Control of gene expression in melanocytes and RPE by conserved distal regulatory elements

Résumé Durant le développement embryonnaire, les cellules pigmentaires des mammifères se développent à partir de deux origines différentes : les melanocytes se développent à partir de la crête neurale alors que les cellules de la rétine pigmentaire (RP) ont une origine neuronale. Un grand nombre de gènes sont impliqués dans la pigmentation dont les gènes de la famille tyrosinase à savoir Tyr, Tyrp1 et Dct. Certaines études ont suggéré que les gènes de la pigmentation sont régulés de manière différentielle dans les mélanocytes et dans la RP. Dans ce travail, les gènes de la famille tyrosinase ont été étudiés comme modèle de la régulation des gènes de la pigmentation par des éléments régulateurs agissant à distance. II a été montré que le promoteur du gène Tyrp1pouvait induire l'expression d'un transgène uniquement dans la RP alors que ce gène est aussi exprimé dans les mélanocytes comme le montre le phénotype des souris mutantes pour Tyrp1. Ce résultat suggère que les éléments régulateurs du promoteur sont suffisants pour l'expression dans la RP mais pas pour l'expression dans les mélanocytes. J'ai donc cherché à identifier la séquence qui régule l'expression dans les mélanocytes. Un chromosome artificiel bactérien (CAB) contenant le gène Tyrp1 s'est avéré suffisant pour induire l'expression dans les mélanocytes, comme démontré par la correction du phénotype mutant. La séquence de ce CAB contient plusieurs régions très conservées qui pourraient représenter de nouveaux éléments régulateurs. Par la suite, j'ai focalisé mon analyse sur une séquence située à -I5 kb qui s'est révélée être un amplificateur spécifique aux mélanocytes comme démontré par des expériences de cultures cellulaire et de transgenèse. De plus, une analyse poussée de cet élément a révélé que le facteur de transcription Sox 10 représentait un transactivateur de cet amplificateur. Comme pour Tyrp1, la régulation du gène tyrosinase est contrôlée par différents éléments régulateurs dans les mélanocytes et la RP. Il a été montré que le promoteur de tyrosinase n'était pas suffisant pour une forte expression dans les mélanocytes et la RP. De plus, l'analyse de la région située en amont a révélé la présence d'un amplificateur nécessaire à l'expression dans les mélanocytes à la position -15 kb. Cet amplificateur n'est toutefois pas actif dans la RP mais agit comme un répresseur dans ces cellules. Ces résultats indiquent que certains éléments nécessaires à l'expression dans les deux types de cellules pigmentaires sont absents de ces constructions. Comme pour Tyrp1, j'ai en premier lieu démontré qu'un CAB était capable de corriger le phénotype albinique, puis ai inséré un gène reporter (lacZ) dans le CAB par recombinaison homologue et ai finalement analysé l'expression du reporter en transgenèse. Ces souris ont montré une expression forte du lacZ dans les mélanocytes et la RP, ce qui indique que le CAB contient les séquences régulatrices nécessaires à l'expression correcte de tyrosinase. Afin de localiser plus précisément les éléments régulateurs, j'ai ensuite généré des délétions dans le CAB et analysé l'expression du lacZ en transgenèse. La comparaison de séquences génomiques provenant de différentes espèces a permis par la suite d'identifier des régions représentant de nouveaux éléments régulateurs potentiels. En utilisant cette approche, j'ai identifié une région qui se comporte comme un amplificateur dans la RP et qui est nécessaire à l'expression de tyrosinase dans ce tissu. De plus, j'ai identifié les facteurs de transcription Mitf et Sox10 comme transactivateurs de l'amplificateur spécifique aux mélanocytes situé à -15 kb. L'identification et la caractérisation des ces éléments régulateurs des gènes tyrosinase et Tyrp1confirme donc que la régulation différentielle des gènes dans les mélanocytes et la RP est liée à des éléments régulateurs séparés. Summary Pigment cells of mammals originate from two different lineages: melanocytes arise from the neural crest, whereas cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain. A large set of genes are involved in pigmentation, including the members of the tyrosinase gene family, namely tyrosinase, Tyrp1 and Dct. Previous studies have suggested that pigmentation genes are differentially regulated in melanocytes and RPE. In this work, the tyrosinase gene family was used as a model for studying the involvement of distal regulatory elements in pigment cell-specific gene expression. The promoter of the Tyrp1 gene has been shown to drive detectable transgene expression only to the RPE, even though the gene is also expressed in melanocytes as evident from Tyrp1-mutant mice. This indicates that the regulatory elements responsible for Tyrp1 gene expression in the RPE are not sufficient for expression in melanocytes. I thus searched for a putative melanocyte-specific regulatory sequence and demonstrate that a bacterial artificial chromosome (BAC) containing the Tyrp1 gene and surrounding sequences is able to target transgenic expression to melanocytes and to rescue the Tyrp1 b (brown) phenotype. This BAC contains several highly conserved non-coding sequences that might represent novel regulatory elements. I further focused on a sequence located at -15 kb which I identified as amelanocyte-specific enhancer as shown by cell culture and transgenic mice. In addition, further functional analysis identified the transcription factor Sox10 as being able to bind and transactivate this enhancer. As for Tyrp1, tyrosinase gene regulation is mediated by different cis-regulatory elements in melanocytes and RPE. It was shown that the tyrosinase promoter was not sufficient to confer strong and specific expression in melanocytes and RPE. Moreover, analysis of tyrosinase upstream sequence, revealed the presence of a specific enhancer at position -15 kb which was necessary to confer strong expression in melanocytes. This enhancer element however failed to act as an enhancer in the RPE, but rather repressed expression. This indicates that some regulatory elements required for tyrosinase expression in both RPE and melanocytes are still missing from these constructs. As for Tyrp1, I first demonstrated that a BAC containing the Tyr gene is able to rescue the Tyr c (albino) phenotype in mice, then I inserted a lacZ reporter gene in the BAC by homologous recombination, and finally analysed the pattern of lacZ expression in transgenic mice. These mice showed strong lacZ expression in both RPE and melanocytes, indicating that the BAC contains the regulatory sequences required for proper tyrosinase expression. In order to localize more precisely these regulatory elements, I have then generated several deletions in the BAC and analysed lacZ expression in transgenic mice. Multi-species comparative genomic analysis then allowed identifying conserved sequences that potentially represent novel regulatory elements. Using this experimental approach, I identified a region that behaves as a RPE-specific enhancer and that is required for tyrosinase expression in the retina] pigment epithelium. In addition, I identified the transcription factors Mitf and Sox l0 as being transactivators of the melanocyte-specific enhancer located at -l5 kb. The identification and characterization of these tyrosinase and Tyrp1 distal regulatory element supports the idea that separate regulatory sequences mediate differential gene expression in melanocytes and RPE.

