O feijão é importante fonte carboidratos, vitaminas, minerais e fibras. No Brasil, a produtividade desta leguminosa é baixa e um dos fatores é a ocorrência de doenças como a antracnose causada pelo Colletothrichum lindemuthianum, que gera perdas de até 100% da produção. Plantas possuem diversos mecanismos de defesa contra patógenos e relatos apontam que o silício é capaz não só de promover mudanças morfológicas nas folhas, mas também de ativar os genes de resistência. O presente trabalho foi dividido em três estudos que tinham como objetivo: (1) entender a resposta de três cultivares de feijoeiro ao silício disponível na solução nutritiva; (2) identificar a contribuição do Si na expressão de genes relacionados à infecção pelo fungo através da construção de duas bibliotecas subtrativas por supressão (SSH), visando selecionar genes diferencialmente representados durante a infecção da planta com a raça 65 de C. lindemuthianum (a) e durante a infeção da planta na presença de uma maior dose silicato de potássio (75 ppm) no substrato (b); (3) identificar a resposta de dez transcritos selecionados no Estudo 2 para tentar entender a resposta dos mesmos em diferentes períodos (0; 6; 42; 72 h) após a inoculação, com ou sem suplemento de Si. Como resultados, foi observado que para as três cultivares avaliadas o Si começa a ser absorvido 14 dias após o transplante. Também foi identificado por de microscopia de varredura (MEV) que não há diferença significativa entre o número de tricomas e cada cultivar, mas que para o número de estômatos a cultivar IAC-Harmonia destacou-se das demais. Além disso, quando as três cultivares foram suplementadas com Si, houve a formação de uma cera epicuticular descrita como mecanismo de defesa da planta contra fungos; e que através de EDX (Energy-dispersive X-ray spectroscopy) foi possível constatar que plantas tratadas com Si apresentam maior teor deste elemento nas folhas. Através de inoculações com a raça 65 do patógeno verificou-se o efeito do mineral na redução da severidade da doença nas cultivares IAC-Harmonia e Pérola. No segundo estudo, duas bibliotecas de hibridização subtrativa por supressão (SSH), foram construídas visando selecionar os genes diferencialmente expressos entre plantas inoculadas e não-inoculadas (A) e entre plantas inoculadas e tratadas ou não com 75 ppm de Si (B). Foram geradas 991 sequências únicas, anotadas através do GeneOntolgy. Quinze genes de cada biblioteca foram selecionados para os experimentos de validação por RT-qPCR. Para a Biblioteca A, 11/15 genes foram positivamente regulados, e em B, 14/15. No terceiro estudo ficou evidenciado que a inoculação com o patógeno alterou positivamente a expressão de sete genes, enquanto que o tratamento com 75 ppm de Si alterou a expressão de oito genes, em pelo menos um dos tempos avaliados / Beans are an important source of carbohydrates, vitamins, minerals and fibers. In Brazil, this legume still has low productivity and one of the factors involved is the occurrence of diseases such as anthracnose, caused by the fungus Colletothrichum lindemuthianum, which causes losses in production of up to 100%. Plants present several defense mechanisms against pathogens and the reports indicate that silicon does not only promote morphological changes in leaves, but also activates resistance genes. This work was divided into three studies aiming: (1) to understand the response of three bean cultivars to a silicon source in a nutrient solution, (2) to identify the contribution of Si in the expression of genes related to the infection by the fungus by constructing two subtractive suppression libraries (SSH), to select genes differentially represented during infection of the plant with race 65 of C. lindemuthianum (a) and during infection of the plant in the presence of higher dose of potassium silicate (75 ppm) in the substrate (b), (3) to identify the response of ten selected transcripts in Study 2 in various periods (0, 6, 42, 72 h) after inoculation, with or without supplemental Si. As a result, it was observed that for all three cultivars Si begins to be absorbed 14 days after transplantation. Was also identified by microscopy (SEM) that there is no significant difference between the number of trichomes among cultivars, but that the number of stomata for the IAC-Harmonia stood out from the rest. Moreover, when the three cultivars were supplemented with Si, thus forming an epicuticular wax described