L’apparition d’une réponse immunitaire contre le Facteur VIII (FVIII) de la coagulation est une complication majeur qui survient chez 30% des hémophile A sévères. Bien que des avancées importantes aient abouti au développement de nouvelles molécules de FVIII thérapeutiques, les mécanismes conduisant à l’apparition d’une réponse immunitaire anti-FVIII restent non élucidés. Des facteurs de risques génétiques et environnementaux ont été identifiés ou suggérés, mais une compréhension complète des processus immunologiques permettant l’initiation de cette réponse au dépend de l’induction de tolérance immune chez 30% des patients restent incomprise. Ma thèse porte sur les aspects fonctionnels et structurels du FVIII et leur rôle dans l’initiation de la réponse immunitaire anti-FVIII chez le modèle murin hémophile A. Le premier rôle du FVIII est sa participation à la cascade de la coagulation, et donc la première partie de ma thèse adresse le rôle du processus de coagulation dans l’initiation de la réponse immunitaire anti-FVIII. La seconde partie de ma thèse se concentre sur l’importance des résidus du domaine C2 impliqué dans la liaison aux phospholipides dans l’endocytose et la présentation du FVIII par les cellules présentatrices de l’antigène in vitro et discute de leur relevance in vivo. / Immunogenicity of Factor VIII (FVIII) is a major hurdle that affects about 30% of severe hemophilia A patients. Though a significant advancement has been accomplished in the development of newer FVIII molecules, the factors that drive FVIII immune responses remain elusive. Many genetic and environmental risk factors have been identified or suggested but a complete understanding of the immunological basis for the antibody formation and the mechanism(s) behind tolerance induction, in the 30% of the patients that never develop anti-FVIII antibodies, are not understood. My thesis involves overlapping aspects important for initiation of an anti-FVIII immune response in a mouse model of hemophilia A. The primary role of FVIII is its participation in coagulation-associated events and thus, the first part of my thesis addresses whether coagulation events per se are implicated in the initiation of anti-FVIII immune responses. The second part of my thesis focuses on the importance of the membrane binding residues within the C2 domain of FVIII in antigen uptake and presentation by antigen presenting cells in vitro and discusses its relevance in vivo.

L’hémophilie A est une maladie hémorragique rare liée au chromosome X qui se traduit par un déficit en facteur VIII (FVIII) fonctionnel. Chez les patients atteints de la forme sévère de l’hémophilie A, l’apparition d’hémorragies incontrôlées augmente la morbidité et affecte leur qualité de vie. Le traitement de choix permettant de prévenir ou de traiter les saignements consiste en l’injection intraveineuse de FVIII thérapeutique. Cependant, chez 5 à 30% des patients, une réponse immunitaire dirigée contre le FVIII se développe. La réponse anti-FVIII est caractérisée par l’apparition d’anticorps qui inhibent l’activité pro-coagulante du FVIII. Ainsi l’apparition de ces anticorps, appelés « inhibiteurs » représente-t-elle la complication majeure du traitement des patients hémophiles A. Plusieurs facteurs de risque ont été suggérés ou proposés comme affectant le développement de la réponse anti-FVIII. Parmi eux, supporté par la théorie du « danger », la présence de médiateurs pro-inflammatoires générés par les épisodes de saignement est considérée comme adjuvant dans la réponse immunitaire anti-FVIII. Cependant, les saignements induisent de nombreux mécanismes pro mais aussi anti-inflammatoires. Ainsi, l’activation de l’endothélium vasculaire induit-elle le recrutement et l’activation des cellules effectrices appartenant aux compartiments innés et adaptatifs du système immunitaire. Au cours de ma thèse, je me suis concentré sur le rôle des saignements dans le développement de la réponse immunitaire dirigée contre le FVIII. Ainsi, ai-je découvert une association entre la présence d’un polymorphisme dans un gène induit par les saignements et la présence d’inhibiteurs du FVIII dans une cohorte de patients hémophiles A sévères. J’ai également étudié le développement de la réponse immunitaire dirigée contre le FVIII thérapeutique dans un modèle murin qui mime les saignements locaux observés chez les patients hémophiles A sévères. Finalement, j’ai caractérisé l’effet