Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.

Characterization of the VHL tumor suppressor gene product: localization, complex formation, and the effect of natural inactivating mutations.

The human VHL tumor suppressor gene has been implicated in the inherited disorder von Hippel-Lindau disease and in sporadic renal carcinoma. The homologous rat gene encodes a 185-amino acid protein that is 88% sequence identical to the aligned 213-amino acid human VHL gene product. When expressed in COS-7 cells, both the human and the rat VHL proteins showed predominant nuclear, nuclear and cytosolic, or predominant cytosolic VHL staining by immunofluorescence. A complicated pattern of cellular proteins was seen that could be specifically coimmunoprecipitated with the introduced VHL protein. A complex containing VHL and proteins of apparent molecular masses 16 and 9 kDa was the most consistently observed. Certain naturally occurring VHL missense mutations demonstrated either complete or partial loss of the p16-p9 complex. Thus, the VHL tumor suppressor gene product is a nuclear protein, perhaps capable of specifically translocating between the nucleus and the cytosol. It is likely that VHL executes its functions via formation of specific multiprotein complexes. Identification of these VHL-associated proteins will likely clarify the physiology of this tumor suppressor gene.

Endolymphatic sac tumor : A report of 2 cases with long term follow-up

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Produtzung : Von medialer zu politischer Partizipation

Wahrscheinlich war es ja nur ein Versuch einer alteingesessenen Institution der Massenmedien, sich bei „den Leuten, die man früher ‚das Publikum‘ nannte“ („the people formerly known as the audience“; Rosen, 2006, n.pag.) wieder einzuschmeicheln. Dennoch, die Anerkennung des kollektiven „You“ in YouTube und anderen kollaborativen Onlineplattformen als Person des Jahres durch das amerikanische Magazin Time (Grossman, 2007) liefert ein weiteres Indiz für die wachsende Bedeutung solcher Projekte zur gemeinschaftlichen Produktion und Distribution von Inhalten. Kreative Websites wie Flickr und YouTube, kollaborative Wissenssammlungen von Wikipedia über Digg bis zu Google Earth, nutzergesteuerte Diskussionen in Slashdot, OhmyNews, und der allgemeinen Blogosphäre, aber auch die Softwareentwicklungsgemeinschaften im Open‐Source‐Bereich – sie alle dienen als Beispiele für diese nun etablierten Trend zur Entwicklung neuer Produktions‐, Geschäfts‐, Gemeinschafts‐ und Selbstlenkungsmodelle, die wesentlich durch zunehmend komplexere Web‐2.0‐Tools unterstützt werden. Hinter diesen Beispielen wird eine allgemeiner Tendenz sichtbar, die aus ökonomischer Sicht bereits von Yochai Benkler als „commons‐based peer production“ (2006), und von Eric von Hippel als „democratizing innovation" (2005) beschrieben worden ist. Henry Jenkins spricht zudem von einer „convergence culture“ (2006), in der solche nutzergesteuerten Projekte operieren, und es können auch Verbindungen zwischen diesen stärker aktive gewordenen Nutzern und Alvin Tofflers professionellen Konsumenten, den „Prosumers“ ziehen (1971).