as a defense mechanism against plant fungi, and that by EDX (Energy-dispersive X-ray spectroscopy) it was found that plants treated with Si have higher content of this element in leaves. Through inoculations with race 65 of the pathogen it was verified the effect of the mineral in reducing disease severity in IAC-Pérola and IAC - Harmonia. In the second study, two libraries from suppression subtractive hybridization (SSH) were constructed in order to select the differentially expressed genes between inoculated and non-inoculated (A) and between plants inoculated and treated or not with 75 ppm of Si (B). In total, 991 unique sequences were generated, those recorded by GeneOntolgy. Fifteen genes from each library were selected for the validation experiments by RT-qPCR. For library A, 11/15 genes were positively regulated, and in B, 14/15. In the third study it is showed that inoculation with the pathogen positively altered expression of seven genes, whereas treatment with 75 ppm of Si changed the expression of eight genes, in at least one of the times analyzed

Os genes homeobox atuam na morfogênese e na diferenciação celular e vêm sendo relacionados a cânceres em humanos. O projeto “Genoma Câncer de Cabeça e Pescoço" encontrou aproximadamente 20 desses genes possivelmente envolvidos com esse tipo de neoplasia e o PITX1 foi um dos genes encontrados. Sabe-se que o gene PITX1 está relacionado ao desenvolvimento das estruturas anteriores do embrião, porém sua participação em neoplasias não está bem estabelecida até o momento. Nesse estudo nos propusemos a verificar a presença de transcritos do gene PITX1 em carcinomas epidermóides de boca e tecidos não tumorais adjacentes, analisando sua possível relação com a morfologia das células neoplásicas. Para tanto, os transcritos do gene foram amplificados por RT-PCR e sua localização celular determinada por hibridização in situ com sondas de mRNA específicas. Os resultados mostraram que o transcrito sofreu amplificação em 86,2% dos casos, sendo que 28% foram somente nos tumores, 16% somente nos tecidos não tumorais e 56% em ambos os tecidos. A análise estatística através do Z-test (teste de diferença de proporção entre duas populações) mostrou que não houve diferença significativa entre a amplificação e o tipo de tecido analisado. Reações de hibridização in situ mostraram marcação predominante no componente epitelial de maneira heterogênea ora intensa ora branda em populações celulares diversas tanto nos tecidos tumorais quantos nos tecidos não tumorais adjacentes à lesão, sem relação aparente com a morfologia celular. De maneira geral, o sinal foi mais intenso no epitélio do tecido não tumoral quando comparado ao neoplásico. Frente aos resultados obtidos sugere-se que o gene PITX1 pode estar envolvido na carcinogênese de boca, sendo que a sua expressão está reduzida na maioria das células do carcinoma epidermóide de boca. / Homeobox genes have functions on morphogenesis and cell differentiation and have been related with cancer in humans. PITX1 was one of the genes found in the Head and Neck Cancer Genome Project. It is known that PITX1 gene is related with anterior structures of the developing embryo, although its relation with neoplasm is not we ll established. In this study we proposed to verify the presence of PITX1 gene transcripts in oral squamous cell carcinoma and adjacent non-tumoral tissues, analyzing its possible relation with the morphology of neoplastic cells. For such study, gene transcripts were amplified by RT -PCR and its cellular localization determined by in situ hybridization with specific riboprobes. Results showed that the transcript was amplified in 86,2% of the cases; 28% were only in the tumors, 16% non-tumoral tissues and 56% were amplified in both tissues. Statistics analysis by Z-test showed there was no significant difference between amplification and the type of tissue analyzed. In situ hybridization reactions showed transcripts mostly expressed in the epithelium component in a heterogenic manner sometimes intense and others discrete in several cells populations in both tumoral and non-tumoral adjacent tissues, with no apparent relationship to cell morphology. In general, signal was more intense in the non-tumoral epithelium when compared to the neoplasia. These results suggest that PITX1 gene can be involved with oral carcinogenesis and that its expression is reduced in most of the oral squamous cell carcinoma.

