GDF3 (growth and differentiation 3), ALCAM (activated leukocyte cell adhesion molecule), ADIPOR1 (adiponectin receptor 1), HMGN1 (high mobility group N1), TXNL1 (thioredoxin like 1), TGM2 (tissue transglutaminase 2), SPARC (secreted protein acidic rich in cystein), et trois gènes codant pour AZIN1 (antizyme inhibitor 1), ODC1 (ornithine decarboxylase 1) et SAT1 (spermidine/spermine N1-acetyltransferase), des enzymes impliquées dans la biosynthèse des polyamines. Le patron de l’expression spatio-temporel de SPARC et d’HMGN1 illustrent spécifiquement un remodelage tissulaire et de la chromatine au niveau utérin à la sortie de la diapause embryonnaire. Ayant mesuré une augmentation des concentrations utérines en polyamines à la reprise du développement embryonnaire, nous avons émis l’hypothèse que les polyamines seraient impliquées dans les événements menant à la sortie de la diapause. L’inhibition de la biosynthèse des polyamines par un traitement à l’ α-difluoromethylornithine (DFMO) a provoqué une diminution significative de la proliferation cellulaire dans les embryons à la réactivation, un retard du moment de l’implantation, mais n’a pas affecté le succès de la reproduction. De manière similaire, nous avons induit un état de dormance dans les cellules de trophoblaste de vison en présence DFMO dans le milieu de culture, et constaté que cet état était réversible. En conclusion, cette étude a non seulement ouvert de nouveaux horizons quant à la compréhension du rôle utérin dans les événements menant à la sortie de la diapause embryonnaire, mais a démontré pour la première fois, l’existence de facteurs utérins indispensables à la réactivation de l’embryon: les polyamines. / Embryonic diapause is characterized by a reversible arrest of blastocyst development prior to implantation and delay in implantation. In the American mink, embryonic diapause is a characteristic of each gestation. Although the mechanisms which control obligate embryonic diapause of this species are well known, the role of the uterus involved in blastocyst reactivation remains elusive. The subject of this doctoral research consisted first in exploring the uterine environment at the emergence of embryonic diapause in order to subsequently determine, the main factors in the uterus that provoke reactivation of the embryo. We have undertaken an analysis of the uterine transcriptome at the emergence of embryonic diapause which has enabled us to set up a library of 123 cDNA uterine sequences differentially expressed at blastocyst reactivation, and homologue gene sequences known in other species. Twenty-five percent of these genes are implicated in genetic expression, 15 % in cell signal transduction, 15 % in cell cycle, 10 % in transport and 9 % in cell structure. All of them reflect significant uterine modifications at blastocyst reactivation. We have validated differential expression of ten genes, identified as: GDF3 (growth and differentiation 3), ALCAM (activated leukocyte cell adhesion molecule), ADIPOR1 (adiponectin receptor 1), HMGN1 (high mobility group N1), TXNL1 (thioredoxine like 1), TGM2 (tissue transglutaminase 2), SPARC (secreted protein acidic rich in cystein), and three genes encoding for AZIN1 (antizyme inhibitor 1), ODC1 (ornithine decarboxylase 1) and SAT1 (spermidine/spermine N1-acetyltransferase), which are enzymes implicated in polyamine biosynthesis. The spatio-temporal expression patterns of SPARC and HMGN1 illustrate tissue and chromatin remodelling in the uterus at the termination of embryonic diapause. Having measured an increase in concentration of polyamines in the uterus at the resumption of blastocyst development, we have hypothetized that polyamines are implicated in the emergence of blastocysts from diapause. We inhibited polyamine biosynthesis in pregnant mink females during early blastocyst reactivation. The inhibition of polyamine biosynthesis through treatment with α-difluoromehtylornithine (DFMO) provoked a major reduction in cell proliferation in blastocysts at reactivation and a delay in the timing of implantation, but did not affect the success of reproduction. Similarly, we induced a reversible dormant state in cultured mink trophoblast cells traited with DFMO. To conclude, not only are results of this study a breakthrough in the understanding of the role of the uterus in stimulating at the emergence of blastocysts from embryonic diapause, but also, for the very first time, they indicate the existence of uterine factors, the polyamines, that are responsible for blastocysts reactivation.

