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.

Systems biology approaches have allowed for comprehensive understanding of complicated biological processes. Here, we’ve developed a global phenotypic profiling method by improving upon transposon mutagenesis methods for identifying genes required for bacterial growth in various conditions. By using the massively parallel power of Illumina sequencing, we precisely redefined the genes required for the growth of Mycobacterium Tuberculosis (Mtb) in vitro. This adapted technique provided more informative data with both increased dynamic range and resolution. As a result, we quantitatively assessed the fitness of individual mutants, as well as identified sub-genic essentiality. Mtb is well adapted to its nutrient-limiting intracellular niche. One important and novel adaptation is its ability to consume cholesterol for both energy and carbon. A combination of this genome-wide phenotypic analysis and global metabolite profiling was used to define the dedicated cholesterol catabolic pathway, as well as important transcriptional and metabolic adaptations required for the consumption of this carbon source. We identified the methylcitrate cycle (MCC) and an unexpected gluconeogenic route as essential pathways. Furthermore, we found that the cholesterol-dependent transcriptional induction of these metabolic enzymes was also essential for growth on this substrate, a function mediated by the Rv1129c regulatory protein. Using a combination of genetic and chemical methods to inhibit these pathways, we show that cholesterol represents a significant source of carbon during intracellular growth in macrophages. Finally, we have begun to define the mechanism by which lipids, such as cholesterol, are imported into the cell by investigating the assembly of the ABC-like lipid transporter, Mce1. The subunits of this system are localized to the cell wall and data is provided to support a novel mechanism for Mce-dependent import of lipids, such as cholesterol. In sum, this global analysis of host cholesterol utilization during infection provides insight into each step of this complicated process; import into the bacterial cell, the degradation of the molecule into primary metabolites, and the transformation of these metabolites into carbon and energy.

No description available.

It is now a matter of routine for the forensic scientist to obtain the genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. Potential contributors of the stain must either be known to investigators (i.e. a developed suspect) or the questioned profile must be searched against a database of DNA profiles such as those maintained in the CODIS National DNA database. However, in those instances where there is no developed suspect and no match is obtained after interrogation of appropriate DNA databases, the DNA profile per se presently provides no meaningful information to investigators, with the notable exception of gender determination. In these situations it would be advantageous to the investigation, if additional probative information could be obtained from the biological stain. A useful biometric that could provide important probative information, and one that may be amenable to molecular genetic analysis, is the biological age of an individual. The ability to provide investigators with information as to whether a DNA donor is a newborn, infant, toddler, child, adolescent, adult, middle-aged or elderly individual could be useful in certain cases, particularly those involving young children such as kidnappings or in providing additional intelligence during terrorist investigations. Currently no validated molecular assays exist for age determination. Biological human ageing can be defined by two distinct processes, degenerative and developmental ageing. The degenerative process of ageing is based on theories which identify an increase or decrease in physiological conditions with increasing age. In contrast, the developmental process of ageing is based on the theory that as individuals increase in chronological age, there will be subtle corresponding molecular based biological changes, each requiring genes to be expressed or silenced, indicative of that particular stage of life. We investigated the degenerative process of chromosomal telomere shortening, as well as the developmental process of gene expression profiling analysis, in an attempt to identify biomarkers of biological age in a self-renewing tissue such as blood. While telomere length analysis was an ineffective method for age determination; gene expression analysis revealed three gene transcripts expressed in an age-dependent physiological manner. These species namely- COL1A2, HBE1 and IGFBP3, were found to be expressed at elevated levels in younger individuals, newborns, or post-pubertal individuals, respectively. The biological process of hemoglobin switching was also investigated for the possibility of determining human age. While experimenting with the potential of using the gamma-hemoglobin chains, as newborn specific gene candidates, we serendipitously discovered four novel truncated transcripts, which we have termed HBG1n1, HBG1n2, HBG2n2 and HBG2n3; whose expression was restricted to whole-blood newborn samples and specific fetal tissues. The molecular origin of these transcripts appears to be at the RNA level, being produced by specific rearrangement events occurring in the standard gamma hemoglobin transcripts (HBG1 and HBG2), which yield these new isoforms that are expressed in a highly regulated tissue specific manner.