Molekularzytogenetische Untersuchungen an Nierenzelltumoren des Menschen

Nierenkarzinome (NZK) des Erwachsenenalters weisen nebenhistologisch-zytologischen Merkmalen typische genetischeCharakteristika auf, die mit dem biologischen Verhalten derTumorzellen korrelieren. Der Nachweis genomischerVeränderungen wird zur Diagnostik und Prognoseeinschätzungvon Tumoren herangezogen. Für die untersuchtenNierentumorerkrankungen wurden lediglich das vonHippel-Lindau Tumorsuppressorgen (VHL) auf Chromosom 3 oderdas MET Proto-Onkogen auf Chromosom 7 als gesichertepathogenetische Faktoren in der Entstehung von NZKidentifiziert. Die vorliegende Arbeit leistet einen Beitragzur molekularen Charakterisierung derNierentumor-Pathogenese. Die gewählten Ansätze umfassen: (A)Die Analyse 91 primärer Nierentumoren mit CGH (comparativegenomic hybridization), (B) die Untersuchung chromosomalerBruchpunkte eines komplex rearrangierten, familiärenNZK-Falles und (C) die Charakterisierung umschriebenerGenomveränderungen im Bereich des VHL-Lokus auf Chromosom 3p25 .(A) Chromosomen-Aberrationen wurden identifiziert undeingegrenzt. Das Auftreten spezifischer chromosomalerAberrationen konnte mit dem dokumentierten Krankheitsverlaufassoziiert werden. Die als progressionsassoziiert oderprognoserelevant identifizierten Chromosomen(bereiche)5(q22-qter), 4, 10, 14q, 17, 6, 8, 9, 12 können alsAusgangspunkte für eine Suche nach subtypspezifischen,relevanten Tumorgenen dienen. (B) Chromosomenanalysen einerFamilie mit Veranlagung für das klarzellige NZK belegteneine Translokation zwischen den Chromosomen 3 und 8 imperipheren Patientenblut. Weitere molekularzytogenetischeUntersuchungen deckten ein komplexes chromosomalesRearrangement t(3;8)(q13.1;p21.1) auf. (C) DieFeinkartierung der Region um den VHL-Genlokus istinsbesondere bei Tumoren ohne molekulargenetischnachzuweisenden VHL-Verlust erforderlich. Zur Identifikationweiterer in dieser Region kartierender, evtl. ko-deletierterTumorsuppressor-Kandidatengene, wurden genomische(PAC)-Sonden isoliert, charakterisiert und zurDeletionskartierung eingesetzt.

Value creation and value capture fro open source software publisher : a new business model based on mutualisation

Key topics: Since the birth of the Open Source movement in the mid-80's, open source software has become more and more widespread. Amongst others, the Linux operating system, the Apache web server and the Firefox internet explorer have taken substantial market shares to their proprietary competitors. Open source software is governed by particular types of licenses. As proprietary licenses only allow the software's use in exchange for a fee, open source licenses grant users more rights like the free use, free copy, free modification and free distribution of the software, as well as free access to the source code. This new phenomenon has raised many managerial questions: organizational issues related to the system of governance that underlie such open source communities (Raymond, 1999a; Lerner and Tirole, 2002; Lee and Cole 2003; Mockus et al. 2000; Tuomi, 2000; Demil and Lecocq, 2006; O'Mahony and Ferraro, 2007;Fleming and Waguespack, 2007), collaborative innovation issues (Von Hippel, 2003; Von Krogh et al., 2003; Von Hippel and Von Krogh, 2003; Dahlander, 2005; Osterloh, 2007; David, 2008), issues related to the nature as well as the motivations of developers (Lerner and Tirole, 2002; Hertel, 2003; Dahlander and McKelvey, 2005; Jeppesen and Frederiksen, 2006), public policy and innovation issues (Jullien and Zimmermann, 2005; Lee, 2006), technological competitions issues related to standard battles between proprietary and open source software (Bonaccorsi and Rossi, 2003; Bonaccorsi et al. 2004, Economides and Katsamakas, 2005; Chen, 2007), intellectual property rights and licensing issues (Laat 2005; Lerner and Tirole, 2005; Gambardella, 2006; Determann et al., 2007). A major unresolved issue concerns open source business models and revenue capture, given that open source licenses imply no fee for users. On this topic, articles show that a commercial activity based on open source software is possible, as they describe different possible ways of doing business around open source (Raymond, 1999; Dahlander, 2004; Daffara, 2007; Bonaccorsi and Merito, 2007). These studies usually look at open source-based companies. Open source-based companies encompass a wide range of firms with different categories of activities: providers of packaged open source solutions, IT Services&Software Engineering firms and open source software publishers. However, business models implications are different for each of these categories: providers of packaged solutions and IT Services&Software Engineering firms' activities are based on software developed outside their boundaries, whereas commercial software publishers sponsor the development of the open source software. This paper focuses on open source software publishers' business models as this issue is even more crucial for this category of firms which take the risk of investing in the development of the software. Literature at last identifies and depicts only two generic types of business models for open source software publishers: the business models of ''bundling'' (Pal and Madanmohan, 2002; Dahlander 2004) and the dual licensing business models (Välimäki, 2003; Comino and Manenti, 2007). Nevertheless, these business models are not applicable in all circumstances. Methodology: The objectives of this paper are: (1) to explore in which contexts the two generic business models described in literature can be implemented successfully and (2) to depict an additional business model for open source software publishers which can be used in a different context. To do so, this paper draws upon an explorative case study of IdealX, a French open source security software publisher. This case study consists in a series of 3 interviews conducted between February 2005 and April 2006 with the co-founder and the business manager. It aims at depicting the process of IdealX's search for the appropriate business model between its creation in 2000 and 2006. This software publisher has tried both generic types of open source software publishers' business models before designing its own. Consequently, through IdealX's trials and errors, I investigate the conditions under which such generic business models can be effective. Moreover, this study describes the business model finally designed and adopted by IdealX: an additional open source software publisher's business model based on the principle of ''mutualisation'', which is applicable in a different context. Results and implications: Finally, this article contributes to ongoing empirical work within entrepreneurship and strategic management on open source software publishers' business models: it provides the characteristics of three generic business models (the business model of bundling, the dual licensing business model and the business model of mutualisation) as well as conditions under which they can be successfully implemented (regarding the type of product developed and the competencies of the firm). This paper also goes further into the traditional concept of business model used by scholars in the open source related literature. In this article, a business model is not only considered as a way of generating incomes (''revenue model'' (Amit and Zott, 2001)), but rather as the necessary conjunction of value creation and value capture, according to the recent literature about business models (Amit and Zott, 2001; Chresbrough and Rosenblum, 2002; Teece, 2007). Consequently, this paper analyses the business models from these two components' point of view.