Validation study of the Spanish Version of the Work-Family Conflict Questionnaire (CCTF)

In this study, a brief Work-Family Conflict (WFC) Questionnaire in the Spanish language is proposed that takes into account the two directions commonly reported in the literature: work interference with family (WIF), and family interference with work (FIW). The results obtained through exploratory factor analyses (EFA) and confirmatory factor analyses (CFA) with two independent samples, carried out for women and men, showed acceptable validity and reliability. A copy of the instrument in Spanish language is provided, together with the Amos 4 syntax to perform the factor invariance analysis for women and men. The suggested Work-Family Conflict Questionnaire (in Spanish, abbreviated as CCTF) may be useful in studies performed in the work setting, considering the special relevance of the concept in this line of research.

How to identify and recruit nurses to a survey 14 and 24 years after graduation in a context of scarce data : lessons learnt from the 2012 nurses at work pilot study on nurses' career paths

Nursing workforce data are scarce in Switzerland, with no active national registry of nurses. The worldwide nursing shortage is also affecting Switzerland, so that evidence-based results of the nurses at work project on career paths and retention are needed as part of the health care system stewardship; nurses at work is a retrospective cohort study of nurses who graduated in Swiss nursing schools in the last 30 years. Results of the pilot study are presented here (process and feasibility). The objectives are (1) to determine the size and structure of the potential target population by approaching two test-cohorts of nursing graduates (1988 and 1998); (2) to test methods of identifying and reaching them 14 and 24 years after graduation; (3) to compute participation rates, and identify recruitment and participation biases.

