Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve the prognosis, novel molecular markers and targets for earlier diagnosis and adjuvant and/or neoadjuvant treatment are needed. Recent advances in human genome research and high-throughput molecular technologies make it possible to cope with the molecular complexity of malignant tumors. With DNA array technology, mRNA expression levels of thousand of genes can be measured simultaneously in a single assay. As several studies using microarrays in PDAC have already been published, this review attempts to compare the published data and therefore to validate the results. In addition, the applied techniques are discussed in the context of pancreatic malignancies. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Cells are the basic units of life. Studying complex tissues and whole organs requires an understanding of cell heterogeneity and responses to stimuli at the single-cell level. Even the cells, which belong to the same cell type, behave differently at a specific moment and contain different amount of mRNA. Quantitative polymerase chain reaction (qPCR) is one the most sensitive methods for the detection of mRNA, however, gene expression profiling in single cells leads to a large amount of missing data due to the fact that the transcript is missing, or is below the level of detection. Therefore, it is necessary to establish a new statistical approach for analysis of single cells. In this thesis the potential of single-cell gene expression profiling using the high throughput instrument Biomark, focusing on data analysis and biological interpretation, is discussed. Data normalization and handling of missing data are two important steps in data analysis that are performed differently at the single-cell level. Single cells are not normalized by reference genes but the number of cells as a normalizer is applied. Missing data are replaced by value, which is equaled one quarter of transcript amount in the cell. Furthermore it is shown how single-cell gene expression data can be viewed and how subpopulations...

O linfoma de células do manto é associado à translocação t(11;14)(q13;q32) e à hiperexpressão da ciclina D1. Os pacientes com linfoma de células do manto apresentam-se com doença avançada ao diagnóstico e a fase leucêmica da doença é observada em cerca de um terço dos casos. Os linfócitos B virgens pré-centro germinativo, que ocupam a zona do manto dos folículos linfóides secundários, constituem a origem celular do linfoma de células do manto. A hiperexpressão da ciclina D1, por si só, não é suficiente para a patogênese da neoplasia, e a elucidação das alterações moleculares adicionais poderá fundamentar novas estratégias terapêuticas. Nesse contexto, os métodos de estudo do perfil de expressão gênica em larga escala têm potencial para auxiliar na descoberta dessas alterações moleculares adicionais. Todavia, nos estudos que empregaram esses métodos até o momento, o material genético foi obtido de amostras de gânglios acometidos pelo tumor, que contêm uma proporção variável de células normais do estroma do tecido linfóide. Por isso, ainda não se sabe quais dos genes identificados como alterados no linfoma de células do manto são específicos das células linfomatosas, e quais são dependentes das células normais que perfazem o estroma do gânglio. Com o objetivo de elucidar as alterações moleculares do linfoma de células do manto em nível celular, realizamos um estudo comparativo entre o perfil de expressão gênica de células linfomatosas purificadas e de linfócitos B virgens, utilizando microarrays de oligonucleotídeos. Células linfomatosas e linfócitos B virgens (IgD+CD38±CD27-) foram purificados em colunas magnéticas a partir do sangue periférico de pacientes com linfoma de células do manto em fase leucêmica e de amígdalas de indivíduos normais, respectivamente (pureza > 95%). Três indivíduos foram selecionados em cada grupo e os experimentos foram realizados em duplicatas usando microarrays comerciais modelo CodeLink Human UniSet I, com 10.000 genes. Foram identificados 106 genes com variação de expressão maior do que três vezes, 