Cellular reprogramming

DNA-methylatie is een epigenetische modificatie. Dit type van modificaties verandert de genetische informatie zelf niet, maar wijzigt de laag erboven. Er worden chemische modificaties toegevoegd of gewijzigd op het DNA of de histonen (waarrond het DNA gewonden is). DNA-methylatie is die modificatie waarbij een methylgroep wordt aangebracht op het cytosine (C) residue en een CG dincucleotide. Als deze wijziging plaats vindt in een zogenaamd CpG eiland (waar deze CG dinucleotiden dens bij elkaar voorkomen) van de promoter van een gen, wordt dit gen niet meer afgeschreven en zal zijn functie verloren gaan. In dit proefschrift worden verschillende aspecten van DNA-methylatie en het belang ervan in de oncologie belicht. In het eerste onderzoeksdeel hebben we verschillende methodes en algoritmes ontwikkeld om methylatiemerkers te identificeren: genen die specifiek in kanker worden gemethyleerd maar niet in normale weefsels of genen die enkel worden gemethyleerd in bepaalde patiëntsubgroepen (bv. die respons vertonen voor een bepaalde chemotherapie). We hebben een databank en methodes gemaakt om kennis uit de literatuur in het snelgroeiende DNA-methylatie veld te halen. Deze databank (PubMeth) laat toe om het verband na te gaan tussen genen en hun methylatiepatroon in verschillende kanker (sub)types. Daarnaast werden verschillende sorterings- en selectiemethodieken ontwikkeld om zo een prioriteit toe te kennen aan de kandidaat DNA-methylatiemerkers. Al deze methodes werden ontwikkeld om data van genoom-wijde experimenten te kunnen verwerken. Nieuwe methylatiemerkers (gemethyleerd in kanker maar niet in normaal) kunnen worden gebruik bij vroegtijdige detectie van kanker. Er werden verschillende computationele oplossingen voorgesteld: - Relaxation ranking: sorteermethodiek gebaseerd op expressie micro-arrays van primair baarmoederhalskanker patiëntmateriaal en re-expressie experimenten op baarmoederhalskanker cellijnen. Deze methode is gebaseerd op lage expressie in kanker en re-expressie na behandeling met het de-methylerend agens DAC en de histondeacetylase inhibitor TSA. De methode gebruikt geen enkele (arbitrair te kiezen) grenswaarde - De deep approach: er werden verschillende DNA-motieven in de promoterregio van genen geïdentificeerd die discrimineren tussen kankerspecifiek gemethyleerde genen en genen die ook gemethyleerd worden in normale weefsels. - De broad approach maakt gebruik van een genoom-wijde alignering van promoterregio’s. Hieruit blijkt dat gekende methylatiemerkers meer dens geclusterd zijn dan verwacht. - Zowel ‘deep’ als ‘broad’ werden gecombineerd met re-expressie studies in cellijnen van verschillende kankertypes. De combinatie van deze experimentele filter en de computationele aanpak verhoogt de succesratio bij het vinden van kankerspecifiek gemethyleerde genen aanzienlijk. - Er werden ook merkers geïdentificeerd die mogelijks de respons op chemotherapie (platinum) kunnen voorspellen in eierstokkanker. Dit opent de weg naar gepersonaliseerde geneeskunde. De methodiek gebruikt een score-schema gebaseerd op zowel primaire kanker stalen (van zowel platinumgevoelige als resistente patiënten) als re-expressie experimenten op kanker cellijnen. De nieuwe identificatiestrategieën werden gevalideerd op primaire kankerstalen en presteren goed: ten opzichte van beschreven priorizatietechnieken is de succesratio verhoogd. Verscheidene nieuwe methylatie biomerkers in verschillende kankertypes (baarmoederhalskanker, eierstok-kanker, hoofd- en nekkanker, neuroblastoom,…) werden succesvol gevalideerd op primaire patiëntenstalen. Vervolgonderzoek op grotere patiëntengroepen zal het mogelijk diagnostische potentieel aantonen. De validatie van sommige studies is momenteel nog lopende. Dit toont de noodzaak aan van snelle en betrouwbare analysetechnieken die geschikt zijn voor validatiedoeleinden. Ten tweede werd er een uniek viraal infectiesysteem gecombineerd met een genoom-wijde methylatiegevoelige detectietechniek om epigenetische ‘herprogrammering’ van humane gastheercellen na infectie met hoge-risico HPV (Humaan Papilloma Virus) te onderzoeken. In dit experi-ment geven we sterke indicaties dat het virus in staat is de gastheercel