La visée d’une société pour tous est souvent remise en question dès qu’il s’agit des personnes présentant un handicap mental. Notre recherche s’empare de cette question et étudie une ingénierie coopérative dans un établissement et service d’aide par le travail (ESAT) du secteur médico-social. Notre cadre théorique s’ancre dans la théorie de l’action conjointe en didactique. Notre méthodologie est qualitative, ethnographique et clinique. Pour donner à voir et à comprendre la construction progressive d’un collectif de pensée, nous nous appuyons sur des exemples emblématiques. Nous les analysons dans un mouvement ascendant et compréhensif. Le dispositif étudié s’appuie sur les compétences de tous dans deux actions complémentaires et liées : un travail d’enquête collectif et une activité didactique dans les ateliers. Nous entendons les savoirs comme des puissances d’agir. Au cours de douze réunions de l’ingénierie coopérative, le collectif (les professionnels et le chercheur) élabore des hypothèses de travail, en réponse à des systèmes de questionnements issus du travail en atelier. Dans les ateliers de l’ESAT, au cours de l’activité ingénierique, ces hypothèses sont éprouvées par un petit groupe spécifique, composé d’un moniteur d’atelier, de travailleurs et du chercheur. De nouveaux problèmes émergent alors. L’analyse de ces actions tend à montrer qu’en travaillant ensemble, le collectif s’institue progressivement en une ingénierie coopérative, avec un style de pensée qui lui est propre. Ce faisant, dans un mouvement du comprendre/transformer, la perception et la conceptualisation de chacun (travailleurs, professionnels, chercheur) se trouvent transformées. / The idea of a society for everyone is often questioned as soon as mentally disabled persons are implied.Our research seizes this matter and studies cooperative engineering in a medico social institution(ESAT: Etablissement Service d’Aide par le Travail: institution for help to disabled adults through work).Our theoretical frame is anchored in the Joint Action Theory in Didactics. Our methodology is qualitative, ethnographic and clinical. To make visible and understandable the progressive construction of thought collective, we draw on emblematic examples. We analyse them in an ascending and understanding movement.The studied device is relying on everyone's skills in two complementary and linked actions: a collective inquiry work and a didactical activity in the workshops. We understand knowledge as power of action.Throughout twelves meetings of the cooperative engineering, the collective (professionals and the researcher) elaborates work hypotheses, in response to questioning systems derived from work in the workshops. In the ESAT workshops, during the engineering activity, these hypotheses are tested by a specific small group, comprised of one workshop instructor, workers and the researcher. New problems then arise.Analysis of these actions tends to demonstrate that by working together, the collective establishes itself as a cooperative engineering, with its own thought style. In doing so, in the understanding/transforming movement, the perception and conceptualisation of everyone (workers, professionals, and researcher)find themselves transformed.

Le travail constitue un levier de rétablissement pour les personnes vivant avec des troubles psychiques ; de nombreux dispositifs existent pour favoriser leur insertion professionnelle. En France, l'accès à l'emploi pour ce public est encore faible et peu de recherches sont réalistes alors que des pratiques existantes sont intéressantes, notamment celle des ESAT de transition de l'association Messidor, qui visent une insertion en milieu ordinaire pour ces personnes. L'objectif de cette thèse était de décrire ce dispositif de transition "par et vers l'emploi" , en le comparant aux pratiques de référence au plan international, et de comprendre comment il peut contribuer à un processus de rétablissement pour les personnes accompagnées.Un volet Accompagnants (étude mixte qualitative puis quantitative) a été consacré à décrire et comparer le dispositif par rapport aux pratiques référencées, et à décrire les métiers des accompagnants, conseiller d'insertion et responsable d'unité de production, afin de voir comment ils contribuent au rétablissement des personnes accompagnées. Un volet Travailleurs (étude quantitative longitudinale, n=160) a été consacré à évaluer la perception des bénéficiaires en début de parcours et à documenter les facteurs prédictifs de leur maintien en emploi dans l'ESAT, d'une évolution positive vers l'insertion en milieu ordinaire et de leur rétablissement.Les ESAT de transition constituent un modèle hybride entre une structure protégée, une entreprise d' économie sociale et un programme d'emploi accompagné. Le binôme d'accompagnants complémentaires, grâce à une posture relationnelle