Les proprotéines convertases (PC) sont d’importantes enzymes impliquées dans l’activation par clivage de précurseurs protéiques dans la voie de sécrétion cellulaire et qui permettent la régulation de la physiologie animale. Toutefois, les PC ont un rôle particulier lors d’infections, puisqu’elles participent à la fois à l’activation de pathogènes et au contrôle de la réponse immunitaire qu’ils induisent. Cette thèse présente le développement d’un inhibiteur peptidique de PC à des fins thérapeutiques contre les pathogènes et se penche sur le rôle de PC1/3, une PC dite neuroendocrinienne, dans le contrôle de la réponse immunitaire au sein des macrophages. Dans un premier temps, l’inhibiteur de PC a été optimisé par peptidomimétique afin de bloquer l’activation de deux pathogènes activés par les PC, l’un viral et l’autre bactérien. Dans un second temps, l’utilisation de shRNA sur un modèle de macrophages en culture NR8383 et du modèle de souris où PC1/3 est inactivée ont permis de déterminer les conséquences physiologiques et moléculaires de l’inactivation de PC1/3 au sein des macrophages grâce au développement d’une approche par spectrométrie de masse. L’approche par spectrométrie de masse s’est avérée être un catalyseur dans cette recherche et a pu être appliquée à l’étude de tissus de patientes atteintes du cancer de l’ovaire, démontrant ainsi tous les avantages de cet outil. En somme, les résultats de cette thèse montrent la faisabilité d’inhiber les PC pour contrôler les infections et ouvrir de nouvelles perspectives sur le contrôle de l’immunité en établissant les bases moléculaires du rôle de PC1/3 dans le maintien de l’homéostasie immunitaire. / The proprotein convertases (PCs) are important enzymes mainly involved in the activation of protein precursors into the cell secretory pathway. This critical activation step that generates various biologically active polypeptides makes the PCs a cornerstone in a variety of biological process, including the neuroendocrine system. However, the PCs are described as very special players during infection, since they both activate various pathogens and control the immune response they induce. This thesis presents the development of a PCs peptide inhibitor for therapeutic purposes against pathogens and examines the role of PC1/3, which is mainly associated with the neuroendocrine system, in the control of the immune response in macrophages. As a first step, the PC inhibitor has been optimized by a peptidomimetic approach to block pathogens activation by PC for both a viral pathogen and a bacterial pathogen. In a second step, gene expression control tools have been used (shRNA) on a macrophage NR8383 cell line combined with the use of PC1/3 inactivated mouse (KO) to determine the physiological and molecular consequences of PC1/3 inactivation in macrophages by the mean of mass spectrometry approaches. The mass spectrometry approaches were proven to be a catalyst in this research and were further applied on ovarian cancer tissues studies, demonstrating the benefits of these tools. Overall, the results of this thesis demonstrate the feasibility of inhibiting PC to control infections and establish new avenues to modulate immunity by laying the foundations of PC1/3 molecular functions in the maintenance of immune homeostasis.

Avian influenza H5N1 causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. NP is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymophcyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physiochemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

Avian influenza H5N1 causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. NP is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymophcyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physiochemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

No description available.

