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L’ingénierie protéique servant autrefois à comprendre les relations structures-fonctions des protéines connait un tournant majeur depuis plusieurs années. L’ingénierie protéique évolue pour créer des nouvelles fonctions protéiques : c’est la naissance de l’ingénierie protéique moderne. L’objectif de ma thèse a consisté à mettre en place et caractériser deux approches indépendantes d’ingénierie protéique dans le domaine du vaccin et du diagnostic. Le premier projet consistait à générer des ligands protéiques à partir d‘échafaudages moléculaires (des alternatifs aux anticorps) en couplant le ribosome display au NGS et en développant des outils d’analyses bio-informatiques. Des sélections contre des cibles protéiques d’origine bactérienne et virale ont conduit à l’identification de ligands Affibodies affins (µM au nM). Leur caractérisation a validé leur potentiel comme outil de recherche et de réactif diagnostique. Ces études ont permis de valider la plateforme de génération des ligands mise en place, en augmentant l’exploration de l’espace de diversité des interactions des ligands. Le second projet portait sur le développement d’une plateforme de présentation et de vectorisation à partir de particules d’encapsuline. Elles ont été génétiquement modifiées pour présenter de manière répétée à leur surface l’ectodomaine de la protéine de matrice M2 (M2e) du virus Influenza A H1N1 tout en encapsulant une protéine hétérologue : l’eGFP. Les nanoparticules modifiées sont correctement formées et encapsulent l’eGFP. Des souris immunisées par ces particules induisent une réponse anticorps spécifique contre l’épitope M2e et l’eGFP. L’utilisation de ces nanoparticules comme plateforme vaccinale de présentation et de vectorisation est prometteuse et ouvre la voie pour d’autres applications en biotechnologie / In the past, protein engineering used to understand function and structure relationship. But since few years, protein engineering was used to create new protein functions: modern protein engineering was born. The aim of my thesis was to set up and characterize two approaches of protein engineering in diagnostic and vaccine field. The first project was to generate artificial binder using protein scaffolds as an alternative to antibodies by coupling ribosome display (RD) to NGS and developing bio-informatics tools. Screening and selection against bacterial and viral targets have led to affibody binder’s identification with an affinity range from µM to nM. Their characterization has validated their potential as research tools and protein reagents for diagnostic assay. Coupling ribosome display to high throughput sequencing as means to directly identify selected binder coding sequences, enormously enhance binder discovery depth. The second project was to generate an innovative nanocarrier based on encapsulin nanoparticle, for customized peptide display and cargo protein vectorization. Encapsulin particles from T.maritima were genetically modified for simultaneous display of the matrix protein 2 ectodomain of the influenza H1N1 A virus and heterologous protein eGFP packaging. Genetically engineered encapsulin nanoparticles were well-formed and abled to efficiently load eGFP. Immunogenicity studies revealed antibody responses against both the surface epitope and the loaded cargo protein. Taken together, this display system is a versatile tool for rational vaccine design and paves the way for new applications in the research fields of vaccine, antimicrobial research and other biotechnological applications

Made available in DSpace on 2016-06-02T20:09:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-13 / In this dissertation the subject of labor safety and health is approached, focusing on prevention of accidents when working in suspended scaffold, based on NR-18 as a preventive tool. The study initially presents information about Civil Engineering distributed in significant subjects for the intended targets is presented. In the second part details of suspended scaffold, pertinent guidelines of the NR-18 and no compulsory standards ABNT to be followed with this equipment are described. In the last part, the fulfillment of rules inserted in the NR-18, focused mainly to the suspended scaffold design, it is studied in the case study in a construction site. The conclusion shows the importance of the global view of the normative guidelines related to suspended scaffold, listed in this research, contributing for the complete fulfillment of the legal guidelines ans its improvement. The result achieved also might be useful as subsidies for the elaboration of the Conditions and Labor Environment Program for the Construction Industry, the project for the production of external covering, for the responsible organizations for monitoring of labor safety and health regulating standards / Esta dissertação aborda o tema da segurança e saúde no trabalho focado na prevenção de acidentes