Neste trabalho é apresentado um método de refinamento para especificações de sistemas embarcados, baseado na linguagem de especificação gráfica UML-RT e na linguagem de especificação formal CSP-OZ. A linguagem UML-RT é utilizada para descrever a arquitetura de sistemas de tempo real distribuídos e esses mapeados para uma especificação formal através de CSP-OZ. A linguagem de especificação formal CSP-OZ é a combinação da linguagem orientada a objetos Object-Z e a algebra de processos CSP, que descreve o comportamento de processos concorrentes. O método de refinamento proposto é baseado na integração de dois métodos: o de bi-simulação, para refinar a parte comportamental da especificação descrita por CSP; e o de equivalência de especificações, para refinar as estruturas de dados descritas por Object-Z, permitindo assim que características de orientação a objetos possam ser utilizadas. Com o método proposto é possível refinar especificações e, conseqüentemente, verificá-las com sua implementação. O desenvolvimento desse método é rigoroso, incluindo a definição formal para um metamodelo da UML-RT. Um exemplo detalhado é apresentado no final deste trabalho. / In this work, a method of refinement of embedded systems specifications based on the graphical specification language UML-RT and the formal specification CSP-OZ is introduced. The UML-RT is used to model real time distributed architecture systems and these are mapped onto formal specifications using CSP-OZ. The CSP-OZ formal specification language is a combination of the state-based object oriented language Object-Z and the CSP process algebra that describes behavioral models of concurrent processes. The rationale of the proposed refinement method is twofold, the use of bisimulation to refine the behavioral part and the specification matching algorithm to refine the state-based part, supporting object-oriented characteristics. Using this result, an equivalence between the specification-matching algorithm and simulation rules is showed. Using the proposed method it is possible to refine CSP-OZ specifications and verify them against their implementations. The development of the proposed refinement method is rigorous, including a formal definition for a UML-RT metamodel. A detailed study case is given at the end of this work.

Os genes homeobox atuam na morfogênese e na diferenciação celular e vêm sendo relacionados a cânceres em humanos. O projeto “Genoma Câncer de Cabeça e Pescoço” encontrou aproximadamente 20 desses genes possivelmente envolvidos com esse tipo de neoplasia e o PITX1 foi um dos genes encontrados. Sabe-se que o gene PITX1 está relacionado ao desenvolvimento das estruturas anteriores do embrião, porém sua participação em neoplasias não está bem estabelecida até o momento. Nesse estudo nos propusemos a verificar a presença de transcritos do gene PITX1 em carcinomas epidermóides de boca e tecidos não tumorais adjacentes, analisando sua possível relação com a morfologia das células neoplásicas. Para tanto, os transcritos do gene foram amplificados por RT-PCR e sua localização celular determinada por hibridização in situ com sondas de mRNA específicas. Os resultados mostraram que o transcrito sofreu amplificação em 86,2% dos casos, sendo que 28% foram somente nos tumores, 16% somente nos tecidos não tumorais e 56% em ambos os tecidos. A análise estatística através do Z-test (teste de diferença de proporção entre duas populações) mostrou que não houve diferença significativa entre a amplificação e o tipo de tecido analisado. Reações de hibridização in situ mostraram marcação predominante no componente epitelial de maneira heterogênea ora intensa ora branda em populações celulares diversas tanto nos tecidos tumorais quantos nos tecidos não tumorais adjacentes à lesão, sem relação aparente com a morfologia celular. De maneira geral, o sinal foi mais intenso no epitélio do tecido não tumoral quando comparado ao neoplásico. Frente aos resultados obtidos sugere-se que o gene PITX1 pode estar envolvido na carcinogênese de boca, sendo que a sua expressão está reduzida na maioria das células do carcinoma epidermóide de boca. / Homeobox genes have functions on morphogenesis and cell differentiation and have been related with cancer in humans. PITX1 was one of the genes found in the Head and Neck Cancer Genome Project. It is known that PITX1 gene is related with anterior structures of the developing embryo, although its relation with neoplasm is not we ll established. In this study we proposed to verify the presence of PITX1 gene transcripts in oral squamous cell carcinoma and adjacent non-tumoral tissues, analyzing its possible relation with the morphology of neoplastic cells. For such study, gene transcripts were amplified by RT -PCR and its cellular localization determined by in situ hybridization with specific riboprobes. Results showed that the transcript was amplified in 86,2% of the cases; 28% were only in the tumors, 16% non-tumoral tissues and 56% were amplified in both tissues. Statistics analysis by Z-test showed there was no significant difference between amplification and the type of tissue analyzed. In situ hybridization reactions showed transcripts mostly expressed in the epithelium component in a heterogenic manner sometimes intense and others discrete in several cells populations in both tumoral and non-tumoral adjacent tissues, with no apparent relationship to cell morphology. In general, signal was more intense in the non-tumoral epithelium when compared to the neoplasia. These results suggest that PITX1 gene can be involved with oral carcinogenesis and that its expression is reduced in most of the oral squamous cell carcinoma.