O agente etiológico da Febre Maculosa das Montanhas Rochosas (RMSF), conhecida no Brasil como Febre Maculosa Brasileira, é a bactéria Rickettsia rickettsii. Essa bactéria é transmitida ao homem pela picada de diferentes espécies de carrapatos ixodídeos. No Brasil, os vetores são Amblyomma cajennense e A. aureolatum. As taxas de prevalência de R. rickettsii nas populações de carrapatos de áreas endêmicas para RMSF são baixas, em geral abaixo de 1%. Essa baixa prevalência parece estar associada a menores taxas reprodutivas e de sobrevivência de linhagens infectadas, sugerindo que R. rickettsii seja patogênica também para os seus vetores. Infecções experimentais demonstraram que 80-100% dos indivíduos de uma colônia de A. aureolatum mantida em laboratório são infectados por R. rickettsii, enquanto apenas 10-60% de A. cajennense adquirem a bactéria. Esses dados indicam que as respostas dessas duas espécies de carrapatos à infecção sejam diferentes, resultando em diferentes taxas de prevalência da bactéria. Dessa maneira, a caracterização molecular das interações entre carrapatos do gênero Amblyomma e a bactéria R. rickettsii é importante, podendo gerar informações não somente para o esclarecimento acerca dos mecanismos de patogenicidade de R. rickettsii para os carrapatos, mas também para um melhor entendimento dos mecanismos responsáveis pela aparente restringência de A. cajennense à infecção. Assim, os objetivos do presente estudo foram: (i) analisar os efeitos da infecção por R. rickettsii sobre o perfil de expressão gênica de carrapatos A. cajennense por hibridação subtrativa por supressão (SSH), (ii) validar os dados de SSH por reação em cadeia de polimerase quantitativa precedida por transcrição reversa (RT-qPCR) e (iii) caracterizar funcionalmente dois genes com expressão induzida pela infecção por RNA de interferência (RNAi). Após a análise bioinformática dos dados de SSH, 44 sequências únicas foram obtidas, das quais 36 representam genes com expressão induzida e 8 genes com expressão reprimida pela infecção. A indução dos genes codificadores da subunidade I da citocromo c oxidase (COX1), da subunidade IV da NADH desidrogenase, de uma proteína com domínio de inibidor de serina-proteases Kunitz-type (papilina-like), identificados por SSH, e de um peptídeo antimicrobiano (hebraeína), foi confirmada por RT-qPCR. O silenciamento gênico da hebraeína e da papilina-like não teve nenhum efeito na aquisição de R. rickettsii pelo vetor, indicando que, isoladamente, não são responsáveis pela proteção de A. cajennense contra a infecção. Os dados gerados pelo presente estudo abrem perspectivas para que outros genes sejam avaliados quanto ao seu papel na aquisição de R. rickettsii, os quais, no futuro, podem ser considerados como alvos para o desenvolvimento de vacinas. / The etiologic agent of the Rocky Mountain Spotted Fever (RMSF), also known as Brazilian Spotted Fever in Brazil, is the bacterium Rickettsia rickettsii. This rickettsia is transmitted to humans by the bite of various tick species. In Brazil, Amblyomma cajennense and A. aureolatum are known as vectors. The prevalence rates of R. rickettsii infected ticks in RMSF endemic areas are low, oscillating around 1%. These low prevalence rates seems to be associated with lower reproductive and survival rates of infected ticks, suggesting that R. rickettsii is also pathogenic to its vectors. Experimental infections with R. rickettsii have demonstrated that 80 to 100% of A. aureolatum ticks from a laboratory colony acquire this bacterium, whereas only 10 to 60% of A. cajennense ticks become infected. These results indicate that the responses of these two tick species against infection are different, resulting in different prevalence rates of the bacterium. Therefore, the elucidation of the interactions between ticks of the genera Amblyomma and the bacterium R. rickettsii at a molecular level is important to provide information to better understand the mechanisms of pathogenicity of R. rickettsii against ticks as well as for the elucidation of the mechanisms responsible for the apparent refractoriness of A. cajennense against infection. Therefore, the objectives of the current study were: (i) analyze the effets of the infection with R. rickettsii on the gene expression of ticks A. cajennense by suppression subtractive hybridization (SSH), (ii) validate SSH data by reverse transcription quantitative polymerase chain reaction (RT-qPCR), and (iii) functionally characterize two genes induced by infection using RNA interference (RNAi). After bioinformatics analysis of SSH data, 44 unique sequences were obtained, among which 36 represent genes with expression induced and 8 repressed genes by infection. The induction of genes encoding subunit I of cytochrome c oxidase (COX1), the NADH dehydrogenase subunit IV, a protein containing Kunitz-type inhibitor domain (papilin-like), identified by SSH, and an antimicrobial peptide (hebraein), was confirmed by RT-qPCR. The effects of knockdown of hebraein and papilin-like encoding genes had no effect on the acquisition of R. rickettsii by the vector. Data of the current study may be used to evaluate the role of other genes in acquisition of R. rickettsii, which, in the future, may be considered as target for vaccine development.

