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La symbiose mycorhizienne entre les champignons du sol et les racines de la plupart des plantes constitue une relation à bénéfice réciproque et joue un rôle majeur dans la productivité des écosystèmes. Les récentes avancées dans le domaine ont abouties à l'identification et à la caractérisation fonctionnelle de nombreux systèmes de transport du partenaire fongique. Le travail présenté s'inscrit dans le cadre de développement d'outils permettant la localisation de gènes d'intérêts du champignon ectomycorhizien Hebeloma cylindrosporum et de leur caractérisation fonctionnelle. Les systèmes de transport candidats ont été identifiés au sein d'une banque EST du champignon et semblent impliqués dans les échanges de phosphate (Pi) et de potassium (K+) entre Hebeloma et la plante hôte Pinus pinaster. Une stratégie de fusion transcriptionnelle utilisant l'EGFP comme gène rapporteur a été développée pour permettre la localisation de deux transporteurs de phosphate, HcPT1 et HcPT2, d'un transporteur de potassium, HcTrk1, et d'un canal potassique de type Shaker, HcSKC1, dans les hyphes en culture pure et au sein de l'ectomycorhize. Les Agrotransformations de la souche h7 d'Hebeloma avec des vecteurs de fusion transcriptionnelle ont montré une expression mycélienne de l'EGFP sous contrôle des promoteurs de nos gènes d'intérêts. Sous contrôle des différents promoteurs, l'expression de l'EGFP apparait comme étant site-spécifique dans les hyphes différenciés des ectomycorhizes. Le promoteur du transporteur de Pi HcPT1 induit l'expression du gène rapporteur au niveau des hyphes extramatriciels et du manteau mycélien entourant la racine. De plus, son expression est stimulée en cas de carence en Pi, indiquant ainsi l'implication de ce transporteur dans la récupération du Pi du sol lorsque celui-ci devient limitant. Pour ce qui est du promoteur de HcTrk1, il permet l'expression de l'EGFP dans les hyphes extraracinaires et dans le manteau, tandis que celui de HcSKC1 permet son expression au niveau du réseau de Hartig et du manteau. Ceci indique, qu'ils semblent respectivement participer à la récupération du K+ du sol et à son excrétion vers la plante. Pour poursuivre la caractérisation fonctionnelle de nos systèmes de transport candidats, un second canal potassique, HcSKC2, a été isolé à partir de la souche h1 et exprimé dans des ovocytes de xénope. Tout comme HcSKC1, HcSKC2 n'a pas été actif en système d'expression hétérologue. Cependant, des fusions traductionnelles avec l'EGFP ont montré que la protéine HcSKC2 est bien dirigée à la membrane. En perspective, la caractérisation fonctionnelle de ce canal issue de la souche h7 récemment séquencée sera tentée. / The mycorrhizal symbiosis made it possible the first plants to conquest emerged lands and is a major biological phenomenon of terrestrial ecosystems. The fungal partner efficiently takes up nutritive ions from the soil solution and transfers them to the host plants in exchange for photosynthetates. However, despite the importance of this symbiosis on ecosystem productivity, our knowledge about molecular processes controlling this symbiotic interaction and solute transports at the membrane level is very scarce. The objective of the project aims at dissecting part of the molecular mechanisms required for a functional ectomycorrhizal symbiosis associated with most of the woody species from boreal and temperate forests, by focusing on K+ exchanges occurring through the continuum soil-hyphae-plant. The general aim of the project is to gain new insights into the molecular mechanisms responsible for the polarization and differentiation of the plasma membrane between the site of nutrient uptake and the site of efflux into the apoplastic space in the ectomycorrhizal root. The team "Canaux Ioniques – Ion channels" has obtained an EST library of the fungus Hebeloma cylindrosporum (1) and has identified and characterized a potassium transporter of the Trk family (2). Also a Shaker-type potassium cannel was identified within the EST library but it is not yet functionally characterized. A second transcript was found from this channel with a longer N-terminus compared to the first transcript isolated in the beginning. Also, a sugar transporter was identified among the ESTs that could participate in the absorption of sugars, coming from the host plant, by the fungus. The objective of the PhD thesis is the functional characterization of these fungal transport systems as well as their localization. The functional characterization of these candidate genes will be accomplished using heterologous expression systems (Xenopus oocytes, COS cells, complementation of yeast mutants) and by the means of electrophysiology. Localization of genes within the fungus being in symbiotic interaction with the host plant, the tree Pinus pinaster, will help to better understand the role of the transport systems. The differentiation of the fungus, when establishing symbiosis, into the specialized interfaces soil-fungus and fungal cell- host plant cell within the ectomycorrhiza (Hartig net) is probably accompanied by a specific expression of transport proteins and ion channels

