Das Bakterium Bradyrhizobium japonicum ist wie andere Rhizobien in der Lage mit Pflanzen der Familie Fabales (Leguminosen) eine Symbiose einzugehen. Im symbiontischen Zustand fixieren die Mikrosymbionten atmosphärischen, molekularen Stickstoff und stellen diesen den Pflanzen in verwertbarer Form zur Verfügung. Im Gegenzug erhalten die Bakterien von den Pflanzen verschiedene Verbindungen als Kohlenstoff- und Energiequelle. Dem geht ein komplexer Signalaustausch voraus um die gegenseitige Erkennung der Partner und die Spezies-spezifische Symbiose zu ermöglichen. Auf Seite der Bakterien erfolgt die Reaktion auf die Gegenwart pflanzlicher Signalmoleküle. In B. japonicum induziert das Flavonoid Genistein die Transkription einer Reihe von Genen. Durch die Expression der nod-Gene erfolgt eine Synthese von Lipochitooligosacchariden. Diese sogenannten Nodulationsfaktoren rufen wiederum eine Reaktion in der Wirtspflanze hervor. Um die zugrunde liegende Genexpression und deren Regulationsmechanismen zu erforschen, standen bis dato nur Methoden für die Untersuchung einzelner Gene zur Verfügung. Die erstmals 1995 publizierte Microarraytechnologie eröffnete die Möglichkeit zu einem Zeitpunkt die Gentranskription eines gesamten Organismus zu untersuchen (Schena et al. 1995). Um mit dieser Technologie die Gentranskription bei B. japonicum zu untersuchen, wurde ein auf PCR-Produkten basierender Microarray hergestellt. Grundlage für die Synthese genspezifischer PCR-Produkte war die symbiontische Region von B. japonicum und andere zu diesem Zeitpunkt bekannte Gensequenzen (Göttfert et al. 2001). Nach der 2002 erfolgten Veröffentlichung des Genoms von B. japonicum erfolgte ein Abgleich der bisher verwendeten Sequenzen mit der vollständigen Sequenzinformation (Kaneko et al. 2002a). Nach Synthese der PCR-Produkte und deren Kontrolle durch Sequenzierung erfolgte die Herstellung des Microarrays unter Einsatz eines Microarrayspotters. Geeignete Techniken der cDNA-Markierung und die Microarray-Hybridisierung wurden mit Total-RNA von B. japonicum-Kulturen ausgetestet und etabliert. Eine differentielle Expremierung war eine Stunde nach Genistein- bzw. Methanolgabe zum Kulturmedium nicht nachweisbar. Zum darauffolgenden Zeitpunkt konnte die bekannte Induktion der nod-Gene durch dass Flavonoid Genistein beobachtet werden. Diese Induktion fiel in den verbleibenden zwei Zeitpunkten stark ab. Dieser Abfall der Induktionsrate ließ sich nicht mit dem Genisteingehalt im Kulturmedium erklären, da dieser Zeitraum konstant blieb. Vier Stunden nach Genisteingabe war die Induktion der Gene nolA und nodD2, deren Produkte sind an der negativen Regulation der nod-Gene beteiligt, nachweisbar. Dies wäre eine mögliche Erklärung für den Abfall der Induktion der nod-Gene. Im gleichen untersuchten Zeitraum wurde die Transkription verschiedener Gene der Stickstofffixierung und Denitrifikation durch das Flavonoid Genistein induziert. Auch für die bekannten Regulatoren dieser Gene war eine Induktion nachweisbar. Nach bisherigen Arbeiten war die Expression dieser Gene auf mikroaerobe und anaerobe Zustände, wie z.B. dem symbiontischen Stadium, beschränkt. Eine Induktion durch Flavonoide ist bisher nicht beobachtet worden. Um die Verwendbarkeit des Microarrays auch für den symbiontischen Zustand von B. japonicum zu testen, wurden Versuche mit Wurzelknöllchen von Sojabohne (Glycine max) durchgeführt. Dafür wurden Keimlinge der Sojabohne (G. max) mit B. japonicum inokuliert und die Total-RNA der entstehenden Wurzelknöllchen isoliert. Jedoch war eine Kreuzhybridisierung mit pflanzlicher Total-RNA zu beobachten. Der auf PCR-Produkten basierende Microarray ist für die Untersuchung des symbiontischen Stadiums von B. japonicum nicht einsetzbar. Im Vergleich dazu wurde ein auf Oligomeren basierender, kommerzieller Microarray für diesen Versuch getestet. Die Kreuzhybridisierung mit pflanzlicher Total-RNA war auf die Oligomere für die 16S rDNA und 23S rDNA reduziert.