des formes réactives de l’oxygène (FRO), qui sont générées au niveau du site de saignement, sur la structure, la fonction et l’immunogénicité du FVIII. Les résultats obtenus au cours de ma thèse démontrent que les saignements ne sont pas associés à un risque plus élevé de développer des inhibiteurs dans le modèle murin largement utilisé d’hémophilie A sévère, contrairement à ce qui est communément admis. De plus, j’ai démontré une association entre un polymorphisme présent dans le promoteur du gène HMOX1 et la survenue d’inhibiteurs dans une cohorte de patients atteints d’hémophilie A sévère. En parallèle, je décris pour la première fois une altération du statut oxydatif chez les souris déficientes en FVIII et je démontre que le contrôle du statut oxydatif in vivo permet de moduler la réponse immunitaire anti-FVIII. Au contraire, l’exposition du FVIII aux FRO augmente son immunogénicité. De fait, mes résultats suggèrent que, bien que les molécules pro-inflammatoires libérées suite aux saignements puissent affecter positivement la réponse immunitaire dirigée contre le FVIII, de puissants médiateurs anti-inflammatoires sont générés in vivo et amenuisent l’effet potentiellement adjuvant des saignements. Également, ces résultats soulignent la balance complexe entre les médiateurs pro- et anti-inflammatoires qui sont générés consécutivement aux saignements ainsi que leurs effets sur la réponse immunitaire dirigée contre le FVIII thérapeutique. / Hemophilia A is a rare X-linked hemorrhagic disease consecutive to the lack of functional pro-coagulant factor VIII (FVIII). In patients with the severe form of hemophilia A, uncontrolled hemorrhages increase the morbidity and affect the quality of life. To prevent or treat bleeding episodes, therapeutic FVIII is administered intravenously. However, an anti-FVIII immune response develops in 5 to 30% of the patients. The anti-FVIII immune response is characterized by the development of anti-FVIII IgG antibodies that inhibit FVIII activity. These antibodies, referred to as “FVIII inhibitors” represent the major complication in the treatment of hemophilia A patients. Several risk factors have been suggested or proposed to predispose to the development of FVIII inhibitors. Amongst them, inspired by the “danger” theory, the presence of inflammatory mediators released upon bleeding episodes is thought to adjuvant the anti-FVIII immune response. Bleeding episodes trigger several pro and anti-inflammatory mechanisms. Thus, damage to the endothelium triggers the recruitment and activation of effector cells from the innate and adaptive compartments of the immune system. During my PhD, I investigated the role of bleedings in the development of the anti-FVIII immune response. Thus, I discovered an association between a polymorphism in the promoter region of a gene that is induced in response to bleedings, and the presence of FVIII inhibitors in a cohort of patients with severe hemophilia A. I followed the development of the immune response to therapeutic FVIII in a mouse model that mimics the localized bleedings found in patients with severe hemophilia A. Ultimately, I characterized the effects of reactive oxygen species (ROS) that are potentially released at the bleeding site in view of the structure, function and immunogenicity of the FVIII molecule. The results I obtained during my PhD demonstrate that bleedings are not associated with a higher risk for FVIII inhibitor development in the commonly used mouse model of severe FVIII deficiency. Additionally, I demonstrated an association between a polymorphism in the promoter of the HMOX1 gene and the presence of FVIII inhibitors. In parallel, I report for the first time an exacerbated oxidative status in FVIII-deficient mice as compared to control mice, and demonstrate that the control of the oxidative status in vivo reduces the anti-FVIII immune response. Conversely, the exposure of FVIII to ROS ex vivo increases the immunogenicity of the FVIII molecule. Taken together, my results suggest that, although the pro-inflammatory molecules released upon bleeding may positively affect the anti-FVIII immune response, several strong anti-inflammatory compounds are generated in vivo that dampen the potential adjuvant effect of bleedings. Ultimately, these results highlight the complex balance between the pro and anti-inflammatory mediators generated upon bleeding and their effect on the anti-FVIII immune response.