Vom Prosumer zum Produser : Ein neues Verständnis nutzergesteuerter Inhaltserschaffung

Alvin Tofflers Bild des Prosumers beeinflußt weiterhin maßgeblich unser Verständnis vieler heutzutage als „Social Media“ oder „Web 2.0“ beschriebener nutzergesteuerter, kollaborativer Prozesse der Inhaltserstellung. Ein genauerer Blick auf Tofflers eigene Beschreibung seines Prosumermodells offenbart jedoch, daß es fest im Zeitalter der Massenmedienvorherrschaft verankert bleibt: der Prosumer ist eben nicht jener aus eigenem Antrieb aktive, kreative Ersteller und Weiterbearbeiter neuer Inhalte, wie er heutzutage in Projekten von der Open-Source-Software über die Wikipedia bis hin zu Second Life zu finden ist, sondern nur ein ganz besonders gut informierter, und daher in seinem Konsumverhalten sowohl besonders kritischer als auch besonders aktiver Konsument. Hochspezialisierte, High-End-Konsumenten etwa im Hi-Fi- oder Automobilbereich stellen viel eher das Idealbild des Prosumers dar als das für Mitarbeiter in oft eben gerade nicht (oder zumindest noch nicht) kommerziell erfaßten nutzergesteuerten Kollaborationsprojekten der Fall ist. Solches von Tofflers in den 70ern erarbeiteten Modells zu erwarten, ist sicherlich ohnehin zuviel verlangt. Das Problem liegt also nicht bei Toffler selbst, sondern vielmehr in den im Industriezeitalter vorherrschenden Vorstellungen eines recht deutlich in Produktion, Distribution, und Konsum eingeteilten Prozesses. Diese Dreiteilung war für die Erschaffung materieller wie immaterieller Güter durchaus notwendig – sie ist selbst für die konventionellen Massenmedien zutreffend, bei denen Inhaltsproduktion ebenso aus kommerziellen Gründen auf einige wenige Institutionen konzentriert war wie das für die Produktion von Konsumgütern der Fall ist. Im beginnenden Informationszeitalter, beherrscht durch dezentralisierte Mediennetzwerke und weithin erhaltbare und erschwingliche Produktionsmittel, liegt der Fall jedoch anders. Was passiert, wenn Distribution automatisch erfolgt, und wenn beinahe jeder Konsument auch Produzent sein kann, anstelle einer kleinen Schar von kommerziell unterstützten Produzenten, denen bestenfallls vielleicht eine Handvoll von nahezu professionellen Prosumern zur Seite steht? Was geschieht, wenn sich die Zahl der von Eric von Hippel als ‚lead user’ beschriebenen als Produzenten aktiven Konsumenten massiv ausdehnt – wenn, wie Wikipedias Slogan es beschreibt, ‚anyone can edit’, wenn also potentiell jeder Nutzer aktiv an der Inhaltserstellung teilnehmen kann? Um die kreative und kollaborative Beteiligung zu beschreiben, die heutzutage nutzergesteuerte Projekte wie etwa die Wikipedia auszeichnet, sind Begriffe wie ‚Produktion’ und ‚Konsum’ nur noch bedingt nützlich – selbst in Konstruktionen wie 'nutzergesteuerte Produktion' oder 'P2P-Produktion'. In den Nutzergemeinschaften, die an solchen Formen der Inhaltserschaffung teilnehmen, haben sich Rollen als Konsumenten und Benutzer längst unwiederbringlich mit solchen als Produzent vermischt: Nutzer sind immer auch unausweichlich Produzenten der gemeinsamen Informationssammlung, ganz egal, ob sie sich dessens auch bewußt sind: sie haben eine neue, hybride Rolle angenommen, die sich vielleicht am besten als 'Produtzer' umschreiben lassen kann. Projekte, die auf solche Produtzung (Englisch: produsage) aufbauen, finden sich in Bereichen von Open-Source-Software über Bürgerjournalismus bis hin zur Wikipedia, und darüberhinaus auch zunehmend in Computerspielen, Filesharing, und selbst im Design materieller Güter. Obwohl unterschiedlich in ihrer Ausrichtung, bauen sie doch auf eine kleine Zahl universeller Grundprinzipien auf. Dieser Vortrag beschreibt diese Grundprinzipien, und zeigt die möglichen Implikationen dieses Übergangs von Produktion (und Prosumption) zu Produtzung auf.