63 deles induzidos e 43 reprimidos no linfoma de células do manto em relação aos linfócitos B virgens. Dez genes (seis induzidos e quatro reprimidos) foram selecionados para quantificação, por RT-PCR em tempo real, em amostras de sangue periférico de 21 pacientes com linfoma de células do manto em fase leucêmica, além de 14 pacientes com outras doenças linfoproliferativas crônicas e de sete indivíduos sem doenças neoplásicas. Os resultados dos experimentos com microarrays foram confirmados pela quantificação por RT-PCR em tempo real em todos os 10 genes selecionados e os valores de expressão do gene TLR1 obtidos por esse método demonstraram correlação significativa com a sobrevida dos pacientes com linfoma de células do manto. Além de identificar vários genes modulados no linfoma de células do manto em nível celular, este estudo também revelou a expressão aberrante de diversos genes relacionados à apoptose e às vias de sinalização PI3K/AKT, WNT e TGFβ. Esses resultados adicionam informações originais à patogênese molecular do linfoma de células do manto e apontam para vias específicas de sinalização molecular como potenciais alvos para novas abordagens terapêuticas. / Mantle cell lymphoma is associated with the translocation t(11;14)(q13;32) and overexpression of cyclin D1. Mantle cell lymphoma is predominantly disseminated at diagnosis and a frank leukemic phase is detected in one third of patients. The pre-germinal-center naive B-cells, which populate the mantle zone of the secondary lymphoid follicles, are the cells of origin of mantle cell lymphoma. Overexpression of cyclin D1 is not sufficient by itself to cause lymphoma, and a better understanding of the additional molecular lesions may provide insights toward new therapeutic approaches. In this context, large scale gene expression studies may be useful in the investigation of such additional molecular lesions. However, the great majority of mantle cell lymphoma cases studied by these methods to date had the genetic material harvested from lymph nodes, which have a variable proportion of normal cells from the lymphoid stroma. It is therefore not known how many genes identified as differentially expressed in mantle cell lymphoma by tumor versus normal experiments are cell-autonomous rather than dependent on the tumor microenvironment. To address this issue, we compared the gene expression profile of mantle cell lymphoma cells and normal naive B-cells using oligonucleotide microarrays. Lymphoma cells and naive B-cells (IgD+CD38±CD27-) were highly purified, by magnetic activated cell sorting, from the peripheral blood of patients with mantle cell lymphoma in the leukemic phase and from tonsils of normal individuals, respectively (purity > 95% in all samples). Three individuals were selected for each group and experiments were performed in replicates using the Amersham CodeLink Human UniSet I Bioarrays, with 10,000 genes. We identified 106 genes differentially expressed with a fold change of at least three-fold, 63 induced and 43 repressed in mantle cell lymphoma in comparison with naive B-cells. Ten genes were selected (six induced and four repressed in lymphoma cells) for quantification by real-time RT-PCR in non-purified peripheral blood samples from 21 patients with mantle cell lymphoma in the leukemic phase, as well as in 14 patients with other chronic lymphoproliferative diseases and seven normal individuals. Microarray results were confirmed by real-time RT-PCR for all selected genes and expression values of the TLR1 gene as quantified by this method showed significant correlation with patient survival in mantle cell lymphoma. In addition to the identification of several modulated genes in mantle cell lymphoma at cellular level, this study revealed that some genes functionally connected through apoptosis or the PI3K/AKT, WNT and TGFβ signaling pathways are aberrantly expressed in mantle cell lymphoma. These results add original data to the molecular pathogenesis of mantle cell lymphoma and point to specific molecular signaling pathways related to inhibition of apoptosis as potential targets for new therapeutic approaches.