epigenetisch te wijzigen. Deze hr-HPV virustypes zijn duidelijk geassocieerd met de ontwikkeling van baarmoederhalskanker gezien meer dan 99 % van de patiënten besmet is. Momenteel is het uitstrijkje een wijdverspreide screening methodiek die wordt gebruikt bij vroegdetectie. Onlangs werden er ook vaccins op de markt gebracht die infectie met de meest voorkomende virustypes (ongeveer 80 % van de infecties) moet voorkomen. De vaccinatie zou moeten gebeuren bij meisjes voor het eerste seksueel contact. De ontdekking van biomerkers met hoge specificiteit en sensitiviteit bij baarmoederhalskanker blijft noodzakelijk voor de niet gevaccineerde groep en gezien de vaccins niet tegen alle virustypes bescherming bieden. DNA-methylatiemerkers zijn uitstekende kandida-ten voor vroegdetectie in een screeningsprogramma: ze kunnen op grote schaal en geautomatiseerd gebeuren. Gezien er aangenomen wordt dat verschillende kankertypes verwant zijn met virusinfecties, verhoogt dit onderzoek de kennis bij dit proces en opent dit de weg naar zeer vroege detectie. Momenteel zijn enkel delen van het ‘kanker epigenoom’ bekend. De komst van methodes die grote hoeveelheden data genereren (zoals volgende-generatie sequenering) zou de bestaande kennis in grote mate kunnen laten toenemen. Dit vereist echter grote collecties primaire kankerstalen (liefst nog in verschillende stadia). Een centraal beheerde, goed geannoteerde bibliotheek van patiëntenmateriaal die verschillende kankertypes bevat, zou het onderzoek in een stroomversnelling plaatsen. Bijkomende uitdaging is dat deze technieken het hoogste niveau van precisie hebben (op baseniveau van een enkele DNA-molecule) en tegelijkertijd een hoog aantal stalen verwerken. Dit betekent dat bij het proefopzet sequentiestukken moeten gekozen worden die aangerijkt zijn aan DNA-methylatie of histonmodificaties. De data-analyse strategie moet snel genoeg zijn maar toch nieuwe kennis extraheren uit de terabytes ruwe data die gegenereerd worden. Ons labo werkt zowel op de ontwikkeling van een goed proefopzet als de verwerking van de gegenereerde data.DNA-methylation is an epigenetic modification. These modifications of the DNA do not change the genetic sequence itself, but affect the level ‘above’ it: chemical modifications of the DNA or the histones (where the DNA is wound around) are added or altered. DNA-methylation is the modification where a methyl group is added on cytosine (C) residues in CG dinucleotides. If this takes place in the so-called CpG islands (where CG dinucleotides occur very densely) within the promoter of a gene, this gene is silenced, and will not be transcribed; its function is lost. In this thesis, different aspects of DNA-methylation and its importance in the field of oncology has been dealt with. First, we created different methodologies and algorithms to identify methylation markers: genes that are specifically methylated in cancer but not in normal tissue or genes, methylated in subgroups of patients (e.g. responders of a certain chemotherapy). We built a database and methodologies to discover existing publications in the fast-growing DNA-methylation field. This database (PubMeth) allows to screen which genes are reported methylated in the selected cancer (sub)types and vice versa. Different ranking, sorting and selection techniques were developed to prioritize promising methylation marker candidates. These methodologies were all developed to deal with data from genome-wide approaches. Novel methylation markers (methylated in cancer while not in normal) could be used as early detection markers. Different computational approaches are described here: - Relaxation ranking: ranking strategy based on expression micro-arrays of primary cervical cancer samples and re-expression experiments on cervical cancer cell lines. This methodology is based on low expression in cancer samples and re-expression after treatment with the demethylation agent DAC and the histone de-acetylase inhibitor TSA. The methodology involves no thresholds. - The deep approach: different DNA-patterns in the promoter region of genes were identified that seem to discriminate between cancer-specifically methylated genes and genes that also are me-thylated in normal