nas tarefas desenvolvidas em andaimes suspensos, utilizando a NR-18 como ferramenta prevencionista. O estudo apresenta inicialmente informações sobre a indústria da construção civil, distribuídas em temas significativos para os objetivos pretendidos. Descrevem-se, a seguir, detalhes dos andaimes suspensos, diretrizes pertinentes da NR-18 e norma não compulsória ABNT, para observação nos trabalhos com estes equipamentos. Finalmente, em estudo de caso, verifica-se em canteiro o cumprimento de disposições inseridas na NR-18, voltadas principalmente ao projeto de andaimes suspensos. A conclusão mostra a importância da visão sistêmica das diretrizes normativas para os trabalhos em andaimes suspensos, elencadas nesta pesquisa, contribuindo para plena observação das diretrizes legais e seu aperfeiçoamento. O resultado alcançado também pode servir de subsídios para a elaboração do PCMAT, do projeto para produção do revestimento externo e por parte dos órgãos com competência para fiscalização das normas regulamentadoras de segurança e saúde do trabalho

Synthèse et utilisation de mimes de quadruplexes contraints pour l'évaluation de ligands II est maintenant bien connu que l'ADN simple brin peut s'associer sous différentes conformations telles que la double hélice, les triplexes, les i-motifs ou bien encore les G-quadruplexes. Ces dernières années les structures de type quadruplexes (G-quadruplexes et i-motif) ont suscité un certain intérêt notamment pour leur implications au niveau cellulaire (maintenance des télomères, activation de gènes…). C'est pourquoi de nombreuses équipes travaillent sur le développement de ligands affins pour ces structures qui pourraient agir en tant qu'anticancéreux. Cependant, le fait que les quadruplexes présentent un polymorphisme important(variabilité du nombre de brins dans la structure, des différents types de boucles et de l'orientation des brins) rend la compréhension des interactions entre un ligand et le quadruplexe plus difficile. Dans ce contexte, l'équipe développe un nouveau concept, « Template Assisted Synthesis of Quadruplexes » (TASQ) dont le but est d'obtenir un quadruplexe ne présentant qu'une topologie de façon contrôlée afin de permettre des études plus précises sur la façon dont un ligand pourrait interagir avec les quadruplexes. La première partie de ce manuscrit reporte l'évaluation par résonnance plasmonique de surface de complexes métalliques en tant que ligands de G-quadruplexe. Ces études reposent sur l'utilisation d'un premier mime de G-quadruplexe parallèle sur lequel deux séries de complexes sont testées : des métalloporphyrines et des ligands de type salphen. La seconde partie du manuscrit décrit la synthèse de mimes de G-quadruplexes antiparallèle. Elle repose sur l'utilisation du gabarit peptidique qui relié aux séquences spécifiques d'oligonucléotides de façon adéquat contraint la structure. Pour se faire, deux réactions chimiosélectives ont été utilisées : la cycloaddition 1,3 dipolaire de Huisgen et la ligation oxime. Les travaux reportés concernent trois types de structure mimant un i-motifs, des G-quadruplexes tétramoléculaires ou bimoléculaires. / Synthesis and use of constrained quadruplexe mimic for the ligand evaluation It has now been shown that single stranded DNA can fold into hairpin, triplex, i-motif and G-quadruplexe structures containing non canonical base pairing. In particular quadruplex structures (G-quadruplexes and i-motif) have generated interest because the formation is considered to have important consequences at the cellular level (telomere maintenance, oncogene activation…). Therefore, the design of small molecules (ligands) targeting quadruplexes is under development, in particular to obtain anticancerous molecules. Nevertheless G-quadruplexes exhibit a wide structural polymorphism (different kind of loop, strand orientation, number of strands) making investigation of ligand tricky. In this context, we have developed a new concept called 'Template Assisted Synthesis of Quadruplex' (TASQ) with aim to constrain the G-quadruplex in a single conformation which enables the study of the interactions of ligand more specifically for a single topology. In the first part of this manuscript, we describe studies by using surface plasmon resonance (SPR) of metal complexes as G-quadruplex ligands. The investigation was performed using a first parallel G-quadruplex as model to interact with two kinds of metal complexes: metalloporphyrins and salphen. The second part of the manuscript reports the work concerning the synthesis of antiparallel quadruplexes. The strategy is based on the use of a peptide scaffold in which the suitable oligonucleotide sequences are anchored with specific orientation. For this purpose two compatible chemistries are used (1,3 dipolar Huisgen cycloaddition and oxime ligation). Three kinds of antiparallel quadruplexe mimics have been investigated: i-motif, tetramolecular G-quadruplexes and loop-constrained G-quadruplexes.