Protein product tumor suppressor PALB2 gene plays a major role in pathway of DNA repair of double-strand breaks throught the homologous recombination mechanism. Significance of its pathogenic variants in hereditary forms of breast cancer in BRCA1/2- negative patients in families with multiple breast cancers may be in the Czech Republic comparable with the BRCA2 gene. A role of the PALB2 gene in sporadic breast cancer occurence, which represent 90 - 95 % of all cancers, is still unknown. This thesis focuses on inactivation pathway of tumor suppressor PALB2 in the sporadic breast cancer by a mechanism of allelic loss detecting by loss of heterozygosity (LOH) of corresponding microsatellite markers and hypermethylation of promoter region as the most common mechanisms of inactivation tumor suppressors in early tumorigenesis. In a group of 51 nonselected patients with sporadic breast cancer we found four samples with PALB2 locus allelic loss. These samples were analyzed for somatic mutations. No mutation was found. There is no evidence of promotor hypermethylation in any of the samples. Our data suggest a role of the PALB2 gene inactivation in a minority group of sporadic breast cancers.

Ces travaux de thèse d’imagerie moléculaire et translationnelle proposent, sur des modèles murins, deux approches innovantes pour les thérapies géniques. La plupart des cancers sont associés à des dérégulations de l’expression génique et certains gènes sont surexprimés. L’utilisation de microARN (miARN) permet d’envisager une réduction de l’expression d’un gène spécifique mais il est nécessaire de limiter cette inhibition au tissu pathologique. L’utilisation des promoteurs thermo-inductibles couplés à un dépôt local de chaleur autorise un contrôle spatial et temporel de l’expression génique in vivo. Notre projet a été de coupler le contrôle spatio-temporel et l’inhibition d’un gène cible. A cette fin, un miARN synthétique a été placé sous contrôle du promoteur thermo-inductible Hsp70B pour induire l’inhibition d’un gène d’imagerie (luciférase firefly) surexprimé dans une tumeur. L’étude a été menée in vitro sur des lignées cellulaires génétiquement modifiées puis in vivo sur un modèle de xénogreffes chez la souris grâce au suivi en imagerie optique de bioluminescence (BLI). Nos résultats montrent la faisabilité d’induire transitoirement l’inhibition génique au sein d’une tumeur. L’induction est modulable par la température. Cette stratégie peut être couplée à des méthodes couramment utilisées en clinique et ouvre des perspectives thérapeutiques intéressantes. Notre travail de thèse s’intéresse également à l’utilisation d’ARN comme molécule thérapeutique pour la thérapie génique. L’électroporation intra-dermique d’ARN codant pour la luciférase permet de suivre et de quantifier in vivo par BLI l’expression génique. Plusieurs types d’ARN ont été utilisés pour comparer les efficacités respectives des différentes voies traductionnelles. Notre travail démontre que les ARN permettent l’expression transitoire, sans risque d’insertion génomique, d’un gène in vivo. Nous montrons ainsi tout le potentiel de l’utilisation des ARN en thérapie génique. / The present thesis work in molecular and translational imaging establishes two innovative approaches for gene therapy in mouse models. Abnormal regulation of gene expression is the hallmark of cancer, and some of them are overexpressed. MicroRNA (miRNA) can be used as tools to reduce specific gene expression but requires inhibition to be limited to the pathological tissue. Thermo-inducibles promoters associated with local hyperthermia allow for spatial and temporal control of gene expression in vivo. The goal of the present study was to achieve gene inhibition with spatio-temporal control of miRNA expression to inhibit a target gene. In our strategy, a synthetic miRNA was placed under transcriptional control of the heat-inducible promoter Hsp70B to induce inhibition of the imaging reporter gene firefly luciferase overexpressed in a tumor. The study was conducted both in vitro using genetically modified cells lines and in vivo using a xenograft model in mice monitored by optical bioluminescence imaging (BLI). Our data show the feasibility of transient induction and heat-modulation of gene inhibition within a tumor. This strategy can be performed with currently clinically available methods and thus, offers interesting therapeutics prospects. Our work also includes a study on RNA as therapeutic vector for gene therapy. The intradermic electroporation of RNA encoding the imaging reporter gene firefly luciferase allows to monitor and quantify gene expression by BLI in vivo. Several types of RNA have been used to investigate efficiency of the different translational mechanisms. Our data clearly demonstrate that RNA allows for transient gene expression in vivo without any risk of insertion into the target cell’s genome. Altogether, our data highlight the potential use of RNA in gene therapy.

