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.

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

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.

Un nombre important d’individus subit des conséquences négatives en lien avec une appartenance à un groupe peu adapté socialement (p. ex., membre d’un gang de rue). Certains parviennent à mettre fin à cette identification, alors que d'autres n’y arrivent pas. Nous proposons que les individus qui réussissent le peuvent grâce à l’intégration d’une nouvelle identité, davantage adaptée, et conflictuelle avec leur identité d’origine. Dans ce mémoire, nous mettons de l’avant l’argument que lors de conflit identitaire majeur entre deux identités, le processus d’intégration identitaire est soustractif. Cinq sous hypothèses ont été testées lors de deux études effectuées avec des participants vivant un conflit identitaire majeur. Un niveau élevé de conflit identitaire prédit un faible niveau d’identification envers l’identité au statut le moins élevé (hypothèse 1). Un lien prédictif est postulé entre le statut perçu d’une identité et le niveau d’identification à cette identité (hypothèse 2). Un niveau d’intégration identitaire élevé de la nouvelle identité prédit un faible niveau d’identification envers l’identité au statut le moins élevé (hypothèse 3). Un niveau d’intégration identitaire élevé de la nouvelle identité prédit un faible niveau de déviance (étude 1) et d’alcoolisme (étude 2) (hypothèse 4). Finalement, un niveau d’intégration identitaire élevé de la nouvelle identité prédit un niveau de bien-être élevé (hypothèse 5). Les résultats de la première étude (N=42), effectuée sur un échantillon de jeunes filles placées en Centre Jeunesse, vont dans le sens des hypothèses 2 et 3. Les résultats de la deuxième étude (N=28), effectuée sur un échantillon d’individus membres des Alcooliques Anonymes, vont dans le sens des hypothèses 2 et 5. / An important number of individuals suffer from negative consequences associated with a negative social identity (i.e., members of street gangs). A number of them are able to get rid of that identity, whereas others continue to belong to a negative group. We theorize that individuals who no longer identify to a negative group are those who integrate a pro-social identity, in conflict with their original identity. In this thesis, we bring forward the argument that in the presence of a strong identity conflict between two identities; the identity integration process follows a subtractive pattern. In order to support this statement, the five following sub-hypotheses have been tested: a high level of identity conflict predicts a low level of identification towards the identity with a perceived lower status (hypothesis 1). The status attributed to an identity predicts of the level of identification toward that identity (hypothesis 2). A high level of identity integration of the new identity predicts a low level of identification towards the identity with the perceived lower status (hypothesis 3). A high level of identity integration of the new identity predicts a low level of deviance (study 1) and alcoholism (study 2) (hypothesis 4). Finally, a high level of identity integration of the new identity predicts a high level of well-being (hypothesis 5). Results from the first study (N=42), conducted on a sample of young girls placed in a rehabilitation center, support hypothesis 2 and 3 whereas results from study 2 (N=28), conducted on a sample of individuals member of Alcoholics Anonymous, support hypotheses 2 and 5.

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

Made available in DSpace on 2015-03-26T13:47:30Z (GMT). No. of bitstreams: 1 texto completo.pdf: 427044 bytes, checksum: 5c1651c701a5ae2b53e04e42782f989e (MD5) Previous issue date: 2007-09-24 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Infectious bursal disease (IBD) has been the major preoccupation for the poultry industry, especially in the past decade with the emergency of high virulent strains. Mutations in the gene that code the VP2 viral protein of pathogenic strains and the amiss use of attenuate vaccines by few passages have been responsible for the springing of new outbreaks. The purpose of this work was to analyze phylogenetically two different isolates of IBDV in Minas Gerais State by the comparison of the nucleotide sequence of the gene that code VP2 viral capside protein. In order to study the pathogen-host interaction, a subtractive library was constructed from VERO cells infected by IBDV (8 hours post-infection) aiming the identification of differential gene expression during this interaction. For the phylogenic analysis of the isolates, fragments of 251 bp amplified by RT-PCR were cloned and sequenced. The comparison of the sequences revealed that these isolates have 98-100% identity with classical vaccinal IBDV strains indicating that these outbreaks might have been caused by the vaccinal virus. To construct the subtractive library, two cDNA populations were hibridizated: one derived by IBDV infected VERO cells and other by non infected VERO cells. The hibridizated products were amplified for the posterior cloning and evaluation of the differential express products. Understanding of the replication characteristics of the IBDV associated to the study of the virus infection effects in the gene expression of the host cell, shown in this work, will allow the elucidation of the mechanisms involved in the pathogen-host interaction contributing, therefore, for the development of methods more effectives in the control and prevention of the IBDV. / A doença infecciosa bursal (IBD) tem sido, há muitos anos, uma grande preocupação para a indústria avícola, especialmente na década passada devido a emergência de cepas virais hipervirulentas. Mutações no gene responsável pela codificação da proteína viral VP2 de linhagens patogênicas e a má utilização de vacinas atenuadas por poucas passagens têm sido responsáveis pelo aparecimento de novos surtos. A proposta deste trabalho foi analisar filogeneticamente dois diferentes isolados de IBDV de Minas Gerais por meio da comparação da seqüência nucleotídica do gene que codifica a proteína do capsídeo viral VP2. Em razão da necessidade de se estudar melhor a interação patógeno-hospedeiro, objetivou-se ainda a construção de uma biblioteca subtrativa a partir de células VERO infectadas pelo IBDV, 8 horas após a infecção, com a finalidade de identificar genes diferencialmente expressos durante essa interação vírus-célula hospedeira. Para a análise filogenética dos isolados, fragmentos de 251 pb amplificados por RT-PCR foram clonados e seqüenciados. A comparação das seqüências revelou que os isolados possuem identidade de 98-100% com linhagens clássicas vacinais de IBDV, indicando que os surtos analisados podem ter sido causados pelo vírus vacinal. Para a construção da biblioteca subtrativa, duas populações de cDNA foram hibridizadas: uma derivada de células VERO infectadas por IBDV e a outra de células VERO não infectadas. Os produtos hibridizados foram amplificados para posterior clonagem e avaliação dos produtos diferencialmente expressos. O conhecimento das características replicativas do IBDV associado ao estudo das conseqüências da infecção pelo vírus na expressão gênica da célula hospedeira, iniciado neste trabalho, permitirá a elucidação dos mecanismos envolvidos na interação patógeno-hospedeiro, contribuindo assim para o desenvolvimento de métodos mais eficazes no controle e prevenção da IBD.

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.

This thesis is devoted to the issue of understanding Romani and the majority languages by Romani preschoolers in order to consider the possibility of introducing Romani language in schools. Also it analyses the influence of using Romani language on the ability to perform age-appropriate tasks by Roma preschoolers. The work is divided into two parts. The first, theoretical part presents a summary of the relevant works on school failure of Romani children with an emphasis on their language handicap. We also pay attention to the theory of language shift, language code, and subtractive bilingualism. In the second, empirical part a primary research is carried out examining the degree of understanding Romani and majority languages in three locations in the Czech Republic and Slovakia. As a methodology, a mixed research design was chosen. It was divided into two phases. During the first phase a field research was conducted, followed by quantitative evaluation of tasks performed by the Romani preschoolers in the second phase. Research has shown, that there are different linguistic situations in the surveyed locations and therefore no generic measure can be introduced. At the same time the outcomes rejected the presumption that children who speak Romani better than the majority language will be more successful in...