Os genes homeobox são uma família de genes reguladores que são vitais para vários aspectos do crescimento e diferenciação celular. Recentemente, implicações dos genes HOXA7, HOXC6 e TGIF na gênese e progressão tumoral vêm sendo verificadas. Entretanto, o envolvimento desses genes em carcinomas epidermóides (CE) de boca ainda não foi demonstrado. A possível presença de transcritos dos genes HOXA7, HOXC6 e TGIF em carcinomas epidermóides de boca e em tecidos não tumorais adjacentes (TN) foi analisada. Os transcritos dos genes foram amplificados por RT-PCR e sua localização celular determinada por hibridização in situ (ISH) com sondas de mRNA específicas. A amplificação do HOXA7 foi observada em 70% dos casos sendo 15% apenas nas amostras TN, 45% somente nos CEs e 10% em ambos tecidos. Nenhuma amplificação do HOXC6 foi observada. O TGIF foi amplificado em 80% dos casos, sendo 5% somente nas amostras TN, 20% nos CEs e 55% em ambos tecidos. Análises estatísticas mostraram que não havia diferença significante entre a amplificação do transcrito HOXA7 ou TGIF e o tipo de tecido analisado. Além disso, nenhuma associação entre a amplificação dos transcritos nas amostras CE e os aspectos clínicos foi observada. O sinal de hibridização in situ foi similar para os transcritos HOXA7 e TGIF. Nas amostras TN o sinal da ISH foi intenso no epitélio, ora disperso sendo mais proeminente na camada espinhosa ora mais proeminente nas camadas basais e suprabasais. Nos CEs os transcritos foram localizados por toda neoplasia sendo que o sinal era menor em áreas menos diferenciadas. Esses resultados mostram que o HOXC6 não está envolvido com a carcinogênese oral enquanto que a presença dos transcritos HOXA7 e TGIF principalmente em regiões bem diferenciadas dos carcinomas epidermóides de boca sugere uma participação desses genes nesta neoplasia / Homeobox genes comprise a family of developmental regulators that are vital for several aspects of growth and differentiation. Recently HOXA7, HOXC6 and TGIF genes have been implicated with carcinogenesis and tumoral progression. However their involvement with oral squamous cell carcinomas (OSCC) has not been demonstrated yet. The possible presence of HOXA7, HOXC6 and TGIF transcripts in OSCC and adjacent non-tumoral tissues (NT) was verified. Transcripts were amplified by RT -PCR and its cellular localization was determined by in situ hybridization with specific riboprobes. Amplification of HOXA7 was seen in 70% of cases, 15% only in NT tissues, 45% only in OSCC samples and 10% in both tissues. No amplification of HOXC6 was observed. TGIF was amplified in 80% of cases, 5% only in NT tissues, 20% only in OSCC samples and 55% in both tissues. Statistical analysis showed that there was no significant difference between amplification of HOXA7 or TGIF and the type of tissue analyzed. Moreover, no association between amplification of transcripts in OSSC and clinical aspects was observed. ISH signal was similar for HOXA7 and TGIF transcripts. In NT tissues there was an intense expression in the epithelium, either in basal and suprabasal layers or disperse and more intense in the spinous layer. In OSCC transcripts were locali zed in all tumoral cells, but in poorly differentiated areas the signal was less intense. These results show that HOXC6 was not involved in oral carcinogenesis while the presence of HOXA7 and TGIF transcripts in OSSC, mostly in well differentiated regions, suggests a participation of these genes in OSCC

Genes da família homeobox têm sido alvo de intensas pesquisas científicas relacionadas ao câncer. Recentemente, mostramos que o gene homeobox TGIF1 está expresso no carcinoma epidermóide de boca na sua forma genérica. Porém, não foi feita uma discriminação entre quais variantes transcricionais, ou subtipos, do TGIF1 estariam expressas. Neste estudo, propusemo-nos a verificar diferenças na freqüência de expressão das variantes transcricionais do TGIF1 em carcinomas epidermóides de boca (CEB) em relação a tecidos morfologicamente não tumorais (TN), avaliar possíveis associações entre essas variantes nos pacientes portadores de CEB e relacionar o grau de expressão dessas variantes com aspectos clínicos, histológicos