Desde a década de 80, o nucleopoliedrovírus Anticarsia gemmatalis (AgMNPV) tem sido utilizado no Brasil como agente de controle biológico no combate à lagarta-da-soja, resultando para o país significativos benefícios econômicos e ecológicos. Este vírus envelopado, pertencente à família Baculoviridae, possui DNA circular de fita dupla (132.239 pb) contido em um capsídeo protéico, que pode estar ocluído em uma matriz para-cristalina. Neste trabalho, analisamos a expressão temporal de seus genes em duas linhagens celulares (UFL-AG-286 e IPLB-SF-9), por PCR em tempo real. Outro objetivo foi o estudo do efeito da multiplicação viral na malha gênica celular (GRN), visando analisar a expressão gênica celular diferenciada durante a infecção, através da técnica de hibridização subtrativa. Verificamos que todas as ORFs (exceto ORFs 64 e 83, que provavelmente não codificam a genes) foram expressas, com diferenças significativas entre as linhagens, principalmente em relação ao nível de expressão. Apesar disso, o grupo de genes ligados a replicação apresentou perfil de expressão similar nas duas linhagens, possivelmente por este ser um processo essencial à replicação viral. De uma forma geral, todos os genes apresentaram um perfil de expressão mais precoce do que o relatado na literatura, o que poderia ser tanto devido à replicação precoce do DNA do AgMNPV quanto até mesmo consequência da sensitividade do método utilizado. O agrupamento dos genes por k-means seguiu, em sua maioria, a hora pós-infecção (p.i.) onde a expressão de cada gene foi detectada, o que é coerente com a expressão gênica em cascata de baculovírus. Entretanto, por esta classificação não foi possível predizer função gênica para os genes pouco caracterizados. Em relação ao efeito da infecção do AgMNPV na GRN da UFL-AG-286, observamos que em 20h p.i., uma grande diversidade de genes e funções celulares foram hipo-expressas. / Since the 80s, the Anticarsia gemmatalis nucleopolyhedroviruses (AgMNPV) has been used in Brazil as a biological control agent against the Anticarsia gemmatalis caterpillar in soybean fields, resulting in considerable economic and ecological benefits. This enveloped virus belongs to the Baculoviridae family. It has circular double-stranded DNA (132239 bp) enclosed in a capsid, which can be occluded in a crystalline matrix. In this work we elucidated the temporal gene expression profile of the AgMNPV-2D in two cell lines (UFL-AG-286 and IPLB-SF-9), using a real time PCR. Another objective was to study the effect of viral replication on the cellular gene regulatory network (GRN), in order to analyze the differential cellular gene expression during infection, using subtractive hybridization method. We found that most ORFs (except 64 and 83 ORFs that probably do not encode genes) were expressed, with significant differences between cell lines, mainly in expression intensity. However, the group of genes associated with viral DNA replication had similar expression profile in both lineages, possibly because replication is an essential process for viral multiplication. In general, most genes had earlier expression than reported in the literature, probably due to the early DNA replication in AgMNPV. Moreover, this could be a consequence of the method sensitivity used herein. We clustered genes with the k-means algorithm according to the time pos infection (p.i.) in which each gene expression was first detected and found it to be consistent with the typical cascade of gene expression known for baculovirus. Nonetheless, following this classification, it was not possible to predict gene function for poorly characterized genes. When looking at the impact of viral replication on the host GRN using subtractive hybridization, we found considerable inhibition of cellular transcription at 20h p.i. Furthermore at this time, a large and diverse set of cellular genes and functions were found to be hypo-regulated, indicative of an extensive effect of AgMNPV infection on the UFL-AG-286 GRN.

A endometriose é uma doença ginecológica benigna, de etiologia complexa e multifatorial, caracterizada pela presença de estroma e tecido glandular tipo endométrio fora da cavidade uterina. Afeta de 10 a 15% da população feminina, que apresentam sintomatologia variada, incluindo dor pélvica e infertilidade. Para elucidar mecanismos potenciais que estejam envolvidos com a fisiopatologia complexa desta doença, analisamos o perfil de expressão gênico diferencial pela metodologia de hibridação subtrativa em tecido eutópico e ectópico (lesões peritoniais e endometrioma ovariano) de 17 mulheres com endometriose, no início da fase proliferativa do ciclo menstrual. Foram identificados 291 genes desregulados nas lesões endometrióticas, considerados como genes candidatos. Para a validação dos dados, utilizamos a metodologia de PCR em tempo real para os genes CTGF e SPARC, indicados como superexpressos; e MYC, MMP3, IGFBP1 e PAEP como menos expressos nas lesões. Diferenças significativas de expressão nas lesões peritoniais foram obtidas para os genes SPARC, MYC, IGFBP1, PAEP e nos endometriomas ovarianos para os genes MMP3 e PAEP. Sugerimos que a desregulação dos genes SPARC, MYC, MMP3, IGFBPI e PAEP seja responsável pela perda da homeostase celular nas lesões endometrióticas, contribuindo para a implantação e sobrevivência do tecido ectópico no ambiente extra-uterino. Este trabalho disponibilizou ao banco de dados da literatura, 291 genes com expressão gênica diferencial em lesões endometriótricas peritoniais e ovarianas como candidatos a investigações futuras. / Endometriosis is a benign gynecological disease, which presents a multifactorial and complex etiology, characterized by the presence of stromal and glandular endometrium tissue outside the uterine cavity. Ten to 15% of the female population is affected by the disease with a wideranging symptomatology including pelvic pain and infertility. To clarify the potential mechanisms involved in the complex physiopathology of this disease, we analyzed the differential gene expression profile by subtractive hybridization in eutopic and ectopic tissue (peritoneal lesions and ovarian endometriomas) from 17 women with endometriosis, in the early proliferative phase of the menstrual cycle. We identified 291 genes deregulated in the endometriotic lesions, considered as candidate genes. For data validation, Real Time PCR was applied for genes CTGF and SPARC, indicated as overexpressed; and for genes MYC, MMP3, IGFBP1 and PAEP, indicated as downregulated in the lesions. Significant differences in the peritoneal lesions expression were obtained for genes SPARC, MYC, IGFBP1, PAEP and in the ovarian endometriomas for genes MMP3 and PAEP. We suggest that the deregulation of genes SPARC, MYC, MMP3, IGFBPI and PAEP is responsible for loss of cellular homeostasis in the endometriotic lesions, contributing for the implantation and maintenance of the ectopic tissue in the extra-uterine environment. This study provided 291 genes with differential gene expression, in peritoneal and ovarian lesions, to the literature database as candidates for future investigations.