Submitted by Daniele Amaral (daniee_ni@hotmail.com) on 2016-09-21T20:30:20Z No. of bitstreams: 1 TeseHWK.pdf: 5259387 bytes, checksum: 72d4bd1fcb3174ede9d168165f46252a (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-09-28T19:23:02Z (GMT) No. of bitstreams: 1 TeseHWK.pdf: 5259387 bytes, checksum: 72d4bd1fcb3174ede9d168165f46252a (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-09-28T19:23:12Z (GMT) No. of bitstreams: 1 TeseHWK.pdf: 5259387 bytes, checksum: 72d4bd1fcb3174ede9d168165f46252a (MD5) / Made available in DSpace on 2016-09-28T19:28:30Z (GMT). No. of bitstreams: 1 TeseHWK.pdf: 5259387 bytes, checksum: 72d4bd1fcb3174ede9d168165f46252a (MD5) Previous issue date: 2015-03-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The present study aimed to evaluate the effect of two different Biosilicate® (P2O5-Na2O-CaO-SiO2 system) presentations - highly porous scaffold and composite material – on a tibial bone defect model in rats. Two studies were performed; the first one aimed at evaluating the effect of highly porous scaffolds on bone regeneration using histopathological analysis, immunohistochemistry and immunoenzymatic assay. In this study, 80 male Wistar rats (12 weeks old and body weight of approximately 300 g) were divided in two groups (control and Biosilicate®) and euthanized after 3, 7, 14 and 21 days post-surgery. The histopathological evaluation revealed that both groups presented similar inflammatory responses after 3 and 7 days. At all time points, the scaffold degradation was observed, mainly in the border of the material, allowing the ingrowth of newly formed bone. The immunohistochemical analysis showed that the Biosilicate® scaffolds induced the synthesis of (i) ciclooxigenase 2 (COX-2), (ii) vascular endothelial growth factor (VEGF) and (iii) runt-related transcription factor 2 (RUNX-2). Additionally, the immunoenzymatic assay indicated that the Biosilicate® group did not presented significant statistical difference in the levels of tumor necrosis factor alpha (TNF-α) in all evaluated periods compared to the control group. In addition, the Biosilicate® group presented a higher concentration of interleukin 4 (IL-4) at day 14 and a lower concentration of interleukin 10 (IL-10) 21 days after the surgery when compared to the control group. The second study aimed at investigating the effects of Biosilicate®/poly lactic-co-glycolic acid (PLGA) composites on the process of bone repair using histopathological, morphometric, immunohistochemical and gene expression (Real-Time PCR, qRT-PCR) analyses. In this study, 80 male Wistar rats were distributed in two groups (Biosilicate® and Biosilicate®/PLGA) and euthanized 3, 7, 14 and 21 days after the material implantation. The main findings showed that the incorporation of PLGA into the Biosilicate® had a significant effect in the material morphological structure, leading to a pH decrease and accelerating the mass loss upon incubation in phosphate buffered saline (PBS). Moreover, the histological evaluation revealed that the Biosilicate®/PLGA group presented a higher material degradation accompanied by a higher bone formation when compared to the plain Biosilicate® after 21 days. The immunohistochemical analysis did not show any difference in the immunolabeling for Runx2, RANKL and OPG between Biosilicate® and Biosilicate®/PLGA. In addition, the qRT-PCR indicated that the Biosilicate®/PLGA induced the osteogenic gene expressions (bone morphogenetic protein 4, Runtrelated transcription factor 2 and osteocalcin) at 21 day after surgery. The results evidenced by the present studies suggest that both materials (highly porous Biosilicate® scaffolds and Biosilicate®/PLGA composites) were effective in inducing the repair of