subtile, favorise un changement de regard sur soi. La mise en situation de travail "réelle", avec ses difficultés productives, conjointement à l'étayage des accompagnants qui soutiennent sans assister, conduit la personne accompagnée à retrouver une estime de soi comme travailleur, un regard sur soi valorisé et un espoir en l'avenir, aspects-clés d'un processus de rétablissement. Les bénéficiaires ont une évaluation positive de leur situation en ESAT de transition et confirment le rôle-clé de leurs accompagnants et de l'estime de soi comme travailleur, pour favoriser aussi bien leur maintien en emploi dans l'ESAT, une évolution vers le milieu ordinaire que leur rétablissement. Les implications pratiques concernent le type d'accompagnement à mettre en oeuvre pour ce public ainsi que la formation et l'encadrement à développer pour les accompagnants, afin d'améliorer l'insertion professionnelle et le rétablissement des personnes vivant avec des troubles psychiques en France. / Work is a key-factor of recovery for people with severe mental illness. Various programs exist to support them to gain and maintain competitive employment. In France, the rate of employment remains low for this population and only scarce researches are developed in this field, although some French practices, such as the Ç ESAT de transition È (transitional workshops) of the MESSIDOR association, appear to be performing well. Theses transitional workshops support people with psychiatric disability to gain a competitive employment on the regular labor market while working in a sheltered activity. The goal of this research was to describe the Ç ESAT de transition È program, comparing it to international evidence-based practices, such as supported-employment programs, and to define how this program can facilitate a recovery process for people with a severe mental illness.The first part of this research is intended to describe the program with a mixed method (qualitative then quantitative) in order to compare it with international evidence-based practices and to describe the roles, tasks and competencies of the two professionals supervising people with a severe mental illness, counselors and supervisors, and to define how they contribute to a recovery process for them. A second part of the research is dedicated to clients of this program, with a longitudinal study (n=160) of clinical and psychosocial variables, at the beginning of the program, and in order to document predictive factors of their job tenure in the program, of their positive evolution to work integration on the regular labor market and of their recovery.These Ç ESAT de transition È are an hybrid form of a sheltered workshop, social enterprise and supported-employment program for people with a severe mental illness. In a workplace close to the context of regular labor market, thanks to a subtle relational posture, the two professionals supervising the workers help them to develop a new positive identity, self-esteem as a worker and hope in the future, which are key-factors of a recovery process. Clients have a positive perception of their situation in this program, on clinical and psychosocial variables, and this perception is stable on the 9 months follow-up of the study. They confirm that self-esteem as a worker, counselors and supervisors are key-factors to ensure their job tenure in the program, their positive evolution to work integration on the regular labor market and their recovery. Practical implications and recommendations from this research are the type of support that should be developed in France for people a with severe mental illness to help their work integration, as well as the type of training and supervision that should be offered to the professionals supporting them.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-13T18:08:23Z No. of bitstreams: 1 carolinedesouzaalmeida.pdf: 1716960 bytes, checksum: 4e2f5cb727250f9b08b6d1633c2a22dc (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T12:58:18Z (GMT) No. of bitstreams: 1 carolinedesouzaalmeida.pdf: 1716960 bytes, checksum: 4e2f5cb727250f9b08b6d1633c2a22dc (MD5) / Made available in DSpace on 2016-10-22T12:58:18Z (GMT). No. of bitstreams: 1 carolinedesouzaalmeida.pdf: 1716960 bytes, checksum: 4e2f5cb727250f9b08b6d1633c2a22dc (MD5) Previous issue date: 2008-04-11 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Um terço da população mundial está infectada com o Mycobacterium tuberculosis. Novos métodos de diagnóstico e a vacinação contra tuberculose (TB) são necessários para o controle da doença. A resposta imunológica contra o M. tuberculosis envolve a produção de várias citocinas e quimiocinas, porém suas interações durante a infecção são bastante complexas. O presente trabalho teve como objetivo o estudo da produção de citocinas e quimiocinas durante a tuberculose pulmonar, frente a estímulos de