Submitted by (ana.araujo@ufrpe.br) on 2017-02-07T16:48:16Z No. of bitstreams: 1 Daniel Oliveira Jordao do Amaral.pdf: 1158857 bytes, checksum: e7a95fb82b8780bf1c190ef328318925 (MD5) / Made available in DSpace on 2017-02-07T16:48:16Z (GMT). No. of bitstreams: 1 Daniel Oliveira Jordao do Amaral.pdf: 1158857 bytes, checksum: e7a95fb82b8780bf1c190ef328318925 (MD5) Previous issue date: 2007-06-14 / To identify tomato plant (Lycopersicon esculentum Mill), cv. BRH, genes which answer to plant pathogen Fusarium oxysporum f. sp. lycopersici and salicylic acid, the carrier molecule for activation of responses of plant defense, it was used the suppression subtractive hybridization (SSH) technique, from leaf cDNAs, 24h after salicylic acid, library denominated AS, and root cDNA, 72h after inoculation with F. oxysporum f. sp. lycopersici, incompatible interaction, library denominated FO. This work represents the first report of global gene expression of tomato plant induced by salicylic acid and F. oxysporum f. sp. lycopersici, using SSH technique; it was identified a total of 307 clones in the two subtractive libraries, being 143 obtained in the AS library and 164 in the FO library. Probable functions for genes were obtained by sequencing of clones and subsequent homology research at datas. These isolated genes are involved in several processes related to resistance against plant pathogen such as: hypersensitive response, programmed cell death, synthesis and transport of antimicrobial metabolites, signal perception and transduction, synthesis of pathogenesis-related proteins, lipid metabolism and selective degradation of proteins. It was identified in FO library a higher number of defense-related genes (26%) than in AS library (24%). In relation to the number of genes encoding antimicrobial proteins, they were only found in FO library (7%). However, genes involved in secondary compound metabolism were higher in AS library (13%) in relation to FO library (4%). These genes related to controlled degradation of proteins were also higher in AS library (3%) than in FO library (1%). The results suggest that the resistance of tomato plant induced by salicylic acid and by plant pathogen occur by distinct mechanisms. / Com o propósito de identificar genes no tomateiro (Lycopersicon esculentum Mill), cv. BRH, que respondem ao fitopatógeno Fusarium oxysporum f. sp. lycopersici e ao ácido salicílico, molécula mensageira na ativação de resposta de defesa em plantas, foi utilizada a técnica de hibridização subtrativa por supressão (HSS), a partir de cDNAs de folhas, 24h após o tratamento com ácido salicílico, biblioteca denominada (AS), e cDNAs de raízes, 72h após a inoculação com Fusarium oxysporum f. sp. lycopersici, interação incompatível, biblioteca denominada (FO). Esse trabalho representa o primeiro relato da expressão gênica global no tomateiro induzido pelo ácido salicílico e pelo F. oxysporum f. sp. lycopersici, utilizando a técnica HSS. Foram identificados um total de 307 clones nas duas bibliotecas subtraídas, sendo 143 clones obtidos na biblioteca (AS) e 164 clones na biblioteca FO. As prováveis funções dos genes foram obtidas pelo sequenciamento dos clones e subseqüente pesquisa de homologia em bancos de dados. Os genes encontrados estão envolvidos em diversos processos relacionados à resistência contra fitopatógenos como: resposta de hipersensibilidade, morte celular programada, síntese e transporte de metabólicos antimicrobianos, percepção e transdução de sinal, síntese de proteínas relacionadas à patogênese, metabolismo de lipídeos e degradação controlada de proteínas. Foram identificados na biblioteca FO um número maior de genes implicados em mecanismos de defesa (26%), do que na biblioteca AS (24%). Em relação ao número de genes codificadores de proteínas antimicrobianas foram encontrados apenas na biblioteca FO (7%). Entretanto, os genes envolvidos no metabolismo de compostos secundários foi maior na biblioteca AS (13%) em relação a biblioteca FO (4%). Os genes relacionados a degradação controlada de proteínas também foi maior na biblioteca AS (3%) do que na biblioteca FO (1%). Os resultados obtidos sugerem que a resistência no tomateiro induzido pelo ácido salicílico e pelo patógeno ocorre por mecanismos distintos.