The aim of the work is to extend the hitherto superficial knowledge of the history and development of the profession of Czech film stuntmen, from the 1960s to the post- revolutionary period with overlapping to the present. Using the oral history method (combined with available sources), the work will focus on professional development. Attention will be paid to the creation of a profession as such, key films or contacts with foreign productions. Other subtopics include stunt training, stunt equipment, or a woman's view of this predominantly male occupation. Topics will be reflected in pre- revolutionary and post-revolutionary years. Subsequently, the stumbling block will be evaluated by the greatest professional differences that have occurred over the years.. Key words: Stuntmen, horses, filming, oral history, dangers

The U.S. Southwest Border is associated with highly politicized topics, yet the lived experience of Border Patrol agents is not one of them. Border Patrol agents face risks to their personal safety and security as they attempt to safeguard the national security of the United States while implementing the policies of their organization, which are sometimes at odds with the beliefs and expectations of agents in the field. The purpose of this phenomenological study was to explore perceptions and lived experiences related to the threats and dangers that Border Patrol agents face as they protect the U.S. Southwest Border. The theoretical framework for this study involved McGregor's organizational behavior theory, Janis's groupthink theory, and the bureaucratic dissonance phenomenon. Data collected through semistructured interviews of 11 former Border Patrol agents with direct experience working along the U.S. Southwest Border were inductively coded and subjected to a thematic analysis procedure. On-duty risks, emotional toll, lack of community support, and separation from family are among the stressors for members of this profession. The key findings regarding threats and dangers included: perceived manpower shortage, fear of assaults, the very nature of the job, political and presidential administration conflicts, and lack of mobility (location and career advancements). The recommendations call for greater policy-and decision-maker understanding of the stresses and conflicts facing Border Patrol agents, which could effect positive social change by encouraging policies and regulations to improve job safety and security, and to inform training programs. The promulgation of the findings may contribute to improvements of the morale and safety of Border Patrol agents and enhance security of the United States.

Syftet var att undersöka föräldrars upplevelser kring barns användning av skärmmedier. Med utgångspunkt i struktureringsteorin, cultural studies och identitetsteorin har uppsatsförfattaren undersökt frågor som berör föräldrar och barns relationer. Några frågor som har undersökts är om föräldrar använder begränsad skärmtid till barn, om föräldrar ansåg att skärmar skapade beteendeförändringar och konflikter och om barn mött faror på nätet. Undersökningen ville belysa föräldrars positiva och negativa bild av barns skärmanvändning. För att besvara frågorna användes en surveyundersökning som delades i föräldragrupper på Facebook där 323 föräldrar svarade. I analysen fick uppsatsförfattaren bland annat reda på att föräldrar engagerar sig i barns skärmmedia, föräldrar spelar spel, ser film och serier med barn och föräldrar upplever att barn införskaffar sig kunskap via media. Föräldrar läser ämnesrelaterad litteratur för barn om faror på nätet med förhoppning om att de ska våga berätta om de möter något obehagligt på nätet. Föräldrar märkte även av en del konflikter och beteendeförändringar hos barn. / The purpose was to investigate parents' experiences of childrens use of screen media. Based on structuring theory, cultural studies and identity theory, the essay author has investigated issues that affect parents and childrens relationships. Some questions that have been investigated are whether parents use limited screen time for children, whether parents considered that screens created behavioral changes and conflicts and whether children encountered dangers online. The survey wanted to shed light on parents' positive and negative image of children's screen use. To answer the questions, a survey was used that was divided into parent groups on Facebook where 323 parents answered. In the analysis, the essay author found out, among other things, that parents get involved in children's screen media, parents play games, watch movies and series with children and parents experience that child acquire knowledge via the media. Parents read subject-related literature for children about dangers online with the hope that they will dare to tell if they encounter something unpleasant online. Parents also noticed some conflicts and behavioral changes in children.