antígenos do M. tuberculosis. Através do método citométrico de esferas ordenadas (CBA-Cytometric Bead Array) foi possível avaliar os níveis plasmáticos das quimiocinas MIG, IP-10, IL-8, RANTES e MCP-1 em pacientes com tuberculose ativa não tratada (TBA), em pacientes sob tratamento (TBST), em pacientes tratados (TBT) e em indivíduos controles sadios (CS). Utilizando esse mesmo método também foi realizada a mensuração das citocinas IFN-γ, TNF-α, IL-6, IL-2, IL-10 e IL-4 e das quimiocinas MIG, IP-10, IL-8, RANTES e MCP-1, produzidas em cultura de células mononucleares do sangue periférico após estímulo com os antígenos ESAT-6/CFP-10 e 16kDa, nos grupos de pacientes com tuberculose ativa não tratada (TBA), de pacientes tratados (TBT), de indivíduos controles sadios (CS) e de indivíduos contatos de pacientes com tuberculose (CT). As quimiocinas plasmáticas MIG, IP-10 e IL-8 estavam elevadas no grupo TBA, em comparação aos demais grupos (CS, TBST e TBT); e ainda os níveis das quimiocinas analisadas no plasma diminuíram após o início do tratamento. Nos ensaios “in vitro” com PBMC humanas, as citocinas IFN-γ, TNF-α, IL-6, IL-2 e as quimiocinas MIG, IP-10, IL-8 e RANTES foram estimuladas em níveis maiores, pelo ESAT-6/CFP-10, nos grupos TBA e CT em relação aos controles e pacientes tratados, enquanto que o estímulo com antígeno 16kDa não permitiu diferenciar entre os grupos estudados quanto a produção dessas citocinas e quimiocinas. Já as citocinas IL-10 e IL-4 e a quimiocina MCP-1 apresentaram níveis elevados nas culturas de PBMC de indivíduos controles e contatos quando estimuladas com os antígenos do M. tuberculosis estudados em relação aos pacientes com tuberculose. O antígeno ESAT-6/CFP-10 diferenciou a tuberculose ativa quanto à produção das citocinas IFN-γ, TNF-α, IL-6 e IL-2, e das quimiocinas MIG, IP-10, IL-8 e RANTES, as quais podem contribuir para uma resposta protetora na tuberculose. O antígeno 16kDa se mostrou um forte estimulador de IL-10 e MCP1, moléculas que podem atuar como reguladoras ou inibidoras da resposta imunológica contra a infecção por M. tuberculosis, sugerindo o papel importante deste antígeno em favorecer a persistência do bacilo no hospedeiro. Os níveis de quimiocinas plasmáticas podem ser eficazes em diferenciar os pacientes com tuberculose ativa de indivíduos controles e ainda pode ser útil em monitorar o efeito do tratamento quimioterápico anti-tuberculose. O entendimento da dinâmica resposta imunológica contra antígenos do M. tuberculosis pode auxiliar no desenvolvimento de novas estratégias de combate à tuberculose. / One-third of the world's population is currently infected with Mycobacterium tuberculosis. The development of new methods of diagnosis and vaccines against tuberculosis (TB) are necessary for disease control. The immune response against M. tuberculosis involves the production of many cytokines and chemokines, but their interaction during the infection is complex. The objective of this work was to study the production of cytokines and chemokines in pulmonary TB under stimulation of M. tuberculosis antigens. The method of Cytometric Bead Array (CBA) was used to evaluate plasma levels of chemokines MIG, IP-10, IL-8, MCP-1 and RANTES in untreated patients with active TB (ATB), in patients during treatment (DTB) and in treated patients (TTB) and healthy controls individuals (HC). We also evaluated, by CBA, the levels of cytokines IFN-γ, TNF-α, IL-6, IL-2, IL-10 and IL-4, and chemokines MIG, IP-10, IL-8, MCP-1 and RANTES, in culture of peripheral blood mononuclear cells (PBMC) after stimulation with the antigens ESAT-6/CFP-10 and 16kDa, in groups of untreated patients with active TB (ATB), in treated patients (TTB), healthy controls individuals (HC) and in contact individuals of patients with TB (CT). The production of plasma chemokines, the levels of MIG, IP-10 and IL-8 were higher in the group ATB, compared to the other (HC, DTB and TTB), and the levels of chemokines analyzed in plasma decreased after the beginning of the treatment. In assays with human PBMC, the cytokine IFN-γ, TNF-α, IL-6, IL-2 and chemokines MIG, IP-10, IL-8 and RANTES were stimulated at higher levels, by the fusion protein ESAT-6 / CFP-10, in groups ATB and CT in relation to the controls and treated patients. The stimulation with antigen 16kDa did not allow for differentiation of these cytokines and chemokines between the groups. IL-10, IL-4 and MCP-1 showed high levels in the cultures of PBMC from healthy control individuals and contacts when stimulated with the studied M. tuberculosis antigens compared to patients with TB. The antigen ESAT-6/CFP-10 differentiated active TB with regard to the production of cytokines IFN-γ, TNF-α, IL-6 and IL-2 and chemokines MIG, IP-10, IL-8 and RANTES, which can contribute to a protective response in TB. The antigen 16kDa was a strong stimulator of IL-4, IL-10 and MCP-1 molecules that can regulate or inhibit the immune response against M. tuberculosis infection, demonstrating the important role of this antigen in promoting the persistence of the bacillus in the host. The plasma levels of chemokines can be effective in differentiating patients with active TB from the healthy control individuals, and can also be useful in monitoring the effect of anti-TB chemotherapy. The comprehension of the dynamics of the immune response against M. tuberculosis antigens can aid the development of new strategies to combat tuberculosis.