No / In recent years, different genes and proteins have been highlighted as potential biomarkers for psoriasis, one of the most common inflammatory skin diseases worldwide. However, most of these markers are not only psoriasis-specific but also found in other inflammatory disorders. We performed an unsupervised cluster analysis of gene expression profiles in 150 psoriasis patients and other inflammatory skin diseases (atopic dermatitis, lichen planus, contact eczema, and healthy controls). We identified a cluster of IL-17/tumor necrosis factor-alpha (TNFalpha)-associated genes specifically expressed in psoriasis, among which IL-36gamma was the most outstanding marker. In subsequent immunohistological analyses, IL-36gamma was confirmed to be expressed in psoriasis lesions only. IL-36gamma peripheral blood serum levels were found to be closely associated with disease activity, and they decreased after anti-TNFalpha-treatment. Furthermore, IL-36gamma immunohistochemistry was found to be a helpful marker in the histological differential diagnosis between psoriasis and eczema in diagnostically challenging cases. These features highlight IL-36gamma as a valuable biomarker in psoriasis patients, both for diagnostic purposes and measurement of disease activity during the clinical course. Furthermore, IL-36gamma might also provide a future drug target, because of its potential amplifier role in TNFalpha- and IL-17 pathways in psoriatic skin inflammation. / Deutsche Forschungsgemeinschaft (DFG) to JW (WE-4428), the René Touraine Foundation (for AD), and the PTJ reference number 0306v12 as part of the Technology and Innovation Program (TIP) North-Rhine Westphalia (gene expression analyses)

No

Die Leber ist das am häufigsten von Metastasen betroffene Organ und kann daher als Modellorgan für metastatische Invasion dienen. Aus diesem Grund war es das Ziel dieser Dissertation Genexpressionsprofile zu verstehen und metastasierungs- sowie invasionsassoziierte Gene zu identifizieren. Differentielle Genexpression wurde in drei Systemen überprüft: Einem syngenen Mausmodell, einem Xenograftmodell sowie in fünf Gewebeproben von Patienten. Genexpressionprofile des syngenen Mausmodells und der Patientenproben zeigten, dass man die Invasionsfront als Ganzes betrachten, um möglichst viele über-lappende Gene zu finden. Globale Genexpressionstudien, die auf den Wirtsteil der Invasionsfront zeigten bemerkenswerte Überrepräsentation z. B. der „GO-terms“ „extrazelluläre Matrix“, Zellkommunikation“, „Antwort auf biotischen Stimulus“, Strukturmolekülaktivität“ und „Zellwachstum“. Marker der Aktivierung hepatischer Sternzellen überrepräsentiert in der invasionsfront, was die Durchführbarkeit einer Analyse differentieller Genexpression im genomweiten Rahmen anzeigt. Globale Genexpressionsstudien, auf den Tumorzellen in der in vitro Situation, in vivo und in der Invasionsfront zeigten insgesamt einen Anstieg zellulärer Spezialisierung von der in vitro zur Invasionsfront. Sezernierte proangiogenetische Chemokine zeigten eine Hochregulation in der Invasionsfront. Das beta catenin Gen war in der Invasionsfront 9.6 fach erhöht im Vergleich zur in vitro Situation. Die Überprüfung der transkriptionellen Aktivierung von beta catenin über die Prüfung der Promotoraktivität zeigte einen 18.4 fachen Anstieg in den Tumorzellen der Invasionsfront. Weiterhin war die Promotoraktivität (an Hand der Aktivität der mRNA des Alkalischen Phosphatase Reportergens) im Tumorinneren 3.5 fach höher als in der Zellkultur, was für einen transkriptionellen Mechanismus der beta catenin Regulation zusätzlich zu den posttranslationalen Mechanismen spricht. / Liver is most frequently populated by metastases and may therefore serve as a model organ for metastatic invasion. So the aim of this thesis is to understand the gene expression profiles and identify metastasis and invasion related genes. Differential gene expression was examined in three systems: A syngeneic mouse model, a xenograft model and five clinical specimens. Gene expression profiles of a syngenic mouse model and human clinical specimen revealed that the invasion front should be considered as a whole to find more overlapping potential target genes. Global gene expression studies on the host part of the invasion front, revealed a pronounced overrepresentation of GO-terms (e.g. “extracellular matrix”, “cell communication”, “response to biotic stimulus”, “structural molecule activity” and “cell growth”). Hepatic stellate cell activation markers were over-represented in the invasion front demonstrating the feasibility of a differential gene expression approach on a genome wide scale. Global gene expression studies of the tumor cells in vitro, in vivo and tumor part of the invasion front revealed an overall increase of cellular specialization from in vitro to the invasion front. Secreted angiogenic cytokines were found to be up regulated in the invasion front. Beta catenin gene of “cell adhesion” GO term was elevated 9.6 fold in invasion front compared to in vitro. Evaluation of transcriptional up-regulation of beta catenin by promoter activity showed an 18.4 fold increase in the tumor cells of the invasion front as compared to those from the faraway tumor. Promoter activity assessed by soluble human placental alkaline phosphatase reporter gene mRNA was 3.5 fold higher in the inner parts of the tumor than in vitro cells indicating a transcriptional mechanism of beta catenin regulation in addition to the posttranslational regulatory mechanisms.