tissues. - The broad approach makes use of a genome-wide alignment of promoter regions. Apparently, genes described as methylation markers, are more densely clustered together. - Both the deep approach and the broad approach were combined with data from re-expression studies in cancer cell lines. The combination of this experimental filter and the computational approaches drastically improves the success rate in finding cancer-specific methylation markers in various cancer types. - There were also markers discovered, that may be able to predict chemotherapy (platinum) response in ovarian cancer, clearing the roads towards personalized medicine. The methodology uses a score-scheme, based on both primary cancer samples (from patients sensitive and resistant to platinum therapies) as re-expression experiments of ovarium cancer cell lines (both cisplatin resistant and sensitive). The novel identification strategies were validated on primary cancer samples and perform well: the success rate was improved in comparison with other prioritization attempts. Several novel methylation markers were discovered in different cancer types (cervical cancer, ovarian cancer, head-and-neck cancer, neuroblastoma, …) and were validated on primary samples. Follow-up research on larger clinical cohorts will demonstrate their potential diagnostic power. For some studies, the validation effort is still in progress; this illustrates the need for fast and accurate analysis techniques, suitable for validation purposes. Secondly, a unique viral infection system, combined with a ge-nome-wide methylation detection methodology was used to investigate the epigenetic ‘reprogramming’ of human host cells after infection with high-risk HPV (Human Papilloma Virus). In this experiment, we prove that a virus is able to epigenetically program their host cell. The high-risk HPV types are clearly related with the development of cervical cancer as more than 99 % of the patients is infected with such a virus type. For the moment, the cytological Pap-smear screening technique is widespread and used for early detection. Recently, vaccines were developed in order to prevent infection with the most prominent virus types (about 80 % of infections covered). The vaccination strategy must be applied in young girls (before sexual contact). The discovery of cervical cancer biomarkers with high sensitivity and specificity remains necessary for the non-vaccinated group and screening programs remain needed as the vaccines do not cover all hr-HPV virus types. DNA-methylation markers that seem to be related with infection with the virus, are ideal candidates for very early detection in a screening program: large scale analysis can be automated. As it is believed that multiple cancers may occur after viral infec-tion, or at least that these infections plays a key role in the development, this broadens the knowledge in this process and opens ways to very early detection. Currently, only fractions of the cancer epigenome are known. The introduction of methods that generate significantly large amounts of data (such as next generation sequencing) might be able to greatly expand the current knowledge. However, large collections of primary cancer samples (preferentially of different stages) will be needed. A centrally managed, well described and annotated library of patient material containing different cancer types would be extremely beneficial. In addition, the highest level of precision (base pair level of single DNA-molecules) will be reached for a high number of samples at the same time with sequencing techniques. The initial set-up of such an experiment must be chosen so that the sequenced parts of the genome are enriched in DNA-methylation or histone modifications. The data analysis pipeline for the interpretation of the generated data must perform fast and extract new knowledge out of the terabytes of raw data generated. Both the experimental set-up and the downstream data analysis, our laboratory is working on

Displaced people for sale? A multi-methodological research project on international refugee organizations' public communication strategies towards the Syrian and Central African displacement crises (2011-2018).