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-16T19:41:01Z No. of bitstreams: 1 anamarciamenezesdemattos.pdf: 1900352 bytes, checksum: 4c5592f85b3e69efcd500c09d135cdcd (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-18T11:49:02Z (GMT) No. of bitstreams: 1 anamarciamenezesdemattos.pdf: 1900352 bytes, checksum: 4c5592f85b3e69efcd500c09d135cdcd (MD5) / Made available in DSpace on 2017-03-18T11:49:02Z (GMT). No. of bitstreams: 1 anamarciamenezesdemattos.pdf: 1900352 bytes, checksum: 4c5592f85b3e69efcd500c09d135cdcd (MD5) Previous issue date: 2016-11-10 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A tuberculose (TB) permanece como um dos maiores problemas de saúde pública no mundo. A identificação de novos antígenos de Mycobacterium tuberculosis e de biomarcadores de infecção, capazes de auxiliar no diagnóstico da doença ativa e da infecção latente são metas importantes para o controle da TB. No presente trabalho foram estudados pacientes TB antes e após receberem o tratamento, e indivíduos contatos TB com diagnóstico de infecção latente (PPD+). Foram investigados os níveis séricos de IgG e IgG1 e a produção de citocinas e quimiocinas contra os seguintes antígenos: 1) antígenos da fase de infecção ativa (ESAT-6/CFP-10, Rv0717 e Rv3353); 2) infecção latente ou DosR (Rv1733, Rv2628, Rv2029 e Rv1737); e, 3) antígenos da fase de reativação da infecção ou Rpf (Rv0867 e Rv2389). A produção de IFN-ꝩ, TNF-α,IL-17, IL-10 e IL-4 foi avaliada em culturas de células mononucleares de sangue periférico (PBMC) estimuladas com antígenos do M. tuberculosis, através de ELISA e/ou CBA-citometria. Níveis séricos de CCL-2, CXCL-8, CXCL-9 e CXCL10 foram mensurados em pacientes TB e controles. Nossos resultados mostram níveis séricos elevados de anticorpos IgG1 contra ESAT-6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2029, Rv2628 e Rv0867 em pacientes com TB ativa em comparação ao grupo controle (p<0.001). Após completada a quimioterapia, a resposta de IgG1 frente aos antígenos ESAT-6/CFP-10, Rv3353, Rv2628, Rv2029, e Rv0867 foi reduzida a níveis de controle (p<0,01). A análise da curva ROC confirmou o bom desempenho dos antígenos Rv0717, Rv1733, Rv3353, Rv2628, Rv2029 e Rv0867 no diagnóstico da TB. Interessantemente, Rv0717 e Rv1733 induziram um pico de resposta de IgG1 após 1-3 meses de quimioterapia (p<0,01). Neste grupo de pacientes (1-3M), uma alta produção de IFN-ꝩ e TNF-α foi observada após estimulação de PBMC com ESAT6/CFP-10, Rv1733 e Rv2029. Os grupos não diferiram na produção de IL-17, IL-10 e IL-4, indicando uma predominante resposta com perfil Th1 nos pacientes estudados. Níveis elevados de CXCL-8, CXCL-9 e CXCL-10 foram detectados no soro de pacientes com TB ativa. Após 6 meses de tratamento os níveis séricos de CXCL-9 e CXCL-10 foram reduzidos a níveis de controle. Níveis séricos elevados de anticorpos IgG e IgG1 específicos para os antígenos ESAT-6/CFP-10 e Rv1733 foram observados em pacientes com TB ativa e em contatos TB, em comparação com o grupo controle (p< 0,05). De forma semelhante, pacientes TB e contatos TB também não diferiram quanto a produção de IFN-ꝩ em resposta ao antígeno ESAT-6/CFP-10, indicando haver dificuldade em diferenciar pacientes com TB ativa de indivíduos com TB latente. Interessantemente, em resposta ao antígeno DosR Rv1733, níveis elevados de IFN-ꝩ foram observados nas culturas de PBMC dos contatos TB, em comparação aos grupos TB ativa e controle sadio (p<0,05). Em conjunto, esses resultados sugerem a existência de biomarcadores da tuberculose, representados por: 1) detecção de níveis elevados de anticorpos IgG1 contra ESAT-6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2628, Rv2029 e Rv0867; 2) alta produção de IFN-ꝩ e TNF-α em resposta a diferentes antígenos do M. tuberculosis; e, 3) níveis séricos aumentados de CXCL-9 e CXCL-10. Esse perfil de resposta imune pode auxiliar no diagnóstico da TB e no monitoramento do tratamento. Além disso, a detecção de níveis elevados de IFN-ꝩ em resposta ao antígeno Rv1733 pode contribuir no diagnóstico da doença em contatos TB que apresentam tuberculose assintomática. / Tuberculosis (TB) remains a major public health problem in the world. The identification of new Mycobacterium tuberculosis antigens and infection biomarkers capable of assisting in the diagnosis