Nosso estudo se caracterizou em descrever o perfil de expressão de nove genes de actina presentes no genoma do Aedes aegypti ao longo do ciclo de vida do mosquito. Experimentos de qRT-PCR confirmaram a expressão estágio-espécifico de dois genes já caracterizados anteriormente como genes com picos de indução em pupas macho (AeAct-3) e pupas fêmeas (AeAct-4). Outros 2 genes já caracterizados também tiveram seus perfis de expressão confirmados. AeAct-1 se mostrou em baixa expressão e AeAct-2 apresentou picos de indução em larvas de quarto instar. Os demais genes de actina se mostraram expressos em todos os intervalos analisados. Os picos de indução para todos os genes se encontravam ou em pupas ou em larvas de quarto instar. Essa característica se deve ao fato da actina ser uma proteína estrutural, justificando a alta transcrição em larvas tardias e principalmente pupas, fase onde ocorre a metamorfose completa do inseto. / Our study was characterized to describe the expression profile of nine actin genes present in the genome of Aedes aegypti throughout the mosquito\'s life cycle. QRT-PCR experiments confirmed the stage-specific expression of two genes as already previously characterized genes with induction peaks of male pupae (AeAct-3) and female pupae (AeAct-4). Two other genes already characterized, also had their expression profiles confirmed. The AeAct-1 showed in low expression and AeAct-2 showed induction peaks in larvae of fourth instar. The remaining actin genes showed expressed in all the analyzed intervals. The induction peaks for all genes were found in pupae or larvae of fourth instar. This characteristic is due to the fact that actin is a structural protein, justifying the high transcription in late larvae and mainly in pupae, stage where the complete metamorphosis of the insect occurs.

xiii, 145 p. : ill. (some col.) / The goal of functional genomics is to elucidate the relationship between an organism's genotype and phenotype. A key characteristic of functional genomics is the use of genome-wide approaches as opposed to more traditional single-gene approaches. Genome-wide expression profiling is used to investigate the dynamic properties of transcriptomes, provides insights into how biological functions are encoded in genomes, and is an important technique in functional genomics. This dissertation describes the use of genome-wide expression profiling and other functional genomics techniques to address a variety of biological questions related to development and disease of the nervous system. Our results reveal novel and important insights into nervous system development and disease and demonstrate the power of functional genomics approaches for the study of nervous system biology. This dissertation also describes a novel technique called TUtagging that facilitates cell type-specific RNA isolation from intact complex tissues. The isolation of RNA from specific cell types within a complex tissue is a major limiting factor in the application of genome-wide expression profiling, and TU-tagging can be used to address a wide array of interesting and important biological questions. This dissertation includes previously published and unpublished co-authored material. / Committee in charge: Dr. John Postlethwait， Chair； Dr. Chris Doe， Advisor； Dr. Bruce Bowerman， Member； Dr. Patrick Phillips， Member； Dr. Tom Stevens， Outside Member

<p>Chicken and Japanese quail are commonly used models in developmental and sex specific neuroendocrine research. There is relatively little known about the mechanisms behind their sex specific brain development, especially regarding the impact of the sex chromosomes (male: ZZ, female ZW) in relation to gonadal hormones. This thesis explores several aspects of these processes. Gene expression analysis with cDNA and Affymetrix arrays on brain tissue from both pre-gonadal embryos and embryos with differentiated gonads indicate a strong sex chromosomal presence in sexual dimorphic somatic tissue development in both chicken and Japanese quail. This sex chromosome pattern seems to remain in adult brain tissue. The data demonstrates that chicken males exhibit a significant level of Z-gene dosage compared to females in both somatic and germ line derived embryonic tissues. Several avian sex determination gene candidates (MHM non-coding RNA, DMRT1, HINTW, and HINTZ) were analyzed by real-time PCR. DMRT1 is dosage