Perdas gestacionais podem ser determinadas por vários fatores, dentre eles a endometriose, doença ginecológica comum caracterizada pela presença e crescimento de glândulas e estroma endometrial fora da cavidade uterina. Os principais sintomas da doença envolvem, além de problemas de fertilidade, dismenorréia, dispaneuria, dor pélvica crônica e irregularidades menstruais. Os locais mais freqüentes das lesões endometrióticas são o peritônio e os órgãos pélvicos, principalmente ovários. A incidência da endometriose é difícil de ser determinada devido à grande variabilidade de sintomas e à dificuldade para confirmação diagnóstica, que requer método cirúrgico. Estima-se que 15% da população feminina em idade reprodutiva seja afetada pela doença, sendo sua patogênese pouco conhecida. A hipótese mais aceita é a da menstruação retrógrada, onde os fragmentos endometriais descamados durante a fase menstrual seriam transportados através das tubas uterinas até a cavidade peritonial com implantação, crescimento e invasão local e de órgãos adjacentes. No entanto, apenas o refluxo não é suficiente para o estabelecimento da doença, sendo necessário que as células endometriais possuam certas características moleculares que favoreçam o aparecimento e a progressão da implantação ectópica. Vários estudos na literatura evidenciam que as principais divergências moleculares entre portadoras e não portadoras de endometriose estão relacionadas a processos envolvidos na apoptose, adesão celular, angiogênese, biosíntese de estrógeno, sistema imune, além de fatores de crescimento e metaloproteinases. Sendo assim, as pesquisas buscam a investigação de genes que se expressem diferencialmente (maior ou menor expressão) nas células de lesões endometrióticas por meio de várias técnicas. A hibridação subtrativa é uma metodologia de screening gênico que compara dois grupos celulares distintos, permitindo isolar moléculas de cDNA representantes do genoma de somente um dos dois grupos, pois remove as seqüências comuns entre eles. A técnica de hibridação subtrativa rápida (RaSH) simplifica e torna mais eficiente esse processo de subtração, identificando grande quantidade de seqüências diferencialmente expressas. Com objetivo de determinar possíveis marcadores moleculares para diagnóstico e tratamento da doença, aplicamos a técnica de RaSH para identificação de genes com expressão diferencial em 11 amostras de lesões endometrióticas (cinco de origem ovariana e seis de origem peritonial) e em 11 amostras de tecido endometrial tópico de mulheres sem a doença. Após análise dos dados referentes a 166 sequências de referência, os genes HTRA1, LOXL1, SPARC e SSAT foram selecionados para validação por meio da técnica de RT-PCR quantitativa em tempo real, sendo que apenas os dois primeiros mostraram diferença significativa entre os dois grupos estudados. Os genes HTRA1 e LOXL1 apresentaram expressão aumentada em lesões endometrióticas de pacientes afetadas, quando comparada aos níveis de expressão no tecido endometrial de mulheres não afetadas. Os resultados obtidos sugerem que os genes HTRA1 e LOXL1 podem ser considerados candidatos a marcadores moleculares para o diagnóstico. / Gestational losses can be determined by a number of factors including endometriosis, a common gynecological disease characterized by endometrial tissue found outside the uterine cavity. The main symptoms of the disease involve dysmenorrhea, dyspaneuria, chronic pelvic pain and menstrual irregularities, besides fertility problems. Endometriotic lesions are more frequently found in the peritoneum and in pelvic organs, mainly ovaries. Endometriosis incidence is difficult to be determined due to the wide variability of the symptoms and the difficult diagnostic confirmation, which requires a surgical method. Nevertheless, it is believed that around 15% of the female population in reproductive age is affected by the disease, although its pathogenesis remains unclear. The most accepted hypothesis is the retrograde menstruation, where endometrial fragments from the menstrual phase are transported through the uterine tubes to the peritoneal cavity, where they undergo implantation, growth, and adjacent tissues invasion. However, only reflux is not enough for the disease establishment, and it is necessary that endometrial cells present molecular characteristics favoring rise and progression of ectopic implantation. In the literature, a number of studies highlight that the main molecular divergences between women with and without endometriosis are related to processes involved in apoptosis, cellular adhesion, angiogenesis, estrogen biosynthesis, immunological system, growth factors and metalloproteinases. In doing so, a number of researches seek for the investigation of genes differently expressed (up or down regulated) in endometriotic lesions cells using a variety of methodologies. The subtractive hybridization is a gene screening methodology that compares two distinct cellular groups, allowing the isolation of cDNA molecules representative from the genome of only one of the two groups, because it removes common sequences among them. The rapid subtractive hybridization methodology (RaSH) makes the process of subtraction easier and more efficient, identifying a large amount of differently expressed sequences. In this study, RaSH was used to identify differently expressed genes in 11 samples of endometriotic lesions (five from ovarian origin and six from peritoneum origin), and in 11 samples from topic endometrial tissue from women without the disease to determine possible molecular markers for diagnosis and treatment. After data analysis related to 166 reference sequences, genes HTRA1, LOXL1, SPARC and SSAT were selected for validation using real time quantitative RT-PCR. Only HTRA1 and LOXL1 presented significant difference between the two studied groups. Genes HTRA1 and LOXL1 were up regulated in endometriotic lesions from affected patients when compared with the expression levels in the endometrial tissue of non-affected women. Our results suggest that the genes HTRA1 and LOXL1 can be considered candidate molecular markers for the diagnosis of endometriosis.