Abstract Background: Type 1 diabetes (T1D) is a multifactorial autoimmune disease. Its incidence in Europe is continuously rising. The highest T1D risk is associated with HLA (human leukocyte antigen) class II genes. HLA class II molecules play a key role in regulation of immune response. They contribute to the selection of T cell repertoire by presenting antigenic peptides to the CD4+ T lymphocytes. HLA class II expression is controlled by regulatory module that is situated 150 - 300 base pairs upstream of the transcription- initiation site in all HLA class II genes. Polymorphisms in this region are linked to some autoimmune diseases. There were identified several promoter alleles (named QAP) in the HLA DQA1 gene promoter region. Most of the polymorphisms appear to be conserved within haplotype. Individual QAP alleles may have a different promoter strength by which they influence expression of HLA DQA1 gene alleles. Promoter strength can be modulated by DNA methylation. Aims:Our aim was to define methylation profile of HLA DQA1 promoters and determine the mRNA expression of individual alleles of HLA DQA1 gene in T1D patients. The mRNA expression level of HLA DQA1 gene alleles was determined using quantitative PCR. Methods: 30 diabetic pacients (age range 21 to 76 years), were included in this pilot...

Las proteínas pequeñas de choque térmico (sHSP) responden a una amplia variedad de estreses ambientales, estabilizando proteínas parcialmente desplegadas y evitando su agregación irreversible, en forma independiente de ATP. En plantas, las sHSPs son especialmente diversas siendo las sHSPs de organelas una característica única de las plantas. La estructura primaria de las sHSP incluye una secuencia N-terminal no conservada de longitud variable, un dominio α-cristalino conservado (ACD) y una secuencia C-terminal corta no conservada. El dominio ACD representa una característica conservada presente en todas las sHSPs, sin embargo, no todas las proteínas que contienen un dominio ACD son sHSP. Las sHSPs pertenecen a una gran superfamilia, siendo su importancia funcional y fisiológica en gran parte desconocida. El objetivo de este trabajo fue dilucidar el rol de sHSPs de localización mitocondrial (sHSPs-M) en Arabidopsis thaliana y Solanum lycopersicum en situaciones de estrés ambiental, y caracterizar probables promotores bidireccionales que regulan la expresión de genes codificantes de proteínas con dominio ACD con orientación cabeza a cabeza en el genoma de Arabidopsis. Este trabajo cubre aspectos desde la organización genómica y la función de sHSPs-M en Arabidopsis hasta el rol de las sHSPs-M en la respuesta al estrés por frío de frutos de tomate. Para ello, se generaron plantas mutantes de Arabidopsis y de tomate utilizando la tecnología de silenciamiento génico por micro ARNs artificiales. Las plantas mutantes fueron analizadas en su proteoma, metaboloma y lipidoma en distintas condiciones de estrés. A continuación, se presenta un resumen de los resultados obtenidos. En la primera parte de este trabajo, se realizó la caracterización funcional de genes con orientación cabeza a cabeza en el genoma de Arabidopsis, que codifican para proteínas con ACD y las regiones intergénicas correspondientes. Se lograron identificar y caracterizar cuatro distintos promotores bidireccionales, entre ellos el promotor del gen At5g51440 que codifica una sHSP de localización mitocondrial (sHSP23.5). Los resultados obtenidos sugieren que el promotor bidireccional contenido en el par At5g51430-At5g51440 es fuertemente inducido por altas temperaturas en una dirección, pero no así en la dirección opuesta. El promotor contenido en el par At1g06460-At1g06470 mostró una actividad alta en ambas direcciones, teniendo por ello, un alto potencial de aplicación en ingeniería genética. Los dos promotores restantes mostraron mayor actividad en una dirección y, por lo tanto, pueden ser considerados como promotores bidireccionales asimétricos. El estudio funcional de los promotores seleccionados reveló el potencial biotecnológico de los mismos ya que pueden ser inducidos específicamente en