Induced Pluripotent Stem cells (iPScs) are artificially generated cells that demonstrate multilineage differentiation potential. These cells demonstrate similar morphology and high differentiation potential to Embryonic Stem Cells (ESCs). Generation of these cells from a terminally differentiated cell line requires activation of the core pluripotency genes Nanog, Oct4, and Sox2 as well as an oncogenic stimulus such as c-Myc. Here we examine the effect of the Human Pappiloma Virus derived proteins E6 and E7 on the ability of a terminally differentiated fibroblast cell line to a more primitive state and examine its multilineage differentiation capacity. In this paper, we attempt to differentiate BJ hTERT fibroblasts into adipogenic and osteogenic lineages with and without the core pluripotency factors Nanog, Oct4, Sox2 and also c-Myc using non-integrative adenoviral infections. We review the potential mechanisms through which changes in differentiation capacity changes occur through examination of the effects of E6 on the tumor suppressor protein p53. We determined that the proteins E6 and E7 when stably infected into BJ hTERT fibroblasts increase induced differentiation into adipogenic and osteogenic lineages. E6 and E7 can be considered components for generating cells with multipotent capacity with the addition of as little as one core pluripotency factor.

Steroid receptor coactivators modulate the final outcome of hormone induced gene transcription by steroid receptors. E6-associated protein (E6-AP), an E3 ubiquitin ligase, acts as a coactivator of steroid receptors, including estrogen receptor (ER). In this study, we elucidated the contribution of E6-AP to ER-dependent gene transcription in breast cancer cells. siRNA-mediated knockdown of E6-AP abrogates transcription of classic ER target genes, GREB1 and pS2, suggesting that E6-AP is essential for normal transactivation function of ER. In order to understand the global influence of E6-AP in ER-dependent gene transcription, we used gene expression microarrays under E6-AP knockdown conditions to identify ER target genes which are regulated by E6-AP. Our microarray analysis revealed 455 genes which are differentially regulated by E6-AP. Pathway analysis revealed that E6-AP regulated genes were involved in cell cycle. Cell cycle profiling at various time points of estrogen treatment reveals that under E6-AP knockdown conditions, breast cancer cells progress slowly through S phase and eventually fail to proliferate. Knockdown of E6-AP has no effect on ovarian and uterine cells, suggesting that E6-AP has cell specific roles. Our analysis suggests that knockdown of E6-AP reduces the levels of early (C-Myc and Cyclin-D1), mid (E2F1, E2F2 and E2F7) and late (BUB1, BUBR1, MAD2, NDC80, NUF2 and CASC5) estrogen-dependent cell cycle genes. Overall our data indicate that E6-AP is a major regulator of cell cycle in breast cancer cells. E6-AP also acts as a coactivator for androgen receptor (AR) and we studied the role of E6-AP in prostate gland development. We report the generation of transgenic mice which specifically over expresses E6-AP in the prostate gland. Prostate glands in these mice are larger when compared with its wild-type litter mates, corroborating our observations that knockout of E6-AP in mice leads to impaired prostate gland development. E6-AP transgenic mice also develop prostatic intra epithelial neoplasia after 18 months of age. In addition to these observations, we also show that over expression of E6-AP in the prostate gland leads to increased Akt signaling. In order to understand the mechanism by which E6-AP regulates prostate gland growth, we generated LNCaP cells that stably overexpress E6-AP protein. Data from these cell lines show that the levels of phosphatidylinositol 3-kinase, total Akt, phosphorylated Akt (active Akt) and its down-stream target protein, GSKβ are elevated, suggesting that E6-AP regulates the PI3K-Akt signaling pathway. We further show that E6-AP modulates PI3K-Akt signaling by regulating the protein levels of RhoA, a small GTPase, which is a negative regulator of the Akt signaling pathway. In addition, we show that stable overexpression of E6-AP in prostate cancer cells results in increased proliferation. Overall our data suggests that E6-AP regulates the PI3K-Akt pathway in prostate cells which results in increased prostate cell growth, proliferation and tumorigenesis.

