Lipoplexes recouverts d’acide hyaluronique pour le ciblage d’ARN interférant à des cellules tumorales surexprimant le récepteur CD44

Recent progresses in the preclinical and clinical use of small interfering RNA (siRNA) have shown their potential as an inhibitor of protein synthesis in many diseases such as cancer. The administration of siRNA encounters a number of problems related to their rapid degradation in biological media, and their difficulty in penetrating targeted cells due to their hydrophilicity and negative charge. A key to improving the therapeutic efficacy of these molecules is based on the use of vectors. In this thesis, lipoplexes that can protect siRNA against degradation and facilitate their transport into target cells were developed and optimized. To do this, lipoplexes covered with HA were formulated for active vectorization of siRNA to tumor cells overexpressing the receptor CD44.In the first part of this thesis, the formation of lipoplexes was studied, and the parameters influencing their supramolecular organization. Insertion of HA within the liposome structure during vesicle formation resulted in the increase in liposome size as a function of HA concentration. Their complexation with siRNA has further increased the size of the particles obtained. The addition of siRNAs when forming lipoplexes caused a displacement of a portion of the HA-DOPE conjugate from the lipoplexes structure, as shown by capillary electrophoresis. The isothermal titration calorimetry and X-ray diffraction studies showed that a rearrangement of the lipid bilayers occur under the effect of electrostatic interactions with siRNAs, leading to the formation of oligolamellar vesicles, which was visually confirmed by cryo-microscopy. Finally, the proper positioning of the HA on the surface of the lipoplexes and its ability to specifically bind to the CD44 receptors has been demonstrated by the surface plasmon resonance technique.In the second part of this thesis, cellular uptake and intracellular localization of HA-lipoplexes were assessed by flow cytometry and fluorescence microscopy, and showed that lipoplexes modified by HA are internalized more rapidly than unmodified lipoplexes, and once in the cells, they are mainly localized within the endosomes. The ability of lipoplexes to transport intact siRNA molecules to the cytoplasm was confirmed by 81% of luciferase in vitro expression inhibition on the lung cancer cell line A549-luc. In vivo, treatment with HA-lipoplexes carrying anti-luciferase siRNA led to a statistically significant decrease in expression of luciferase, which was confirmed by reducing the mRNA expression of luciferase in lungs of animals treated with HA-lipoplexes. The analysis of the distribution of lipoplexes in the lungs showed that lipoplexes modified with HA are distributed more evenly in the lung tissue than unmodified lipoplexes.In the third part of this thesis, the movement of siRNA HA-lipoplexes in the mucus was studied to assess the feasibility of administering these particles directly to the lungs. Studies using the technique of "multiple particle tracking (MPT)" showed that the presence of HA combined with the addition of siRNA allowed the preparation of two lipoplexes formulations with efficient mucus-penetration, HA-lipoplexes and PEG/HA-lipoplexes.In conclusion, an efficient siRNA lipoplex system for inhibiting gene expression targeted TO the CD44 receptorS has been developed. The results confirm that the HA-lipoplexes are able to effectively release in vitro and in vivo the siRNA molecules in the cytoplasm of cells.Des progrès récents dans l’utilisation préclinique et clinique des petits ARN interférents (siRNA) ont montré leur potentiel en tant qu’inhibiteur de la synthèse protéique dans de nombreuses pathologies comme le cancer. L’administration des siRNA rencontre un certain nombre de problèmes liés à leur dégradation rapide dans les milieux biologiques, ainsi qu’à leur difficulté à pénétrer au sein des cellules cibles en raison de leur hydrophilie et de leur charge négative. Une des clés de l’amélioration de l’efficacité thérapeutique de ces molécules repose sur l’emploi de vecteurs. Au cours de cette thèse, des lipoplexes capables de protéger les siRNA contre la dégradation et de favoriser leur transport jusqu’aux cellules cibles ont été développés et optimisés. Pour ce faire, des lipoplexes recouverts d’HA ont été formulés pour la vectorisation active de siRNA vers des cellules tumorales surexprimant le récepteur CD44. Dans la première partie de cette thèse, la formation des lipoplexes a été étudiée, ainsi que les paramètres influençant leur organisation supramoléculaire. L'insertion de l'HA dans la structure du liposome au moment de la formation des vésicules a entraîné une augmentation de la taille des liposomes en fonction de la concentration d’HA. Leur complexation avec les siRNA a encore augmenté la taille des particules obtenues. L’ajout des acides nucléiques lors de la formation des lipoplexes a provoqué un déplacement d'une partie du conjugué HA-DOPE de la structure des lipoplexes, comme montré par électrophorèse capillaire. La titration calorimétrique isotherme et les études de diffraction des rayons X ont démontré que, sous l’effet des interactions électrostatiques avec les siRNA, un réarrangement des bicouches lipidiques a lieu, conduisant à la formation de vésicules oligolamellaires, confirmé visuellement par cryo-microscopie. Enfin, le positionnement convenable de l’HA sur la surface des lipoplexes et sa capacité de se lier spécifiquement aux récepteurs de CD44 ont été démontrés par la technique de résonance plasmonique de surface. Dans la deuxième partie de cette thèse, la capture cellulaire et localisation intracellulaire des lipoplexes-HA ont été évalués par cytométrie en flux et microscopie de fluorescence, et ont montré que les lipoplexes modifies par l’HA sont internalisés plus rapidement que les lipoplexes non modifiés, et une fois dans des cellules, ils sont localisés principalement à l’intérieur des endosomes. La capacité des lipoplexes à transporter des molécules de siRNA intactes au cytoplasme a été confirmée par 81 % d'inhibition d'expression de luciferase in vitro sur la lignée cellulaire de cancer du poumon A549-luc. In vivo, le traitement avec les lipoplexes-HA transportant un siRNA anti-luciferase a mené à une diminution statistiquement significative de l’expression de luciferase, ce qui a été confirmé par la réduction de l'expression d’ARNm de la luciferase dans le poumon des animaux traités avec les lipoplexes-HA. L'analyse de la distribution des lipoplexes dans les poumons a montré que les lipoplexes modifiés par l’HA sont distribués de façon plus importante et plus homogène dans le tissu pulmonaire que les lipoplexes non modifiés. Dans la troisième partie de cette thèse, la diffusion des lipoplexes-HA de siRNA dans le mucus a été étudié, afin d’évaluer la faisabilité de l’administration pulmonaire de ces particules. Les études utilisant la technique de « multiple particle tracking (MPT) » ont montré que la présence d’HA combinée à l'ajout de siRNA ont permis l’obtention de deux formulations de lipoplexes présentant une pénétration efficace dans le mucus, les lipoplexes-HA and les lipoplexes-PEG/HA. En conclusion, un système efficace de lipoplexes utilisant siRNA pour l'inhibition de l'expression génique ciblant les récepteurs CD44 a été développé. Les résultats obtenus confirment que les HA-lipoplexes sont capables de libérer efficacement le siRNA dans le cytoplasme des cellules in vitro et in vivo