This research study was conducted to determine if there were significant differences or relationships between teachers' perceptions of knowledge of Internet dangers to students and factors such as demographics, self-reported comfort levels with Internet use, and with knowledge of policies and laws regarding Internet safety. Three hundred seventy-two teachers from three central Florida school districts were surveyed using an adapted survey developed by authors Patchin and Hinduja (2006) and Willard (2006, 2009). The world of the Internet is constantly changing, and students will encounter harassment and dangers while pursuing their interests on line. Recent research on the types of Internet dangers involving youth (Hinduja & Patchin, 2009; Juvonen & Gross, 2008, Leichtling, 2008; Lenhart, Madden & Hitlin, 2005; Li, 2007; Willard, 2009), and evidence supporting limited adult knowledge of Internet dangers to youth (Finkelhor, Mitchell & Wolak, 2000; Hinduja & Patchin, 2009; Patchin & Hinduja, 2006; Willard, 2006,2009) provided the conceptual framework for this study. Descriptive and inferential statistics were performed to investigate each research question. These statistics included one-way ANOVA, Scheffe post-hoc analysis, chi-square tests of independence, independent T-tests, and Kruskal-Wallis tests. These findings demonstrated that the null hypotheses were rejected for each research question. Significant differences were found between teachers' knowledge of Internet dangers and age, years of experience, level of school taught, and personal comfort with the Internet. Other significant relationships were found between personal Internet comfort and knowledge of laws and policies, specifically the Jeffrey Johnston Stand Up for All Students Act, and between male teachers and knowledge of victim behaviors. Gender was the only demographic variable found to be not significantly related to a teachers' knowledge of Internet dangers to students. The implications of these results validate the importance of more teacher training to increase knowledge of Internet dangers, policies and programs, as well as increase teachers' ability to identify victims and provide them with assistance. As technology expands, Internet dangers for children online expand and are a growing concern for parents, teachers, and administrators. Since technology will continue to grow, adults play a major role in educating children concerning the dangers of being online. However, adults struggle to play catch-up to the young digital natives, and are not really present to intervene when needed. We cannot cross the digital divide and help our students if we are not seeking out the information ourselves (Willard, 2009).

Citocinas geradas durante a I/R-i promovem lesão pulmonar aguda e podem causar imunossupressão. Investigamos se a inflamação pulmonar após dias de reperfusão intestinal está relacionada com alterações no sistema imune. Ratos machos Wistar foram submetidos a oclusão da artéria mesentérica superior por 45 min e a 2 h, 3, 5 ou 20 dias de reperfusão. Observamos que após 5 dias de reperfusão intestinal enterobactérias estão presentes nos linfonodos mesentéricos e que os neutrófilos circulantes estão mais ativos no combate a patógenos, enquanto leucócitos da cavidade peritoneal não. Notamos aumento de IL-1b, TNF-a, IL-17, IL-4 e diminuição de IL-10 no explante pulmonar e aumento de IL-1b, IFN-g e LDH no soro. Já os níveis de nitritos, corticosterona, HMGB-1, ácido úrico, AMPc e a mecânica respiratória não se alteraram. Os resultados apontam para a I/R-i como promotora de modificação temporais no organismo. Alterações nos mecanismos regulatórios da inflamação e do sistema imune contribuem para a inflamação pulmonar e sistêmica, sem alterar a função mecânica pulmonar / Cytokines generated during intestinal I/ R promote acute lung injury and may cause immunosuppression. We investigate if the pulmonary inflammation observed after several days of reperfusion is related to changes in the immune system. Male Wistar rats were subjected to occlusion of the superior mesenteric artery for 45 min and reperfused for 2 h , 3, 5 or 20 days. We observed that after 5 days of reperfusion enterobacteraceae are present in mesenteric lymph nodes and that circulating neutrophils are more effective against pathogens, whereas leukocytes from the peritoneal cavity are not. We noted increase of IL-1b, TNF-a, IL-17, IL -4 and decrease of IL-10 in lung explant and increase of IL-1b, IFN-g and LDH in serum. Furthermore, the levels of nitrites, corticosterone, HMGB -1, uric acid, cAMP, as well as respiratory function, did not change. Based on the foregoing, we observed that alterations in regulatory mechanisms of inflammation and immune system after contribute to pulmonary and systemic inflammation, but do not alter pulmonary mechanical function.