of active disease and latent infection are important targets for TB control. In the present study, TB patients before and after receiving treatment and contacts TB diagnosed with latent infection (PPD +), were studied. We investigated the levels of serum IgG and IgG1 and the production of cytokines and chemokines against the following antigens: 1) antigens from the active phase of infection (ESAT-6/CFP-10, Rv0717 and Rv3353); 2) latent infection antigens or DosR (Rv1733, Rv2628, Rv2029 and Rv1737); and 3) antigens from the reactivation phase of infection or Rpf (Rv0867 and Rv2389). The production of IFN-ꝩ, TNF-α, IL-17, IL-10 and IL-4 was evaluated in peripheral blood mononuclear cell (PBMC) cultures stimulated with M. tuberculosis antigens, by using ELISA and/or CBA-cytometry. Serum levels of CCL-2, CXCL-8, CXCL-9 and CXCL-10 were measured in TB patients and controls. Our results show elevated serum levels of IgG1 antibodies against ESAT6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2628, Rv2029 and Rv0867 in patients with active TB, in comparison with the control group (p<0.001). After completion of the chemotherapy, the IgG1 response to ESAT-6/CFP-10, Rv3353, Rv2628, Rv2029 and Rv0867 was reduced to control levels (p <0.01). The ROC curve analysis confirmed the good performance of Rv0717, Rv3353, Rv1733, Rv2628, Rv2029 and Rv0867, and antigens in the diagnosis of TB. Interestingly, Rv0717 and Rv1733 induced an IgG1 peak response after 1-3 months of chemotherapy (p<0.01). In this patient group (1-3m), a high IFN-ꝩ and TNF-α response was observed after stimulation of PBMC with ESAT-6/CFP-10, Rv1733 and Rv2029. The groups did not differ in IL-17, IL-10 and IL4, indicating a predominant Th1 response in the patients studied. High levels of CXCL8, CXCL-9 and CXCL-10 were detected in the serum of patients with active TB. After 6 months of treatment, levels of both CXCL-9 and CXCL-10 were reduced to control levels. Elevated serum levels of IgG and IgG1 specific for both ESAT-6/CFP-10 and Rv1733 were observed in patients with active TB and in TB contacts, in comparison with the control group (p<0.05). Similarly, TB patients and TB contacts also did not differ in the production of IFN-ꝩ in response to ESAT-6/CFP-10, indicating that the discrimination between patients with active TB and individuals with latent TB is difficult. Interestingly, in response to the DosR Rv1733 antigen, high levels of IFN-ꝩ were observed in PBMC cultures of TB contacts in comparison to both active TB and healthy control groups (p<0.05). Together, these results suggest the existence of tuberculosis biomarkers represented by: 1) elevated serum levels of IgG1 antibodies against ESAT6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2628, Rv2029 and Rv0867; 2) high IFN-ꝩ and TNF-α production in response to different M. tuberculosis antigens; and 3) increased serum levels of CXCL-9 and CXCL-10. This profile of immune response may assist in the diagnosis of TB and monitoring drug therapy. Furthermore, detection of high levels of IFN-ꝩ in response to the Rv1733 antigen may contribute to the diagnosis of TB disease in contacts presenting asymptomatic tuberculosis.

This study explores political parallelism in the context of diaspora-based transnational media through the experience of the Ethiopian Satellite Television and Radio (ESAT). The station is conceived as a party media outlet and transformed into a diaspora-based, non-profit and mainly diaspora funded institution. It has been operating from its three studios in Amsterdam, London and Washington, D.C., until recently. ESAT has emerged as one of the most influential media outlets in the political landscape of Ethiopia in the last ten years. The research, through qualitative and in-depth case study interviews, examines the underlying ideological, political and organizational affiliations that defined ESAT’s position in the media landscape. The study concluded that political parallelism, as an indicator of the dynamics between media and politics, can be used in the diaspora-based transnational media context. However, the study also validated critics on the inapplicability of the two preconditions of political parallelism, namely the existence of competitive system and patterns. The analysis confirms a high level of political parallelism in ESAT in all the five indicators selected for the study. The indicators considered are Ownership, Organizational connections, Party or ideological loyalty, Media personnel’s political involvement, and Journalists’ role orientation. Each of them demonstrated a level of parallelism in ideological orientations or party connection with Ginbot 7 Movement for Democracy and Justice. The study concluded that the salient features of political parallelism should further be studied in the context of the transnational media space of diaspora-based media.