A evolução da vida na terra foi possível graças ao desenvolvimento de mecanismos temporais precisos capazes de ajustar processos fisiológicos que ocorriam no interior do organismo com os ciclos ambientais, promovendo assim, ganhos na capacidade adaptativa e comportamental desses indivíduos. A retina exerce função de suma importância nesse processo através da percepção da informação fótica que possibilita o ajuste dos ritmos circadianos. Nesse tecido, o glutamato apresenta um importante papel tanto na transmissão da informação fótica direcionada ao processo de formação de imagem quanto nos ajustes dos relógios biológicos. O objetivo desse trabalho foi avaliar como o glutamato, aplicado por períodos diferentes (6 e 12h), é capaz de modular a expressão dos genes de relógio na retina de Gallus gallus. Através de diferentes protocolos que envolveram a administração de glutamato na concentração de 100μM por 6 e 12 horas e em diferentes repetições (1 e 3 pulsos) avaliou-se através de PCR quantitativo a expressão dos genes Clock, Per2 e Bmal1. Os diferentes genes de relógio na retina de Gallus gallus apresentam diferentes respostas frente às trocas de meio e frente ao tratamento com o glutamato. O gene Clock responde com ativação da transcrição para ambos os tratamentos, de forma dependente da repetição dos estímulos. Já para o gene Per2 o tratamento com glutamato impõe uma oscilação de expressão com um ritmo ultradiano, enquanto que as trocas de meio não determinam alterações na transcrição. A expressão do gene Bmal1 não é afetada nem por trocas de meio, nem por glutamato. Novos estudos devem ser fomentados no sentido de se elucidar as vias pelas quais o glutamato leva ao perfil de oscilação observado e qual o mecanismo pelo qual a repetição de trocas de meio atua como sinalizador para o estabelecimento da sincronização celular / The evolution of life on earth was possible thanks to the development of precise temporal mechanisms to adjust physiological processes to environmental cycles, thus promoting gains in the individual adaptive and behavioral ability. The retina plays a very important role of paramount importance in this process through the perception of photic information that allows the adjustment of circadian rhythms. In this tissue, glutamate functions in the transmission of photic information directed to both image formation and biological clock entrainment. The aim of this study was to evaluate how glutamate, applied for different periods (6 and 12h), is able to modulate the expression of the clock genes in the retina of Gallus gallus. Using different protocols involving the administration of 100μM glutamate for 6 and 12 hours and with different repetitions (1 and 3 pulses) the expression of Clock, Per2 and Bmal1 genes was evaluated by quantitative PCR. Clock gene responds with activation of transcription to both treatments depending on the repetition of the stimulus. As for Per2 gene, glutamate treatment imposes an oscillation with an ultradian expression rhythm, whereas medium changes do not affect its transcription. The expression of Bmal1 gene is not affected by either medium changes or glutamate. Further studies should be encouraged in order to elucidate the pathways by which glutamate leads to observed oscillation profile, and which mechanism triggered by the repetition of medium changes acts as signal to establish cell synchronization

Perdas gestacionais podem ser determinadas por vários fatores, dentre eles a endometriose, doença ginecológica comum caracterizada pela presença e crescimento de glândulas e estroma endometrial fora da cavidade uterina. Os principais sintomas da doença envolvem, além de problemas de fertilidade, dismenorréia, dispaneuria, dor pélvica crônica e irregularidades menstruais. Os locais mais freqüentes das lesões endometrióticas são o peritônio e os órgãos pélvicos, principalmente ovários. A incidência da endometriose é difícil de ser determinada devido à grande variabilidade de sintomas e à dificuldade para confirmação diagnóstica, que requer método cirúrgico. Estima-se que 15% da população feminina em idade reprodutiva seja afetada pela doença, sendo sua patogênese pouco conhecida. A hipótese mais aceita é a da menstruação retrógrada, onde os fragmentos endometriais descamados durante a fase menstrual seriam transportados através das tubas uterinas até a cavidade peritonial com implantação, crescimento e invasão local e de órgãos adjacentes. No entanto, apenas o refluxo não é suficiente para o estabelecimento da doença, sendo necessário que as células endometriais possuam certas características moleculares que favoreçam o aparecimento e a progressão da implantação ectópica. Vários estudos na literatura evidenciam que as principais divergências moleculares entre portadoras e