In Vivo Biodistribution of Respirable Solid Lipid Nanoparticles Surface-Decorated with a Mannose-Based Surfactant: A Promising Tool for Pulmonary Tuberculosis Treatment?

The active targeting to alveolar macrophages (AM) is an attractive strategy to improve the therapeutic efficacy of ‘old’ drugs currently used in clinical practice for the treatment of pulmonary tuberculosis. Previous studies highlighted the ability of respirable solid lipid nanoparticle assemblies (SLNas), loaded with rifampicin (RIF) and functionalized with a novel synthesized mannose-based surfactant (MS), both alone and in a blend with sodium taurocholate, to efficiently target the AM via mannose receptor-mediated mechanism. Here, we present the in vivo biodistribution of these mannosylated SLNas, in comparison with the behavior of both non-functionalized SLNas and bare RIF. SLNas biodistribution was assessed, after intratracheal instillation in mice, by whole-body real-time fluorescence imaging in living animals and RIF quantification in excised organs and plasma. Additionally, SLNas cell uptake was determined by using fluorescence microscopy on AM from bronchoalveolar lavage fluid and alveolar epithelium from lung dissections. Finally, histopathological evaluation was performed on lungs 24 h after administration. SLNas functionalized with MS alone generated the highest retention in lungs associated with a poor spreading in extra-pulmonary regions. This effect could be probably due to a greater AM phagocytosis with respect to SLNas devoid of mannose on their surface. The results obtained pointed out the unique ability of the nanoparticle surface decoration to provide a potential more efficient treatment restricted to the lungs where the primary tuberculosis infection is located