Bien que la persistance du virus H5N1 dans l'environement soit possible, il n'existe aucune méthode bien définie, standardisée, pour détecter du virus à partir de l'eau, du sol ou de la boue. De plus, il n'y a que très peu de données relatives au rôle du virus H5N1 dans l'environnement en pays tropical. Dans ce travail, des méthodes de concentration, d'identification, et de quantification du virus influenza dans l'eau et dans la boue ont été développées, validées puis utilisées pour l'analyse de prélèvements de l'environnement collectés au cours d'investigations d'épidémies de virus H5N1 au Cambodge et pour l'étude de la survie du virus influenza aviaire dans des biotopes aquatiques artificiels reproduisant le plus possible les conditions naturelles observées dans les mares ou les lacs au Cambodge. L'ARN du virus H5N1 été détecté dans 19% des échantillons environnementaux de terrain collectés au décours des épidémies. Des particules virales infectieuses ont été isolées dans un échantillon d'eau d'une ferme. Dans des systèmes expérimentaux, le virus H5N1 infectieux persiste seulement 4 jours dans l'eau de pluie. Mais l'ARN viral peut encore être détecté jusqu'à 20 jours dans l'eau de pluie et 7 jours dans l'eau de mare ou de lac. Dans la boue, les particules virales infectieuses ne semblent pas pouvoir survivre bien que l'ARN puisse persister au moins 2 semaines. La faune et la flore aquatique n'ont aucune influence sur la persistance du virus infectieux dans l'eau. Ces organismes semblent être essentiellement des concentrateurs et des transporteurs passifs du virus plutôt que des hôtes autorisant la réplication du virus. Nos résultats montrent que l'environnement au cours d'épidémies est fortement contaminé par le virus H5N1 et pourrait constituer une source potentielle de contamination humaine et/ou animale. Une restriction de l'accès à l'eau potentiellement contaminée doit être recommandée autour des foyers épidémiques. La surveillance de l'environnement doit être intégrée dans les programmes de lutte contre la grippe aviaire qui doivent par conséquent prendre en considération des mesures de désinfection de l'environnement. / Although the persistence of the H5N1 virus in the environment is possible, there is no well-defined and standardized method for the detection of viruses from water, soil or mud. In addition, there is very little data available regarding the role of H5N1 virus in the environment in the tropics. In this work methods of concentration, identification, and quantification of influenza viruses in water, mud and soils have been developed, validated and used to test environmental samples collected following H5N1 outbreaks in Cambodia and to analyze samples obtained during experiments in artificial aquatic biotopes aiming to reproduce as faithfully as possible the characteristics observed in ponds and lakes in Cambodia. The H5N1 viral RNA was detected in 19% of environmental samples. Among these, infectious viral particles were isolated in a single water sample. In experimental systems, the infectious H5N1 virus survived only 4 days in the rain water. But viral RNA persisted up to 20 days in rain water and 7 days in pond and lake water. In mud, infectious viral particles did not survive even viral RNA could persist for at least 2 weeks. Aquatic flora and fauna have no influence on infectious H5N1 virus persistence in water. These organisms seem to concentrate and to passively carry the virus but do not allow virus replication. Our results showed that following outbreaks, the environment is widely contaminated by H5N1 virus and therefore can act as a potential source of human and/or animal contamination. Restricted access to potentially contaminated water should be recommended during outbreak episodes. Monitoring the environment is recommended in the effort to fight against avian influenza and measures including environment disinfection should also be considered.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Filosofia e Ciências Humanas. Programa de Pós-Graduação em Sociologia Política. / Made available in DSpace on 2012-10-23T03:25:42Z (GMT). No. of bitstreams: 1 248376.pdf: 623101 bytes, checksum: a7887c8e8a25d571d42e7efaa3330f08 (MD5) / O presente trabalho analisa sob uma perspectiva sociológica as medidas preventivas tomadas frente aos riscos da Gripe Aviária no Estado de Santa Catarina (Brasil). Ainda que a doença não esteja presente no Brasil, consideramos importante entender como tais medidas são formuladas e adotadas e que articulações sociais, políticas e econômicas são desencadeadas frente a um tipo de risco que pode ser de graves conseqüências na alta modernidade. O principal foco de estudo foi como o Estado e as agroindústrias em Santa Catarina (entre as mais importantes do país) estão percebendo e agindo com relação à Influenza Aviária. A dissertação inicia com a definição do que é a Gripe Aviária, traçando uma incipiente perspectiva histórica da doença, para em seguida contextualizá-la internacional, nacional e regionalmente. A luz da teoria da estruturação (Anthony Giddens) e da sociedade de risco (Ulrich Beck), consideramos como a ciência lida com as contingências e ambivalências na sociedade de risco ou alta modernidade. No trabalho de campo foram realizadas entrevistas junto aos membros do Comitê Estadual de Sanidade Avícola do Estado de Santa Catarina, espaço social onde ocorre a interação entre Estado e Agroindústria. Este Comitê desenvolve as medidas preventivas. A partir das entrevistas foram