Mycobacterium tuberculosis (Mtb) is the pathogen causing tuberculosis (TB), a disease most often affecting the lung. 1.5 million people die annually due to TB, mainly in low-income countries. Usually considered a disease of the poor, also developed nations recently put TB back on their agenda, fueled by the HIV epidemic and the global emergence of drug-resistant Mtb strains. HIV-coinfection is a predisposing factor for TB, and infection with multi-drug resistant and extremely drug resistant strains significantly impedes and lengthens antibiotic treatment, and increases fatality. Mtb is transmitted from a sick individual via coughing, and resident macrophages are the first cells to encounter the bacterium upon inhalation. These cells phagocytose intruders and subject them to a range of destructive mechanisms, aiming at killing pathogens and protecting the host. Mtb, however, has evolved to cope with host pressures, and has developed mechanisms to submerge macrophage defenses. Among these, inhibition of phagosomal maturation and adaptation to the intracellular environment are important features. Mtb profoundly alters its phenotype inside host cells, characterized by altered metabolism and slower growth. These adaptations contribute to the ability of Mtb to remain dormant inside a host during latent TB infection, a state that can last for decades. According to recent estimates, one third of the world’s population is latently infected with Mtb, which represents a huge reservoir for active TB disease. Mtb is also intrinsically tolerant to many antibiotics, and adaptation to host pressures enhances tolerance to first-line TB drugs. Therefore, TB antibiotic therapy takes 6 to 9 months, and current treatment regimens involve a combination of several antibiotics. Patient noncompliance due to therapeutic side effects as well as insufficient penetration of drugs into TB lesions are reasons for treatment failure and can lead to the rise of drug-resistant populations. In view of the global spread of drug-resistant strains, new antibiotics and treatment strategies are urgently needed. In this thesis, we studied the interplay of the primary host cell of Mtb, human macrophages, and different Mtb phenotypes. A low-burden infection resulted in restriction of Mtb replication via phagolysosomal effectors and the maintenance of an inactive Mtb phenotype reminiscent of dormant bacteria. Macrophages remained viable for up to 14 days, and profiling of secreted cytokines mirrored a silent infection. On the contrary, higher bacterial numbers inside macrophages could not be controlled by phagolysosomal functions, and intracellular Mtb shifted their phenotype towards active replication. Although slowed mycobacterial replication is believed to render Mtb tolerant to antibiotics, we did not observe such an effect. Mtb-induced macrophage cell death is dependent on ESAT6, a small mycobacterial virulence factor involved in host cell necrosis and the spread of the pathogen. Although well-studied, the fate of ESAT6 inside infected macrophages has been enigmatic. Cultivation of Mtb is commonly carried out in broth containing detergent to avoid aggregation of bacilli due to their waxy cell wall. Altering cultivation conditions revealed the presence of a mycobacterial capsule, and ESAT6 situated on the mycobacterial surface. Infection of macrophages with this encapsulated Mtb phenotype resulted in rapid ESAT6-dependent host cell death, and ESAT6 staining was lost as bacilli were ingested by macrophages. These observations could reflect the earlier reported integration of ESAT6 into membranes followed by membrane rupture and host cell death. In conclusion, the work presented in this thesis shows that the phenotype of Mtb has a significant impact on the struggle between the pathogen and human macrophages. Taking the bacterial phenotype into account can lead to the development of drugs active against altered bacterial populations that are not targeted by conventional antibiotics. Furthermore, deeper knowledge on Mtb virulence factors can inform the development of virulence blockers, a new class of antibiotics with great therapeutic potential.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The causative agent of human tuberculosis, Mycobacterium tuberculosis, is an intracellular pathogen that secretes virulence factors, namely ESAT-6 and CFP-10, as substrates of the ESX-1 secretion system. It is hypothesised that these substrates interact with host proteins in a targeted manner in order to elicit a required immune response, and they have been shown to be involved in processes related to pro-inflammatory responses, necrosis, apoptosis, membrane lysis and cytolysis. However, the biological function of ESX-1 substrates during host-pathogen interactions remains poorly and incompletely understood. Therefore, the present study was designed to gain insight into the role of the ESX-1 secretion system substrates in host-pathogen interactions and to identify how M. tuberculosis mediates