Este trabalho descreve a ocorrência da doença meningocócica na região de abrangência da Divisão Regional de Saúde de Sorocaba-SP, no período de 1999 a 2008. Fundamentado em dados fornecidos pelo Instituto Adolfo Lutz e pelo Grupo de Vigilância Epidemiológica, a incidência e a letalidade foram calculadas, para toda a população e por faixa etária. Os valores obtidos foram comparados com os dados do Estado de São Paulo e do Brasil. Além disso, foram obtidas as distribuições de ocorrência por manifestação clínica e de critérios diagnósticos utilizados, permitindo a análise da situação epidemiológica da doença meningocócica, na região em estudo. Ao verificar os resultados relativos aos sorogrupos, sorotipos e soross ubtipos identificados, foi possível estabelecer o fenótipo das cepas que, predominantemente, causam a doença na região. Em relação à incidência, conclui-se que, durante praticamente todo o período em estudo, é maior do que os valores endêmicos encontrados nos países desenvolvidos. A faixa etária mais atingida, tanto do ponto de vista da incidência como da letalidade é a de 0 a 4 anos, indicando a necessidade de incremento e continuidade dos programas de vacinação relativos a esse grupo populacional. Em relação às cepas circulantes, os fenótipos B:4,7:P1.19,15 e C:23:P1.14-6 predominam, fato coerente com os resultados obtidos para a Grande São Paulo e Baixada Santista / This work describes the meningococcical disease occurrence in the area related to the Health Regional Division of Sorocaba-SP, between 1999 and 2008. Based on data available at Adolfo Lutz Institute and Epidemiological Vigilance Group, the incidence and the lethality were calculated, for the whole population and for age groups. The obtained values were compared with the data related to São Paulo State and Brazil. Besides, the clinical manifestation and diagnosis criteria distributions were obtained, allowing the analysis of the meningococcical disease epidemiological situation in the studied region. Verifying the results related to the identified serogroups, serotypes and serosubtypes, it was possible to establish the phenotype of the strains that, primarily, cause the disease in the region. Related to the incidence, it can be concluded that, in almost all the study period, it is greater than the endemic values found for developed countries. The more affected age group, concerning either incidence or lethality, is the 0 to 4 years old, indicating the necessity of increment and continuity of the vaccination programs. Related to the circulating strains, the phenotypes B:4,7:P1.19,15 e C:23:P1.14-6 are the more frequent, coherently with the obtained results for the Great São Paulo and Baixada Santista

Tesis presentada a la Universidad de Chile para optar al grado de Magíster en Química área de Especialización en Química Medicinal y Memoria para optar al Título de Química Farmacéutica / El presente trabajo de investigación informa sobre la preparación de dos familias de compuestos: cationes lipofílicos derivados del ácido cafeico y bromoésteres derivados de ácido cafeico como compuestos citotóxicos mitocondriotrópicos selectivos para células tumorales. Para el diseño de las moléculas, se consideró: (1) Largo de cadena entre los componentes farmacofóricos. (2) Presencia o ausencia de carga que permita o no dirigirlas hacia la mitocondria (3) Incorporación de grupos funcionales con capacidad antioxidante Las moléculas fueron ensayadas en las líneas celulares: HCT-116, H1975, HeLa, MCF-7, HL60 y Vero, con MTT y Rojo Neutro, dando como resultado mayor citotoxicidad para los cationes lipofílicos fosforados, mejores resultados para ésteres bromados y cationes fosforados con cadena de 10 carbonos y mayor selectividad para HeLa con 9-P / This research work provides information on the preparation of families of compounds: lipophilic cations derived from caffeic acid and bromoesters derived from caffeic acid as selective cytotoxic mitochondrotropic compounds for tumor cells. For the design of the molecules, it was considered: (1) Chain length between the pharmacophoric moieties. (2) Presence or absence of charge that allows or does not direct them towards the mitochondria (3) Presence of antioxidant groups The molecules were tested in the cell lines: HCT-116, H1975, HeLa, MCF-7, HL60 and Vero, with MTT and Neutral Red, resulting in greater cytotoxicity for the phosphorus lipophilic cations, better results for brominated esters and phosphorus cations with chain of 10 carbons and greater selectivity for HeLa with 9-P / Conicyt; Fondecyt