não portadoras de endometriose estão relacionadas a processos envolvidos na apoptose, adesão celular, angiogênese, biosíntese de estrógeno, sistema imune, além de fatores de crescimento e metaloproteinases. Sendo assim, as pesquisas buscam a investigação de genes que se expressem diferencialmente (maior ou menor expressão) nas células de lesões endometrióticas por meio de várias técnicas. A hibridação subtrativa é uma metodologia de screening gênico que compara dois grupos celulares distintos, permitindo isolar moléculas de cDNA representantes do genoma de somente um dos dois grupos, pois remove as seqüências comuns entre eles. A técnica de hibridação subtrativa rápida (RaSH) simplifica e torna mais eficiente esse processo de subtração, identificando grande quantidade de seqüências diferencialmente expressas. Com objetivo de determinar possíveis marcadores moleculares para diagnóstico e tratamento da doença, aplicamos a técnica de RaSH para identificação de genes com expressão diferencial em 11 amostras de lesões endometrióticas (cinco de origem ovariana e seis de origem peritonial) e em 11 amostras de tecido endometrial tópico de mulheres sem a doença. Após análise dos dados referentes a 166 sequências de referência, os genes HTRA1, LOXL1, SPARC e SSAT foram selecionados para validação por meio da técnica de RT-PCR quantitativa em tempo real, sendo que apenas os dois primeiros mostraram diferença significativa entre os dois grupos estudados. Os genes HTRA1 e LOXL1 apresentaram expressão aumentada em lesões endometrióticas de pacientes afetadas, quando comparada aos níveis de expressão no tecido endometrial de mulheres não afetadas. Os resultados obtidos sugerem que os genes HTRA1 e LOXL1 podem ser considerados candidatos a marcadores moleculares para o diagnóstico. / Gestational losses can be determined by a number of factors including endometriosis, a common gynecological disease characterized by endometrial tissue found outside the uterine cavity. The main symptoms of the disease involve dysmenorrhea, dyspaneuria, chronic pelvic pain and menstrual irregularities, besides fertility problems. Endometriotic lesions are more frequently found in the peritoneum and in pelvic organs, mainly ovaries. Endometriosis incidence is difficult to be determined due to the wide variability of the symptoms and the difficult diagnostic confirmation, which requires a surgical method. Nevertheless, it is believed that around 15% of the female population in reproductive age is affected by the disease, although its pathogenesis remains unclear. The most accepted hypothesis is the retrograde menstruation, where endometrial fragments from the menstrual phase are transported through the uterine tubes to the peritoneal cavity, where they undergo implantation, growth, and adjacent tissues invasion. However, only reflux is not enough for the disease establishment, and it is necessary that endometrial cells present molecular characteristics favoring rise and progression of ectopic implantation. In the literature, a number of studies highlight that the main molecular divergences between women with and without endometriosis are related to processes involved in apoptosis, cellular adhesion, angiogenesis, estrogen biosynthesis, immunological system, growth factors and metalloproteinases. In doing so, a number of researches seek for the investigation of genes differently expressed (up or down regulated) in endometriotic lesions cells using a variety of methodologies. The subtractive hybridization is a gene screening methodology that compares two distinct cellular groups, allowing the isolation of cDNA molecules representative from the genome of only one of the two groups, because it removes common sequences among them. The rapid subtractive hybridization methodology (RaSH) makes the process of subtraction easier and more efficient, identifying a large amount of differently expressed sequences. In this study, RaSH was used to identify differently expressed genes in 11 samples of endometriotic lesions (five from ovarian origin and six from peritoneum origin), and in 11 samples from topic endometrial tissue from women without the disease to determine possible molecular markers for diagnosis and treatment. After data analysis related to 166 reference sequences, genes HTRA1, LOXL1, SPARC and SSAT were selected for validation using real time quantitative RT-PCR. Only HTRA1 and LOXL1 presented significant difference between the two studied groups. Genes HTRA1 and LOXL1 were up regulated in endometriotic lesions from affected patients when compared with the expression levels in the endometrial tissue of non-affected women. Our results suggest that the genes HTRA1 and LOXL1 can be considered candidate molecular markers for the diagnosis of endometriosis.