Työturvallisuusriskien tunnistaminen, arviointi ja hallinta pelastuslaitoksissa

Firefighters work contains numerous hazards. In rescue missions there are always hazards, which are unavoidable. Risk assessments and near-miss –situation investigation increases firefighters risk awareness and decreases accidents. This thesis concerns occupational risk management in fire department. This thesis introduces methods for identifying occupational hazards and risk assessment. In this thesis is introduced risk assessment method developed whit fire departments. This thesis also concerns a model of fire departments safety management system. In addition thesis contains a short introduction of how occupational health care cold be used and what kind of information system would support fire departments daily occupational safety activities.

Ethnic sensitive substance abuse work : services for non-Finnish speaking clients in substance abuse treatment units in Finland today

The purpose of this study was to examine the current situation in substance abuse treatment units in Finland in taking non-Finnish speaking clients into consideration. The initiative for this research came from the Development of Alcohol and Drugs Intervention group at Stakes (National Research and Development Centre for Welfare and Health). Their aim was to gather information about the functioning and relevance of the quality assessment forms based on the quality recommendations for substance abuse work, filled in by substance abuse treatment units. The ethnic issue was chosen as the main approach in the study. The aim of this research was to answer the following questions: what is the readiness and competence in substance abuse treatment units in Finland to receive and encounter non-Finnish speaking clients, how is the quality of these services assessed and/or developed in the units, and what has been the role and functioning of the quality recommendations and quality assessment forms in working with non-Finnish speaking clients. The research methods used in the study were both quantitative and qualitative. The information concerning language services provided in the units was gathered from the quality assessment forms and basic information forms found in the database maintained by Stakes. The total amount of units found in the database was 267. In addition to that, semi-structured theme-interviews were carried out in four substance abuse treatment units in order to get a more deep understanding of how the services function in practice. The few number of non-Finnish speaking clients in the units may explain to a certain degree the results of the research. The results however showed that there is still space for improving the services. In the light of quality recommendations, the degree of language options provided in substance abuse treatment units in Finland today is low. Also the quantity of interpreter services provided in the units is scarce. There could also be unified guidelines specially tailored for substance abuse treatment units on how to work with ethnic minorities, as the knowledge is currently adopted from several different instances. The quality recommendations as well as quality assessment forms were valued and applied in the units appropriately and were also perceived to have an effect on the functioning, and quality, in the units.

Opening The Fortress : the work of public gaze on the Swiss asylum reality

The Fortress (La Forteresse) is a 2008 documentary film by Fernand Melgar that reports the Swiss asylum reality from a distant but committed point of view. The documentary describes the life of asylum seekers awaiting in a federal centre the decision to grant them-or not-refugee status. It subtly raises the issue of the role that "textual realities", grasped from the spectator's point of view, play in the production of public discourses. Most of all, it subtly poses the question of the (Swiss) spectator as an actor of the asylum policy, in the context of a semi-direct democracy. After evoking the notion of sensible experience for linking spectatorship to politics, we look at how the documentary invites its model spectator to accept the film's moral premises. Furthermore, focusing on the Swiss public sphere, we deliver an account of the reception by empirical spectators, notably by a group of leftist activists that tend to subvert Melgar's intentions. This two-fold analysis leads us to exhibit that, in a context of discursive struggles, The Fortress generates an original space of deliberation and experience, which appeals to the public to exercise their political agency on asylum policy without being constricted by an antagonist framework.

The Challenges Of Implementing High Performance Work Practices In The Nonprofit Sector

The last economic crisis raised huge challenges for nonprofit organizations. It is now critical for nonprofit organizations to show not only their social legitimacy but also their efficiency and competency to claim for grants (Kearns, Bell, Deem, & McShane, 2012). High Performance Work Practices (HPWP) are a way to foster performance and thus to answer challenges nonprofit organizations are currently facing. However, such practices have until then only been considered for the corporate world. The entire philosophy behind nonprofit organizations contrasts radically from the for-profit sector. Human resources management in particular may differ as well. The aim of this article is precisely to analyze the challenges of implementing HPWP in nonprofit organizations. In order to explore those challenges, we study the HR practices of a nonprofit organization based in UK that struggles against poverty. Discussion of results highlights good practices that should be applied along the nonprofit sector.