tibial bone defects in rats, demonstrating that these materials are promising alternatives for treating bone fractures. / O presente estudo teve como objetivo principal avaliar a ação de duas diferentes apresentações (scaffold altamente poroso ou compósito contendo PLGA) de uma vitrocerâmica bioativa do sistema quaternário P2O5-Na2O-CaO-SiO2 (Biosilicato®) sobre o processo de reparação óssea em um modelo de defeito ósseo tibial em ratos. Para isto, foram realizados dois estudos, sendo que o primeiro teve como objetivo avaliar os efeitos dos scaffolds altamente porosos de Biosilicato® sobre o processo de regeneração óssea por meio da avaliação histopatológica, imunohistoquímica e ensaio imunoenzimático. Neste estudo, 80 ratos machos Wistar (12 semanas de idade e peso corporal de aproximadamente 300 g) foram divididos em dois grupos (controle e Biosilicato®) e eutanasiados após 3, 7, 14 e 21 dias do procedimento cirúrgico. A avalição histopatológica revelou que ambos os grupos apresentaram uma resposta inflamatória similar no período de 3 e 7 dias após a cirurgia. Durante todos os períodos experimentais, a degradação dos scaffolds de Biosilicato® foi observada principalmente na região periférica do material, o que possibilitou o desenvolvimento do tecido ósseo neoformado para o interior destes materiais. A Análise imunohistoquímica demonstrou que os scaffolds de Biosilicato® estimularam a síntese da ciclooxigenase 2 (COX-2), fator de crescimento endotelial vascular (VEGF) e fator de transcrição relacionado a runt-2 (Runx2). Além disso, o ensaio imunoenzimático revelou que o grupo Biosilicato® não apresentou diferença estatística significativa nos níveis do fator de necrose tumoral alfa (TNF-α) em todos os períodos avaliados quando comparado ao grupo controle. Ainda, o grupo Biosilicato® apresentou uma maior concentração da interleucina 4 (IL-4) 14 dias e uma menor concentração da interleucina 10 (IL-10) 21 dias após a cirurgia, quando comparado ao grupo controle. O segundo estudo teve como objetivo investigar os efeitos do compósito de Biosilicato® e ácido poli-láctico-co-glicólico (PLGA) sobre o processo de reparo ósseo através das análises histopatológica, morfomética, imunohistoquímica e de expressão gênica (PCR em tempo real, qRT-PCR). Neste estudo, 80 ratos machos Wistar foram distribuídos em dois grupos (Biosilicato® e Biosilicato®/PLGA) e eutanaziados após 3, 7, 14 e 21 dias do processo de implantação dos materiais. Os achados principais mostraram que a incorporação do PLGA na vitrocerâmica Biosilicato® teve um efeito significativo na estrutura morfológica do material, levando a diminuição do pH e acelerando a perda de massa após incubação do material em solução tampão fosfato (PBS). Além disso, a avaliação histológica revelou que o grupo Biosilicato®/PLGA apresentou uma maior degradação do material, acompanhada pela maior formação de osso quando comparado ao grupo somente com Biosilicato® no período de 21 dias. Na analise imunohistoquímica nenhuma diferença na imunomarcação de Runx2, receptor ativador do ligante nuclear fator kappa-B e osteoprotegerina foram observadas entre o grupo Biosilicato® e Biosilicato®/PLGA. Ainda, a análise de qRT-PCR demonstrou que o grupo Biosilicato®/PLGA induziu a expressão de genes osteogênicos (proteína morfogenética óssea 4, fator de transcrição relacionado a runt-2, e osteocalcina) 21 dias após cirurgia. Diante dos resultados encontrados nos dois estudos, é possível concluir que ambos os materiais utilizados neste estudo, scaffold de Biosilicato® altamente poroso e compósito de Biosilicato® e PLGA, foram eficazes em estimular o reparo de um defeito ósseo tibial em ratos, demonstrando serem alternativas promissoras para tratamento de fraturas ósseas.