The member countries of the enlarged European Union show large differences in the structure of their tax systems. While consumption taxes have been largely harmonized over the past decades, differences remain in taxes on factor incomes, in particular on capital income. Also, effective tax rates on capital income in Central and Eastern European countries (CEEC) have been falling substantially over the last decade- a trend that may suggest that some tax competition has taken place in the enlarged European Union. The contribution of this paper is twofold. First, it presents and contrasts effective tax rates of Western European countries with those of the CEEC. Second, from a theoretical aspect, it presents a model framework within which a quantitative macroeconomic analysis of tax competition between the two regions can be conducted. In addition the model suggests that part of the large real exchange rate appreciation and current account deficits that CEE countries have experienced during the last decade might be attributed to effects from tax competition. (author's abstract) / Series: Discussion Papers SFB International Tax Coordination

A study has been conducted into the causes of cycle by cycle variations in combustion within a spark ignition engine, the best measured engine parameter to use for its characterization, and the effects that: ignition timing, equivalence ratio, fuel type, throttle position and knock, have upon it. A Ricardo E6 single cylinder variable compression ratio research engine was instrumented to allow measurement of: cylinder pressure, temperatures, speed, load, fuel flow and air flow. The engine was also fitted with an optical slice that allowed optical access to the combustion chamber and enabled measurement of the early flame speed (up to 10 mm from spark plug gap) using a laser schileren system. Cylinder pressure data were collected on a dedicated HP1000 computer for every degree of crank angle rotation for up to 300 successive cycles. A phenomenological model was developed for turbulent combustion that split the combustion process into three phases: early laminar burn, turbulent combustion, and final burn. The model allowed the study of the physical phenomena occurring within the combustion chamber and enabled insights to be gained into their effects on combustion and cyclic variations. The study showed: The variation in mixture strength has a far greater effect on the average and Coefficient of Variation (COV) values of all the combustion performance parameters, than does changing the fuel type. Cycle by cycle variations in combustion are best characterized by COV of imep. The onset of knock has no discernible effect on the COVs of the measured parameters. The part throttle results show higher COVs than at Wide Open Throttle (WOT) due to slower burn, supporting the theory that faster initial flame speeds reduce cyclic variations. The combustion model was used to support the hypothesis that cycle by cycle variations are caused by movement of the flame kernel by turbulence within the combustion chamber.