the response of the human host. In this study, a cDNA yeast two-hybrid library was constructed from human lung mRNA, to identify mycobacterial-host protein-protein interactions that occur within the lung alveoli. The ESX-1 secretion system substrates, ESAT-6 and CFP-10, were cloned in-frame into the pGBKT7 vector, which was used in the yeast two-hybrid system to screen the lung cDNA library in Saccharomyces cerevisiae. The ESAT-6 and CFP-10 screens identified 79 and 19 positive colonies, respectively. Of the total number of clones characterised, only two in-frame inserts were identified with the ESAT-6 screen, corresponding to the human proteins filamin A and complement component 1, q subcomponent, A chain (C1QA). In addition, the screen with CFP-10 also identified C1QA as binding partner. Subsequent in vitro and in vivo experiments were unable to confirm the putative interactions of C1QA with ESAT-6 and CFP-10. However, the interaction between filamin A and ESAT-6 was demonstrated and confirmed by both in vivo co-localisation and co-immunoprecipitation. Furthermore, the degradation of filamin A in the presence of ESAT-6 was shown to be reflective of cytoskeleton remodelling and the induction of cell death. The work presented here suggests that as ESAT-6 gains access to the cytosol, it initiates cell death by inducing destabilisation of the cytoskeleton cell structure. This may possibly be driven by the interaction of ESAT-6 and filamin A. Finally, we also initiated an investigation of the identified putative binding partners (filamin A and C1QA) as possible genetic markers for genetic susceptibility studies to tuberculosis. A case-control analysis was performed involving 604 cases, of which 109 were Tuberculous Meningitis (TBM), and 486 were controls from the South African Coloured (SAC) population within the Ravensmead-Uitsig catchment area. The results of this analysis demonstrated a novel association of a regulatory variant (rs587585) located upstream of the C1QA gene and demonstrated an increasing trend towards increased values in tuberculosis patients with the associated genotype. This study has contributed significantly to our understanding of human-mycobacterial hostpathogen protein-protein interactions and has opened the way for future studies further exploring the consequences and function of the identified ESAT-6-filamin A interaction. It has also led to the identification of a novel genetic association with tuberculosis. Finally, it demonstrates the usefulness of the yeast two-hybrid system to identify potential proteinprotein (host-pathogen) interactions that can lead to additional important and exciting research. / AFRIKAANSE OPSOMMING: Die organisme wat tuberkulose veroorsaak, Mycobacterium tuberculosis, is `n intrasellulȇre patogeen wat virulensie faktore afskei, naamlik ESAT-6 en CFP-10, as substrate van die ESX-1 sekresiesisteem. Daar word vermoed dat hierdie substrate met gasheerproteïene in „n teiken wyse interaksie het om `n vereiste immuunreaksie voort te bring. Hierdie substrate is betrokke by prosesse soos pro-inflammatoriese reaksies, nekrose, apoptose, membraanlise en sitolise. Die biologiese funksie van die ESX-1 substrate tydens gasheer-patogeen interaksies word egter tans swak en onvolledig verstaan. Daarom was die huidige studie ontwerp om insig te bekom oor die rol hiervan in gasheer-patogeen interaksies en om te identifiseer hoe M. tuberculosis die reaksie teenoor die gasheer bemiddel. In hierdie studie was `n komplementȇre deoksiribonukleïensuur (kDNS) gis twee-hibried biblioteek gemaak vanaf long boodskapper ribonukleïensuur (bRNS) om proteïen-proteïen interaksies wat in die long plaasvind, te identifiseer. Die substrate van die ESX-1 sekresiesisteem, ESAT-6 en CFP-10, is in volgorde gekloneer in die pGBKT7 vektor en is gebruik om die long kDNS biblioteek in Saccharomyces cerevisiae te ondersoek. In die soeke na interaksies met ESAT-6 and CFP-10, was 79 en 19 positiewe kolonies onderskeidelik geïdentifiseer. Van die aantal klone, was slegs twee volgordes in-leesraam geïdentifiseer met ESAT-6. Hierdie proteïene het ooreengestem met filamin A en “complement component 1, q subcomponent, A chain” (C1QA). Bykomend hiertoe, is C1QA ook geïdentifiseer as „n bindende vennoot met CFP-10. Daaropvolgende in vitro and in vivo eksperimente kon nie die vermeende interaksie van C1QA met ESAT-6 en CFP-10 bevestig nie. Maar die interaksie tussen filamin A en ESAT-6 kon wel gedemonstreer word deur die gebruik van mede-lokalisering en medeimunopresipitasie. Die afbreek van filamin A in die teenwoordigheid van ESAT-6 is ook aangetoon en blyk „n weerspieëling te wees van sitoskelet hermodellering en die induksie van seldood. Die werk wat hier aangebied word, dui daarop dat soos ESAT-6 toegang kry tot die sitosol, inisieër dit seldood deur die destabilisaisie van die sitoskelet selstruktuur. Dit word moontlik aangedryf deur die interaksie van ESAT-6 met filamin A. Laastens het ons `n ondersoek van die geïdentifiseerde bindingsvennote (filamin A and C1QA) as moontlike genetiese merkers vir genetiese vatbaarheidsstudies vir tuberkulose uitgevoer. `n Pasiënt-kontrole studie is gedoen waarby 604 individue ingesluit is, waarvan 109 gediagnoseer is met Tuberculosis Meningitis (TBM), en die ander 486 kontrole individue was van die Suid Afrikaanse Kleurling (SAC) bevolking binne die Ravenmead-Uitsig opvanggebied. Die resultate het „n nuwe assosiasie van „n regulerende variant (rs587585) wat stroomop van die C1QA geen gelokaliseer is, getoon. Hierdie variant het `n verhoogde neiging in tuberkulose pasiënte met die geassosieërde genotipe getoon. Hierdie studie het `n beduidende bydrae gemaak tot ons begrip van menslike-mikobakteriese gasheer-patogeen proteïen-proteïen interaksies. Hierdie resultate het die weg oopgemaak om die gevolge en funksie van die geïdentifiseerde ESAT-6-filamin A interaksie verder te ondersoek. Dit het ook aanleiding gegee tot die identifikasie van `n genetiese assosiasie met tuberkulose. Om saam te vat, hierdie werk bewys die bruikbaarheid van die gis twee-hibriede sisteem, om potensiële proteïen-proteïen interaksies te ontdek wat die moontlikheid het om aanleiding te gee tot addisionele navorsingsvrae. / The National Research Foundation, / Harry Crossley Foundation / Medical Research Council of South Africa / Stellenbosch University Postgraduate bursary / Prof. Paul van Helden

Στην παρούσα διπλωματική εργασία αναζητείται ένας αυτόματος τρόπος ελέγχου, του επιπέδου της εκπεμπόμενης ισχύος προς το εσωτερικό του ανθρωπίνου σώματος. Εκεί μέσα βρίσκεται κάποιο ενεργό ιατροτεχνολογικό εμφύτευμα. Αυτό το εμφύτευμα στην περίπτωση της εργασίας αυτής, ήταν μία κάψουλα που καταγράφει με φωτογραφίες το γαστρεντερικό σύστημα καθώς οι περισταλτικές κινήσεις του εντέρου προωθούν την κάψουλα προς την έξοδο. Οι φωτογραφίες μεταδίδονται προς καταγραφικό που βρίσκεται έξω από το σώμα με ασύρματο τρόπο. Όπως καταλαβαίνουμε η κάψουλα αυτή αλλά και οποιοδήποτε άλλο ενεργό ιατροτεχνολογικό εμφύτευμα έχει ενεργειακές ανάγκες για την απρόσκοπτη λειτουργία του. Αυτές οι ανάγκες καλύπτονται με ασύρματη μετάδοση ενέργειας. Οι καινοτομίες που υπάρχουν σ’ αυτήν την εργασία είναι οι εξής: 1. Όσον αφορά το εξωτερικό τροφοδοτικό χρησιμοποιήθηκε ένας αντιστροφέας συντονισμού κλάσης D 2. Το πιο καινοτόμο στοιχείο είναι η δημιουργία κλειστού βρόχου ελέγχου μεταξύ του εξωτερικού τροφοδοτικού και του εμφυτεύματος ώστε αυτό να λαμβάνει την ποσότητα της ενέργειας που χρειάζεται κάθε στιγμή. 3. Επίσης σημαντικό είναι ότι η μετάδοση πληροφορίας από το εμφύτευμα προς τα έξω δεν γίνεται με μία ξεχωριστή συχνότητα αλλά χρησιμοποιώντας αρχές παθητικής τηλεμετρίας. Η εργασία αυτή πέρα από την θεωρητική προσέγγιση υλοποιήθηκε και πρακτικά σε εργαστήρια του πανεπιστημίου KUL (ESAT MICAS) στο Βέλγιο. Ο Βρόγχος ελέγχου λειτούργησε και πολλά συμπεράσματα εξάχθηκαν για περεταίρω βελτιώσεις. Η δομή του παρόντος πονήματος είναι ως εξής: Μετά την αρχική εισαγωγή το δεύτερο κεφάλαιο μας δίνει ένα θεωρητικό υπόβαθρο για την ασύρματη μετάδοση ενέργειας. Στη συνέχεια τα διάφορα μέρη των ηλεκτρονικών κυκλωμάτων που αναπτύχθηκαν αναλύονται διεξοδικά στα επόμενα κεφάλαια. Τέλος καταγράφονται τα συμπεράσματα και προτείνονται πιθανές βελτιώσεις για το μέλλον. / In this diploma thesis a way to have an automated control of the transmitted power level into the human body is sought. Inside the body there is an active medical implant. This implant in the case of this project is a swallowable capsule-camera that captures images along the GI tract as the peristaltic propulusion of the bowel push the capsule towards the exit. The photos are transmitted wirelessly to a special recording device that is located out of the body. It is obvious that not only this capsule but any other active medical implant needs energy to operate uninterrupted. This necessary energy is given through inductive power transmission. Innovations in this project are these: 1. The power supply outside the body is realized with Class-D resonant inverter topology. 2. The most innovative is the effectuation of closed control loop between the outer power supply and the implant in order to be received from the implant the exact amount of power that is needed every instant. 3. Also significant is that the transmission of data from the implant to the controlled power supply is not be done with a different carrier but using passive telemetry principles. Beyond the theoretic approximation that was made for this project, it was also realized in KUL university laboratories (ESAT MICAS) in Belgium. The closed control loop functioned properly and conclusions for further development are inferred. The structure of this diploma thesis is as follows: After the starting introduction the theoretic background for wireless inductive power transmission is given in chapter 2. Following, the different parts of the electronic circuits that were developed are analyzed comprehensively in next chapters. Finally conclusions are registered and future improvements are proposed.