Spiroplasma citri est une bactérie phytopathogène transmise par la cicadelle Circuliferhaematoceps. L'absence de symptômes malgré la multiplication de S. citri dans l'hémolymphe, suggèreque le système immunitaire joue un rôle important dans la tolérance de la cicadelle vis-à-vis duspiroplasme.Le but de cette thèse a donc été d'étudier la réponse immunitaire de C. haematoceps aucours de l'infection par S. citri.Notre étude sur le système immunitaire de la cicadelle a montré la présence dans le plasma d'uneactivité antibactérienne et d'une activité phénoloxidase. Parmi les principaux types d'hémocytes unephagocytose des bactéries par les granulocytes et les plasmatocytes a été observée. Les gènessusceptibles d'être impliqués dans ces processus ont été recherchés par une approche par hybridationsoustractive. De manière étonnante, aucun gènes codant des récepteurs ni d'effecteurs connus del'immunité n'ont été identifiés. En revanche certains gènes (23 en tout) codent des protéines ayantpotentiellement un rôle immunitaire. Six de ces 23 gènes ont été retenus pour suivre leur expressionau temps précoce d'une infection bactérienne. Les résultats ont montré que les gènes codantI'Hexamérine, la DDBPl et la Thiorédoxine peroxydase étaient surexprimés lors de l'infection par 5.citri. Une approche fonctionnelle d'interférence par ARN a montré d'une part que I'Hexamérine étaitimpliquée dans l'activité phénoloxidase et d'autre part qu'elle jouait un rôle important dans la surviede C. haematococeps au cours de l'infection par 5. citri. En parallèle, le suivi de l'activité phénoloxidaseet de la phagocytose au cours de l'infection a montré que 5. citri était capable de s'adapter à laréponse immunitaire de l'insecte et d'y échapper. Ces résultats rejoignent ceux obtenus chez ladrosophile concernant S. poulsonii. / Spirop/asma citri is phytopathogenic bacteria transmitted by the leafhopper Circuliferhaematoceps. The absence of symptoms despite the multiplication of S. citri in the hemolymph,suggests that the immune system plays an important role in the tolerance of the leafhopper towardsthe spiroplasma infection. The purpose of this thesis was to study the immune response of C.haematoceps during the infection by 5. citri.The characterization of the immune system of the leafhopper showed that an antibacterial activity anda phenoloxidase activity were present in the plasma. The main types of hemocytes were identified.Among them, granulocytes and plasmatocytes are capable to phagocyte bacteria. The genes involvedin these immune processes were searched using subtractive hybridization method. lnterestingly, noneof the genes known to encode receptors or effectors of the immune system were identified. On theother hand 23 putative immune genes were identified. Six of these genes were retained to follow theirexpression in the early time of a bacterial infection. The results showed that the genes encodingHexamerin, DDBPl and Thioredoxin peroxidase were up-regulated during the infection by 5. citri. Afunctional approach by gene silencing showed that Hexamerin was involved in the phenoloxidaseactivity and played an important role in the survival of C. haematoceps during the infection by S. citri.Finally, the follow-up of the phenoloxidase activity and phagocytosis by hemocytes showed anadaptation and an evasion of S. citri from the immune response of the insect, according to the resultsobtained for 5. pou/sonii-infected drosophila.Keywords : 5piroplasma citri, phenoloxidase, phagocytosis, hemocytes, gene silencing, Hexamerine,subtractive hybridization.