Le blé tendre, Triticum aestivum, est une des céréales les plus cultivées dans le monde. Le changement climatique qui se développe actuellement contraint fortement les cultures et altère leur rendement. La compréhension des mécanismes de réponse du blé tendre aux stress abiotiques est donc une problématique d’actualité. Plusieurs grandes familles de facteurs de transcription, dont la famille NAC,interviennent dans le développement de la plante et dans sa réponse aux stress environnementaux. Cette thèse, structurée en 3 volets, est ciblée sur l’étude de la famille NAC chez le blé tendre : les TaNAC. Dans un premier temps, nous avons étudié la structuration génomique et phylogénétique des 488 membres de la famille TaNAC, recensés à partir de la base de données la plus récente du blé tendre.Nous avons aussi étudié l’histoire évolutive de cette famille, qui a été marquée par des événements de duplication et de rétroposition. Enfin, une analyse de sa diversité allélique a permis d’identifier des gènes qui présentent des SNP montrant une forte association avec des paramètres d’accumulation des protéines de réserve dans le grain. Le deuxième chapitre de cette thèse a porté sur l’étude de l’expression de ces 488 gènes TaNAC dans plusieurs organes et en réponse aux stress thermique et sécheresse. Une analyse globale a été réalisée à partir de données bio-informatiques, suivie d’une étude in planta de l’expression d’une sélection de 23 gènes. Les profils d’expression obtenus ont révélé l’existence de 4 gènes TaNAC, encore jamais décrits dans la littérature et qui interviennent dans le développement du grain de blé tendre mais aussi dans sa réponse adaptative à plusieurs stress abiotiques. Le troisième volet de cette thèse a donc porté sur la caractérisation génétique, moléculaire et physiologique de ces 4 facteurs de transcription TaNAC. Ils appartiennent à un clade rassemblant des séquences présentant des similitudes génomique et structurale. De plus, ils sont localisés dans le noyau et leurs profils d’expression sont similaires, avec toutefois un niveau variable entre gènes et entre homéologues pour chaque gène. En réponse à un stress thermique modéré, ce profil d’expression est accéléré au cours du développement du grain ; le stade 120°Cj étant le stade clé qui montre la plus grande différence d’expression de ces gènes entre les conditions contrôle et stressée. Pour des raisons techniques, la production de plantes transgéniques sur- et sous-exprimant ces gènes n’a pas permis de valider l’implication de ces 4 TaNAC dans le développement du grain et en réponse à la température. Une analyse de génétique d’association a toutefois permis de mettre en évidence un lien entre des marqueurs moléculaires situés dans ces gènes et l’accumulation des protéines de réserve.Globalement, les résultats obtenus ont montré que des membres de la famille TaNAC sont impliqués dans le développement du blé tendre et dans sa réponse aux stress abiotiques. Plus particulièrement, 4 facteurs de transcription TaNAC semblent jouer un rôle clé dans l’accumulation des protéines dans le grain en réponse à un stress thermique modéré. / Bread wheat, Triticum aestivum, is one of the most cultivated cereal in the world. The climate change that is currently developing strongly constrains crops and impairs their yield. Understanding the wheat response mechanisms to abiotic stresses is therefore a current issue. Several major families of transcription factors, including the NAC family, are involved in the plant development and its response to environmental stresses. This thesis, structured in three parts, is focused on the study of the NAC family in bread wheat (TaNAC).First, we studied the genomic and phylogenetic structure of the 488 members of the TaNAC family identified from the latest database of bread wheat. We also studied the evolutionary history of this family, which was marked by duplication and retroposition events. Finally, an analysis of its allelic diversity allows us to identify genes with SNP showing a strong association with storage protein accumulation parameters in the grain. In a second part, we studied the expression of these 488 TaNAC genes in several organs and in response to heat and drought. An overall analysis was performed using bioinformatic data, followed by an in planta study of the expression of a selection of 23 genes. The expression profiles revealed that four TaNAC genes, never described in the literature, are involved in the wheat grain development but also in its adaptive response to several abiotic stresses. In a third part, we focused on the genetic, molecular and physiological characterization of these four TaNAC transcription