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

Nach der Eradikation der humanen Pockenerkrankung stellen zoonotische Orthopockenvirus-(OPV-)Infektionen heute eine mögliche Bedrohung der öffentlichen Gesundheit dar. Hierbei sind insbesondere Kuhpocken-(CPXV), Affenpocken-(MPXV) und Vaccinia Viren (VACV) von Bedeutung. In dieser Arbeit wurde das Genexpressionsprofil humaner (HeLa) Zellen nach Infektion mit CPXV, MPXV oder VACV untersucht. Es wurden sowohl zelluläre Gene identifiziert, welche generell von allen verwendeten Viren reguliert wurden, als auch Gene, die eine Virus-spezifische Regulation durch individuelle OPV aufwiesen. Gemeinsamkeiten zeigten sich insbesondere zwischen CPXV und MPXV, welche, im Gegensatz zu VACV, die Expression zahlreicher Cytokine und Chemokine induzierten. Insbesondere für Interleukin-6, -8 und CXCL1 konnte auch auf Proteinebene eine gesteigerte Sekretion durch CPXV-infizierte Zellen nachgewiesen werden. Vermutlich aufgrund dieser Induktion, trat in vitro eine verstärkte Rekrutierung von Monozyten und Makrophagen in Folge einer CPXV-, nicht aber einer VACV-Infektion auf. Makrophagen spielen eine kontroverse Rolle im Rahmen einer OPV-Infektion und sind sowohl für deren Bekämpfung, als auch, im infizierten Zustand, für die Ausbreitung der Viren im Organismus von Bedeutung. Daher wurde die Replikationsfähigkeit von CPXV und VACV in Makrophagen charakterisiert. Der Virulenzfaktor p28, welcher von den meisten VACV Stämmen nicht kodiert wird, konnte als essentiell für die Replikation von CPXV in einer murinen Makrophagen-Zelllinie, primären peritonealen Makrophagen der Ratte und in Makrophagen aus primären humanen PBMCs identifiziert werden. In Anbetracht der Bedeutung der Replikationsfähigkeit in Makrophagen für die Ausbreitung einer OPV-Infektion im Wirtsorganismus, deuten diese Ergebnisse darauf hin, dass CPXV, im Fall einer weiteren Adaption an den Menschen, ein höheres Bedrohungspotential im Vergleich zu VACV aufweisen könnten. / Today, following the eradication of human smallpox, zoonotic infections caused by orthopoxviruses (OPV) are emerging as a potential human health threat. Especially cowpox viruses (CPXV), vaccinia viruses (VACV), and monkeypox viruses (MPXV) are gaining importance as a cause of infectious disease of man and livestock. This study aimed to analyse and compare the gene expression profile of human (HeLa) cells following infection with CPXV, MPXV or VACV. Cellular genes were identified which were either commonly modulated by infection with any of the three viruses, or which were specifically modulated by one individual OPV. Particularly similar effects on cellular gene expression were observed in the case of CPXV and MPXV infection, which both induced the expression of several cytokine and chemokine genes. Especially interleukin-6, -8, and CXCL1 were strongly secreted by CPXV-infected cells but not by VACV-infected cells. Consequently, CPXV infection also induced a strong chemotactic recruitment of monocytes and macrophages in vitro in contrast to VACV infection. Especially macrophages are known to play a controversial role during OPV infection. On the one hand, macrophages are of importance for the control of the infection. On the other hand, infected macrophages also facilitate virus spread across the organism. Therefore, the capability of CPXV and VACV to replicate in macrophages was analysed. Thereby, the poxviral virulence factor p28, which is absent from most strains of VACV, was identified as an essential factor, allowing CPXV replication in a murine macrophage cell line, primary peritoneal rat macrophages and in human PBMC-derived macrophages. Concerning the importance of productively infected macrophages for OPV spread, these results suggest that CPXV, if further adapted to human beings as host species, may harbor a greater threat to human health when compared to VACV.

Durch Anwendung Array-basierter Transkriptanalyse auf mikrodissezierte Prostatagewebe konnten eine Vielzahl differenziell exprimierter Gene des Prostatakarzinoms identifiziert werden. Innerhalb dieser wurden CD166 und CD24 für die weiterführende Analyse ausgewählt. CD24 ist ein kleines Zelloberflächenmolekül, das ursprünglich in B-Zellen und malignen hämatologischen Erkrankungen beschrieben wurde. Eine CD24 Expression wurde auch in verschiedenen soliden Tumoren gefunden. Da CD24 ein Ligand von P-Selektin ist, könnte CD24 den Tumorzellen pro-metastatische Eigenschaften verleihen. Ziel der Studie war, die CD24 Expression in unseren Tumorkollektiven von Mamma- (n=201), Prostata- (n=102), Nicht-kleinzelligen Lungen- (n=89), Ovarial- (n=56) und Pankreaskarzinomen (n=95) immunhistologisch zu bestimmen. Diese Ergebnisse wurden mit klinisch-pathologischen Daten einschliesslich der Überlebensdaten korreliert. Die differenzielle Expression von CD166 wurde ebenfalls immunhistochemisch validiert. Eine CD24 Expression fand sich in 85% der Mammakarzinome, 48% der Prostatakarzinome, 45% der NSCLC, in 84% der Ovarialkarzinome und 72% der Pankreaskarzinome. CD24 Expression korrelierte univariat und multivariat signifikant (p / Applying array based transcript analysis to microdissected prostate tissues, a variety of differentially expressed genes of prostate cancer were identified. Among these, CD166 and CD24 were selected for further analysis. CD24 is a small cell surface molecule that has originally been described in B-cells and hematologic malignancies. Expression of CD24 was also found in various solid tumours. Being a ligand of P-selectin, CD24 might confer pro-metastatic properties to tumour cells. We aimed to clarify the expression of CD24 in our collectives of breast cancer (n=201), prostate cancer (n=102), non-small cell lung cancer (NSCLC, n=89), epithelial ovarian cancer (EOC, n=56) and pancreatic cancer (n=95) by immunohistochemistry. These results were correlated to clinicopathological parameters including survival data. The differential expression of CD166 was validated immunohistochemically as well. Expression of CD24 was found in 85% of breast cancer, 48% of prostate cancer, 45% of NSCLC, 84% of EOC and 72% of pancreatic cancer. CD24 expression correlated significantly (p

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

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Colon cancer causes approximately seven percent of all cancer-related deaths in the world and presumably due to modern lifestyle, it is also one of the most frequently diagnosed cancers. The inefficiency of standard treatment indicates the need for intensive research of molecular mechanisms of cancer development. The canonical Wnt signaling pathway is essential for maintenance of the progenitor phenotype of stem cells in crypts of the intestine and controls repopulation of the epithelia, in physiological conditions. However, aberrant activation leads to tumor formation. Although Wnt signaling in cancer has been subjected to thorough investigation, there is still a lot of questions concerning further branching of the pathway. As a model of Wnt/β-catenin triggered colorectal cancer, we use mice with mutated APC, which is the tumor suppressor involved in this pathway. Previous expression profiling of the intestinal tumors from relevant mice revealed two transcription factors: ETV4 and MSX1 which are significantly overexpressed in cancer cells. In this project we elucidate whether the overexpression is really tumor restricted and Wnt dependent or there is a crosstalk with another signal transduction pathway. We investigate the function and regulation of these transcription factors by synthetic reporter assays,...

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