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Arrière-plan: les cellules tumorales circulantes (CTC) sont détectables dans de nombreux cancers et peuvent être utiles cliniquement pour le pronostic de la maladie, pour mesurer la récidive et pour prédire la sensibilité aux medicaments chimiothérapeutiques. Au cours des dernières années, l’études des CTC dans de nombreux cancers tels que le cancer du sein, du poumon, du côlon et de la prostate a grandement évolué. Alternativement, il y peu d'études à ce sujet concernant le cancer du col de l’utérus (CCU). Objectifs: Notre objectif est d’optimiser le processus d'enrichissement des CTC dans le CCU et la détection moléculaire des biomarqueurs E6 et E7. Matériel et Méthodes: Dans l’optique de mimer la présence de CTC dans le sang, nous avons dilué des cellules cancéreuses CaSki VPH16-positif provenant d’un CCU dans du sang humain prélevé sur des volontaires sains. Les CaSki ont été collectées suite à une centrifugation par densité avec le Ficoll, la lyse des globules rouges (RBC) et la lyse des RBC combinée avec un enrichissement positif et négatif à l’aide de marqueurs de surface cellulaire. Les CTC ont été détectées par la mesure d’expression des oncogènes E6 et E7 du virus du papillome humain (VPH), de la cytokératine 19 (CK19) et de la cycline p16INK4 en utilisant la technique quantitative en temps réel de Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). Pour valider notre méthode de détection des CTC in vivo, nous avons recruté dix patientes atteintes d’un CCU VPH16 positif et six contrôles sains. Résultats: Dans le modèle de dilutions de cellules CaSki, la lyse des RBC seule ou combinée avec l'enrichissement négatif ou positif suggèrent des limites de détection de 1 CTC par mL de sang pour tous les biomarqueurs moléculaires utilisés. La sensibilité de détection est accrue lors de l'utilisation de l’enrichissement positif et négatif en réduisant le bruit de fond causé par les monocytes sanguins. Contrairement aux oncogènes E6 et E7, les marqueurs CK19 et p16INK4A ont été détectés chez des individus sains, les niveaux d'expression de base appropriés doivent donc être déterminés avec précision par rapport aux patientes CCU. Le gradient de densité par Ficoll a une limite de détection de seulement environ 1000 cellules par mL de sang. Enfin, les CTC ont été détectées dans 2/10 patientes en utilisant le marqueur CK19. Cependant, ces patientes étaient négatives pour les oncogènes E6/E7. Le marqueur p16INK4A était exprimé au même niveau dans tous les échantillons (CCU et normaux). Conclusion: Notre étude suggère que les oncogènes E6 et E7 du VPH16 sont les marqueurs biologiques les plus sensibles et spécifiques en qRT-PCR pour détecter les CTC dans le modèle de dilution de cellules de CCU dans le sang. Chez les patientes atteintes d’un CCU de stade précoce, seulement CK19 a révélé la présence potentielle de CTC, ce qui suggère que ces cellules sont rares à ce stade de la maladie. Mots clés: cancer du col de l’utérus, cellules tumorales circulantes, RT-qPCR, E6 et E7, CK19, p16INK4A, enrichissement immunomagnétique, détection moléculaire. / Background: Circulating tumor cells (CTCs) have been detected in many cancers and are used in multiple clinical applications including disease prognosis, tumor recurrence prediction and prediction of tumor sensitivity to chemotherapeutic drugs. Studies in most major solid cancer(s) (breast, lung, colon and prostate) are progressing rapidly, but there has been very little progress concerning uterine cervical cancer (UCC).Objective: our aim is to optimize enrichment processes and the molecular biomarker-based detection of human circulating tumor cells (CTCs) in uterine cervical cancer (UCC). Material & Methods: To mimic CTCs in patients, we designed an experimental spiking model where the CaSki HPV16-positive UCC cell line was serially diluted and spiked into human blood collected from healthy volunteers. CaSki CTCs were enriched using either Ficoll density centrifugation, red blood cell (RBC) lysis or RBC lysis combined with cell surface markers negative or positive enrichment. CTCs were detected using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure the gene expression of human papillomavirus (HPV) viral oncogenes (E6 and E7), cytokeratin 19 (CK19), or the cyclin dependent kinase inhibitor p16INK4A. Finally, ten HPV16- positive UCC patients and six healthy controls were recruited to validate CTCs detection in vivo. Result: In the spiking model, RBC lysis alone or combined with negative or positive enrichment suggests detection limits close to 1 CTC per mL of blood for all molecular biomarkers used. The sensitivity of detection increased when using positive and negative enrichment probably by reducing the peripheral blood mononuclear cell-derived RNA background. Unlike HPV oncogenes, CK19 and p16INK4A were detected in normal individuals, thus appropriate basal expression levels need to be accurately determined compared to cancer patients. Alternatively, Ficoll density gradient had a detection limit of only about 1000 cells per mL of blood. Finally CTCs were detected in 2/10 patients using CK19. None of the patients had E6/E7 transcripts and p16INK4A was expressed at similar level across all samples (cancer and healthy). Conclusion: qRT-PCR of HPV16 E6 and E7 is the most sensitive and specific biomarker used to detect CTCs in the spiking model. In early disease UCC patients, only CK19 revealed the presence of CTCs suggesting that these cells are rare at that stage of the disease. Keywords: uterine cervical cancer, circulating tumor cells, qRT-PCR, E6 and E7 oncoprotein, CK19, p16INK4A, immune-magnetic enrichment, molecular detection.

L’ oncoprotéine E6 - qui joue un rôle crucial dans le processus d’oncogenèse induit par les papillomavirus a longtemps résisté à toute analyse. Depuis 1995 l’équipe Oncoprotéines a concentré ses efforts sur cette problématique. Ce qui a permis la résolution par RMN de la structure du domaine C-terminal de E6 en 2006. C’est dans ce cadre que j’ai commencé ce Doctorat en 2008, avec objectif de continuer la quête de données structurales sur E6 tout en acquérant des informations sur ses modes d’interaction avec ses cibles cellulaires. Les travaux de cette thèse ont permis l’obtention de la structure cristallographique de E6 (HPV16) en complexe avec un peptide de E6AP, en utilisant une approche originale capable de produire des protéines E6 stables et solubles. Cette structure constitue la première information structurale publiée sur des protéines E6 entières, attendue depuis plus de 20 ans par la communauté scientifique. J’ai effectué également durant cette thèse une analyse du système d’interaction de la protéine E6 basée sur une large étude d’interaction entre les protéines E6 (7 types) et 93 peptides porteurs de motif LxxLL. / The oncoprotein E6 which plays a crucial role in the process of carcinogenesis induced by HPV, has withstood all tests for a long time. Since 1995 the team « oncoproteins » has focused on this issue. This allowed the resolution of structure of C-terminal domain of E6 by NMR analysis, in 2006. In this context, i started my PhD in 2008 with aim to continue the pursuit of structural data on E6 while also acquiring information about its modes of interaction with its cellular targets. The work of this thesis has enabled us to obtain the crystal structure of E6 (HPV16) in complex with a peptide of E6AP, using an original approach capable of producing stable and soluble proteins E6. This structure represents the first structural information on full-length E6, awaited for over 20 years by the scientific community. I also performed during this thesis an analysis of the interaction system of the E6 protein based on a large study of interaction between proteins E6 (7 types) and 93 peptides bearing LxxLL motif.