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Our intention is to find similarity among the time series expressions of the genes in microarray experiments. It is hypothesized that at a given time point the concentration of one gene’s mRNA is directly affected by the concentration of other gene’s mRNA, and may have biological significance. We define dissimilarity between two time-series data set as the variance of Euclidean distances of each time points. The large numbers of gene expressions make the calculation of variance of distance in each point computationally expensive and therefore computationally challenging in terms of execution time. For this reason we use autoregressive model which estimates nineteen points gene expression to a three point vector. It allows us to find variance of difference between two data sets without point-to-point matching. Previous analysis from the microarray experiments data found that 62 genes are regulated following EGF (Epidermal Growth Factor) and HRG (Heregulin) treatment of the MCF-7 breast cancer cells. We have chosen these suspected cancer-related genes as our reference and investigated which additional set of genes has similar time point expression profiles. Keeping variance of difference as a measure of distance, we have used several methods for clustering the gene expression data, such as our own maximum clique finding heuristics and hierarchical clustering. The results obtained were validated through a text mining study. New predictions from our study could be a basis for further investigations in the genesis of breast cancer. Overall in 84 new genes are found in which 57 genes are related to cancer among them 35 genes are associated with breast cancer.

Due to the current rise of exposure to natural and synthetic compounds in our daily life, the debate concerning the safety of many substances is becoming increasingly relevant. The estrogenic activity of various compounds, described as xenoestrogens, is the major part of this debate. Humans beings are exposed to these substances from different environmental contaminations ranging from conscious intake of estrogenic substances, as in contraception or in hormone replace therapy (HRT), to unconscious exposure, from food, the use of synthetic material in daily life and air and water pollution. At this point the need for methods to investigate the activity and the safety of these substances is becoming increasingly important. Classical methods for the analysis of the estrogenic activity of substances, like batteries of in vivo test systems on the rat uterotrophic assay are not able to describe the different pathways of action of recently discovered estrogenic substances. This evidence was already shown by the Organization for Economic Cooperation and Development (OECD), introducing new test guidelines for the investigation of effects of endocrine disruptors (according to enhanced Test Guideline 407). As reviewed by Nilsson (Nilsson et al., 2001), after the interaction of the estrogens with the Estrogen Receptor (ER) in the cells, the mechanism of activation possible is not only via direct binding of the ER to the Estrogen Responsive Elements (EREs) present in the promoter region of the target gene, very well described for many target genes, but that also other mechanisms are used: the interaction of the ER with the AP 1, Sp 1 and NFkB modes, that are discovered but not yet comprehensively described. The aim of my work is to produce a microarray DNA chip for the investigation of the estrogenic activity of different compounds present in the environment. The chip will consist of a selection of 100 genes that are estrogen responsive and it will cover the spectrum of activities of estrogenic compounds in various organs of the body. In the gene selection, genes were chosen that are estrogen responsive in the classical target tissues of estrogens, linked to reproduction, like uterus and mammary gland, and also in tissues not related to reproduction like liver, bones and capillars. In addition, other genes are included to monitor different pathways that are related to disease states; control of cell proliferation, apoptosis or cancer related genes. Currently these kinds of investigations are already in process, but by other methods which are more time consuming and with a lower throughput e.g. the gene expression profiling using the real time RT-PCR. The use of microarray’s satisfies the need for a less time consuming, high throughput method, to obtain a fast characterization of the gene expression finger print of the candidate substances and their mechanism of action in the organism. In my work I investigated the estrogenic potency of different Xenoestrogens that commonly occur in our daily life, in rat cells and tissue using well known estrogen sensitive genes like C3, Clu, IGFBP1 and CaBP9k. I focused on their effect on cell proliferation, studying PCNA expression. For the first time sensitivity of the gene CA2 was proofed in liver and uterus. A new identified mRNA sequence, r52, was characterized for its sensitivity to estrogenic exposure. This sequence was investigated at the molecular level expanding the known nucleic sequence. I produce a microarray chip with 16 genes to investigate the estrogenic potency of different compounds. As proof of principle of the microarray method completely produced in house I compared the result of gene expression obtained by the chip to that obtained by real time RT PCR finding a similarity of results. This new established method is less sensitive than the real-time RT PCR but allows a high throughput of gene expression analysis producing at the end a more complete picture of the expression signature of a compound.