factors. They belong to a clade gathering sequences with genomic and structural similarities. Moreover, they are localized in the nucleus and their expression profiles are similar, with a variable level between genes and between homeologs for each gene. In response to moderate heat stress, this expression profile is accelerated during grain development and a key stage at 120°Cj was identified, it shows the greatest difference in genes expression level between control and stressed conditions. For technical reasons, the production of transgenic plants over- and under-expressing these genes did not validate the involvement of these 4 TaNAC in grain development and in its temperature response. An association genetic analysis, however, showed a link between molecular markers located in these genes and the storage proteins accumulation. Overall, the results showed that members of the TaNAC family are involved in the bread wheat development and its response to abiotic stresses. In particular, four TaNAC transcription factors appear to play a key role in grain protein accumulation in response to a moderate heat stress.

L'effet stimulateur de la croissance et de la nutrition des plantes exercés par les PGPR (Plant Growth-Promoting Rhizobacteria) a longtemps été étudié en s'intéressant à la bactérie. Cependant, les voies de signalisations impliquées dans la réponse de la plante à l'inoculation restent mal étudiées. A cet effet, notre étude entre dans le cadre des recherches visant les réponses physiologiques et moléculaires de la plante induites par une PGPR. Dans notre équipe de recherche, nous avons choisi la PGPR Phyllobacterium brassicacearum STM196 isolée de la rhizosphère de Colza et nous l'avons inoculée à la plante modèle Arabidopsis thaliana. Cette PGPR a montré sa capacité à stimuler l'allongement des racines latérales et des poils racinaires ainsi que d'augmenter la production de biomasse par la plante. Une forte surexpression de deux gènes de la famille de transporteurs de nitrate NRT2, NRT2.5 et NRT2.6, a été observée chez les plantes inoculées avec STM196. La fonction des produits de ces deux gènes n'est pas connue. Cependant, les données de transcriptomiques accumulées dans l'équipe font ressortir ces deux gènes comme des candidats intéressants dans les réponses moléculaires à l'interaction avec STM196. D'autre part, des études précédentes dans l'équipe ayant montré des effets antagonistes de la bactérie et du nitrate sur le développement racinaire, il est important de considérer la relation entre les effets de la nutrition nitrique et de la bactérie. Le principal transporteur responsable de l'absorption de NO3- étant NRT2.1, nous nous sommes intéressés à son rôle dans les réponses de la plante à la bactérie et à sa relation éventuelle avec NRT2.5 et NRT2.6. Nous avons réalisé une approche de génomique inverse avec les trois simples mutants ko nrt2.1, ko nrt2.5 et ko nrt2.6 dont nous disposions au départ, et avec les trois doubles mutants nrt2.5xnrt2.6, nrt2.1xnrt2.6 et nrt2.1xnrt2.5 que nous avons généré. Nous avons démontré que les gènes NRT2.5 et NRT2.6 sont impliqués dans les réponses de stimulation de croissance de la plante et de modification d'architecture racinaire à la PGPR STM196. Cette voie de régulation est indépendante des contrôles exercés par le statut azoté de la plante.Mots clés: Interaction plante-microorganisme, Phyllobacterium brassicacearum STM196, Arabidopsis thaliana, transporteurs de nitrate, NRT2.1, NRT2.5, NRT2.6, Activité nitrate réductase, NR1, expression des gènes. / AbstractThe promotion of plant growth and nutrition by some rhizospheric bacteria (Plant Growth Promoting Rhizobacteria, PGPR) is well known for a long time. However, the signaling pathways involved in the plant responses to these bacteria still remain essentially obscure. Our study aims at identifying molecular factors of plant physiological and developmental responses induced by PGPR. For this goal, we used the PGPR strain Phyllobacterium brassicacearum STM196, which has been isolated from rape rhizosphere, and the plant model Arabidopsis thaliana. This PGPR stimulates lateral root and root hair elongation and induce an increase of plant biomass production. Two genes of the NRT2 family of nitrate transporters, namely NRT2.5 and NRT2.6, are strongly overexpressed upon inoculation of Arabidopsis with STM196. The function of NRT2.5 and NRT2.6 is not known. However, transcriptomic data obtained in our team show that these two genes are promising candidates of the molecular responses to STM196. In addition, previous work in our team showed antagonistic effects of STM196 and exogenous nitrate on root development, showing