Les papillomavirus humains (HPV) constituent l’archétype des virus à ADN oncogènes. Les HPV muqueux à haut risque (principalement HPV16) sont les principaux agents étiologiques du cancer du col utérin. Les protéines virales E6 et E7 sont des acteurs cruciaux de la cancérogenèse induite par HPV. Nous avons construit une ressource composée de 600 ADNc codant les effecteurs humains du système ubiquitine-protéasome (UPS) et identifié de nouvelles cibles potentielles des protéines E6 et E7 en utilisant une méthode basée sur la complémentation protéique de la Gaussia princeps luciférase (GPCA). HPV16 E6 lie les motifs LxxLL présents dans E6AP et IRF3. Nous avons résolu la structure cristallographique des complexes E6/LxxLL de E6AP/p53 et E6/LxxLL de IRF3. Par ailleurs, nous avons montré que les HPV ciblent une nouvelle voie suppresseur de tumeurs, la voie Hippo, avec ses deux médiateurs clef YAP et TAZ. Nous avons généré une banque d’ADNc codant 265 domaines PDZ humains et identifié de nouveaux partenaires potentiels des protéines YAP/TAZ en utilisant la méthode GPCA. Les résultats obtenus permettent de mieux comprendre la biologie des HPV et leur pouvoir oncogène. / The human papillomavirus (HPVs) are the archetype of DNA oncogenic viruses. High-risk mucosal HPVs (mainly HPV16) are the main causative agents of cervical cancer and are also involved in other cancers. HPV oncogenic properties are mainly due to the expression of E6 and E7 proteins. We built a resource composed of 600 cDNA encoding the human ubiquitin-proteasome system (UPS) effectors and identified novel E6 and E7 potential targets by using a method based on the complementation of the Gaussia princeps luciferase (GPCA). HPV16 E6 binds to specific LxxLL motifs present in E6AP and IRF3. We have solved the crystallographic structure of the E6/E6AP LxxLL/p53 and E6/IRF3 LxxLL complexes. Furthermore, HPV may target a novel tumour suppressor pathway, the Hippo signalling pathway with its two main mediators YAP and TAZ. We have built a cDNA library dedicated to the 265 human PDZ domains and identified news potential partners of YAP and TAZ proteins by using the GPCA. The results provide novel insights on HPV biology and their oncogenic property.

The overarching purpose of the studies presented in this report is the exploration of the uses of information theory and Bayesian inference applied to neural codes. Two approaches were taken: Starting from first principles, a coding mechanism is proposed, the results are compared to a biological neural code. Secondly, tools from information theory are used to measure the information contained in a biological neural code. Chapter 3: The REC model proposed by Harpur and Prager codes inputs into a sparse, factorial representation, maintaining reconstruction accuracy. Here I propose a modification of the REC model to determine the optimal network dimensionality. The resulting code for unfiltered natural images is accurate, highly sparse and a large fraction of the code elements show localized features. Furthermore, I propose an activation algorithm for the network that is faster and more accurate than a gradient descent based activation method. Moreover, it is demonstrated that asymmetric noise promotes sparseness. Chapter 4: A fast, exact alternative to Bayesian classification is introduced. Computational time is quadratic in both the number of observed data points and the number of degrees of freedom of the underlying model. As an example application, responses of single neurons from high-level visual cortex (area STSa) to rapid sequences of complex visual stimuli are analyzed. Chapter 5: I present an exact Bayesian treatment of a simple, yet sufficiently general probability distribution model. The model complexity, exact values of the expectations of entropies and their variances can be computed with polynomial effort given the data. The expectation of the mutual information becomes thus available, too, and a strict upper bound on its variance. The resulting algorithm is first tested on artificial data. To that end, an information theoretic similarity measure is derived. Second, the algorithm is demonstrated to be useful in neuroscience by studying the information content of the neural responses analyzed in the previous chapter. It is shown that the information throughput of STS neurons is maximized for stimulus durations of approx. 60ms.