compensated in male brain tissue, in contrast to its reported gene dosage in male gonads. Early embryonic ethinylestradiol (EE2) exposure did not affect male or female neural gene expression patterns during later development. A peptidomics analysis on quail embryonic day 12 (ed12) and ed17 diencephalon by LC-MS identified over 60 endogenous peptides and analyzed the expression patterns for 38 of them with regard to age, sex and early EE2 exposure. There was a general upregulation between ed12 and ed17, but no clear sex effects were detected. Multivariate analysis indicates that EE2 exposed individuals differ from control individuals in a gender independent manner, and that Gonadotropin-inhibiting hormone related peptide 2 (GnIH-RP2) is a candidate for EE2 induced peptidomic alterations in male embryonic brain.</p>

Each organ of a multicellular organism is unique at the level of its tissues and cells. Furthermore, responses to environmental stimuli or developmental signals occur differentially at the single cell or tissue level. This underlines the necessity of precise investigation of the “building block of life” -the individual cell. Although recently large amount of data concerning different aspects of single cell performance was accumulated, our knowledge about development and differentiation of individual cell within specialized tissue are still far from being complete. To get more insight into processes that occur in certain individual cell during its development and differentiation changes in gene expression during life cycle of A. thaliana leaf hair cell (trichome) were explored in this work. After onset of trichome development this cell changes its cell cycle: it starts endoreduplication (a modified cell cycle in which DNA replication continues in the absence of mitosis and cytokinesis). This makes trichomes a suitable model for studying cell cycle regulation, regulation of cell development and differentiation. Cells of interest were sampled by puncturing them with glass microcapillaries. Each sample contained as few as ten single cells. At first time trichomes in initial stage of trichome development were investigated. To allow their sampling they were specifically labelled by green fluorescent protein (GFP). In total three cell types were explored: pavement cells, trichome initials and mature trichomes. Comparison of gene expression profiles of these cells allowed identification of the genes differentially expressed in subsequent stages of trichome development. Bioinformatic analysis of genes preferentially expressed in trichome initials showed their involvement in hormonal, metal, sulphur response and cell-cycle regulation. Expression pattern of three selected candidate genes, involved in hormonal response and early developmental processes was confirmed by independent method. Effects of mutations in these genes on both trichome and plant development as well as on plant metabolism were analysed. As an outcome of this work novel components in the sophisticated machinery of trichome development and cell cycle progression were identified. These factors could integrate hormone stimuli and network interactions between characterized and as yet unknown members of this machinery. I expect findings presented in this work to enhance and complement our current knowledge about cell cycle progression and trichome development, as well as about performance of the individual cell in general. / Jedes Organ eines vielzelligen Organismus weißt einzigartige Merkmale auf seiner Gewebe und Zellebene auf. Darüber hinaus, werden entwicklungsabhängige sowie aus der Umwelt empfangene Signale zelltypspezifisch interpretiert. Aus dieser Spezialisierung einzelner Zellen ergibt sich somit unmittelbar die Notwendigkeit einzelne Zellen, als Bausteine komplexer Organe, individuell zu untersuchen. Obwohl in den letzten Jahrzehnten große Datenmengen über verschiedene Aspekte einzelner Zellen akkumuliert wurden, ist das Gesamtbild der Differenzierung und Entwicklung individueller Zellen in einem vielzelligen Organismus weitgehend unbekannt. Um der Frage nachzugehen, welche Prozesse sich in einer einzelnen Zelle während ihrer Differenzierung und Entwicklung abspielen, wurden Genexpressionsprofile einzelner Blatthaarzellen der Pflanze Arabidopsis thaliana in verschiedene Entwicklungsstadien erstellt. Nach dem Beginn der Entwicklung einer Protodermalzelle in ein