e com a sobrevida dos pacientes. Adicionalmente, procuramos analisar a expressão da proteína do TGIF1. Foram analisadas 48 amostras congeladas de CEB e 12 de TN. O RNA total de cada amostra foi extraído utilizando-se solução de TRizol®. Os transcritos do TGIF1 foram amplificados por RT-PCR para cada caso de CEB e TN utilizando-se inicialmente um par de iniciadores genéricos para todas as suas variantes. Após essa triagem, os casos positivos foram amplificados utilizando-se pares de iniciadores específicos para cada variante. Não houve diferença estatística entre a freqüência de expressão das variantes do TGIF1 nos grupos CEB e TN, porém as variantes 4 e 8 foram as que apresentaram p-valores menores. Dentro do grupo de CEB, houve associação significativa entre algumas variantes entre si (sete dos vinte e um cruzamentos), de forma que as variantes 1 e 4 foram as que mais tiverem associação com outras variantes. Dessa forma, optamos por prosseguir o estudo utilizando somente as variantes 1, 4 e 8. Não houve correlação entre a expressão das variantes selecionadas e variáveis clinicas e histológicas propostas. Em relação à sobrevida, a expressão das variantes 1 e 8 mostraram correlação univariada com o desfecho óbito, de maneira que o grupo de indivíduos que mais expressou ambas as variantes foi o que apresentou menor risco de óbito. Através da análise multivariada das variantes 1 e 8 e aspectos clínicos e histológicos, somente o tamanho da lesão e a invasão vascular sanguínea tiveram significado estatístico. A expressão protéica do TGIF1 foi analisada em 46 amostras parafinadas considerando-se a diferenciação celular, a graduação imunoistoquímica e o compartimento celular. Os resultados obtidos mostraram que as variantes do TGIF1 estão expressas diferentemente no grupo dos pacientes com CEB e sugerese que a expressão das variantes 1 e 8 estejam relacionadas, de maneira semelhante, a um menor risco de óbito para pacientes portadores de CEB. Adicionalmente, sugere-se que o tamanho da lesão e a invasão vascular sanguinea representem fatores de risco relevantes para o óbito de pacientes portadores de CEB. Sugere-se também que a expressão simultânea da proteína do TGIF1 tanto no núcleo quanto no citoplasma da célula esteja correlacionada a lesões pobremente diferenciadas. / Genes of the homeobox family have been the subject of intense scientific research related to cancer. Recently, we show that this gene is expressed in oral squamous cell carcinoma in its generic form. However, it was not made a discrimination between which transcript variants, or subtypes\", of TGIF1 were expressed. In this study, we aim to verify differences in the expression of the eight transcript variants described for the homeobox gene TGIF1 in oral squamous cell carcinomas (OSCC) related with morphologically non-tumoral tissues (NT), evaluate possible associations between these variants in patients with OSCC and relate the degree of expression of these variants with clinical, histological and survival aspects of patients. Additionally, we analyzed the expression of TGIF1 protein. It was analyzed 48 frozen samples of OSCC and 12 of NT. The total RNA from each sample was extracted using TRizol solution. TGIF1 transcripts were first amplified by RT-PCR for each case of OSCC and NT using a generic pair of primers. After these screening, the positive cases were amplified using specific pair of primers for each variant. There was no statistical difference between the frequency of expression of TGIF1 transcript variants in OSSC and NT groups, but the variants 4 and 8 had the lowest p-values. Within the OSCC group, there was a significant association between some variants among themselves (seven of the twenty-one associations), in a way that the variants 1 and 4 were the ones that had most association with other variants. Thus, we chose to continue the study using only variants 1, 4 and 8. There was no correlation between the expression of the selected variants with the clinical and histological aspects. Regarding survival, the expression of variants 1 and 8 showed statistical correlation with the outcome death, in a way that the group of patients that most expressed both