This data article contains data related to the research article entitled, "in vitro characterization of bone marrow stromal cells from osteoarthritic donors" [1]. Osteoarthritis (OA) represents the main indication for total joint arthroplasty and is one of the most frequent degenerative joint disorders. However, the exact etiology of OA remains unknown. Bone marrow stromal cells (BMSCs) can be easily isolated from bone marrow aspirates and provide an excellent source of progenitor cells. The data shows the identification of pivotal genes and pathways involved in osteoarthritis by comparing gene expression patterns of BMSCs from osteoarthritic versus healthy donors using an array-based approach.

La phosphorylation du domaine C-terminal de l’ARN polymérase II permet à ce complexe protéique d’exécuter la transcription des gènes, en plus de coupler à la transcription des événements moléculaires comme la maturation des ARNm. Mes résultats montrent que même si cette phosphorylation suit un patron similaire à l’ensemble des gènes, il existe des exceptions pouvant être dues à des mécanismes alternatifs de phosphorylation du CTD. Le présent ouvrage s’intéresse également au rôle qu’occupe la variante d’histone H2A.Z dans l’organisation de la chromatine. Des études précédentes on montré que le positionnement de certains nucléosomes le long de l’ADN serait influencé par H2A.Z et aurait une influence sur la capacité de transcrire les gènes. Par une approche génomique utilisant les puces à ADN, j’ai cartographié l’impact de la délétion de H2A.Z sur la structure des nucléosomes. Enfin, des résultats intéressants sur la dynamique d’incorporation de H2A.Z à la chromatine ont été obtenus. / RNA Polymerase II is the molecular complex responsible for the transcription of class II genes. Proper transcription and associated events such as mRNA processing are thought to require the phosphorylation of its C-terminal domain. Here I show that this phosphorylation follows a similar pattern for most of the genes, althought some exceptions exist. These exceptions could be explained by alternative phosphorylation mechanisms. Also, this work provides data on how the variant histone H2A.Z influences chromatin structure. Previous studies have shown a role for H2A.Z in the positioning of some nucleosomes along the DNA, which would impact the ability to transcribe genes. Here I used a microarray technology to profile nucleosome positions in a genome-wide manner. My data provide further evidence that H2A.Z influences nucleosome positioning. Interesting results regarding the dynamics of H2A.Z incorporation into chromatin are also shown.

Os papilomavírus são pequenos vírus de DNA dupla-fita, não envelopados, mucoepiteliotrópicos, capazes de infectar inúmeros vertebrados superiores de maneira espécie-específica. A infecção por estes vírus está associada a uma série de desordens proliferativas que levam desde de a formação de verrugas comuns até a do carcinoma invasivo. Aproximadamente 200 tipos de papilomavírus humano (HPVs) foram identificados, sendo que cerca de 40 deles infectam o trato genital. Dentre estes, os chamados HPVs de alto-risco estão associados etiologicamente ao carcinoma de colo de útero, enquanto que os de baixo-risco estão relacionados às lesões epiteliais benignas. A infecção por HPVs de alto-risco é muito comum, no entanto, a maioria destas é transitória e somente uma pequena proporção de mulheres desenvolvem o carcinoma. Entretanto, algumas mulheres são incapazes de eliminar esta infecção, levando a persistência viral e o conseqüente desenvolvimento da neoplasia. Para que a infecção pelo HPV persista é necessário um mecanismo de escape ao sistema imune do hospedeiro. O mecanismo de escape à resposta imune inata parece ser característico da infecção pelo HPV, pois o ciclo infeccioso