Notre travail qui sinscrit dans le cadre général de létude du développement embryonnaire de Phaseolus a pour objectif principal disoler et de caractériser des gènes différemment exprimés chez les embryons en voie davortement, et donc nécessaires au développment normal des embryons. Des embryons en cours de dégénérescence issus des hybridations interspécifiques et de la mutagenèse induite ont été analysés. Des ADNc différemment exprimés chez ces embryons ont été identifiés par les techniques de lHybridation Soustractive Suppressive (HSS) et de la dot blot. Les hybridations interspécifiques ont été réalisées entre lespèce P. vulgaris L. utilisée comme parent mâle et les espèces P. coccineus L. et P. polyanthus Greenm. utilisées comme parents femelles (formes sauvages et cultivées). La mutagenèse induite à lEthyl Méthyl Sulfonate (EMS) a été appliquée sur le génotype BAT93 de P. vulgaris, une variété améliorée du CIAT. Dans les croisements P. coccineus x P. vulgaris, 938 hybridations ont été effectuées et le taux de gousses avortées au-delà de 8 JAP est denviron 12%. Quatre gousses supposées hybrides ont été obtenues. Pour les croisements P. polyanthus x P. vulgaris, 733 hybridations ont été réalisées. Le taux de gousses avortées au-delà de 8 JAP est denviron 18% et une seule gousse supposée hybride a été produite. Les caractères hybrides dune plante de chacune des deux combinaisons interspécifiques ont été mis en évidence au moyen de caractères morphologiques des fleurs et des graines, mais aussi grâce à lutilisation dun marqueur moléculaire, le microsatellite BM160. La mise en évidence et la caractérisation des embryons en voie davortement ont été effectuées à partir de matériels issus des hybridations interspécifiques et de la mutagenèse à lEthyl Méthyl Sulfonate (EMS). Les observations, faites sur des embryons extraits et sur des coupes histologiques dovules, révèlent des malformations au niveau du suspenseur et des cotylédons et des retards de croissance. Les plantes issues de la mutagenèse et produisant des graines avortant avant la maturité ont été croisées avec des plantes normales. Lanalyse de la F2 effectuée sur 96 plantes révèle une proportion mendélienne 3:1 de plantes avec des graines normales et de plantes avec graines qui avortent. Ce résultat suggère un contrôle du caractère « avortement des graines » par une paire dallèles récessifs. La technique de lHSS a permis disoler des fragments dADNs complémentaires différemment exprimés dans les graines en voie davortement. Lanalyse des séquences de ces ADNs complémentaires montre quils codent pour plusieurs protéines intervenant dans les développements cellulaire et embryonnaire. Les principales protéines sont le cytochrome P450, la myo-inositol 1-phosphate synthase, la peroxydase cationique, le voltage-dependent anion channel et la sucrose synthase. A lexception du cytochrome P450, les niveaux dexpression des autres gènes sont plus faibles dans les graines en voie davortement issues de la mutagenèse par rapport aux graines normales. The main objective of this study was to isolate and to characterize cDNAs differentially expressed in Phaseolus degenerating embryos. Aborting embryos from interspecific hybridizations and induced mutation were analysed. cDNAs differentially expressed in these embryos were isolated using the Suppressive Subtractive Hybridization (SSH) and the dot blot techniques. The interspecific hybridizations were performed between P. vulgaris L. used as male parent and P. coccineus L. and P. polyanthus Greenm. used as female parents (wild and cultivated forms). The induced mutation was performed whith Ethyl Methyl Sulfonate (EMS) applied on the genotype BAT93 of P. vulgaris, a breeding line from CIAT. A total number of 938 crosses P. coccineus x P. vulgaris and 733 crosses P. polyanthus x P. vulgaris were carried out. In the crosses P. coccineus x P. vulgaris, the rate of pod abortion after 8 days after pollination (DAP) is 12%. Four putative hybrid pods were obtained. The rate of pod abortion after 8 DAP in the crosses P. polyanthus x P. vulgaris is 18% and one putative hybrid pod was produced. The hybrid nature of one plant from each interspecific combination was confirmed using morphological characters of flowers and seeds and molecular marker (microsatellite BM160). The isolation and the characterization of degenerating embryos were realised with materials from interspecific hybridizations and from chemical mutagenesis with EMS. The observations of these two materials revealed abnormalities mainly in suspensor and cotyledons; and the embryos failed to grow normally. Plants from mutagenesis which produce degenerating seeds were crossed with normal plants. Genetic analysis on 96 F2 plants revealed a 3:1 Mendel ratio of plants with normal seeds and plants with degenerating seeds. This result suggests the control of the seed abortion trait by a single recessive gene. The SSH technique was used to isolate cDNAs fragments differentially expressed in aborting seeds. Analysis of the cDNAs sequences revealed that these cDNAs encode for proteins involved in cellular and embryonic development. The main proteins are cytochrome P450, myo-inositol 1-phosphate synthase, cationic peroxidase, voltage-dependent anion channel and sucrose synthase. All the genes showed a reduction of their expression in developing seeds of the mutagenized plants, compared to those observed in wild-type plants.