variants was that one with the lower risk of death. By multivariate analysis of variants 1 and 8 and clinical and histological aspects, only the size of the lesion and vascular invasion blood were statistically significant. The protein expression of TGIF1 was analyzed in 46 paraffin-embedded tissues considering the cell differentiation, the immunohistochemistry graduation and the cell compartment. The results showed that the variants of TGIF1 are differently expressed in the OSCC group of patients and it is suggested that the expression of variants 1 and 8 may be related, in a similar way, to a lower risk of death for patients with OSCC. Additionally, it is suggested that the size of the lesion and the vascular invasion of blood represent relevant risk factors for death in patients with OSCC. It is also suggested that the simultaneous expression of TGIF1 protein not only in the nucleus but also in the cytoplasm of the cell is correlated with the poorly differentiated lesions of OSCC.

Le bassin d’Arcachon est une lagune semi-fermée qui concentre de forts enjeux économiques grâce à la pratique de l’ostréiculture. Cette activité est affectée depuis une trentaine d’années par des phénomènes de mortalités estivales, et plus récemment par des surmortalités du naissain. Ces travaux se sont intéressés au rôle des polluants majeurs du bassin d’Arcachon dans ce contexte de crise en étudiant leurs effets sur la biologie de Crassostrea gigas. Une approche in situ a d’abord été adoptée afin d’identifier les contaminants les plus présents dans le milieu naturel. Des opérations de transplantation d’huîtres et des prélèvements de sédiments ont permis de quantifier divers contaminants et d’associer leur présence à des réponses biologiques. Les polluants ainsi identifiés ont ensuite été employés en conditions contrôlées au laboratoire. Trois expérimentations ont été réalisées : la première concerne l’étude des voies de contamination par le tributylétain ; la seconde concerne les effets des pesticides et du cuivre ; la troisième concerne l’effet des HAP sur des huîtres diploïdes et triploïdes. Nos résultats indiquent que les organismes transplantés au coeur de la lagune sont plus exposés aux polluants, en lien avec les caractéristiques hydrodynamiques du système. La plupart des paramètres biologiques étudiés sur le terrain ont par ailleurs montré une saisonnalité liée aux processus de gamétogenèse. Au laboratoire, la plupart des contaminants testés ont induit une réponse adaptative chez les huîtres exposées. Notre étude souligne l’importance de coupler approche de terrain et approche expérimentale pour comprendre le fonctionnement des écosystèmes côtiers. / The Arcachon Bay is a semi-enclosed lagoon and represents the core of strong economic stakes through the practice of oyster-farming. This activity has been affected for around thirty years by summer mortality events, and more recently by abnormally high death rates of juveniles. This work focused on the role of the Arcachon Bay’s main contaminants in this crisis, by studying their effects on the cupped oyster’s biology. Firstly, an in situ approach was adopted in order to identify the major pollutants of the bay: caged oysters were transplanted and sediments were sampled. The presence of contaminants in the samples was associated to biological responses. Secondly, the contaminants identified in situ were used in controlled conditions at the laboratory. Three experimentations were performed; the first dealt with the contamination pathways of tributyltin; the second focused on the biological effects of pesticides and copper; the third concerned the effects of PAH towards diploid and triploid oysters. Our results indicate that the inner stations present higher accumulation of metals and PAH, in accordance with the hydrodynamic features of the bay. The bioindicators used in situ exhibited seasonal trends related to the oysters’ gametogenesis. In the laboratory, most of the contaminants used at environmental levels induced an adaptive response of the exposed oysters. Our study highlights the importance of coupling in situ and laboratory approaches in order to understand the functioning of coastal ecosystems.