Introdução: O diagnóstico precoce de câncer de próstata (CaP) é essencial para aumentar a sobrevida dos pacientes, mas os marcadores e métodos atuais não possuem sensibilidade e especificidade suficientes, tornando o diagnóstico ainda muito impreciso. Recentemente, as células tumorais circulantes (CTCs) têm surgido não como método de rastreio do CaP, mas sim como marcadores de prognóstico utilizando um arsenal de diversos alvos para a captura dessas células. Contudo, a busca por um método ou marcadores comuns para o rastreio, diagnóstico, prognóstico e monitoramento da doença ainda se apresenta com um dos principais objetivos técnico-científicos a ser alcançado. Objetivo: apresentar um novo marcador, o aptâmero A4 selecionado previamente por 3DCell SELEX na linhagem PC3, e avaliar sua capacidade de detectar CTCs por citometria de fluxo no sangue de pacientes com CaP virgens de tratamento e sob diferentes regimes terapêuticos. Material e métodos: o estudo avaliou 34 homens com CaP e 16 homens sem alterações prostáticas. Foi coletado o sangue em tubo com EDTA, e após proceder a lise de hemácias, as células nucleadas de cada paciente foram incubadas com o aptâmero A4 conjugado à biotina, e em seguida lavadas e incubadas com estreptoavidina-FITC para posterior análise em citometria de fluxo. Os percentuais de CTCs foram comparados entre os dois grupos de pacientes e correlacionados com idade, níveis de PSA, estadiamento e procedimentos terapêuticos adotados (bloqueio hormonal, radioterapia e cirurgia). O limite de detecção acima de 1% de CTCs foi considerado positivo, utilizando como base o percentual observado em todos os 16 controles negativos. Resultados: todos os pacientes foram diagnosticados como positivos independentemente do tempo de terapia ou do estadiamento, exceto um paciente sob bloqueio hormonal que não apresentou CTCs. O percentual de CTCs apresentou alta correlação com idade (R=0,75) e com os níveis de PSA (R=0,80) de forma exponencial, embora seis pacientes com altos índices de células circulantes apresentaram PSA<0,02ng/mL, considerados como falha bioquímica. Conclusão: nossos resultados preliminares indicam uma acurácia elevada de 98% e demonstra um grande potencial de aplicação dessa nova tecnologia diagnóstica tanto no rastreamento, quanto no monitoramento do tratamento do CaP, o qual deverá ser melhor investigado em população de risco. / Introduction: prostate cancer (PCa) early diagnosis is essential to boost patients’ life expectance. Although, current biomarkers and diagnosis methods do not present reliable sensibility and specificity, making the diagnosis rather imprecise. Recent methodologies have been using circulating tumor cells (CTCs), not for screening of PCa, but as prognosis indicators, employing a vast array of techniques to capture those cells. However, the search for a new biomarkers or diagnosis methods able to screen, diagnosis, assist in prognosis and in the disease monitoring still one of the major technical and scientific objectives to be achieved. Objective: To present a new biomarker for PCa, the aptamer A4, previous screened in the prostate cancer cell line PC3, using 3DCell SELEX. And to able to detect, by flow cytometry, CTCs in blood samples of PCa patients undergoing various treatment regimen. Material and methods: the study evaluated 34 PCa patients and 16 health controls. Blood samples were collected in EDTA tubes, and after erythrocytes lysis, nucleated cells were incubated with A4 aptamer conjugated with biotin, them the cells were washed and incubated with streptavidin-FITC for later flow cytometer analysis. Percentage of CTCs were compared between patient’s groups and correlated against age, PSA levels, staging and treatment regimen (hormonal blockade, radiotherapy and surgery). Detection limit above 1% of CTCs was considered positive, based on the percentage observed on all of the 16 negative controls. Results: all patients were positively diagnosed independently of therapy time or staging, except for one patient undergoing hormonal blockade therapy, which does not present detectable CTCs. CTCs percentage presented high correlation against age (R=0.75) and with PSA levels (R=0.80) with exponential behavior, although, six patients with high CTCs count presented PSA levels <0.02 ng/mL, and were considered was biochemical errors. Conclusion: Our preliminary results indicated high accuracy (98%) and demonstrate a potential application of this technology for diagnosis and screening, as well as in the monitoring of PCa evolution, which should be better investigated in the risk population. / Dissertação (Mestrado)