La diapause embryonnaire se manifeste par un arrêt réversible du développement embryonnaire durant la période de préimplantation et induit un retard de l’implantation. Chez le vison américain, une diapause embryonnaire obligatoire caractérise chaque gestation. Si les mécanismes de contrôle de la diapause embryonnaire obligatoire chez cette espèce sont bien connus, le rôle utérin impliqué dans la réactivation de l’embryon demeure, quant à lui, encore inconnu. Le sujet de ce doctorat a consisté dans un premier temps à explorer l’environnement utérin à la sortie de la diapause embryonnaire afin de caractériser, dans un deuxième temps, les principaux acteurs utérins qui provoquent la réactivation de l’embryon. Nous avons effectué une analyse du transcriptome utérin à l’émergence de la diapause embryonnaire ce qui a permis de construire une librairie de 123 séquences d’ADNc utérines différentiellement exprimées à la réactivation de l’embryon et homologues à des séquences de gènes connues chez d’autres espèces. Ces gènes sont impliqués dans la régulation du métabolisme (25 %), de l’expression génique (21 %), de la transduction de signal (15 %), du cycle cellulaire (15 %), du transport (10 %) et de la structure cellulaire (9 %), reflétant ainsi d’importantes modifications utérines à la réactivation embryonnaire. Nous avons validé l’expression différentielle de dix gènes ainsi identifiés : GDF3 (growth and differentiation 3), ALCAM (activated leukocyte cell adhesion molecule), ADIPOR1 (adiponectin receptor 1), HMGN1 (high mobility group N1), TXNL1 (thioredoxin like 1), TGM2 (tissue transglutaminase 2), SPARC (secreted protein acidic rich in cystein), et trois gènes codant pour AZIN1 (antizyme inhibitor 1), ODC1 (ornithine decarboxylase 1) et SAT1 (spermidine/spermine N1-acetyltransferase), des enzymes impliquées dans la biosynthèse des polyamines. Le patron de l’expression spatio-temporel de SPARC et d’HMGN1 illustrent spécifiquement un remodelage tissulaire et de la chromatine au niveau utérin à la sortie de la diapause embryonnaire. Ayant mesuré une augmentation des concentrations utérines en polyamines à la reprise du développement embryonnaire, nous avons émis l’hypothèse que les polyamines seraient impliquées dans les événements menant à la sortie de la diapause. L’inhibition de la biosynthèse des polyamines par un traitement à l’ α-difluoromethylornithine (DFMO) a provoqué une diminution significative de la proliferation cellulaire dans les embryons à la réactivation, un retard du moment de l’implantation, mais n’a pas affecté le succès de la reproduction. De manière similaire, nous avons induit un état de dormance dans les cellules de trophoblaste de vison en présence DFMO dans le milieu de culture, et constaté que cet état était réversible. En conclusion, cette étude a non seulement ouvert de nouveaux horizons quant à la compréhension du rôle utérin dans les événements menant à la sortie de la diapause embryonnaire, mais a démontré pour la première fois, l’existence de facteurs utérins indispensables à la réactivation de l’embryon: les polyamines. / Embryonic diapause is characterized by a reversible arrest of blastocyst development prior to implantation and delay in implantation. In the American mink, embryonic diapause is a characteristic of each gestation. Although the mechanisms which control obligate embryonic diapause of this species are well known, the role of the uterus involved in blastocyst reactivation remains elusive. The subject of this doctoral research consisted first in exploring the uterine environment at the emergence of embryonic diapause in order to subsequently determine, the main factors in the uterus that provoke reactivation of the embryo. We have undertaken an analysis of the uterine transcriptome at the emergence of embryonic diapause which has enabled us to set up a library of 123 cDNA uterine sequences differentially expressed at blastocyst reactivation, and homologue gene sequences known in other species. Twenty-five percent of these genes are implicated in genetic expression, 15 % in cell signal transduction, 15 % in cell cycle, 10 % in transport and 9 % in cell structure. All of them reflect significant uterine modifications at blastocyst reactivation. We have validated differential expression of ten genes, identified as: GDF3 (growth and differentiation 3), ALCAM (activated leukocyte cell adhesion molecule), ADIPOR1 (adiponectin receptor 1), HMGN1 (high mobility group N1), TXNL1 (thioredoxine like 1), TGM2 (tissue transglutaminase 2), SPARC (secreted protein acidic rich in cystein), and three genes encoding for AZIN1 (antizyme inhibitor 1), ODC1 (ornithine decarboxylase 1) and SAT1 (spermidine/spermine N1-acetyltransferase), which are enzymes implicated in polyamine biosynthesis. The spatio-temporal expression patterns of SPARC and HMGN1 illustrate tissue and chromatin remodelling in the uterus at the termination of embryonic diapause. Having measured an increase in concentration of polyamines in the uterus at the resumption of blastocyst development, we have hypothetized that polyamines are implicated in the emergence of blastocysts from diapause. We inhibited polyamine biosynthesis in pregnant mink females during early blastocyst reactivation. The inhibition of polyamine biosynthesis through treatment with α-difluoromehtylornithine (DFMO) provoked a major reduction in cell proliferation in blastocysts at reactivation and a delay in the timing of implantation, but did not affect the success of reproduction. Similarly, we induced a reversible dormant state in cultured mink trophoblast cells traited with DFMO. To conclude, not only are results of this study a breakthrough in the understanding of the role of the uterus in stimulating at the emergence of blastocysts from embryonic diapause, but also, for the very first time, they indicate the existence of uterine factors, the polyamines, that are responsible for blastocysts reactivation.