Im ersten Teil der Arbeit wurden Strategien zur Analyse von Transkripten erarbeitet. Die ersten Versuche zielten darauf ab, in mit Glaskapillaren genommenen Einzelzellproben verschiedener Gewebeschichten RT-PCR durchzuführen, um spezifische Transkripte nachweisen zu können. Dies gelang für eine Reihe von Genen aus verschiedenen Pflanzenspezies. Dabei konnten sowohl Transkripte stark wie auch schwach exprimierter Gene nachgewiesen werden. <br /> Für die Erstellung von Gewebe-spezifischen Expressionsprofilen war es notwendig, die in vereinigten Zellproben enthaltene mRNA zunächst zu amplifizieren, um eine ausreichende Menge für Arrayhybridisierungen zu erhalten. Vor der Vermehrung wurde die mRNA revers transkribiert. Es wurden daran anschließend verschiedene Amplifikationsstrategien getestet: Die neben Tailing, Adapterligation und anderen PCR-basierenden Protokollen getestete Arbitrary-PCR hat sich in dieser Arbeit als einfache und einzige Methode herausgestellt, die mit so geringen cDNA-Mengen reproduzierbar arbeitet. Durch Gewebe-spezifische Array-hybridisierungen mit der so amplifizierten RNA konnten schon bekannte Expressionsmuster verschiedener Gene, vornehmlich solcher, die an der Photosynthese beteiligt sind, beobachtet werden. Es wurden aber auch eine ganze Reihe neuer offensichtlich Gewebe-spezifisch exprimierter Gene gefunden. Exemplarisch für die differentiell exprimierten Gene konnte das durch Arrayhybridisierungen gefundene Expressionsmuster der kleinen Untereinheit von Rubisco verifiziert werden. Hierzu wurden Methoden zum Gewebe-spezifischen Northernblot sowie semiquantitativer und Echtzeit-Einzelzell-RT-PCR entwickelt.<br /> Im zweiten Teil der Arbeit wurden Methoden zur Analyse von Metaboliten einschließlich anorganischer Ionen verwendet. Es stellte sich heraus, daß die multiparallele Methode der Gaschromatographie-Massenspektrometrie keine geeignete Methode für die Analyse selbst vieler vereinigter Zellinhalte ist. Daher wurde auf Kapillarelektrophorese zurückgegriffen. Eine Methode, die mit sehr kleinen Probenvolumina auskommt, eine hohe Trennung erzielt und zudem extrem geringe Detektionslimits besitzt. Die Analyse von Kohlenhydraten und Anionen erfordert eine weitere Optimierung. Über UV-Detektion konnte die K+-Konzentration in verschiedenen Geweben von A. thaliana bestimmt werden. Sie lag in Epidermis und Mesophyll mit ca. 25 mM unterhalb der für andere Pflanzenspezies (Solanum tuberosum und Hordeum vulgare) publizierten Konzentration. Weiter konnte gezeigt werden, daß zwölf freie Aminosäuren mittels einer auf Kapillarelektrophorese basierenden Methode in vereinigten Zellproben von Cucurbita maxima identifiziert werden konnten. Die Übertragung der Methode auf A. thaliana-Proben muß jedoch weiter optimiert werden, da die Sensitivität selbst bei Laser induzierter Fluoreszenz-Detektion nicht ausreichte.<br /> Im dritten und letzten Teil der Arbeit wurde eine Methode entwickelt, die die Analyse bekannter wie unbekannter Proteine in Gewebe-spezifischen Proben ermöglicht. Hierzu wurde zur Probennahme mittels mechanischer Mikrodissektion eine alternative Methode zur Laser Capture Microdissection verwendet, um aus eingebetteten Gewebeschnitten distinkte Bereiche herauszuschneiden und somit homogenes Gewebe anzureichern. Aus diesem konnten die Proteine extrahiert und über Polyacrylamidgelelektrophorese separariert werden. Banden konnten ausgeschnitten, tryptisch verdaut und massenspektrometrisch die Primärsequenz der Peptidfragmente bestimmt werden. So konnten als Hauptproteine im Mesophyll die große Untereinheit von Rubisco sowie ein Chlorophyll bindendes Protein gefunden werden.