deste vírus não promove inflamação. A infecção por HPV promove a liberação de citocinas, tal como o fator de necrose tumoral (TNF). Esta citocina possui um potente efeito citostático em queratinócitos normais e imortalizados com HPV, enquanto que em queratinóctos imortalizados com HPV18 este efeito não é observado. Do mesmo modo, observamos que a expressão do oncogene E6 de HPV16 ou 18 é suficiente para promover resistência ao efeito antiproliferativo do TNF em culturas em monocamada e organotípica. A expressão aumentada e contínua destes ocogenes é sabidamente o principal evento favorável ao desenvolvimento do câncer de colo de útero. Estas proteínas são essenciais na indução da transformação celular, visto que interferem na regulação do ciclo celular e apoptose. O produto dos genes E6 e E7 se liga ao produto dos genes supressores de tumor p53 e pRb, respectivamente, levando a sua degradação pela via de proteólise dependente de ubiquitina. As bases moleculares desta resistência ao TNF ainda são pouco conhecidas. Neste estudo, comparamos o efeito desta citocina em queratinócitos normais e que expressam E6 ou E7. Observamos através de cDNA Microarray a expressão de um grupo de genes, entre eles TCN1, DEK, HMGB2, INHBA, MCM2, MCM5 e MMP9, com expressão diferencial entre as células sensíveis e as resistentes ao TNF. / Papillomaviruses are small, non-enveloped, epitheliotropic, double-stranded DNA viruses that infect mucosal and cutaneous epithelia in a wide variety of higher vertebrates in a species-specific manner. Papillomavirus infections are associated to a series of proliferative disorders that range from common warts to invasive carcinomas. Almost 200 types of human papillomaviruses (HPVs) have been identified and approximately 40 of them infect the genital tract. Only the so-called high-risk HPV types mediate human carcinogenesis, whereas the low-risk HPVs have been linked to benign epithelial lesions. High-risk genital HPV infection is very common, and the majority of individuals clear their infection with time. However, a proportion of women cannot effectively clear the virus, and the persistence of a high-risk HPV is the major risk factor for the development of anogenital malignancies. To persist, HPV must escape the host immune system. Effective evasion of innate immune recognition seems to be the hallmark of HPV infections, since the infectious cycle is one in which viral replication and virion release is not associated with inflammation. Furthermore, HPV infections promote cytokine release, as tumor necrosis factor-alpha (TNF). This cytokine has a potent cytostatic effect on normal and HPV16 immortalized keratinocytes, while it does not affect HPV18 immortalized keratinocytes proliferation. In addition, we have observed that expression of HPV 16 or 18 E7 oncogene is sufficient to overcome TNF antiproliferative effect in monolayer and organotypic cell cultures. The increased and sustained expression of HPV oncogenes, E6 and E7, is the main contributor to the development of cervical cancer. Both E6 and E7 proteins are essential to induce and maintain cellular transformation, due to their interference with cell-cycle and apoptosis regulation. The most manifest function of the E6 protein is to promote the degradation of p53, while E7 is known to bind to and promote the proteasomal degradation of the retinoblastoma tumor suppressor gene product, pRb, and its family members. The molecular basis of TNF resistance is not well understood. In this study we compared the effect of TNF between normal and HPV16 E6 or E7 expressing keratinocytes. We observed by cDNA Microarray the differential expression of a common set of genes in TNF-sensitive cell lines, including TCN1, DEK, HMGB2, INHBA, MCM2, MCM5 and MMP9, that differs from those modulated in TNF-resistant cells.