Les cellules tumorales circulantes (CTC) représentent une source de matériel tumoral accessible de manière non invasive, susceptible de fournir des informations cliniques et fondamentales. Ces cellules issues de tumeurs primitives ou métastatiques représentent une population hétérogène d’éléments très rares circulant dans le sang. La personnalisation des traitements en oncologie repose sur la caractérisation moléculaire de biopsies tumorales mais celles-ci peuvent être difficiles à réaliser ou peu informatives. De ce fait, la caractérisation moléculaire et fonctionnelle des CTC présente un double intérêt, clinique pour identifier des biomarqueurs de sensibilité à des traitements, et fondamental pour étudier les mécanismes qui sous-tendent leur potentiel à initier des tumeurs.Les objectifs de ma thèse ont été d’une part de caractériser par séquençage de l’exome (WES) les CTC à l’échelle de cellule unique de patients atteints de cancers de la prostate (PCa) métastatiques et d’autre part d’établir puis caractériser des modèles de xénogreffes dérivés de CTC (CDX) chez des patients atteints de cancers bronchiques non à petites cellules (CBNPC) ou de PCa.Pour répondre au premier objectif, nous avons développé une méthode expérimentale globale incluant trois approches technologiques permettant d’enrichir et d’isoler des CTC individuelles de différents phénotypes (épithélial, épithélio-mésenchymateux et mésenchymateux), d’amplifier la totalité du génome (WGA) et de le séquencer. Le WES a été réalisé pour 34 échantillons de CTC sélectionnés sur des critères de qualité du WGA, ainsi que pour les biopsies de métastases correspondantes chez sept patients. Deux patients présentant une hétérogénéité phénotypique de leurs CTC, ont été analysés en profondeur. Nous avons mis en évidence des mutations partagées entre les CTC et les biopsies tumorales correspondantes ainsi que des mutations uniquement retrouvées dans les CTC. Ces mutations spécifiques aux CTC sont présentes dans tous les phénotypes et affectent particulièrement les gènes impliqués dans le remodelage du cytosquelette, la réparation de l’ADN ou l’invasion. L’existence de mutations communes entre les CTC de différents phénotypes suggère une relation phylogénique entre ces cellules mais une évolution divergente pendant le processus métastatique. Ce travail est soumis pour publication.Dans la seconde partie de ma thèse, nous avons implantés les CTC de 67 patients atteints de CBNPC et 24 patients atteints de PCa chez des souris immunodéprimées. Nous avons établis quatre CDX de CBNPC et un CDX de PCa. La caractérisation de ces modèles, des biopsies tumorales, des CTC collectées au moment de la xénogreffe, des CDX et des lignées cellulaires établies à partir du CDX, ont révélé que les CTC, le CDX et les lignées cellulaires « miment » le phénotype et le profil mutationnel des biopsies tumorales. La caractérisation plus approfondie de l’une des lignées cellulaires montre la présence d’un stress réplicatif et d’une instabilité génomique élevée. Ce résultat nous oriente sur l’hypothèse d’un rôle éventuel de l’instabilité génomique dans la tumorigénicité des CTC.Dans ce travail, nous avons montré que le profil mutationnel des CTC présente de fortes similitudes avec les biopsies tumorales des patients dans les patients atteints de PCa étudiés. De plus, nous avons observé l’existence de mutations spécifiques aux CTC, non détectées dans les biopsies tumorales. Également, nous montrons que des CTC issues de CBNPC et de PCa sont tumorigéniques in vivo et qu’elles reflètent le profil mutationnel des biopsies tumorales des patients. Ces modèles constituent des outils originaux et intéressants pour identifier de nouvelles cibles thérapeutiques et stratégies anti-cancéreuses, et comprendre les mécanismes qui supportent le potentiel des CTC à initier des tumeurs. / Circulating tumor cells (CTCs) represents an non invasive source of tumor material which may provide clinical and basic information. These cells derived from primary or metastatic tumors represents an heterogeneous population of very rare events which circulates in the blood. Oncology personnalized medicine is based on biopsies molecular characterization but these are sometimes which difficult to realize and poorly informative. Thereby molecular and functional characterization of CTCs presents a double interest, clinical to identify treatments biomarkers sensitivity and basic to study mechanisms underlying their tumor inititiating cell (TIC) potential. The two goals of my thesis were on the one hand to characterize by whole-exome sequencing (WES) at the single level the CTCs from patients with metastatic prostate cancers (mPCa) and on the other hand to establish and characterize CTC-derived xenografts (CDX) from patients with non-small-cell lung cancer (NSCLC) or mPCa. For the first goal we developped a global workflow which include three technological approaches to enrich and isolate individual CTCs from different phenotype (epithelial, epithelial and mesenchymal, mesenchymal), to perform whole genome amplification (WGA) and to sequence them. WES was performed on 34 CTC samples selected according to WGA quality and on corresponding metastasis biopsies from seven patients. Two patients with phenotypic heterogeneity of CTCs were deeply analyzed. We highlighted shared mutations between CTCs and matched biopsies as well as mutations only detected in CTCs. These private CTC mutations are detected in all phenotype and particularly affect genes invlved in cytoskeleton remodeling, DNA repair