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-05-09T18:09:49Z No. of bitstreams: 1 DELANO HÉLIO OLIVEIRA - DISSERTAÇÃO PPGCC 2015..pdf: 994388 bytes, checksum: 3aafc37c8cabe33b27414e66db934ccb (MD5) / Made available in DSpace on 2018-05-09T18:09:49Z (GMT). No. of bitstreams: 1 DELANO HÉLIO OLIVEIRA - DISSERTAÇÃO PPGCC 2015..pdf: 994388 bytes, checksum: 3aafc37c8cabe33b27414e66db934ccb (MD5) Previous issue date: 2015 / O desenvolvimento de aplicações corporativas modernas para web baseia-se na utilização de padrões e ferramentas para viabilizar a rápida prototipagem, garantindo a separação entre modelo de negócio e interface gráfica de usuário (GUI, do inglês Graphical User Interface). As plataformas de Scaffold, por exemplo, permitem um aumento da produtividade dos desenvolvedores ao gerarem código a partir dos elementos do modelo conceitual. Porém,o código fonte de GUI gerado apresenta muita replicação, devido ao acoplamento ainda existente entre os componentes das telas de interface gráfica e as propriedades inerentes ao modelo conceitual da aplicação, dificultando a manutenção do software. Os padrões de renderização propostos por Welick et al. se apresentam como uma solução conceitual para este problema, através do mapeamento de metadados do modelo conceitual em componentes gráficos, organizando o código de GUI e reduzindo a replicação de código. Neste trabalho, tem-se como objetivo a criação de um arcabouço para o desenvolvimento de aplicações corporativas com arquitetura web moderna, com foco em GUI, baseado em padrões de renderização. O arcabouço permite que o desenvolvedor construa componentes de GUI sem acoplá-los aos elementos do modelo conceitual. A associação da GUI com o modelo conceitual é feita através de regras de renderização, que podem ser alteradas facilmente. O arcabouço proposto foi validado através de um estudo de caso, no qual foi demonstrada uma redução significativa na duplicação do código quando comparada às plataformas de Scaffold. / The modern enterprise web application development is based on the use of patterns and tools to enable rapid prototyping, ensuring separation between the business model and graphical user interface (GUI). The Scaffold frameworks, for example, allows an increase in productivity of developers to generate code from the elements of domain model. However, the GUI source code generated presents a lot of replications due to coupling still extant between GUI components and properties inherent to the domain model of the application, making it difficult to maintain the software. The rendering patters proposed by Welick et al. are presented as a conceptual solution to this problem by mapping domain model metadata to graphical components, organizing the GUI code and reducing code duplication. In this Work, we have aimed to create a framework for enterprise applications development with web modern architecture, focusing on GUI, based on rendering patterns. This framework allows the developer to build GUI components without engage to elements of domain model. The GUI link with domain model is made by rendering rules, which can be changed easily. The proposed framework was validated by a case study in which it was demonstrated a significant reduction in code duplication when compared to Scaffold frameworks.

The aim of the thesis was development of an innovative treatment of bone defects. Human multipotent mesenchymal stromal cells (MSC) play a crucial role in bone healing. Clinical applications of MSC require large amount of cells, which could be obtained by autologous expansion of MSC harvested from bone marrow. As a first step, the standard protocol of MSC expansion based on αMEM medium and fetal bovine serum (FBS) was used. Experiments replacing FBS by pooled human serum (HS) in the culture medium concluded in patenting of a new MSC cultivation protocol (EU 1999250, CR 301141). This one-step cultivation protocol and xenogeneic protein-free cultivation medium is based on CellGro® for Hematopoietic Cells' Medium, HS, human recombinant growth factors, dexamethasone, insulin and ascorbic acid. The preclinical in vitro and in vivo experiments with MSC from both expansion protocols were carried out. Fibrillar polylactic scaffolds were seeded with MSC, cultured, differentiated and implanted in immunodeficient mice (NOD/LtSz-Rag1-). Bone-like mineralized tissue containing vessels was observed. The MSC cultured according to patented method were classified as Advanced-therapy Medicinal Product and has to fulfil the European Medicines Agency regulations to enter the clinical trials. Nevertheless the use of MSC seems...