Blatthaar (Trichom) kommt es zu einem Umschalten des Zellzyklus; Endoreduplikation setzt ein. Dies bedeutet, dass DNA repliziert wird, aber keine Zellteilung mehr stattfindet. Aus diesem Grunde eignen sich heranwachsende Trichome besonders gut Mechanismen zu erforschen, die in Verbindung mit der Zellzyklusregulation und Zellentwicklung stehen. Die Inhalte ausgewählter Einzelzellen wurden mit Glasmikrokapillaren extrahiert. Jeweils zehn derartige Einzelzellextrakte wurden daraufhin vereint. Als besonders hervorzuheben gilt, dass es uns in dieser Studie zum ersten mal überhaupt gelang die Inhalte einzelner Trichomzellen in ganz frühen Entwicklungsstadien zu extrahieren und anschließend zu analysieren. Um die Extraktion der Inhalte dieser frühen Zellstadien überhaupt zu ermöglichen, war es erforderlich diese mit dem grün fluoreszierenden Protein (GFP) zu markieren. Neben den Trichominitialzellen wurden ausgewachsene Trichomzellen sowie Epidermiszellen (Pavementzellen) mittels der Einzelzelltechnik untersucht. Ein Vergleich der erstellten Genexpressionsprofile dieser drei Zelltypen ermöglichte es Gene zu identifizieren, die in den ausgewählten Entwicklungsstadien der Trichombildung differentiell induziert wurden. Mittels bioinformatischer Analysemethoden gelang es, Gruppen von Genen zu identifieren, die exklusiv in Trichominitialzellen exprimiert sind und den Kategorien, Hormonregulation, Metallhomeostase, Schwefelstoffwechesol sowie Zellzyklusregulation zuzuordnen sind. Weiterhin wurde das Expressionsmuster dreier ausgewählter Kandidatengene mit alternativen Techniken verifiziert. Die ausgewählten Kandidatengene gehörten den Katergorien, Hormonrespons sowie frühe Entwicklungsprozesse, an. Darüber hinaus wurden Mutanten in allen drei Gene erzeugt und der Einfluss dieser Mutationen auf die Trichomentwicklung analysiert. Ein weiterer Aspekt der Mutantenanalyse lag in der Erstellung von Metabolitenprofilen ausgewählter Mutanten. Als ein wesentliches Ziel dieser Arbeit gelang es mir bisher unbekannte Komponenten in der Trichomentwicklung und damit der Zellzyklusregulation zu identifizieren. Diese neu identifizierten Komponenten führen zu einer Integration der hormonellen Kontrolle der Zellteilung und Entwicklung mit bisher unbekannten Faktoren. Ich erwarte, dass die von mir erbrachten Ergebnisse zu einem tieferen Verständnis der Prozesse, die an der Trichomentwicklung sowie an der Zellzyklusregulation beteiligt sind, beitragen. Insbesondere, zu einem erweiterten Verständnis des Verhaltens individueller Zellen in einem vielzelligen Organismus.

Chicken and Japanese quail are commonly used models in developmental and sex specific neuroendocrine research. There is relatively little known about the mechanisms behind their sex specific brain development, especially regarding the impact of the sex chromosomes (male: ZZ, female ZW) in relation to gonadal hormones. This thesis explores several aspects of these processes. Gene expression analysis with cDNA and Affymetrix arrays on brain tissue from both pre-gonadal embryos and embryos with differentiated gonads indicate a strong sex chromosomal presence in sexual dimorphic somatic tissue development in both chicken and Japanese quail. This sex chromosome pattern seems to remain in adult brain tissue. The data demonstrates that chicken males exhibit a significant level of Z-gene dosage compared to females in both somatic and germ line derived embryonic tissues. Several avian sex determination gene candidates (MHM non-coding RNA, DMRT1, HINTW, and HINTZ) were analyzed by real-time PCR. DMRT1 is dosage compensated in male brain tissue, in contrast to its reported gene dosage in male gonads. Early embryonic ethinylestradiol (EE2) exposure did not affect male or female neural gene expression patterns during later development. A peptidomics analysis on quail embryonic day 12 (ed12) and ed17 diencephalon by LC-MS identified over 60 endogenous peptides and analyzed the expression patterns for 38 of them with regard to age, sex and early EE2 exposure. There was a general upregulation between ed12 and ed17, but no clear sex effects were detected. Multivariate analysis indicates that EE2 exposed individuals differ from control individuals in a gender independent manner, and that Gonadotropin-inhibiting hormone related peptide 2 (GnIH-RP2) is a candidate for EE2 induced peptidomic alterations in male embryonic brain.