una determinada condición (como altas temperaturas) en una o en ambas direcciones cuando sea necesario. En la segunda parte, se presenta la caracterización funcional de sHSP mitocondriales en condiciones de estrés y durante el desarrollo de Arabidopsis. Se identificaron tres genes parálogos en el genoma de Arabidopsis (At5g51440, At4g25200, y At1g52560), y se diseñaron microARN artificiales específicos para la obtención de plantas mutantes por silenciamiento (amiR simple, doble y triple). Las plantas amiR simples y dobles (para sHSP23.5 y sHSP23.6) no mostraron un fenotipo evidentemente afectado, probablemente debido a la compensación o redundancia funcional de las sHSP mitocondriales. Por otro lado, las plantas triple mutante amiR23.5/23.6/26.5 muestran un fenotipo alterado en las etapas vegetativa y reproductiva. Estas plantas presentan hojas pequeñas, células epidérmicas con áreas reducidas, pero no reducción en el número de células epidérmicas por hoja. Además, exhiben hojas cloróticas, raíces cortas y menor producción de semillas en comparación con las plantas Col-0. Las plantas triple amiR son considerablemente pequeñas debido a una alteración en el proceso de expansión celular, pero no en la proliferación celular, lo que indica una profunda alteración en el programa de desarrollo de la planta. En el análisis proteómico de las mutantes amiR se observó un aumento significativo de proteínas implicadas en el metabolismo, y alteración en la abundancia de varias proteínas relacionadas con la traducción, y con el funcionamiento y la estructura de los ribosomas. La triple mutante exhibió un mayor número de proteínas con abundancia alterada involucradas en estos procesos en comparación con las mutantes simples y doble amiR23.5/23.6. Estos cambios tan amplios observados en proteínas relacionadas con el funcionamiento de los ribosomas sugieren una posible alteración en la función normal de los mismos. Los resultados presentados en este trabajo proporcionan evidencias sobre el importante rol de las sHSPs-M no sólo en la respuesta a las altas temperaturas, sino también durante el desarrollo de la planta de Arabidopsis. Los resultados indican que una compensación funcional podría ser responsable del fenotipo observado en las plantas mutantes con niveles reducidos de cada sHSPs-M individual. Sin embargo, la reducción simultanea de las tres sHSPs-M causó una profunda alteración en la función normal de mitocondrias y ribosomas, afectando gravemente el metabolismo energético y la homeostasis celular, lo que llevó a alteraciones en el desarrollo correcto de la planta. En la última parte de este trabajo, las consecuencias de la disminución de la proteína sHSP23.8 de localización mitocondrial en frutos de tomate fueron investigadas. Los frutos fueron analizados en su fenotipo y en la susceptibilidad al daño por frío. Los frutos de las plantas mutantes amiR23.8 que fueron conservados en frío mostraron mayor pérdida de agua y de electrolitos en el tejido pericárpico en comparación con frutos WT. El deterioro observado en los frutos amiR23.8 indica que estos frutos mutantes son mayormente susceptibles al estrés por frío desarrollando síntomas de daño por frío. El lipidoma de los frutos amiR23.8 frigoconservados mostró cantidades alteradas de glicerolípidos, y los niveles de lípidos saturados en amiR23.8 disminuyeron luego del tratamiento con frío, pero no por debajo de los niveles encontrados en frutos WT en condiciones normales. Lo opuesto se encontró en el porcentaje relativo de lípidos insaturados, con niveles significativamente más bajos de insaturaciones en frutos amiR23.8 en condiciones normales y después del enfriamiento. Los resultados presentados indican una degradación diferencial de lípidos extraplastídicos y plastídicos en frutos amiR23.8, y alteraciones en la remodelación del lipidoma luego del estrés por frío, lo que podría conducir a una mayor sensibilidad al daño por frío. Los resultados discutidos aquí indican que la proteína sHSP23.8 podría estar directamente involucrada en los mecanismos de protección contra el estrés por frío en frutos de tomate.