Impact of public health and higher education policies on the profile of final-year Brazilian dental students : challenges and future developments

Introduction: Brazil has experienced transformations in higher education and health services, including launching more inclusive public policies focused on these two areas. Objective: To evaluate the profile of final-year dental students from a Brazilian public university from 2010 to 2019, accompanied by changes in public health and higher education policies. Methods: A prospective observacional study was carried out with final-year dental students. A self-applicable semi-structured questionnaire was applied. Result: Six-hundred and seventy-seven students participated, of which 71.5% were women, 72.9% aged between 21 and 25 years, 96.2% single, and 96.4% were without children. Over ten years, it was possible to identify trends in the profile explained by implementing public policies toward more inclusive access to Brazilian higher educa tion by socioeconomically disadvantaged individuals. Students who completed the course between 2018 and 2019 did not have the state capital city (the wealthiest area) as their origin city and had lower parental education and income levels than dental students graduating between 2010 and 2011. Moreover, working as a primary care dentist in the Brazilian National Health System was considered a professional possibility by 61.4% of the students, and has gained prominence significantly over time, ranging from 21.1% in 2010 to 72.9% in 2019 (p< .05). Conclusion: Over the study period, concomitantly to advances in public health and higher education policies in Brazil, more diverse access to public dental education was observed, allowing students from low socioeconomic positions to take the education. Changes have also impacted the students' perspectives regarding the need for post graduate training and a career in public health dentistry. However, these trends need to be consolidated, and public policies continued and strengthened

Fatores que influenciam na qualidade de vida de homens com neoplasias malignas: uma revisão integrativa Factors influencing the quality of life of men with malignant neoplasms: an integrating review

Objetivo: Revisar fatores que influenciam na qualidade de vida de homens com neoplasias malignas. Métodos: Trata-se de uma revisão integrativa da literatura realizada nas bases de dados SCOPUS, CINAHL, LILACS e PUBMED. Resultados e discussão: Após a aplicação dos critérios de inclusão e exclusão obteve-se como resultado, uma amostra de oito artigos. Logo, emergiram três categorias: Atividade física como melhoria para a qualidade de vida de homens com neoplasias malignas; O funcionamento sexual e o estado civil da população masculina como fatores para a qualidade de vida; Autoestima e bem-estar: fatores condicionantes para a qualidade de vida do homem com câncer. Conclusão: Observou-se que estudos voltados para a população masculina com neoplasias malignas são insuficientes e necessitam de atenção para este grupo pouco estudado.Descritores: Qualidade de vida, neoplasia, saúde do homem

Fatores que influenciam na qualidade de vida de homens com neoplasias malignas: uma revisão integrativa Factors influencing the quality of life of men with malignant neoplasms: an integrating review

Objetivo: Revisar fatores que influenciam na qualidade de vida de homens com neoplasias malignas. Métodos: Trata-se de uma revisão integrativa da literatura realizada nas bases de dados SCOPUS, CINAHL, LILACS e PUBMED. Resultados e discussão: Após a aplicação dos critérios de inclusão e exclusão obteve-se como resultado, uma amostra de oito artigos. Logo, emergiram três categorias: Atividade física como melhoria para a qualidade de vida de homens com neoplasias malignas; O funcionamento sexual e o estado civil da população masculina como fatores para a qualidade de vida; Autoestima e bem-estar: fatores condicionantes para a qualidade de vida do homem com câncer. Conclusão: Observou-se que estudos voltados para a população masculina com neoplasias malignas são insuficientes e necessitam de atenção para este grupo pouco estudado. Descritores: Qualidade de vida, neoplasia, saúde do homem

Perfil epidemiológico de sífilis adquirida diagnosticada e notificada em hospital universitário materno infantil