The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain. / Dissertation

In dieser Arbeit wurden Genexpressionsprofile diffuser und intestinaler Magenadenokarzinome mittels Microarray-Technik erstellt. Der intestinale Typ konnte als stark proliferierender Tumor mit signifikanter Überexpression von zellzyklusrelevanten Genen definiert werden, während der diffuse Typ als stark stromaabhängig mit signifikanter Überexpression von Genen der extrazellulären Matrix hervortrat. Thrombospondin 4 (THBS4) wurde dabei als das am stärksten differentiell exprimierte Gen identifiziert, wobei seine mRNA in diffusen Tumoren eminent überexprimiert wird. Immunhistochemische Studien bestätigten diese starke Überexpression auf Proteinebene und zeigten, dass THBS4 eine übermäßig angereicherte extrazelluläre Komponente des Tumorstromas ist. Kolokalisierungsstudien zeigten zudem, dass THBS4-positive Zellen auch positiv für Vimentin und Smooth muscle actin (alpha) sind. Diese Ergebnisse belegen, dass THBS4 von Tumor-assoziierten Fibroblasten (TAF) exprimiert wird. Dies konnte durch zusätzliche in vitro Experimente bestätigt werden, die aufzeigten, dass TAF von diffusen Tumoren eine stärkere THBS4-mRNA Expression aufweisen als normale Fibroblasten des Magens. Abschließend konnten in vitro Kokultur-Studien aufdecken, dass die THBS4-Expression in Fibroblasten durch Tumorzellen diffuser Magentumore transkriptionell stimuliert wird. Metastasenbefall regionaler Lymphknoten (N+) ist bei den meisten Magenadenokarzinomdiagnosen bereits vorhanden. Dieser ist der stärkste derzeit verfügbare Parameter zur Abschätzung der Prognose, reicht aber für eine eindeutige Vorhersage nicht aus. Um ergänzende molekulare Prognoseindikatoren zu identifizieren, wurden aus den Microarray-Daten Gene, deren Expression mit dem klinischen Verlauf von N+ Patienten korreliert, extrahiert. Einige dieser Gene, z.B. RAN binding protein 17 und ras-related associated with diabetes, konnten mittels quantitativer real-time PCR als Marker für verkürztes progressionsfreies Überleben validiert werden. / In this work, gene expression profiles of diffuse and intestinal-type gastric adenocarcinomas were established using the microarray technique. The intestinal type was identified to be a highly proliferative entity with significant overexpression of cell cycle-relevant genes, whereas the diffuse type was proven to be strongly stroma-dependent with significant overexpression of extracellular matrix genes. Thrombospondin 4 (THBS4) was identified as the gene most differentially expressed between the two types with vast mRNA overexpression in diffuse-type tumors. Immunohistochemical studies proved overexpression on protein level and elucidated that THBS4 is a heavily accumulated extracellular constituent of the tumor stroma. Colocalization studies uncovered that THBS4-positive cells are also positive for vimentin and alpha-smooth muscle actin. These data signify that THBS4 is expressed by subpopulations of cancer-associated fibroblasts (CAFs). This was further evidenced by in vitro experiments demonstrating that THBS4 mRNA expression is increased in CAFs of diffuse-type tumors compared to normal gastric fibroblasts. Finally, in vitro coculture studies revealed that transcriptional THBS4 expression in fibroblasts is stimulated by diffuse-type gastric tumor cells. Metastatic involvement of regional lymph nodes (N+) usually accompanies diagnosis of gastric adenocarcinoma and is currently considered the most important parameter for assessment of prognosis. However, estimation of prognosis based on this parameter alone is not sufficiently reliable. In order to identify additional molecular prognosis markers, genes whose expression correlates with clinical outcome of N+ patients were extracted from the microarray data. Via quantitative real-time PCR, several genes, e.g. RAN binding protein 17 and ras-related associated with diabetes, were successfully validated to allow an expression-based stratification of patients with respect to disease-free survival.