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 microM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and > 50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and > 37.5 microM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2'-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5'-oxygen in the transition state. We suggest structural reasons why the Mg(2+)-La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.

Board monitoring and judgement as processes of sensemaking

When organizational scandals occur, the common refrain among commentators is: 'Where was the board in all this?' 'How could the directors not have known what was going on?''Why didn't the board intervene?' The scandals demonstrate that board monitoring or oganizational performance is a matter of great importance. By monitoring, we mean the act of keeping the organization under review. In many English-speaking countries, directors have a legal duty of care, which includes duties to monitor the performance of their organizations (Hopt and von Hippel 2010). However, statutory law typically merely states the duty, while providing little guidance on how that duty can be met.

Is stereotype threat a useful construct for organizational psychology research and practice?

Stereotypes about different groups persist in organizations. Employees from such groups may experience stereotype threat, or the concern that they are being judged on the basis of demeaning stereotypes about groups to which they belong. The goal of this focal article is to discuss whether stereotype threat is a useful construct for organizational psychology research and practice. To this end, we focus on consequences other than acute performance deficits in laboratory settings. In particular, we examine studies that highlight the effects of stereotype threat on intrapersonal outcomes (e.g., job attitudes), interpersonal outcomes (e.g., negotiation), and on the relationship between employees and their organization. The research reviewed suggests that stereotype threat is a potentially important phenomenon in organizations, but it also highlights the paucity of research in an organizational context. We provide suggestions for future research directions as well as for the prevention and amelioration of stereotype threat in the workplace.

A new biophysics approach using photoacoustic spectroscopy to study the DNA-ethidium bromide interaction

We have examined the binding processes of ethidium bromide interacting with calf thymus DNA using photoacoustic spectroscopy. These binding processes are generally investigated by a combination of absorption or fluorescence spectroscopies with hydrodynamic techniques. The employment of photoacoustic spectroscopy for the DNA-ethidium bromide system identified two binding manners for the dye. The presence of two isosbestic points (522 and 498 nm) during DNA titration was evidence of these binding modes. Analysis of the photoacoustic amplitude signal data was performed using the McGhee-von Hippel excluded site model. The binding constant obtained was 3.4 x 10(8) M(bp)(-1), and the number of base pairs excluded to another dye molecule by each bound dye molecule (n) was 2. A DNA drug dissociation process was applied using sodium dodecyl sulfate to elucidate the existence of a second and weaker binding mode. The dissociation constant determined was 0.43 mM, whose inverse value was less than the previously obtained binding constant, demonstrating the existence of the weaker binding mode. The calculated binding constant was adjusted by considering the dissociation constant and its new value was 1.2 x 10(9) M(bp)(-1) and the number of excluded sites was 2.6. Using the photoacoustic technique it is also possible to obtain results regarding the dependence of the quantum yield of the dye on its binding mode. While intercalated between two adjacent base pairs the quantum yield found was 0.87 and when associated with an external site it was 0.04. These results reinforce the presence of these two binding processes and show that photoacoustic spectroscopy is more extensive than commonly applied spectroscopies.

Singular Value Decomposition and Ligand Binding Analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Übersicht über die bisher bekannten Arten der Gattung Thunbergia L. f.

Von G. Lindau