Tunnuslukujärjestelmän rakentaminen pelastuslaitokselle

Tutkimuksen tavoitteina oli rakentaa pelastuslaitoksen tuloksellisuuden arvioimiseen tasapainotettu mittaristo sekä valita mittareita, joiden avulla voidaan vertailla pelastuslaitoksia keskenään. Tutkimuksessa selvitetään, miten julkisen sektorin palveluiden tuloksellisuutta arvioidaan erityisesti kuntasektorilla, mitä tarkoitukseen soveltuvia mittausjärjestelmiä on olemassa, miten tasapainotetun mittausjärjestelmän rakentamisprosessi etenee, miten henkilöstön aikaansaannoskyky voidaan ottaa huomioon kuntien tuloksellisuuden arvioinnissa, minkälaisilla mittareilla voidaan parhaiten arvioida pelastuslaitoksen toimintaa ja mitkä mittareista soveltuvat pelastuslaitosten vertailuun. Teoriaosuudessa käsitellään julkisten palvelujen erityispiirteitä, suositusta kunnallisten palvelujen tuloksellisuuden arvioimisesta sekä henkilöstön aikaansaannoskyvyn mittaamista. Työssä esitellään joitakin tasapainotetun mittaamisen malleja sekä kuvataan mittariston rakentamisprosessin vaiheita. Teorian pohjalta rakennettiin tasapainotettu tunnuslukujärjestelmä Etelä-Karjalan pelastuslaitokselle. Kunnalliselle organisaatiolle voidaan rakentaa tasapainotettu mittaristo, mutta yksittäisen pelastuslaitoksen osalta on lähdettävä sen omista kriittisistä menestystekijöistä ja noudatettava kunnallisen sektorin suosituksia. Yhteisiä mittareita pelastuslaitosten käyttöön löytyy, mutta pääosin yksikön tuloksellisuutta ohjaavat mittarit soveltuvat vain sisäiseen käyttöön.

The contribution of individual and professional characteristics to general and work-related well-being

Cette thèse porte sur la contribution des caractéristiques individuelles et des situations professionnelles au bien-être. En combinant différentes perspectives théoriques, notamment la théorie de la construction de la carrière, la théorie de la justice organisationnelle, les modèles du bien-être au travail, et les conceptualisations de l'incivilité au travail, un certain nombre d'hypothèses sont proposées concernant le lien entre certaines caractéristiques individuelles et situationnelles et le bien-être général et professionnel. Les deux premières études se focalisent sur la validation d'une échelle d'adaptabilités de carrière et sur le rôle médiateur de cette adaptabilité dans la relation entre des dispositions et le bien-être. La troisième étude évolue l'hypothèse d'un possible effet de médiation de l'adaptabilité mais cette fois de la relation entre insécurité professionnelle et charge de travail d'une part et le bien-être d'autre part. La quatrième étude adopte une perspective longitudinale et analyse les associations entre les dimensions de la personnalité, l'adaptabilité de carrière et le bien-être dans quatre parcours professionnels différents. La cinquième étude porte sur une autre caractéristique individuelle, à savoir la croyance en un monde juste. Cette étude illustre comment la croyance en un monde juste influence les perceptions de justice organisationnelle une année après, qui ont une incidence importante sur le bien-être. Enfin, la dernière étude se concentre sur une population spécifique, les immigrants en Suisse, et souligne qu'être la cible d'incivilité sur le lieu de travail est généralement liée au pays d'origine. Globalement, cette thèse met en évidence que les caractéristiques individuelles ont des effets tant directs qu'indirects sur le bien-être et que ces mêmes caractéristiques explique en partie, les relations entre la situation professionnelle et le bien-être. Plus spécifiquement, des situations professionnelles peuvent influencer l'expression de certaines caractéristiques individuelles, soit en contribuant à leurs activations ou à leurs inhibitions. De plus, l'impact des caractéristiques individuelles sur le bien-être semble dépendre de la situation professionnelle. Il est donc important de considérer les influences simultanées et réciproques des caractéristiques individuelles et de la situation contextuelle et professionnelle pour rendre compte du bien-être général et professionnel. -- This thesis explores how individual characteristics and professional situations correspond to well-being. Drawing from various theoretical backgrounds, such as career construction theory, justice theory, models of job strain, and theories on subtle discrimination, a number of specific hypotheses are put forward pertaining to a selection of individual and professional aspects as well as general and work-related well-being. The six studies presented in this thesis focus on specific aspects and adopt different methodological and theoretical approaches. The first two studies concern the validation of the career adapt-abilities scale and test the potential of career adapt-abilities to mediate the relationship between dispositions and outcomes. The third study extends the hypothesis of career adapt-abilities as a mediator and finds that it mediates the effects of job insecurity and job strain on general and professional well-being. The fourth study adopts a longitudinal approach and tests the associations between personality traits and career adaptability and well-being in four different professional situations. Study five concerns another individual characteristic, belief in a just world, and illustrates how justice beliefs drive perceptions of organizational justice, which in turn impact, on well-being outcomes one year later. The final study focuses on the professional experiences of a specific population, immigrants in Switzerland, and confirms that being a target of incivilities is related to national origin. Globally, this thesis finds that individual characteristics have direct and indirect influences on well-being and that these characteristics may also mediate the associations between professional situations and outcomes. In particular, the professional situation may alter the display of individual characteristics, either by contributing to their activation or their depletion, and the ways in which individual factors influence well-being does seem to depend on the professional situation. It is thus necessary to adopt a "both...and" perspective when studying the impact of individual and professional characteristics as these factors mutually influence each other.