observadas diferenças quanto ao discurso político e científico; a emergência de seis pontos de tensão articulados ao risco; e algumas questões específicas dos fatores sociais e políticos da Influenza Aviária. A partir deste material e de uma extensa pesquisa bibliográfica, a Influenza Aviária é analisada na dinâmica da globalização, levantando-se três tipos de ambivalências: a ambivalência da Influenza Aviária, a do risco em si, e a dos meios de comunicação. No final foram avaliados os diferentes tipos de conhecimento envolvidos no debate, no qual estão presentes os peritos, os meios de comunicação e os leigos. Uma conclusão desta dissertação aponta para a heterogeneidade de conhecimentos envolvida nos conflitos e o debate sobre o controle da Gripe Aviária, abrangendo diferentes racionalidades, como aspecto central para entender as medidas tomadas para seu controle. Em termos gerais, procuramos mostrar como a constituição e atividades do Comitê Estadual expõem as complexas relações entre riscos globais e ações regionais. The present work analyzes through a sociological perspective the preventive measures taken in Santa Catarina State (Brazil) in relation to possible risks of the Avian Flu. Even if the illness is not present in Brazil, we consider important to understand how such measures are formulated and adopted and what economic, political, and social articulations are triggered due to a type of risk that can be of serious consequences in the high modernity. The main focus of study was how the State and the agro-industries in Santa Catarina (between the most important of the country) perceive and acte regarding the Avian Flu. The dissertation initiates with the definition of the Avian Flu, drawing an incipient historical perspective of the illness, for right away put it into international, national and regional contexts. Concerning the theory of structuration (Anthony Giddens) and of the risk society (Ulrich Beck), we consider how science deals with the contingencies and ambivalences in high modernity. In the fieldwork were carried out interviews with the members of the State Committee for the Avian Sanitary Control of Santa Catarina State, social space where take place the interaction between State and agro-industries. This Committee develops the preventive measures. From the interviews we observed differences regarding the scientific and political discourse; the emergency of six tension points articulated to the risk; and some specific social and politics factors. From this material and an extensive bibliographical research, the Avian Flu is analyzed in the dynamic of globalization, raising three kinds of Ambivalences: the one related to the Avian Flu, to the risk itself, and to the media. At the end we evaluate the different kinds of knowledge involved in the debate, in which are present the experts, the media and the laymen. A conclusion of this dissertation emphasized the importance of considering the heterogeneous knowledge involved in the conflicts and the debate about the Avian Flu, including different rationalities, to the understanding of its control. In general terms, we considered that the constitution and activities of the State Committee expose the complex relations between global risks and regional actions.

Suite à la survenue des cas humains de grippe aviaire au Cambodge, il est devenu nécessaire en termes de santé publique d'évaluer l'ampleur de la transmission dans la population et d'identifier les sources potentielles et les modes de transmission à l'homme afin de pouvoir mettre en place des mesures de contrôle et de prévention efficaces. Les travaux de cette thèse ont pour objectif principal d'étudier la transmission du virus A(H5N1) et les facteurs de risque d'infection humaine. Cette thèse rapporte des résultats des études au Cambodge listées ci-dessous. (1) Des études sérologiques et sur les facteurs de risque de l'infection par le virus A(H5N1) ont été conduites en 2006. (2) Une autre étude sérologique a été faite en 2007. (3) Les investigations séro-épidémiologiques ont été menées en 2008-2010 avec une analyse des données cumulées sur cinq années (2006-2010). (4) Une évaluation des comportements et pratiques en lien avec le risque de transmission du virus A(H5N1) dans la population rurale cambodgienne a été menée entre 2006 et 2007. (5) Les caractérisations du virus A(H5N1) isolés au Cambodge durant 2004-2007 et l'exploration de l'origine de l'introduction du virus au Cambodge ont été réalisées à l'aide des méthodes moléculaires. / Following the occurrence of A (H5N1) human cases in Cambodia, it became necessary in terms of public health to evaluate the extend of the transmission in the population and to identify the potential sources and routes of transmission to human in order to set up efficient control and preventive measures. The main objective of this work is to study the transmission and risk factors for infections in human. The thesis reports the results the studies listed below. (1) Serology studies and studies on risk factors for A (H5N1) infections were conducted in 2006. (2) Another serology study was done in 2007. (3) Sero-epidemiological investigations were also conducted during 2008-2010 with analysis of pooled data from 5 years (2006-2010). (4) An assessment of behaviors and practices with regard to the risk of A (H5N1) transmission in Cambodian rural population was done in 2006-2007. (5) Characterizations of A (H5N1) virus isolated in Cambodia were also carried out using molecular methods.