In the studies this thesis is based upon, microarrays were applied to profilemRNA populations in biological samples to gain insights into transcriptionalpatterns and their relation to inflammatory disease.Rheumatoid arthritis (RA) is a chronic inflammatory disease, which leads todegradation of cartilage and bone. RA is characterized by synovial inflammationwith varying levels of tissue heterogeneity. This was confirmed by microarrayanalyses of multiple biopsies from the joints of 13 patients, which showed interindividualvariation in transcript populations to be higher than intra‐individualvariationTherapeutic antibodies targeting TNF‐α have revolutionized treatment of RA,although some patients do not respond well. Identification of non‐responders isimportant, not only because anti‐TNF treatment elevates the risk of infections,but also because of the cost of treatment. A proof‐of‐concept study to investigatetranscriptional effects of anti‐TNF treatment demonstrated that differencesbetween response groups could be identified and that these differences revealedbiological themes related to inflammatory disease.A subsequent study was therefore initiated with a larger cohort of 62 patients toinvestigate gene expression patterns in the synovium prior to anti‐TNFtreatment. Here, the heterogeneity was even more pronounced, thetranscriptional patterns were confounded by the presence of synovial aggregatesand only a weak therapy‐correlated signature was detected. The presence oflymphocyte aggregates was found to correlate to response to therapy, which isconsistent with previous findings indicating a higher level of inflammation ingood responding patients.Periodontitis is an inflammatory disease with many similarities to RA. Both areincurable chronic auto‐immune diseases, characterized by tissue destructionwith common genetic associations. Individuals with RA are at higher risk ofaccumulating significant periodontal problems than the general population. PGE2(prostaglandin E2) is known to stimulate inflammation and bone resorption inperiodontitis. In further studies, microarrays were applied in a time seriesdesign on human gingival fibroblats to explore the signal transduction pathwayscontrolling TNF‐α induced PGE2 synthesis in order to identify novel therapeutictargets. The JNK and NF‐kb pathways were identified as being differentiallyaffected by TNF‐a treatment. The transcriptional patterns were further verifiedusing antibodies against phosphorylated JNK/NF‐kb molecules and specificinhibitors of the JNK and NF‐kb signaling cascades. / QC 20100820

Regulation of gene expression occupies a central role in the control of the flow of genetic information from genes to proteins. Regulatory events on multiple levels ensure that the majority of the genes are expressed under controlled circumstances to yield temporally controlled, cell and tissue-specific expression patterns. The combined set of expressed RNA transcripts constitutes the transcriptome of a cell, and can be analysed on a large-scale using both sequencing and microarray-based methods. The objective of this work has been to develop tools for analysis of the transcriptomes (methods), and to gain new insights into several aspects of the stem cell transcriptome (applications). During recent years expectations of stem cells as a resource for treatment of various disorders have emerged. The successful use of endogenously stimulated or ex vivo expanded stem cells in the clinic requires an understanding of mechanisms controlling their proliferation and self-renewal. This thesis describes the development of tools that facilitate analysis of minute amounts of stem cells, including RNA amplification methods and generation of a cDNA array enriched for genes expressed in neural stem cells. The results demonstrate that the proposed amplification method faithfully preserves the transcript expression pattern. An analysis of the feasibility of a neurosphere assay (in vitro model system for study of neural stem cells) clearly shows that the culturing induces changes that need to be taken into account in design of future comparative studies. An expressed sequence tag analysis of neural stem cells and their in vivo microenvironment is also presented, providing an unbiased large-scale screening of the neural stem cell transcriptome. In addition, molecular mechanisms underlying the control of stem cell self-renewal are investigated. One study identifies the proto-oncogene Trp53 (p53) as a negative regulator of neural stem cell self-renewal, while a second study identifies genes involved in the maintenance of the hematopoietic stem cell phenotype. To facilitate future analysis of neural stem cells, all microarray data generated is publicly available through the ArrayExpress microarray data repository, and the expressed sequence tag data is available through the GenBank. / QC 20100927