Os resíduos químicos gerados em instituições de ensino e pesquisa trazem grande risco ao meio ambiente e para as pessoas envolvidas com seu manuseio, mesmo sendo produzidos em menor quantidade do que em setores industriais. Isto se deve ao grau de periculosidade e diversidade de seus componentes. Atualmente, é exigido por lei que as instituições da área da saúde se mantenham atualizadas sobre os resíduos perigosos gerados em suas unidades e tenham atitudes para a redução desde a geração destes resíduos. Entretanto, as legislações vigentes não demonstram como tratar misturas de resíduos de complexidade diversificada. Assim, o objetivo do presente trabalho visa elaborar estratégias de tratamento dos resíduos químicos gerados nos laboratórios de ensino e pesquisa. Análises de ações preventivas de minimizar a geração destes resíduos e tratá-los sempre que possível também é o foco desta pesquisa. Para tanto, foram coletadas informações sobre o descarte mensal realizado por cada laboratório gerador de resíduos químicos da Faculdade de Ciências Farmacêuticas da Universidade de São Paulo (FCF/USP) no período de janeiro de 2014 a agosto de 2016. Com esse levantamento, observou-se que existe a possibilidade de tratar in loco alguns dos resíduos produzidos na FCF/USP, como os resíduos classificados em ácidos, bases e soluções aquosas salinas não tóxicas, que totalizam 10% dos resíduos produzidos no período do estudo. Ademais, testes de recuperação do solvente acetonitrila (ACN) a partir de uma mistura ACN-água (60:40) foram realizados demonstrando a possibilidade de recuperação de aproximadamente 50% da acetonitrila inicial com pureza superior a 99% após analise através das técnicas de Karl Fischer e de índice de refração. Alguns dos tratamentos propostos favoreceram a diminuição de custos com a disposição posterior destes resíduos, reduzindo, assim, danos ambientais e proporcionando à instituição, visibilidade perante a sociedade sobre os cuidados e preocupação com o gerenciamento de seus resíduos químicos perigosos. / Chemical waste generated by educational and research institutions lead to a great risk to the environment and people involved with its handling, even though they are produced in less quantity than in industrial sectors. This is due to the level of danger and diversity of their components. Currentely the law requires that institutions from the Health area keep up-to-date about the hazardous waste that they generate and promote actions to reduce their production from waste generation to treatment and disposal. However, the current laws do not demonstrate how to handle with mixtures of wastes of diversified complexity. Thus, the objective of this study is to develop strategies for the treatment of chemical waste generated in teaching and researching laboratories. Analyzes of preventive actions to minimize the generation of these wastes and treat them whenever possible are also the focus of this research. Information were collected every month about waste discharged by each laboratory that generated chemical residues of the School of Pharmaceutical Sciences of the University of São Paulo (FCF / USP) from january 2014 to august 2016. After performing this survey, it was observed that there is the possibility of treating some of the residues produced in FCF/USP in loco, such as acid, bases and salt solutions which correspond to 10% of the waste generated during through the study period. Furthermore, recovery tests concerning solvent acetonitrile (ACN) from ACN-water mixture (60:40) were carried out and showed the possibility of recover about 50 % from the initial ACN with purity above 99% after analysis techniques of Karl Fischer and refractive indices. Some of the proposed treatments encouraged a reduction of costs with further disposal of waste, and thus lessen environmental damage while providing the institution with visibility to society about care and concern with management of its chemical waste.

Os resíduos químicos gerados em instituições de ensino e pesquisa trazem grande risco ao meio ambiente e para as pessoas envolvidas com seu manuseio, mesmo sendo produzidos em menor quantidade do que em setores industriais. Isto se deve ao grau de periculosidade e diversidade de seus componentes. Atualmente, é exigido por lei que as instituições da área da saúde se mantenham atualizadas sobre os resíduos perigosos gerados em suas unidades e tenham atitudes para a redução desde a geração destes resíduos. Entretanto, as legislações vigentes não demonstram como tratar misturas de resíduos de complexidade diversificada. Assim, o objetivo do presente trabalho visa elaborar estratégias de tratamento dos resíduos químicos gerados nos laboratórios de ensino e pesquisa. Análises de ações preventivas de minimizar a geração destes resíduos e tratá-los sempre que possível também é o foco desta pesquisa. Para tanto, foram coletadas informações sobre o descarte mensal realizado por cada laboratório gerador de resíduos químicos da Faculdade de Ciências Farmacêuticas da