With a case fatality rate higher than the 1918 Spanish Flu pandemic, H5N1 highly pathogenic avian influenza represents a threat to global public health. Efforts to identify locations with the greatest potential for pandemic emergence, as well as how the virus is spreading, may help minimize this threat. First detected in Egypt in 2006, H5N1 viruses have resulted in the deaths of millions of birds in both commercial and backyard poultry flocks, and more than 350 human infections, the most of any country, have been confirmed. Human outbreaks have been so far constrained by poor viral adaptation to non-avian hosts. There are two evolutionary mechanisms by which the H5N1 avian influenza virus could acquire pandemic potential: 1) via reassortment as a result of coinfection with another subtype (such as low pathogenic avian influenza H9N2); and/or 2) via antigenic drift and the accumulation of randomly occurring genetic changes found to improve viral fitness, herein called key substitutions (KS). Both mechanisms were investigated using geospatial methods including ecological niche modeling and hot spot analyses to predict locations with elevated potential for pandemic emergence. Using ecological niche modeling environmental, behavioral, and population characteristics of H5N1 and H9N2 niches within Egypt were identified, with niches differing markedly by subtype. Niche estimates were combined using raster overlay to estimate co-infection potential, with known occurrences used for validation. Co-infection was successfully predicted with high accuracy (area under the receiver operating characteristic (ROC) curve (AUC) 0.991). 41 distinct KS in H5N1 were detected in Egyptian isolates, including 17 not previously reported in Egypt. Phenotypic consequences of detected KS were varied, but the majority have been implicated in improving mammalian host adaptation and increasing virulence. Statistically significant spatial clustering of high KS rates was detected in the northwestern portion of the Nile River delta in the governorates of Alexandria and Beheira. To investigate how the virus spreads between poultry farms, landscape genetics techniques were employed. Viral genetic sequences were evaluated using phylogenetics to determine viral relatedness between samples, then distance models representing competing diffusion mechanisms were created using road networks and a least-cost path model designed to approximate wild waterbird travel using niche modeling and circuit theory. Spatial correlations were evaluated using Mantel tests, Mantel correlograms, and multiple regression of distance matrices within causal modeling and relative support frameworks. Samples from backyard farms were most strongly correlated with least cost path distances, implicating wild bird diffusion, while samples from commercial farms were most strongly correlated with road network distances, implicating human-mediated diffusion. Results were largely consistent across gene segments. Identifying areas at risk of co-infection can help target spaces for increased surveillance. Similarly, detecting spatial hot spots of KS highlight areas of concern for pandemic emergence from antigenic drift. Demonstration of different diffusion mechanisms by farm type should inform both surveillance and biosecurity practices. Knowledge of where to focus intervention efforts, both spatially and strategically, allows limited public health resources to be targeted most effectively. By detecting where in the country pandemic influenza is likely to emerge and identifying how the virus is spreading between farms, this work contributes to efforts to predict and prevent the next influenza pandemic.