Objective: To characterize the epidemiologic profile of users with acquired syphilis.Method: This was an exploratory and descriptive study of quantitative approach, through secondary data collected in investigation forms/syphilis notification in women assisted in a Maternal-Child University Hospital in Santa Cruz-RN in 2012. The study was approved by the Research Ethics Committee under report number 772.884.Results: 67% of mothers reported they had only attended elementary school, 33% were single, 42% had undergone less than six prenatal consultations, 58% of them had performed previous treatment of syphilis, but only 25% of their partners had joined and performed it.Conclusion: This study shows progress regarding diagnosis of syphilis during prenatal care, but it evidences the non-accomplishment of treatment by pregnant women before delivery, as well as by their partners. Therefore, innovative strategies are needed aiming at an early and proper treatment of pregnant women and their partnersObjetivo: Caracterizar el perfil epidemiológico de las usuarias portadoras de sífilis adquirida.Método: Se realizó un estudio exploratorio y descriptivo, con abordaje cuantitativo, a través de datos secundarios recogidos en los formularios de investigación/notificación de la sífilis de las mujeres atendidas en Hospital Universitario materno infantil, en Santa Cruz-RN en 2012. El estudio fue aprobado por el Comité Ético de Investigación con el número 772.884.Resultados: 67% de las madres asistieron a la escuela primaria, 33% son solteras, 42% tenía menos de 06 consultas prenatales, 58% de estas realizaron tratamiento previo de la sífilis, pero sólo 25% de los esposos fueron examinados.Conclusión: El estudio muestra el progreso en relación con el diagnóstico de la sífilis durante la atención prenatal, señalándose la falta de funcionamiento del tratamiento de las mujeres embarazadas antes del parto, así como de sus esposos. Por lo tanto, se necesitan estrategias innovadoras con el objetivo de un tratamiento precoz y adecuado de las mujeres embarazadas y de sus parejas.Objetivo: Caracterizar o perfil epidemiológico das usuárias portadoras da sífilis adquirida.Método: Realizou-se um estudo exploratório e descritivo de abordagem quantitativa, através de dados secundários coletados nas fichas de investigação/notificação de sífilis de mulheres assistidas em Hospital Universitário Materno Infantil, no município de Santa Cruz-RN, em 2012. O estudo foi aprovado pelo Comitê de Ética em Pesquisa sob o parecer nº 772.884.Resultados: Indicaram que 67% das puérperas notificadas cursaram apenas o ensino fundamental, 33% são solteiras, 42% realizaram menos de 06 consultas de pré-natal, 58% delas realizaram tratamento prévio da sífilis, mas apenas 25% dos esposos aderiram e o realizaram.Conclusão: O estudo aponta avanços quanto ao diagnóstico da sífilis durante o pré-natal, porém ressaltou-se a não realização do tratamento das gestantes antes do parto, como também do esposo. Logo, estratégias inovadoras são necessárias visando o tratamento precoce e adequado das gestantes e seu(s) parceiro(s)

Bioética e o Compromisso com o Ambiente Natural

Questões ecológicas e éticas são recorrentes nos debates dos últimos tempos. Ambas refletem a crise civilizacional que enfrentamos nos dias de hoje. O aumento da consciência e preocupação às questões ambientais resultou em discussões políticas internacionais e acordos globais. No atual modelo econômico a natureza recebe o “título” de recurso natural, servindo apenas para alimentar o sistema e satisfazer a demanda, desconsiderando a sua resiliência, sua habilidade de regeneração e sua capacidade produtora de substância de valor econômico. O capitalismo se faz predominante, e com o advento da globalização a moeda, o capital, foi imbuído de ainda mais força no controle sobre a ordem social, regendo a vida das pessoas, aumentando a desigualdade social, originando, posteriormente, as lutas de classes, que persistem nos dias atuais. Ele peca ao desconsiderar os processos ambientais dentro do processo econômico e a globalização atenua o pensamento capitalista da concentração das riquezas e o desconhecimento da outridade. Nossa espécie evoluiu e se libertou das amarras impostas pelas condições ambientais, e involuiu ao passo que desenvolveu um pensamento de posse sobre a natureza e se excluindo dos processos naturais. Torna-se de grande valia, portanto, uma reflexão quanto à bioética e o meio ambiente, a forma como “pensamos a natureza”, a consciência da outridade, e o resultado do pensamento contemporâneo sobre estes fatores