A metilação do DNA é uma alteração epigenética que atua na regulação da expressão gênica. A deficiência de vitaminas (cobalamina, B6 e folato) pode interferir na taxa de metilação. O efeito da deficiência destas vitaminas foi determinado em estudos com cultura de células e em animais. No entanto, são raros os estudos realizados com seres humanos. Os objetivos deste trabalho foram: avaliar a taxa de metilação do DNA de leucócitos na região promotora dos genes Interferon gama (IFNγ), Serpin B5 e Stratifin; verificar se existe associação entre as concentrações das vitaminas e dos metabólitos com a taxa de metilação do DNA dos 3 genes; e analisar quais são os fatores de predição para a taxa de metilação do DNA (variável dependente) considerando como variáveis independentes os valores séricos das vitaminas e metabólitos, em mulheres com idades gestacionais de 16, 28 e 36 semanas. Cento e oitenta e três mulheres foram convidadas a participar desse estudo, porém apenas 96 completaram o estudo prospectivo. Foram determinadas as concentrações séricas da cobalamina (Cbl), folato, vitamina B6, S-adenosilmetionina (SAM), S-adenosilhomocisteína (SAH), ácido metilmalônico (MMA), homocisteína total (tHcy) e folato eritrocitário. A taxa de metilação nos 3 genes foi determinada por qMSP (Quantitative Methylation Specific - Polimerase Chain Reaction). Várias mulheres estavam fazendo uso de suplementação com ácido fólico e/ou polivitamínicos. Diante deste fato foram formados 4 subgrupos: Grupo 1 (constituído por mulheres que não usaram suplementação), Grupo 2 (mulheres que usaram suplementação em todas as idades gestacionais estudadas - 16, 28 e 36 semanas), Grupo 3 (mulheres que usaram suplementação no início da gestação até a 16ª semana) e Grupo 4 (mulheres que usaram suplementação na 16ª e 28ª semana gestacional). Não houve diferença entre as taxas de metilação do DNA dos genes IFNγ, Serpin B5 e Stratifin durante o período gestacional estudado. As taxas de metilação no gene IFNγ do grupo 1 foram maiores, quando comparadas as taxas dos demais grupos. Em modelos de regressão linear múltipla, considerando o período gestacional como um todo, apenas a vitamina B6 e a tHcy foram diretamente associadas aos valores da taxa de metilação do gene IFNγ. No entanto, a vitamina B6 foi inversamente associada, enquanto que tHcy esteve diretamente associada aos valores da taxa de metilação do gene Stratifin. A taxa de metilação não sofre alterações durante a gestação; a vitamina B6 e a tHcy foram os fatores de predição para as taxas de metilação do DNA na região promotora dos genes IFNγ e Stratifin. / DNA methylation is an epigenetic modification that regulates gene expression. Cobalamin, vitamin B6 and folate deficiencies can impair the DNA methylation index. Studies involving cultured cells and animals have evaluated the effect of vitamin deficiencies in DNA methylation. However, few studies were conducted in humans. The goals of this study were: to evaluate the leukocyte DNA methylation index in the promoter region of interferon gamma (IFNγ), Serpin B5 and Stratifin genes; to assess the association between vitamins and metabolites concentrations and DNA methylation index in three genes; and to examine the predictive factors for DNA methylation index using as independent variables: serum folate, serum cobalamin, serum vitamin B6, erythrocyte folate, methylmalonic acid (MMA), total homocysteine (tHcy) in three gestational ages (16th, 28th and 36,th weeks). A hundred eighty three women were included, but only 96 completed the prospective study. The serum concentrations of cobalamin, folate, vitamin B6, S-adenosylmethionine (SAM), Sadenosylhomocysteine (SAH), MMA, tHcy were determined. The erythrocyte folate concentration was also evaluated. The DNA methylation index was determined in three genes by qMSP (Quantitative Methylation Specific - Polymerase Chain Reaction). Several women were taking folic acid