Influenza viruses cause significant mortality and morbidity worldwide due to seasonal oubreaks as well as occassional, and sometimes devastating, pandemics. Estimates state that approximately 5% of the adult and 20% of the child population is infected yearly, leading to approximately a half-million deaths and three million severe infections in non-pandemic years. Aside from globally-circulating strains, zoonotic outbreaks caused by avian strains are a constant threat. In 1997, the first human cases of H5N1 infections occurred and since then strains of this subtype have killed approximately 700 people causing a severe disease with as high as 60% lethality rate. In March 2013, a strain of the H7N9 subtype started an epizootic in China causing a severe respiratory disease reminiscent of H5N1 infections and with a 20% case fatality rate. In this thesis, we have studied the host responses as well the viral replication kinetics of H5N1 and H7N9 strains and compared then to those of mild H1N1 seasonal and 2009 pandemic strains. During early infections of A549 cells, we have shown that the H5N1 virus induced a more profound and functional change to the host proteome. All viruses induced the NRF2-mediated oxidative stress responses and the H7N9 and H5N1 strains downregulated fibronectin, a host protein vital to infection for human strains. Using mathematical modeling and extensive growth kinetic analysis, we showed that the H5N1 and H7N9 strains had higher peak titers and faster growth kinetics. This was due to an higher infection rate for the H7N9 strain and an higher production rate for the H5N1 strain, compared to the human viruses. Conversely, the 2009 pandemic H1N1 strain had the poorest replication kinetics, longest eclipse phase and lowest infection rates. These results point towards the higher level of cellular disruption during infection with highly pathogenic strains of influenza, which may be indicative of the more profound changes required to support growth of viruses with faster kinetics to higher titers. Furthermore, the greater changes in the cellular proteome that we have characterized in vitro may be connected to the significantly greater virulence associated with infection by avian viruses in vivo, opening a novel and productive avenue of investigation to understand viral virulence mechanisms. / October 2015

Those who are interested in the public mood, including politicians and economists, comment that the public are becoming ever more sceptical about many things, but health risk information should not be one of them. If health risk information is perceived by the public as ‘just another scary story’, or ‘more of the same we heard last month’, then the ability of risk messages to convey urgency and recommend action could be greatly diminished; the ‘cry wolf’ scenario becomes more real every time a threat appears in the media but fails to materialise. This thesis explores how avian influenza, (H₅N₁), as a health risk category, has been reported and represented in the New Zealand media. By analysing avian influenza-related items in four New Zealand newspapers over a six-year period, 2002-2008, and by comparing results with those found in a U.S. study by Dudo, Dahlstrom & Brossard (2007), this thesis explores the dominant themes and discourses the media drew upon when reporting the health threat of avian influenza. In addition, data from four focus groups sessions was analysed for the purpose of exploring public perceptions of health risk messages and the influence of the media on those perceptions. This thesis was situated within a constructionist epistemology, and employed a mixed-methods methodology with content, thematic and textual analyses. Risk communication theories and models, media conventions of agenda-setting and framing, and sociological concepts informed how the topic of health risk communication was operationalised. The analysis of the focus group data explored how the participants discussed the threat of H₅N₁; how they constructed concepts of personal and community risk, what role, if any, they attributed to the media in their construction and how they positioned themselves in regards to illness and contagion. The focus group analysis revealed that three dominant themes - risk, media and ‘othering’ – represented how the focus group participants talked about the risk of avian influenza. These and several sub-dominant themes shared similarities to those found in the newspaper analysis. Whilst initial discussions seemed to indicate a nonchalant attitude towards the risk of avian influenza, the many topics and themes that characterised the way the participants discussed the risk between them, showed that they had thought about the personal consequences of a possible health risk, and had formed strong opinions about many facets of that risk. Results from the newspaper analysis largely mirrored those of the above U.S. study, and showed that the New Zealand media favoured episodic over thematic framing; sensationalising the reporting of avian influenza, whilst providing little in the way of scientific and contextual information. Moreover, the analysis showed that, when reporting health risks, media templates are well established. The analysis of the focus group data revealed that the participants wanted media health risk messages to be clear, concrete and factual. However, this desire for messages that communicate certainty about risk, which is, by definition inherently uncertain, raises questions about the very nature of risk communication. Findings of this thesis suggest that future risk communication research should focus, not on how the media are reporting health risks, but how the public conceptualise risk, construct it in times of crisis and evaluate their ability to control it.