A endometriose é uma doença ginecológica benigna, de etiologia complexa e multifatorial, caracterizada pela presença de estroma e tecido glandular tipo endométrio fora da cavidade uterina. Afeta de 10 a 15% da população feminina, que apresentam sintomatologia variada, incluindo dor pélvica e infertilidade. Para elucidar mecanismos potenciais que estejam envolvidos com a fisiopatologia complexa desta doença, analisamos o perfil de expressão gênico diferencial pela metodologia de hibridação subtrativa em tecido eutópico e ectópico (lesões peritoniais e endometrioma ovariano) de 17 mulheres com endometriose, no início da fase proliferativa do ciclo menstrual. Foram identificados 291 genes desregulados nas lesões endometrióticas, considerados como genes candidatos. Para a validação dos dados, utilizamos a metodologia de PCR em tempo real para os genes CTGF e SPARC, indicados como superexpressos; e MYC, MMP3, IGFBP1 e PAEP como menos expressos nas lesões. Diferenças significativas de expressão nas lesões peritoniais foram obtidas para os genes SPARC, MYC, IGFBP1, PAEP e nos endometriomas ovarianos para os genes MMP3 e PAEP. Sugerimos que a desregulação dos genes SPARC, MYC, MMP3, IGFBPI e PAEP seja responsável pela perda da homeostase celular nas lesões endometrióticas, contribuindo para a implantação e sobrevivência do tecido ectópico no ambiente extra-uterino. Este trabalho disponibilizou ao banco de dados da literatura, 291 genes com expressão gênica diferencial em lesões endometriótricas peritoniais e ovarianas como candidatos a investigações futuras. / Endometriosis is a benign gynecological disease, which presents a multifactorial and complex etiology, characterized by the presence of stromal and glandular endometrium tissue outside the uterine cavity. Ten to 15% of the female population is affected by the disease with a wideranging symptomatology including pelvic pain and infertility. To clarify the potential mechanisms involved in the complex physiopathology of this disease, we analyzed the differential gene expression profile by subtractive hybridization in eutopic and ectopic tissue (peritoneal lesions and ovarian endometriomas) from 17 women with endometriosis, in the early proliferative phase of the menstrual cycle. We identified 291 genes deregulated in the endometriotic lesions, considered as candidate genes. For data validation, Real Time PCR was applied for genes CTGF and SPARC, indicated as overexpressed; and for genes MYC, MMP3, IGFBP1 and PAEP, indicated as downregulated in the lesions. Significant differences in the peritoneal lesions expression were obtained for genes SPARC, MYC, IGFBP1, PAEP and in the ovarian endometriomas for genes MMP3 and PAEP. We suggest that the deregulation of genes SPARC, MYC, MMP3, IGFBPI and PAEP is responsible for loss of cellular homeostasis in the endometriotic lesions, contributing for the implantation and maintenance of the ectopic tissue in the extra-uterine environment. This study provided 291 genes with differential gene expression, in peritoneal and ovarian lesions, to the literature database as candidates for future investigations.

Newts have the incredible ability to regenerate many different organs and tissues as adults, including the limbs. Limb regeneration occurs via the dedifferentiation of stump tissue and the formation of a blastema, which provides the majority of cells for the regenerate. Despite all that we have learned about dedifferentiation and blastema formation, the cellular and molecular mechanisms underlying these processes are still poorly understood. We used representational difference analysis (RDA) to identify genes involved in the early dedifferentiation process in newt forelimb regeneration. Our analysis identified approximately 410 unique genes that were differentially regulated during this process. Microarray analysis was used to determine the expression profile of these genes throughout limb and tail regeneration. We used quantitative PCR (qPCR) to validate the expression of a subset of these genes [β-catenin, wntless, dapper, thymosin-β 4 (Tβ4), and thymosin-β 10/15 (Tβ10/15)] in regenerating limb and tail tissue, as well as in differentiating newt myoblasts. We also verified the expression of these genes in the regenerating newt limb using immunohistochemistry (IHC) and in situ hybridization (ISH). Finally, we performed a functional analysis on β-catenin, wntless, dapper, and Tβ4 by overexpressing these genes in mouse myoblasts to examine their effects on differentiation and potential roles in dedifferentiation. Quantitative PCR verified the expression of β-catenin, wntless, dapper, and Tβ4 during limb regeneration and IHC/ISH localized the β-catenin and Tβ4 proteins to the blastema during regeneration. Tβ10/15 was shown by qPCR to be expressed in the tail during regeneration. Overexpression of newt β-catenin, wntless, dapper, and Tβ4 in mouse myoblasts showed that each of these genes has an inhibitory effect on the differentiation of myoblasts into myotubes and, therefore, may play a role in promoting or maintaining the dedifferentiated state. Our work has identified a large number of genes with potential roles in regulating the dedifferentiation process during newt forelimb regeneration. We have also laid a framework from which much more work can be done by drawing on the genes we have identified and the microarray data, which indicate ideal follow-up candidates. Our analysis of specific genes has also increased our understanding of the molecular events occurring during the dedifferentiation process in the regenerating newt limb.