supplementation and/or multivitamins. Four groups were formed according to supplementation use: Group 1 (women who take no supplementation), Group 2 (women who took supplements at 16th, 28th and 36th weeks of pregnancy), Group 3 (women who took supplements in early pregnancy and up to 16 weeks) and Group 4 (women who took supplements in the 16th and 28th week of pregnancy). There was no difference between the DNA methylation index in the IFNγ, Serpin B5 and Stratifin genes during the gestational periods studied. The DNA methylation index in the IFNγ gene in group 1 was higher than those indexes from other groups. In multiple linear regression models considering the gestational periods as a whole, only vitamin B6 and tHcy were directly associated to DNA methylation index in IFNγ gene. However, vitamin B6 was inversely associated, whereas tHcy was directly associated with values of DNA methylation in Stratifin. The DNA methylation index does not change during pregnancy, vitamin B6 and tHcy were the predictors of DNA methylation in the promoter region of IFNγ and Stratifin genes.

O amadurecimento dos frutos é um processo caracterizado pela ocorrência de diversas alterações bioquímicas que ocorrem em um curto intervalo de tempo e que são importantes para a qualidade desses alimentos. Na banana uma das características mais importantes é o adoçamento do fruto, que ocorre como resultado da degradação do amido e acúmulo de sacarose. Resultados do nosso grupo apontam a β-amilase como uma enzima importante no processo de mobilização do amido, o que também é visto em estudos recentes utilizando Arabidopsis thaliana como modelo, os quais mostram que a principal via de degradação do amido transitório presente nas folhas ocorre pela ação da β-amilase. Entretanto, em bananas, faltam evidências quanto à funcionalidade de um gene de β-amilase, parcialmente isolado da polpa do fruto, e que é expresso durante o amadurecimento e que parece ser modulado por hormônios vegetais. Em vista disso, esse trabalho objetivou realizar a caracterização funcional desse gene, a qual permitiu constatar que esse gene codifica, de fato, para uma proteína capaz de ser endereçada aos cloroplastos. Também foi observado que o promotor desse gene contém motivos regulatórios para os mesmos hormônios previamente relacionados com a modulação da expressão desse gene em bananas. Essas novas evidências reforçam a idéia de que o produto desse gene de β-amilase tem um importante papel no processo de degradação do amido durante o amadurecimento da banana. / Fruit ripening is characterized by several biochemical changes that occur in a short time. These changes account for the color, taste and texture of the edible fruits, which are important postharvest characteristics for the fruit commercialization. In bananas, one of the most important features is the fruit sweetness which is the result of the starch degradation and sucrose accumulation. Results of our research group point β-amylase as an important enzyme in starch degradation process in bananas which is in agreement to recent studies using Arabidopsis thaliana as plant model. These studies show that the main degradation pathway of the transitory starch present in leaves on Arabidopsis plants occurs due β-amylase action. However, in bananas there are no evidences about the functionality of the expression product of a β-amylase gene, which was demonstrated to be modulated by plant hormones and expressed during ripening. In view of this, the aim of this work was to proceed the functional characterization of this gene which was showed to encode for an protein targeted to chloroplasts. It was also observed that its promoter region contains regulatory motifs related to the same plant hormones previously reported to modulate β-amylase expression. These new evidences support the idea that expression of β-amylase gene has an important role in starch degradation during banana ripening.