Background: Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). Methods: Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-β on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. Results: Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. Conclusions: Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

Introdução: A tomografia das artérias coronárias (ANGIO-TC) tem o potencial de medir as dimensões dos vasos e pode ser opção, aos métodos invasivos, para análises quantitativas em intervenções coronárias com suportes vasculares bioabsorvíveis (SVB) poliméricos radiolucentes. Objetivos: Medidas quantitativas pela ANGIO-TC do lúmen de segmentos coronários de pacientes submetidos a implante de um SVB com eluição de novolimus (DESolve®) foram comparadas às do ultrassom intracoronário (USIC). Os objetivos primários foram a comparação da área mínima e do volume do lúmen do SVB. Outros objetivos incluíram medidas nas margens do dispositivo, de referências do vaso e dos percentuais de estenose do SVB. A precisão de identificação do local de menor dimensão foi estimada pela distância entre este e a borda proximal do SVB. Método: Vinte e um pacientes submetidos a implante de um SVB DESolve e que foram reestudados após 6 meses com cinecoronariografia e USIC realizaram, também, ANGIO-TC. Sem conhecimento dos valores um do outro, um operador, em cada método, efetuou as medidas de volume, área e diâmetro mínimos do lúmen do SVB, de áreas e diâmetros mínimos do lúmen nas margens proximal e distal do SVB, de diâmetros e áreas de referência luminais e dos percentuais de estenose de diâmetros e áreas do SVB. Diferenças entre as médias foram significativas quando testes resultaram o valor de p< 0,05. Coeficientes de correlação foram calculados e a concordância foi analisada pelo método de Bland-Altman. Resultados: Os métodos não se mostraram correlacionados ao medirem área mínima do lúmen do SVB e a ANGIO-TC subestimou significativamente os valores em relação ao USIC (diferença de médias= -1,27 mm2; p= 0,004). As medidas do volume do lúmen do SVB mostraram correlação (r= 0,58; p= 0,006) e foram equivalentes (diferença de mediana= 5,4 mm3; p= 0,14). Em ambas, houve ampla variabilidade entre as medidas (variação percentual do erro de 128% para a área e de 119% para o volume). Os métodos mostraram correlações significativas para todas as demais variáveis. As médias das medidas de diâmetros, pela ANGIO-TC, não mostraram diferenças significativas em relação ao USIC. A ANGIO-TC subestimou significativamente as medidas da área mínima do lúmen no segmento distal ao SVB (diferença= -1,09 mm2; p = 0,017) e da área de referência dos vasos (diferença = -1,34 mm2; p = 0,008). Apesar do viés mínimo, os métodos mostraram ampla variação ao identificar o ponto de menor dimensão do SVB (erro percentual = 186%). A ANGIO-TC, assim como o USIC, não identificou casos de reestenose. Os métodos mostraram melhor nível de concordância ao medirem diâmetros e maiores discrepâncias ao estimarem percentuais de estenose. Conclusões: Em segmentos coronários com SVB polimérico, a ANGIOTC não obteve correlação e subestimou a área mínima do lúmen em relação ao USIC. Quantificações do volume do lúmen foram equivalentes e correlacionadas. Independentemente do nível de correlação, o padrão de concordância das medidas evidenciou um nível de acurácia insatisfatório para a ANGIO-TC substituir o USIC para quantificações de lumens em estudos com SVB radiolucentes, embora permaneça útil para análises visuais na prática clínica. / Computed tomography coronary angiography (CTA) is able to quantify vessel dimensions and might potentially be an alternative to substitute invasive methods for quantitative analysis in percutaneous coronary interventions with bioresorbable vascular scaffolds (BVS). This study compared quantitative measurements derived from CTA images to intravascular ultrasound (IVUS) in coronary segments implanted with radiolucent DESolve(TM) novolimuseluting BVS. Primary objectives were comparisons of BVS minimal luminal area and luminal volume in BVS. Secondary objectives included comparisons of minimal luminal areas and diameters in proximal and distal segments to the BVS, luminal vessel reference areas and diameters and BVS percent area and diameter stenosis. Precision of identifying