that the effects of the bacteria must be considered together with those of nitrate nutrition. Since NRT2.1 is the major transporter for NO3- uptake, we looked at its role in the plant response to STM196 and its possible relationship with NRT2.5 and NRT2.6. We carried out a reverse genetic approach using the single mutants ko nrt2.1, ko nrt2.6 and ko nrt2.5 available at the moment this thesis work began and the double mutants nrt2.5xnrt2.6, nrt2.1xnrt2.6 and nrt2.1xnrt2.5 we generated. We demonstrated that NRT2.5 and NRT2.6 are involved in plant growth stimulation by STM196 and the root architecture changes elicited by this bacterium. This NRT2.5/NRT2.6-dependent pathway is independent from the regulations exerted by N nutritional status. Key words: Plant-microorganism interaction, Phyllobacterium brassicacearum STM196, Arabidopsis thaliana, nitrate transporter, NRT2.1, NRT2.5, NRT2.6, nitrate reductase activity, NR1, genes expression.

Master of Science / Department of Anatomy and Physiology / Masaaki Tamura / Previous studies have shown that both human and rat umbilical cord matrix mesenchymal stem cells (UCMSC) possess the ability to control the growth of breast carcinoma cells. Comparative analysis of two types of UCMSC suggest that rat UCMSC-dependent growth regulation is significantly stronger than that of human UCMSC. Accordingly, the present study was designed to clarify their different tumoricidal abilities by analyzing gene expression profiles in two types of UCMSC. Gene expression profiles were studied by microarray analysis using Illumina HumanRef-8-V2 and RatRef-12 BeadChip for the respective UCMSC. The gene expression profiles were compared to untreated naïve UCMSC and those co-cultured with species-matched breast carcinoma cells; human UCMSC vs. MDA-231 human carcinoma cells and rat UCMSC vs. Mat B III rat carcinoma cells. The following selection criteria were used for the screening of candidate genes associated with UCMSC-dependent tumoricidal ability; 1) gene expression difference should be at least 1.5 fold between naive UCMSC and those co-cultured with breast carcinoma cells; 2) they must encode secretory proteins and 3) cell growth regulation-related proteins. These analyses screened 17 common genes from human and rat UCMSC. The comparison between the two sets of gene expression profiles identified that two tumor suppressor genes, adipose-differentiation related protein (ADRP) and follistatin (FST), were specifically up-regulated in rat UCMSC, but down-regulated in human UCMSC when they were co-cultured with the corresponding species’ breast carcinoma cells. The suppression of either protein by the addition of a specific neutralizing antibody in co-culture of rat UCMSC with Mat B III cells significantly abrogated UCMSC ability to attenuate the growth of carcinoma cells. Over-expression of both genes by adenovirus vector in human UCMSC enhanced their 4 ability to suppress the growth of MDA-231 cells. In the breast carcinoma lung metastasis model generated with MDA-231 cells, systemic treatment with FST-over-expressing human UCMSC significantly attenuated the tumor burden. These results suggest that both ADRP and FST may play important roles in exhibiting stronger tumoricidal ability in rat UCMSC than human UCMSC and imply that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression.

Diplomová práce Abstract - Iveta Brabcová Abstract Ischemia-reperfusion heart injury is one of the most significant diseases affecting mankind and therefore current research pays more attention to its prevention and knowledge of the possible mechanisms which protect the heart. Adaptation to hypoxia has been known for several decades as a cardioprotective intervention but the main issues of protective mechanisms which are induced by the adaptation are still not completely understood. An important role of mitochondria as the main producers of energy and reactive oxygen species which can play a signalizing role in these mechanisms is confirmed in many studies. For this reason a special conplastic strain SHR/OlaIpcv-mtBN/Crl was created. This strain carries the nuclear genome of spontaneously hypertensive rat (SHR) and the mitochondrial genome of normotensive, highly resistant strain Brown Norway (BN). The aim of this study was to compare the expression of selected gene transcripts in the area of energy metabolism, of genes which are related to mitochondrial biogenesis and signaling and antioxidant systems. Comparing the expression was analyzed between strains and after chronic hypoxia adaptation, which cause cardioprotective phenotype in both of these strains. Our results showed a different expression HIF-1α...