O colágeno XVIII é um componente das membranas basais com diversos domínios funcionais, como a endostatina e o domínio frizzled, que têm importante papel em processos celulares como proliferação e diferenciação. COL18A1 possui dois promotores alternativos: o promotor 1, que regula a síntese da variante NC11-303, e o promotor 2 responsável pelas variantes NC11- 728 e NC11-493, expressas por hepatócitos. Existe uma variação interindividual da endostatina circulante e da expressão do colágeno XVIII no fígado. A expressão do colágeno XVIII/endostatina foi correlacionada com a progressão tanto do hepatocarcinoma (HCC), quanto da fibrose/cirrose hepática. Elucidar a regulação da expressão de COL18A1 pode auxiliar na compreensão dessa variação interindividual e da progressão dessas doenças. Neste trabalho demos início a caracterização do promotor 2 do COL18A1. Identificamos na seqüência predita como promotora cinco regiões conservadas entre humanos e camundongos. A análise in silico e funcional dessas regiões revelou que os fatores de transcrição, Sp1, Sp3, YY1, Oct-1, C/EBPα e C/EBPβ, interagem com as mesmas. Demonstramos que C/EBPβaumenta a taxa de transcrição do promotor 2 em hepatócitos, e que existe uma correlação positiva da expressão de NC11-493 com C/EBPαe C/EBPβem tecido hepático cirrótico e tumoral. As expressões de C/EBPαem tecido hepático cirrótico e tumoral estão diretamente correlacionadas, enquanto que os níveis de NC11-493 nos tumores estão inversamente correlacionados com o tamanho dos mesmos. Mostramos a existência de diversos SNPs no promotor 2. O SNP-700T/G, funcional in vitro, afeta a interação de Sp3 e YY1 com essa região regulatória. A deleção da região do SNP indicou que ela possui elementos importantes para a transcrição em hepatócitos, apesar deste SNP não estar relacionado com o nível de expressão do colágeno XVIII em fígado fibrótico ou com susceptibilidade a HCC. O SNP- 700T/G está em desequilíbrio de ligação com o SNPc.1135C/T, no domínio frizzled do colágeno XVIII. Não foi possível elucidar a funcionalidade do SNPs c.1135C/T in vitro, mas os haplótipos formados por esses dois SNPs têm diferentes frequências entre descendentes de europeus e de africanos. Nosso trabalho traz importantes contribuições e abre novas perspectivas para a compreensão da regulação do colágeno XVIII em fígado humano, tanto em situações fisiológicas, quanto em processos fibrogênicos e tumorigênicos¶ / Collagen XVIII is a basal membrane component with several funcional domains, such as endostatin and frizzled domains, which have important roles in cellular processes such as proliferation and differentiation. COL18A1 has two promoter regions: promoter 1, that regulates the synthesis of NC11-303 isoform, and promoter 2, localized in intron 2, responsible for NC11-728 and NC11-493 isoforms expressed by hepatocytes. There is a large interindividual variation in circulating endostatin and in collagen XVIII liver expression. Collagen XVIII/endostatin levels were correlated with hepatocellular carcinoma (HCC) progression, as well as liver fibrosis/cirrhosis, conditions that precede HCC. Elucidating the mechanisms that regulate COL18A1 expression in hepatocytes may help understanding its variation among individuals and liver disease stages, as well as contribute to new treatment strategies. In this work we began to characterize COL18A1 promoter region 2. We identified in the predicted promoter sequence five conserved regions between human and mouse. The in silico and functional analysis of these regions revealed that transcription factors Sp1, Sp3, YY1, Oct-1, C/EBPα and C/EBPβ interact with them. We have demonstrated that C/EBPβ increases promoter 2 transcription rate in hepatocytes, and that there is a positive correlation of NC11-493 expression with that of C/EBPα and C/EBPβ in cirrhotic and tumor liver samples. Non-tumor and tumor C/EBPα expressions positively correlate between themselves, while NC11-493 tumor expression inversely correlates with tumor size. We also showed that there are several SNPs in COL18A1 promoter 2 region. SNP-700T/G, functional in vitro, affects Sp3 and YY1 interaction with the promoter 2 region and deletion of the SNP region indicated that this sequence has important hepatocyte regulatory elements. Our results suggest that this SNP does not significantly affects COL18A1 expression in fibrotic/cirrhotic liver and is not associated with HCC susceptibility. SNP-700T/G is in linkage disequilibrium with SNPc.1135C/T, at collagen XVIII frizzled domain. We could not elucidate SNPc.1135C/T functionality in vitro, but the haplotypes formed by these two SNPs have different frequencies in European and African descendants. In conclusion, our work brings important contributions and opens new perspectives for the comprehension of collagen XVIII regulation in human liver in physiological situations, as well as in fibrotic/cirrhotic and tumorigenic process.