BVS luminal minimal area were assessed by measuring distance from this point to proximal BVS border. Twenty-one patients underwent both CTA and IVUS, six months after BVS deployment. Each method was performed by an experienced operator, blinded to other\'s quantifications. Correlation coefficients were calculated and mean differences with 95% limits of agreement were assessed by Bland-Altman analysis. A p-value less than 0.05 were considered statistically significant. CTA did not show correlation to IVUS and significantly underestimated minimal luminal area in BVS (mean differences = -1.27 mm2; p = 0.004). Quantitative measurements of luminal volume in BVS were equivalent (median difference = 5.4 mm3; p = 0.14) and showed modest correlation (r= 0.58; p= 0.006). Both variables showed wide limits of agreement (percent error = 128% in minimal luminal area and 119% in luminal volume). Correlations were significant in all other variables. Both methods did not show significant differences quantifying all-segment diameters, and percent area and diameter stenosis. CTA significantly underestimated measurements of minimal luminal area in distal segment after BVS (mean difference = -1,09 mm2; p = 0,017) and luminal reference area (mean difference = -1,34 mm2; p = 0,008). CTA and IVUS showed nonsignificant bias to identify BVS luminal minimal area, but very wide limits of agreement (percent error= 186%). Both methods agreed in showing no cases of binary restenosis. Regardless of correlations or mean differences, all measures showed high variability, caracterized by wide limits of agreement. The least variations resulted from diameter quantifications, whereas estimated percent stenosis presented more disparities. These discrepancies between both methods showed that CTA analysis is still not fully developed to replace IVUS in the assessment of quantitative measurements in vessels treated with BVS. It remains, however, clinically useful for visual qualitative analysis.

Le Myélome Multiple (MM) est une hémopathie maligne caractérisée par la prolifération de plasmocytes tumoraux médullaires. MYC occupe un rôle central dans l'oncogenèse du MM car son activation est responsable de la progression du stade précurseur de MGUS en MM symptomatique. Dans ce travail, nous rapportons que l’expression de LIN28B est corrélée à celle de MYC et est associée à un mauvais pronostic dans le MM. Nous montrons que l'axe LIN28B/let-7 module l'expression de l’ARNm de MYC, lui-même cible de let-7. De plus, la perturbation de l'axe LIN28B/let-7 induit une régulation la prolifération des lignées cellulaires de MM in vitro et in vivo. L'analyse par séquençage d’ARN de modèles de KO par utilisation de la technologie CRISPR a montré que l'axe LIN28B/let-7 régule les voies de signalisation de MYC et du cycle cellulaire dans MM. Nous avons de plus établi une preuve de principe thérapeutique de la possibilité de cibler MYC par l’emploi de LNA-GapmeR contenant une séquence analogue à let-7b. Dans un modèle de xénogreffe murin, nous montrons que des niveaux élevés d'expression de let-7, par administration de LNA-GapmeR let-7b, répriment la croissance tumorale en régulant l’expression de MYC. Ces résultats révèlent un nouveau mécanisme de ciblage thérapeutique de MYC via l'axe LIN28B/let-7 dans MM. Nous nous sommes ensuite intéressés à évaluer de nouvelles formes de biomarqueurs moléculaires dans le MM par étude des miARN contenus dans les exosomes circulants. Nous avons examiné le rôle pronostique des miARN exosomaux dans une cohorte de 156 échantillons de patients uniformément traités pour un MM au diagnostic. Après analyse du profil de miARN exosomaux par séquençage de nouvelle génération, nous avons utilisé technique de qRT-PCR pour étudier la corrélation entre le niveau d’expression de 22 miARN et la survie sans progression (SSP) et la survie globale (SG). Deux miARN, à savoir let-7b et miR-18a, étaient significativement associés à la SSP et SG en analyse univariée, et étaient statistiquement significatifs après ajustement pour le système international de stratification du risque (ISS) et les marqueurs cytogénétique en analyse multivariée. Nos résultats confirment le niveau d’expression des miARN let-7b et miR-18a au sein des exosomes circulants permettent d’améliorer la stratification du risque