Bactérias do gênero Methylobacterium são encontradas em associação com espécies vegetais, onde são capazes de promover o crescimento, aumentar a atividade fotossintética e reduzir o ataque de patógenos ao hospedeiro. Além de conferir estas vantagens para a planta hospedeira, estas bactérias podem também produzir biopolímeros (PHA e PHB). Desta forma, o objetivo deste trabalho foi anotar o genoma de Methylobacterium mesophilicum SR1.6/6 e avaliar o seu transcriptoma em estágios iniciais de interação com Citrus sinensis. A análise do genoma mostrou que SR1.6/6 pode produzir auxina, reduzir o estresse da planta alterando os níveis de etileno, apresenta sistema de monitoramento de populacional pelo sistema quorum sensing (QS) e um metabolismo metilotrófico completo. A análise do transcriptoma evidenciou que os exsudatos radiculares de C. sinensis induzem a expressão de genes de resposta ao estresse oxidativo, seguido da indução de genes de adesão e biofilme durante a colonização da planta hospedeira. A interação entre M. mesophilicum SR1.6/6 e a planta hospedeira envolve mecanismos de reconhecimento e adaptação ao estresse, antes mesmo de ocorrer o primeiro contato físico entre a célula bacteriana e a planta hospedeira, seguido da indução de genes de biofilme bacteriano. Além disso, foi estudada uma metodologia para a realização de mutações genéticas em Methylobacterium spp. que permitirá a obtenção de mutantes relacionados com a interação com a planta. / Methylobacterium genus are found in association with plant species, where they are able to promote plant growth, increase the photosynthetic activity and reduce the incidence of pathogens to the host. In addition to providing these benefits to the host plant, these bacteria can also produce biopolymers (PHA and PHB). Thus, the aim was to annotate the genome of Methylobacterium mesophilicum SR1.6 / 6 and assess their transcriptome in the early stages of interaction with Citrus sinensis. Genome analysis showed that SR1.6 / 6 can produce auxin, reduce plant stress by altering ethylene levels, presents population monitoring system (QS) and a complete methylotrophic metabolism. The transcriptomic analysis showed that C. sinensis exudates induce the expression of genes related to oxidative stress followed by induction of adhesion and biofilm genes during colonization of the host plant. The interaction between M. mesophilicum SR1.6 / 6 and the host plant involves recognition mechanisms and adaptation to stress, even before the first physical contact occurs between the bacterial cell and the host plant, followed by the induction of bacterial biofilm genes. Furthermore, a method has been studied for carrying genetic mutations in Methylobacterium spp. allowing the obtaining of mutants related to interaction with the plant.

Les fibroses anormales sont responsables de plus de 40% des décès pour raison médicale ; elles se développent suite à une inflammation chronique. Les fibroses hépatiques causées par les schistosomes et par le virus HCV sont en grande partie déterminées par la génétique du malade. Notre thèse a consisté à poursuivre le travail de caractérisation du déterminisme génétique des fibroses hépatiques et cutanées.La première partie de notre thèse, est l’étude informatique et statistique des données de génotypage GWAS de Brésiliens qui présentent une fibrose hépatique bilharzienne grave sur plus de 2,5 millions de SNPs. 180 SNPs qui montraient une association suggestive avec les fibroses graves ont été sélectionnés, dont certains affectent les gènes des voies Wnt. Ces SNPs ont été testés sur une cohorte de 460 pêcheurs ougandais exposés à S.mansoni et nous avons confirmé l’association avec la fibrose de 4 SNPs.La deuxième partie de notre thèse est l’analyse transcriptômique (RNA-Seq) des mécanismes responsables des fibroses anormales de la peau de sujets affectés par des fibroses chéloïdes de 20 tissus chéloïdes, 7 tissus sains et 7 tissus affectés par des cicatrices hypertrophiques. Cette analyse montre que le développement des chéloïdes est la conséquence d’une stimulation anormale des voies de la cicatrisation en partie due à l’activation de la voie Wnt βcatenin et Wnt PCP. Pour conforter cette proposition, nous avons effectué une analyse génétique de la voie Wnt dans deux cohortes indépendantes. L’analyse statistique montre que des SNPs dans 6 gènes de la voie Wnt βcatenin contribuent