Die Transkription der Chloroplasten ist erstaunlich komplex. Ihr relativ kleines Genom kodiert für Komponenten der Photosynthese und der eigenen Genexpressionsmaschine. Es wird mindestens durch zwei RNA Polymerasen transkribiert. Neben der plastidär kodierten RNA Polymerase (PEP), existiert mindestens eine weitere kernkodierte RNA Polymerase (NEP). Die PEP spielt eine wichtige Rolle bei der Expression der Photosynthesegene und ist essentiell für die Biogenese der Chloroplasten und schließlich für das Überleben der Pflanzen. In der vorliegenden Arbeit wird erstmals die spezifische Interaktion der PEP mit ihren Transkriptionseinheiten unter verschiedenen Lichtbedingungen in vivo auf plastomweiter Ebene gezeigt. Darunter befinden sich hauptsächlich DNA Fragmente, die Photosynthesegene repräsentieren. Außerdem zeigt die PEP eine eindeutige Präferenz zu den rRNA Genen und einigen tRNA Genen. Eine Reduktion dieser Assoziation der PEP in Dunkelperioden lässt einen lichtabhängigen Prozess bei der PEP-DNA Assoziation vermuten. Außerdem wurde die PEP Aktivität an den bestimmten DNA Regionen in vivo ermittelt durch die Analyse naszierender Transkripte. Ein Großteil der plastidären RNA Spezies kopräzipitiert mit der PEP, aber sie scheint präferentiell mit tRNAs zu interagieren. Die Analyse der Verteilung der PEP bewies erstmals, dass sie hauptsächlich mit den Membranen assoziiert. Dort befindet sich auch das transkriptionsaktive Chromosom (TAC). Im TAC wurden vor kurzem neben der PEP auch Mitglieder von RNA Prozessierungsfaktoren identifiziert, was auf eine mögliche Kopplung von Transkription und posttranskriptionellen Prozessen hindeutet. Für einen Einblick in dieses Interaktionsnetzwerk wurden Transkriptanalysen des TACs ausgeführt. Mit wenigen Ausnahmen assoziieren alle plastidären RNA Spezies mit dem TAC. Einige RNAs liegen bereits prozessiert vor, was eine Verbindung zwischen Transkription und posttranskriptionellen Prozessen noch im TAC vermuten lässt. / Chloroplast transcription is astonishingly complex. The relative small genome of plastids, which codes for components of the photosynthetic machinery as well as for components of their own gene expression machinery, is transcribed by at least two different RNA polymerases. Beside the plastid-encoded plastid RNA polymerase (PEP) a nuclear-encoded plastid RNA polymerase (NEP) exists as well. PEP plays a major role in the expression of photosynthesis genes and is essential for chloroplast biogenesis and thus for plant survival. This work shows the specific in vivo interaction between PEP and its transcription units under different light conditions on a plastome-wide scale. Among them are DNA fragments that represent photosynthetic genes. In addition, PEP shows clear preferences to rRNA and tRNA genes. Furthermore, the association of PEP with photosynthesis-related genes was reduced during the dark period, indicating that PEP-DNA association is a light-dependent process. To survey PEP activity in vivo on plastid DNA regions, the association to nascent transcripts was analyzed. The majority of plastid RNA species could be found in PEP precipitates, but PEP seems to interact more strongly with tRNAs. Analysis of the suborganellar distribution of PEP shows that PEP is preferably associated with chloroplastmembranes. The transcriptionally active chromosome (TAC) was also found to be membrane-attached. Beside PEP different RNA processing factors were identified within the TAC and related purifications, indicating a possible coupling of transcription and posttranscriptional processes. To gain more insights into this interaction network, transcripts of TAC were analyzed. It is shown that nearly all plastid RNA species with only a few exceptions are associated to the TAC and that at least selected transcripts are already processed. This indicates that there is a link between transcription and posttranscriptional processes already within the TAC.

Camundongos AIRmax e AIRmin homozigotos para os alelos R e S do gene Slc11a1 foram avaliados para susceptibilidade à artrite induzida por pristane (PIA). A presença do alelo S aumentou a incidência e a gravidade nos AIRmax, sugerindo que o gene Slc11a1, ou outro próximo, esteja interagindo com os loci de resposta inflamatória na modulação de PIA. Para identificar estes loci foram realizados estudos de associação genótipo-fenótipo e de expressão gênica global. Os RNAs das patas dos animais foram isolados após 180 dias da indução por pristane. As análises de expressão gênica global foram realizadas usando a plataforma Codelink (36k genes), cujos resultados foram validados por PCR em tempo real. Os estudos de associação foram realizados através da análise de polimorfismo de microssatélites pelo programa MapManager. Foram identificadas duas regiões nos cromossomos 1 e 11. Um número grande de genes diferencialmente expressos foi verificado nos animais AIRmax SS cujos temas biológicos significativamente sobre-representados foram a resposta inflamatória e quimiotaxia. Os camundongos AIRmax SS também possuem uma ativação maior dos genes Ccl3, Ccl7, C3ar1, Il10, Stat3, Tirap, Trem 1, Trem 3, Mefv, Ptx3, Chi3l3 e Kras. Alguns desses genes co-localizam com regiões previamente mapeadas nos cromossomos 1 e 11. / AIRmax and AIRmin mice homozygous for Slc11a1 R and S allele were evaluated for pristane-induced arthritis (PIA) susceptibility. The presence of S allele increased the incidence and the arthritis severity in ARmax mice, suggesting that Slc11a1 or other closed-linked gene interacts with inflammatory loci to modulate PIA. In order to identify inflammatory modifier loci modulating experimental arthritis development, genotype-phenotype association studies and global gene expression analyses were performed. Mice received i.p. injections of pristane and the paw RNAs were isolated at day 180. Global gene expression analysis was performed on Codelink bioarrays (36k genes) and validated by real time PCR. The microsatellite polymorphism analyses were performed using MapManager program. Two regions on chromosomes 1 and 11 were identified. Higher number of differentially-expressed genes were detected in AIRmax SS subline, which significant over-represented biological themes were related to inflammatory response and chemotaxis. Susceptible AIRmax SS mice also display high up-regulation of Ccl3, Ccl7, C3ar1, Il10, Stat3, Tirap, Trem 1, Trem 3, Mefv, Ptx3, Chi3l3 e Kras genes. Some of them co-localize with previously identified regions mapped on chromosomes 1 and 11.