Die einzellige Grünalge C. reinhardtii dient als Modellorganismus und ist aufgrund des GRAS-Status, des schnellen, kostengünstigen Wachstums und der Möglichkeit posttranslationaler Modifikationen im Kerngenom, als Expressionssystem für beispielsweise orale Impfstoffe sehr interessant. Herausforderungen sind die im Vergleich zu konventionell verwendeten Expressionssystemen sehr geringen Expressionsraten im Kerngenom. Daher sollten in dieser Arbeit neuartige, teils induzierbare, Promotorkonstrukte verwendet und mittels Luciferase-Reporter auf ihre Expressionssteigerung hin getestet werden. Zunächst wurden ausgewählte Promotoren des Chlorella-Virus-1 (PBCV-1) gewählt, diese führten allerdings zu keiner Expression. Außerdem wurden synthetische, aneinandergereihte Hitzeschockelemente mit dem endogenen RBCS2-Promotor fusioniert und die Expressionsraten analysiert. Dabei ergab sich bei der Kombination aus dem synthetischen Hitzeschockelement in achtfacher Wiederholung (HSE8x) mit RBCS2 nach der Hitzeinduktion eine Steigerung der Expressionsrate um das bis zu dreifache. Die Basalexpression war hierbei bei HSE1x-RBCS2 am höchsten und erreichte Expressionslevels, welche um das fünffache höher lagen als die Positivkontrolle HSP70A-RBCS2. Mittels Chromatinimmunopräzipitation mit dem Antikörper gegen HSF1 konnte gezeigt werden, dass eine Bindung an das synthetische Hitzeschockelement vorliegt und deshalb die Expression über die konventionelle Hitzeschockantwort in Chlamydomonas reinhardtii funktioniert. Im Gegensatz zur Luciferase benötigen Fluoreszenzproteine als Reporter kein Substrat. Infolge dieses Vorteils und der Möglichkeiten der FACS-Analyse und Fluoreszenzmikroskopie wurde tagRFP als neuer Reporter in C. reinhardtii etabliert. Die Erhöhung der Expressionsrate durch neue Promotorkombinationen und die Anwendung von tagRFP als neuen Fluoreszenzreporter bedeuten wichtige Schritte in der Etablierung von C.reinhardtii als Expressionssystem für Produkte in der Biotechnologie. / The unicellular green alga C.reinhardtii which is used as a model organism could be an interesting expression system for oral vaccines. This is because the alga is generally regarded as safe, it shows fast growth rates and culturing is cheap. Furthermore it offers the possibility of posttranslational modifications. Challenges lie in the low expression rates in the nuclear genome when compared to other expression systems. Therefore the first step in this work was to test whether selected Chlorella virus PBCV-1 promoters, do lead to enhanced expression rates, but there was no detectable expression. Furthermore synthetic repeats of heat shock elements were used in combination with the endogenous RBCS2-promoter and analysed for expression rates via reporter measurements. The combination of synthetic heat shock elements in eightfold repeats in combination with RBCS2 enhanced expression rates of luciferase after heat shock up to threefold in comparison with the up to now strongest known promoter combination HSP70A-RBCS2. Basalexpression turned out to be best for the HSE1x-RBCS2 promoter and reached expression levels fivefold higher compared to HSP70A-RBCS2. To examine if the artificial heat shock elements (HSEs) are bound by the heat shock factor 1 in C. reinhardtii a ChIP assay with HSE8x-RBCS2 and the antibody of HSF1 of C. reinhardtii was done. It could be shown that HSF1 binds HSEs and therefore one can explain the heat shock inducibility of HSEs is regulated via the conserved heat shock response. In contrast to luciferase fluorescence reporters do not need substrate. Because of this advantage and the possibility of FACS analyses and fluorescence microscopy tagRFP was established as new reporter in C.reinhardtii. Enhancement of expression rates through new constitutive and inducible promoter combinations and the possible use of tagRFP as new fluorescence reporter are significant steps in establishing C.reinhardtii as expression system for products in biotechnology.