Universidade de São Paulo (FCF/USP) no período de janeiro de 2014 a agosto de 2016. Com esse levantamento, observou-se que existe a possibilidade de tratar in loco alguns dos resíduos produzidos na FCF/USP, como os resíduos classificados em ácidos, bases e soluções aquosas salinas não tóxicas, que totalizam 10% dos resíduos produzidos no período do estudo. Ademais, testes de recuperação do solvente acetonitrila (ACN) a partir de uma mistura ACN-água (60:40) foram realizados demonstrando a possibilidade de recuperação de aproximadamente 50% da acetonitrila inicial com pureza superior a 99% após analise através das técnicas de Karl Fischer e de índice de refração. Alguns dos tratamentos propostos favoreceram a diminuição de custos com a disposição posterior destes resíduos, reduzindo, assim, danos ambientais e proporcionando à instituição, visibilidade perante a sociedade sobre os cuidados e preocupação com o gerenciamento de seus resíduos químicos perigosos. / Chemical waste generated by educational and research institutions lead to a great risk to the environment and people involved with its handling, even though they are produced in less quantity than in industrial sectors. This is due to the level of danger and diversity of their components. Currentely the law requires that institutions from the Health area keep up-to-date about the hazardous waste that they generate and promote actions to reduce their production from waste generation to treatment and disposal. However, the current laws do not demonstrate how to handle with mixtures of wastes of diversified complexity. Thus, the objective of this study is to develop strategies for the treatment of chemical waste generated in teaching and researching laboratories. Analyzes of preventive actions to minimize the generation of these wastes and treat them whenever possible are also the focus of this research. Information were collected every month about waste discharged by each laboratory that generated chemical residues of the School of Pharmaceutical Sciences of the University of São Paulo (FCF / USP) from january 2014 to august 2016. After performing this survey, it was observed that there is the possibility of treating some of the residues produced in FCF/USP in loco, such as acid, bases and salt solutions which correspond to 10% of the waste generated during through the study period. Furthermore, recovery tests concerning solvent acetonitrile (ACN) from ACN-water mixture (60:40) were carried out and showed the possibility of recover about 50 % from the initial ACN with purity above 99% after analysis techniques of Karl Fischer and refractive indices. Some of the proposed treatments encouraged a reduction of costs with further disposal of waste, and thus lessen environmental damage while providing the institution with visibility to society about care and concern with management of its chemical waste.

Citocinas geradas durante a I/R-i promovem lesão pulmonar aguda e podem causar imunossupressão. Investigamos se a inflamação pulmonar após dias de reperfusão intestinal está relacionada com alterações no sistema imune. Ratos machos Wistar foram submetidos a oclusão da artéria mesentérica superior por 45 min e a 2 h, 3, 5 ou 20 dias de reperfusão. Observamos que após 5 dias de reperfusão intestinal enterobactérias estão presentes nos linfonodos mesentéricos e que os neutrófilos circulantes estão mais ativos no combate a patógenos, enquanto leucócitos da cavidade peritoneal não. Notamos aumento de IL-1b, TNF-a, IL-17, IL-4 e diminuição de IL-10 no explante pulmonar e aumento de IL-1b, IFN-g e LDH no soro. Já os níveis de nitritos, corticosterona, HMGB-1, ácido úrico, AMPc e a mecânica respiratória não se alteraram. Os resultados apontam para a I/R-i como promotora de modificação temporais no organismo. Alterações nos mecanismos regulatórios da inflamação e do sistema imune contribuem para a inflamação pulmonar e sistêmica, sem alterar a função mecânica pulmonar / Cytokines generated during intestinal I/ R promote acute lung injury and may cause immunosuppression. We investigate if the pulmonary inflammation observed after several days of reperfusion is related to changes in the immune system. Male Wistar rats were subjected to occlusion of the superior mesenteric artery for 45 min and reperfused for 2 h , 3, 5 or 20 days. We observed that after 5 days of reperfusion enterobacteraceae are present in mesenteric lymph nodes and that circulating neutrophils are more effective against pathogens, whereas leukocytes from the peritoneal cavity are not. We noted increase of IL-1b, TNF-a, IL-17, IL -4 and decrease of IL-10 in lung explant and increase of IL-1b, IFN-g and LDH in serum. Furthermore, the levels of nitrites, corticosterone, HMGB -1, uric acid, cAMP, as well as respiratory function, did not change. Based on the foregoing, we observed that alterations in regulatory mechanisms of inflammation and immune system after contribute to pulmonary and systemic inflammation, but do not alter pulmonary mechanical function.