Since 2011, the world has faced a major increase in forced displacement (UNHCR, 2017). Moreover, displaced people are nowadays confronted with hostility, xenophobia and an increasing popularity of the far-right. In those contexts, refugee organizations play a vital role to protect the rights and well-being of these people. In recent decades, however, several states have become more reluctant to cooperate with refugee organizations (Loescher, 1996; Betts, Loescher & Milner, 2012) and/or have tightened their asylum policies (Betts & Loescher, 2011; Johnson, 2011). Consequently, public communication – as a tool for informing, sensitization and agenda-setting (Atkin & Rice, 2013) – is crucial for refugee organizations’ operations (Dijkzeul & Moke, 2005). Within this constellation, we will present the objectives and research design of a recently started project on refugee organizations’ public communication strategies and this from a multi-method perspective. Despite the significant contribution of said communication to the public perception of displaced people and displacement crises (Chouliaraki, 2012), few studies have examined how refugee organizations attempt to influence the media agenda and broader public opinion in terms of “what to think” (“second-level agenda setting”) (Sallot & Johnson, 2006, p. 152, original italics). Therefore, the general objective of this research project is to critically investigate if and how the textual, production and reception dimensions involved in international refugee organizations’ public communication strategies towards the recent Syrian and Central African crises relate to and interact with each other. This overarching objective is operationalized through an empirical focus on three key actors (the Office of the United Nations High Commissioner for Refugees, International Rescue Committee and Norwegian Refugee Council) following three interrelated research objectives. First, we will study the textual dimension of refugee organizations’ public communication strategies. Our main objective is to gain insights in the applied strategies regarding the representation of displaced people in public communication. For this purpose, we will conduct a critical discourse analysis (Machin & Mayr, 2012) and quantitative content analysis (Riff, Lacy & Fico, 2014) on the selected organizations’ press releases, news articles, videos and photos of 2011 till 2018. Considering the magnitude of the dataset and the time-consuming nature of our discursive research, we will take a sample that consists of the data of the years 2011, 2014, 2016 and 2018. Second, we will focus on the production dimension. Central are the political, economic and socio-cultural forces and motivations behind the public communication strategies. How does this context helps us to explain their communication practices? To reconstruct and analyze these practices, we will conduct 18 in-depth expert interviews with the selected refugee organizations’ heads of communication and press and regional officers responsible for the communication around the Syrian and Central African crises. Finally, we will examine how various intended publics actually perceive refugee organizations’ public communication strategies. For reasons of pragmatics and feasibility, we look at the reception by a sample of citizens, displaced people and journalists living in Flanders. How do these publics interpret the various strategies and how is this related to political, economic and socio-cultural contexts and motivations? For this analysis, eight focus group interviews consisting of 7 Flemish citizens (N=56) will be organized. Just as in the other reception analyses, the respondents will be selected with attention to diversity in terms of gender, age (18+) and socio-economic background, and the interviews will focus on the participants’ reception of the presented public communication and on their general attitude. Further, we will conduct in-depth interviews, given the research subject’s sensitivity, with displaced people. We will interview ten recognized Syrian refugees and ten recognized Central African refugees, both living in Flanders (N=20). These actors’ (mostly largely ignored) opinions about this subject seem highly relevant, regarding their unique involvement in the crises. Considering the importance of media attention, we also focus on journalists’ opinions about refugee organizations’ public communication. How do they perceive the displayed communication and which role does it play in the news production process? We plan expert interviews with 21 journalists who regularly report about the Syrian and/or Central African crisis. This includes audiovisual news media (public broadcaster VRT and commercial broadcaster VTM), printed news media (quality newspapers De Morgen and De Standaard and popular newspapers Het Laatste Nieuws and Het Nieuwsblad) and one alternative news medium (MO* magazine). Apart from its topical nature and relevance for a better understanding of the political, economic and cultural dimensions involved in international refugee organizations' public communication strategies, this project will provide a significant and original empirical contribution to key debates in non-profit studies and the research field of international communication