au développement des fibroses chéloïdes. / Abnormal fibrosis is responsible for more than 40% of medical deaths. They develop as a result of chronicinflammation. Hepatic fibroses caused by schistosomes andHCV virus are largely determined by the genetic background of the patient. Our thesis consisted of continuing thework of characterizing the genetic determinism of liver and skin fibrosis.The first part of our thesis is the computer and statistical study of GWAS genotyping data of Brazilians whohave severe bilharzeal liver fibrosis on more than 2.5 million SNPs. 180 SNPs that showed suggestive associationwith severe fibrosis were selected, some of which affect the Wnt pathway. These SNPs were then tested on a cohortof 460 Ugandan fishers exposed to S.mansoni and the results confirmed the association of 4 SNPs with fibrosis.The second part of our thesis is the genomic analysis (transcriptome and genetics) of the mechanismsresponsible for the abnormal skin fibrosis with subjects affected by keloid scars. We performed an analysis of genes(RNASeq) expressed differently between 20 keloids, 7 healthy tissues and 7 tissues affected by hypertrophic scars.This analysis shows that the development of keloids is the consequence of an abnormal stimulation of cicatrizationpathways with strong activation of the Wnt βcatenin and Wnt PCP pathway. To support this proposal, we performeda genetic analysis of the Wnt pathway in two independant cohorts.The statistical analysis of the results shows that polymorphisms in 6 genes of the Wnt βcatenin pathway contributeto the development of keloid fibrosis.

Bactérias do gênero Methylobacterium são encontradas em associação com espécies vegetais, onde são capazes de promover o crescimento, aumentar a atividade fotossintética e reduzir o ataque de patógenos ao hospedeiro. Além de conferir estas vantagens para a planta hospedeira, estas bactérias podem também produzir biopolímeros (PHA e PHB). Desta forma, o objetivo deste trabalho foi anotar o genoma de Methylobacterium mesophilicum SR1.6/6 e avaliar o seu transcriptoma em estágios iniciais de interação com Citrus sinensis. A análise do genoma mostrou que SR1.6/6 pode produzir auxina, reduzir o estresse da planta alterando os níveis de etileno, apresenta sistema de monitoramento de populacional pelo sistema quorum sensing (QS) e um metabolismo metilotrófico completo. A análise do transcriptoma evidenciou que os exsudatos radiculares de C. sinensis induzem a expressão de genes de resposta ao estresse oxidativo, seguido da indução de genes de adesão e biofilme durante a colonização da planta hospedeira. A interação entre M. mesophilicum SR1.6/6 e a planta hospedeira envolve mecanismos de reconhecimento e adaptação ao estresse, antes mesmo de ocorrer o primeiro contato físico entre a célula bacteriana e a planta hospedeira, seguido da indução de genes de biofilme bacteriano. Além disso, foi estudada uma metodologia para a realização de mutações genéticas em Methylobacterium spp. que permitirá a obtenção de mutantes relacionados com a interação com a planta. / Methylobacterium genus are found in association with plant species, where they are able to promote plant growth, increase the photosynthetic activity and reduce the incidence of pathogens to the host. In addition to providing these benefits to the host plant, these bacteria can also produce biopolymers (PHA and PHB). Thus, the aim was to annotate the genome of Methylobacterium mesophilicum SR1.6 / 6 and assess their transcriptome in the early stages of interaction with Citrus sinensis. Genome analysis showed that SR1.6 / 6 can produce auxin, reduce plant stress by altering ethylene levels, presents population monitoring system (QS) and a complete methylotrophic metabolism. The transcriptomic analysis showed that C. sinensis exudates induce the expression of genes related to oxidative stress followed by induction of adhesion and biofilm genes during colonization of the host plant. The interaction between M. mesophilicum SR1.6 / 6 and the host plant involves recognition mechanisms and adaptation to stress, even before the first physical contact occurs between the bacterial cell and the host plant, followed by the induction of bacterial biofilm genes. Furthermore, a method has been studied for carrying genetic mutations in Methylobacterium spp. allowing the obtaining of mutants related to interaction with the plant.