chez les patients atteints de MM. Enfin, pour mieux comprendre le programme oncogénique piloté par MYC, nous avons étudié l’efficacité thérapeutique d’une librairie de petites sur des lignées cellulaires avec une forte expression de MYC, dans le MM. Les résultats ont permis d’identifier les rocaglates, une famille de composés inhibant l’initiation de la traduction, comme étant les plus actifs. L’étude du profil transcriptionnel par séquençage de l’ARN de lignées cellulaires de MM traitées par CMLD010509 ou DMSO a révélé l’activation d’un programme de transcription et l’inhibition d’un programme traductionnel, caractéristique de l’inactivation de HSF1 secondaire à l’inhibition de la traduction. Le profile traductionnel était étudié par spectrométrie de masse quantitative, permettant d’identifier un ensemble de protéines, tels que MYC, MDM2, CCND1, MAF et MCL-1, spécifiquement affectées par l’inhibition de la traduction liée au composé CMLD010509 dans le MM. Nous avons confirmé l’efficacité thérapeutique des rocaglates dans plusieurs modèles murins de MM. Ces résultats démontrent la possibilité de cibler le programme de traduction oncogénique lié à MYC dans MM. / MYC is a major oncogenic driver of Multiple Myeloma (MM) and yet almost no therapeutic agents exist that target MYC in MM. Here we report that the let-7 biogenesis inhibitor LIN28B correlates with MYC expression in MM and is associated with adverse outcome. We also demonstrate that the LIN28B/let-7 axis modulates the expression of MYC, itself a let-7 target. Further, perturbation of the axis regulates the proliferation of MM cells in vivo in a xenograft tumor model. RNA-sequencing and gene set enrichment analyses of CRISPR-engineered cells suggested that the LIN28/let-7 axis regulates MYC and cell cycle pathways in MM. We provide proof of principle for therapeutic regulation of MYC through let-7 with an LNA-GapmeR (locked nucleic acid-GapmeR) containing a let-7b mimic in vivo, demonstrating that high levels of let-7 expression repress tumor growth by regulating MYC expression. These findings reveal a novel mechanism of therapeutic targeting of MYC through the LIN28B/let-7 axis in MM. We next sought to establish new biomarkers in MM, enable to capture the molecular alterations of the disease. For this purpose, we examined the prognostic significance of circulating exosomal microRNAs (miRNAs) in a cohort of 156 patients with newly diagnosed MM, uniformly treated and followed. Circulating exosomal miRNAs were isolated and used to perform small RNA sequencing analysis on 10 samples and a qRT-PCR array on 156 samples. We studied the relationship between miRNA levels and patient outcomes including progression-free survival (PFS) and overall survival (OS). We identified miRNAs as the most predominant small RNAs present in exosomes isolated from the serum of MM patients and healthy controls by small RNA sequencing of circulating exosomes and used a qRT-PCR assay to measure the expression of 22 exosomal miRNAs. Two of them, namely let-7b and miR-18a, were significantly associated with both PFS and OS in the univariate analysis, and were still statistically significant after adjusting for the International Staging System (ISS), and adverse cytogenetics in the multivariate analysis. Our findings support the use of circulating exosomal let-7b and miR-18a improves the identification of patients with newly diagnosed MM with poor outcomes. Finally, to better understand the oncogenic program driven by MYC and investigate its potential as a therapeutic target, we screened a chemically diverse small molecule library for anti-MM activity in cell lines with high expression of MYC. The most potent hits identified were rocaglate-scaffold inhibitors of translation initiation. Expression profiling of MM cells revealed reversion of the oncogenic MYC-driven transcriptional program by CMLD010509, the most promising rocaglate. Proteome-wide, reversion correlated with selective depletion of short-lived proteins that are key to MM growth and survival, most notably MYC, MDM2, CCND1, MAF, and MCL-1. The efficacy of CMLD010509 in several mouse models of MM confirmed the therapeutic relevance of these findings in vivo and supports the feasibility of targeting the oncogenic MYC-driven translation program in MM with rocaglates.