INTRODUÇÃO: De acordo com a estimativa de 2012 do INCA, eram esperados 52.680 novos casos de câncer de mama no Brasil, com um risco estimado de 52 casos a cada 100 mil mulheres. Estes dados mostram a necessidade da identificação de biomarcadores efetivos para rastreamento precoce e seguimento destas mulheres durante seu tratamento. Neste trabalho, para a avaliação de potenciais biomarcadores desta doença, foi idealizado um modelo laboratorial específico que avalie tanto a capacidade de um dado biomarcador rastrear um tumor inicial de mama, bem como testar o seu potencial valor para o seguimento de mulheres já diagnosticadas durante seu tratamento. Este modelo baseia-se na avaliação de células tumorais circulantes e perfilamento gênico tumoral. MÉTODOS: Amostras biológicas (sangue periférico e tumor) de 167 pacientes diagnosticadas com carcinoma mamário estadios I, II e III com indicação de quimioterapia adjuvante para: a) avaliação da presença de células tumorais circulantes através da expressão de CK19 e HER2 na Fração Mononuclear do Sangue Periférico (FMNSP) por RT-PCR quantitativo e b) perfilamento gênico tumoral através da análise da expressão de 21 genes relacionados a importantes processos de carcinogênese mamária em amostras de tecido parafinado por ensaio multiplex de RT-PCR quantitativo utilizando o sistema Plexor®. RESULTADOS: Foi observada uma correlação significativa entre CK19 e HER2 na primeira coleta e queda da concentração de HER2 no SP durante o tratamento; porém, não foi percebida queda significativa do CK19 ao longo do estudo. A expressão de HER2 na segunda coleta de pacientes positivas para HER2 na primeira coleta tendeu a se correlacionar significativamente com um pior Intervalo Livre de Doença (ILD). Através da padronização da pontuação em quartis das análises realizadas em multiplex pelo sistema Plexor, foi percebido que o quartil superior apresentava ILD significativa pior do que a de pacientes nos demais quartis. Também foi observada uma estratificação do estadio clínico II em pior ou melhor prognóstico de acordo com o quartil de pontuação do teste de perfilamento proposto neste estudo; além disso, verificou-se que pacientes submetidas a tratamento neoadjuvante com pontuações inferiores tenderam a responder melhor à quimioterapia. CONCLUSÃO: Pelas características do comportamento evolutivo no presente estudo, HER2 parece ser melhor como possível biomarcador de células tumorais circulantes do que o CK-19. Até o presente momento do seguimento das pacientes incluídas neste estudo, não foi possível criar um modelo com diversas variáveis para prever o prognóstico de pacientes com câncer de mama. Isto ocorreu principalmente pelas características preditivas prognósticas superiores do perfilamento genético do tumor que desloca fatores de prognóstico tais como células circulantes e estadio clínico, expressão hormonal do tumor e idade de um modelo multivariado. Por outro lado, foi padronizada uma tecnologia genômica complexa que poderá viabilizar seu uso para a população se estudos posteriores confirmarem seu valor em outras coortes de pacientes com câncer de mama / BACKGROUND: According to the estimate of 2012 INCA, were expected 52,680 cases of breast cancer in Brazil, with an estimated risk of 52 cases per 100 000 women. These data show the need for effective identification of biomarkers for early screening and follow-up of these women during their treatment. In this work, for the evaluation of potential biomarkers of this disease, a model laboratory was designed to evaluate both the specific capacity of a given biomarker trace an initial breast tumor, as well as test its potential value for the follow-up of women already diagnosed during their treatment. This model was based on the evaluation of circulating tumor cells and tumor gene profiling. METHODS: Biological samples (peripheral blood and tumor) of 167 patients diagnosed with breast cancer stages I, II and III with an indication for adjuvant chemotherapy: a) to evaluate the presence of circulating tumor cells through the expression of HER2 and CK19 in Peripheral Blood Mononuclear fraction (PBMN) by quantitative RT-PCR and b) tumor profiling gene by analyzing the expression of 21 genes related to important processes of mammary carcinogenesis in paraffinized tissue samples by multiplex assay for quantitative RT-PCR using the Plexor ® System. RESULTS: Was observed a significant correlation between HER2 and CK19 in the first collection and decrease in concentration of HER2 in PB during the treatment, but were not perceived significant decrease of CK19 along the study. The expression of HER2 in the second collection of patients positive for HER2 in the first test tended to correlate with a significantly worse disease-free interval (DFI). Through standardization of the scores in quartiles of the analyzes performed at multiplex Plexor system was seen that the upper quartile ILD had significantly worse than patients in the other quartiles. Also stratification was observed in clinical stage II in better or worse prognosis according to quartiles of test score profiling proposed in this study, in addition, it was found that patients submitted to neoadjuvant treatment with lower scores tended to better respond to chemotherapy. CONCLUSION: HER2 seems to be better as possible biomarker of circulating tumor cells than the CK-19. So far the monitoring of patients included in this study, it was not possible to create a model with multiple variables to predict the prognosis of patients with breast cancer. This occurred primarily due to the characteristics predictive prognostic upper genetic profiling of tumor that displaces prognostic factors such as circulating cells and clinical stage, tumor hormone expression and age in a multivariate model. In the other hand, was standardized complex genomic technology that may enable their use for the population if further studies confirm its value in other cohorts of patients with breast cancer