<br /> Die in dieser Arbeit entwickelten und auf die Modellpflanze Arabidopsis thaliana angewandten Einzelzellanalysetechniken erlauben es in Zukunft, physiologische Prozesse besser sowohl räumlich als auch zeitlich aufzulösen. Dies wird zu einem detaillierteren Verständnis mannigfaltiger Vorgänge wie Zell-Zell-Kommunikation, Signalweiterleitung oder Pflanzen-Pathogen-Interaktionen führen. / The subject of this thesis was the analysis of single plant cells in respect to their contents of i) transcripts, ii) inorganic cations and anions, iii) metabolites like amino acids and carbohydrates as well as iv) proteins. One task was the transfer of existing methods to single cell analysis on leaf tissues of the model plant Arabisopsis thaliana L., the second one was the refinement and the development, respectively, of new protocols for the analysis of such picoliter samples. For cell type specific sampling two different complimentary methods were applied: Using micro glass capillaries specific single cell contents could be harvested from intact plants, whereas typical sample volumes were in the picoliter range. Even the sampling of inner cell types such as companion cells could be demonstrated. Using mechanical micro dissection of embedded tissue a larger amount of homogenous tissue could be collected.<br /> Because single cell samples contain only femtogram amounts of mRNA, direct detection of transcripts is impossible. Therefore, two amplification protocols were applied to the cell samples: The first procedure makes use of specifically primed RT-PCR for amplification. Several genes derived from different plants and tissues could be detected after successful RT-PCR, including high as well as low expressed genes. The second method was developed to monitor the activity of many genes in parallel using array hybridisation with filters containing the cDNA of as many as 16.000 ESTs. For this purpose, unspecific RT-PCR as it is applied in the differential display was used to amplify different transcripts in just one reaction. However, in these tissue specific array hybridisations the expression patterns of several hundreds genes could be monitored. These included known tissue specific expression patterns (of mainly photosynthesis related genes) as well as a couple of unknown expression patterns. To verify the tissue specificity of gene activity some results were reconsidered using tissue specific northern blot hybridisations and real time RT-PCR, respectively. <br /> Secondly, metabolites (including inorganic ions) were investigated: Because gas chromatography-mass spectrometry does not reveal the sensitivity which in necessary for the analysis of even multiple pooled single cell samples capillary electrophoresis was applied for these studies. This method has a high potential as it needs only small amounts of starting material, has uncomparable low detection limits and exhibits a high number of theoretical plates.<br /> The analysis of inorganic anions and carbohydrates needs further optimisations. Using UV absorption-detection potassium could be detected in different cell types whereas the concentrations in mesophyll and epidermis were found around 25 mM each. These concentrations are lower than in other species as Solanum tuberosum or Hordeum vulgare. For investigations of amino acids the cell samples were derivatized to make the use of laser induced fluorescence-detection capable. In samples derived from pumpkin (Cucurbita maxima) mesophyll twelve amino acids could be detected and identified. The transfer of this method to A. thaliana derived samples exhibited no results which may be due to the low concentration of free amino acids in these plants.<br /> Finally, a method was developed with which the existence of known and unknown proteins in tissue specific samples could be monitored. For this, mechanical micro dissection was used to: After embedding and sectioning the tissue of interest was cut out by an vibrating steel chisel to get homogenous samples. The proteins contained in these tissue pieces were extracted and separated by one dimensional SDS polyacrylamid gel electrophoresis. Several protein bands could be detected after staining with either silver or coomassie blue stain. These bands were cut out and sequenced by mass spectrometry. The large subunit of rubisco as well as one chlorophyll binding protein could be identified as the major proteins within the mesophyll.<br /> The single cell analysis methods which were developed and applied to the model plant A. thaliana in this thesis allow a better spatial as well as temporal resolution of analysis. This will lead to a more detailed understanding of physiological processes like cell to cell communication, signalling or plant-pathogen interactions.

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