Glicose e oxigênio são moléculas essenciais para a maioria dos organismos vivos. Além de sua importância nos processos de produção de energia - glicose como fonte de carbono e energia e oxigênio como aceptor dos elétrons doados por NADH e FADH2 - estes dois compostos funcionam como efetuadores, modulando vários processos metabólicos e fisiológicos nas células. Visto que a mitocôndria é um dos alvos afetados pelas disponibilidades destas duas moléculas, nós isolamos e seqüenciamos o genoma mitocondrial de Trichoderma reesei, um fungo multicelular empregado neste trabalho como sistema modelo. Foi estudado o efeito da variação de concentração de glicose e oxigênio sobre a expressão de transcritos do genoma mitocondrial, bem como sua implicação no metabolismo de glicose. São apresentadas análises da expressão gênica de aproximadamente 2000 transcritos de T. reesei submetido a concentrações limitantes de oxigênio dissolvido, realizadas com o emprego da técnica de microarrays de cDNA. Pelo menos 330 transcritos foram diferencialmente expressos em função da disponibilidade de oxigênio. Aqueles envolvidos nos processos de síntese protéica e divisão celular foram regulados negativamente, enquanto transcritos relacionados com funções de defesa celular e síntese de RNA foram positivamente regulados. Uma fração substancial de outros genes afetados pela baixa disponibilidade de oxigênio não possui, atualmente, funções celulares conhecidas. Esta observação deve contribuir para a posterior anotação funcional do genoma de T. reesei. Também foram identificados reguladores transcricionais diferencialmente expressos em baixas tensões de oxigênio. O perfil de expressão destes reguladores aponta-os como potenciais candidatos ao envolvimento com a expressão de genes afetados pela disponibilidade de oxigênio. / Glucose and oxygen are essential molecules in most of living organisms. In addition to their importance in production of energy - glucose as a carbon and energy source and oxygen as an acceptor of electrons donated by NADH and FADH2 - both molecules function as effectors modulating various metabolic and physiological processes in the cell. Because one of the targets affected by both molecules is the mitochondrion, we isolated and sequenced the mitochondrial genome of Trichoderma reesei, a multicellular fungus that is used in this study as a model system. The effect of varying the concentration of glucose and oxygen on the expression of the transcripts of the mitochondrial genome, and its implication on the metabolism of glucose, was studied. Gene-wide expression analyses of nearly 2000 transcripts of T. reesei under limited concentration of dissolved oxygen, using cDNA microarry technique, are presented. At least 330 transcripts were differentially expressed with respect to oxygen availability. Those involved in protein synthesis and cell division processes were downregulated, while transcripts involved in cell defense and RNA synthesis were upregulated. A substantive fraction of other anaerobically affected genes have currently unknown cellular roles, and these results should therefore contribute to further functional annotation of the genome. ln addition, we have identified transcriptional regulators that are differentially expressed at a low oxygen tensions. The expression profile of these regulators points them out as potential candidates involved in the expression of genes affected by oxygen availability.