Representations of work and worker in the three generations of knowledge management. Critical Discourse Analysis of selected texts.

The thesis studies the representations of different elements of contemporary work as present in Knowledge Management (KM). KM is approached as management discourse that is seen to affect and influence managerial practices in organizations. As representatives of KM discourse four journal articles are analyzed, using the methodology of Critical Discourse Analysis and the framework of Critical Management Studies, with a special emphasis on the question of structure and agency. The results of the analysis reveal that structural elements such as information technology and organizational structures are strongly present in the most influential KM representations, making their improvement also a desirable course of action for managers. In contrast agentic properties are not in a central role, they are subjugated to structural constraints of varying kind and degree. The thesis claims that one such constraint is KM discourse itself, influencing managerial and organizational choices and decision making. The thesis concludes that the way human beings are represented, studied and treated in management studies such as KM needs to be re-examined. Pro gradu-tutkielmassa analysoidaan työhön ja sen tekijään liittyviä representaatioita Tietojohtamisen kirjallisuudessa. Tietojohtamista tarkastellaan liikkeenjohdollisena diskurssina, jolla nähdään olevan vaikutus organisaatioiden päätöksentekoon ja toimintaan. Tutkielmassa analysoidaan neljä Tietojohtamisen tieteellistä artikkelia, käyttäen metodina kriittistä diskurssianalyysiä. Tutkielman viitekehyksenä on kriittinen liikkeenjohdon tutkimus. Lisäksi työssä pohditaan kysymystä rakenteen ja toimijan välisestä vuorovaikutuksesta. Tutkielman analyysi paljastaa, että tietojohtamisen vaikutusvaltaisimmat representaatiot painottavat rakenteellisia tekijöitä, kuten informaatioteknologiaa ja organisaatiorakenteita. Tämän seurauksena mm. panostukset em. tekijöihin nähdään organisaatioissa toivottavana toimintana. Vastaavasti representaatiot jotka painottavat yksilöitä ja toimintaa ovat em. tekijöille alisteisessa asemassa. Tapaa, jolla yksilöitä kuvataan ja käsitellään Tietojohtamisen diskurssissa, tulisikin laajentaa ja monipuolistaa.

Police work and new 'security devices' : a tale from the beat

Mobile technologies have brought about major changes in police equipment and police work. If a utopian narrative remains strongly linked to the adoption of new technologies, often formulated as 'magic bullets' to real occupational problems, there are important tensions between their 'imagined' outcomes and the (unexpected) effects that accompany their daily 'practical' use by police officers. This article offers an analysis of police officers' perceptions and interactions with security devices. In so doing, it develops a conceptual typology of strategies for coping with new technology inspired by Le Bourhis and Lascoumes: challenging, neutralizing and diverting. To that purpose, we adopt an ethnographic approach that focuses on the discourses, practices and actions of police officers in relation to three security devices: the mobile digital terminal, the mobile phone and the body camera. Based on a case study of a North American municipal police department, the article addresses how these technological devices are perceived and experienced by police officers on the beat.