or invasion. The existence of common mutations between CTCs from various phenotype suggests a phylogenic link between these cells but a divergent evolution during metastatic process. This work is submitted for publication. For the second goal, we implanted CTCs from 67 NSCLC patients and 28 mPCa patients in immunocompromised mice. We established four NSCLC CDX and one mPCa CDX. The characterization of tumor biopsies, CTCs collected at the time of xenograft, CDX and CDX-derived cell lines revealed that CTCs, CDX and cell lines miror the phenotype and mutational landscape of tumor biopsies. The more deeply characterization of one cell line show the presence of a high replicative stress and genomic instability. This result directs us to the hypothesis of a possible role of the genomic instability in CTC tumorigenicity.We demonstrated in this work that CTCs mutational landscape harbors high similairities with patients tumor biopsies in mPCa. Furthermore we observed CTC private mutations not detected in tumor biopsies. Also we showed that some CTCs from NSCLC and mPCa are tumorigenic in vivo and that these CTCs mirror mutational profile of patients tumor biopsies. These models are original and interesting tools to identify new therapeutic targets and anti-tumoral strategies and understand mechanisms underlying the TIC potential of CTCs.

La neuroradiologie Interventionnelle est devenue une spécialité médicale à part entière en évolution permanente avec l'amélioration des technologies radiologiques et endovasculaires. Néanmoins, de trop nombreuses complications liées à la technique elle-même sont encore rapportées. Basés sur notre expérience clinico-radiologique, nous nous sommes plus particulièrement intéressés aux complications thromboemboliques grâce à de nouvelles technologies biologiques permettant d'envisager une analyse des lésions vasculaires à l'échelle cellulaire (Cellsearch®). Ainsi au cours de différents examens diagnostiques et thérapeutiques nous avons analysé les taux artériels et veineux de cellules endothéliales circulantes à différents temps de la procédure. Nous avons démontré l'agression du matériel endovasculaire sur les parois artérielles induisant un relargage immédiat de CECs unitaires et en amas de taille variable, parfois géants (300 µm). Les amas présentant une taille supérieure aux diamètres des microcapillaires ne peuvent pas migrer dans le compartiment veineux et sont donc potentiellement à l'origine d'une occlusion artérielle très distale responsable de lésions microischémiques qualifiées de silencieuses car asymptomatique dans l'ensemble des cas observés. Les lésions pariétales artérielles induites sont ensuite probablement réparées par un mécanisme dynamique mettant en jeu une augmentation lente et progressive des CEPs jusqu'à l'obtention d'une réparation de l'endothélium. Au delà des facteurs mécaniques endovasculaires, l'analyse cellulaire de cette cinétique destruction-régénération des parois artérielles pourrait s'avérer intéressante dans l'évaluation de l'endothélialisation des endoprothèses intracrâniennes. / Permanents Improvements in radiological and endovascular devices made the Interventional Neuroradiology considered as a complete medical specialty. However, too many procedural complications due to the devices remain observed in all reported series. On the basis of our clinical center experience we decided to analyze thromboembolic complications by using new biological tests allowing to the detection of circulating endothelial cells. During cerebral diagnostic or therapeutic angiography, arterial and venous CECs rates were analyzed before, during, and after endovascular procedures. Thus, we demonstrated the potential arterial wall injury following catheterization that induced unit and cluster of CECs, some of them were giants (300 µm). Clusters presenting with a size up than those described from microcapillary lumen cannot go through venous compartment, and potentially block into a distal artery inducing microischemic stroke (silent embolism) as observed in this work. In addition, the induced arterial wall injury is probably regenerated by a dynamic mechanism involving a delayed increase of CEPs rates, until the new endothelialization. Over endovascular mechanism factor analysis, the CECs-CEPs investigations could be an interesting strategy in the monitoring of intracranial endoprothesis endothelialization.

Les cellules tumorales circulantes (CTC) sont des cellules présentes en très faible proportion (une cellule pour un million de cellules normales) dans la circulation sanguine, et qui jouent un rôle important dans le processus de métastase responsable de la majorité des décès de patients atteints de cancer. La détection du cancer à un stade précoce augmente les chances de survie des patients. Le but de ce travail a été de développer un ensemble de technologies permettant de mieux caractériser et détecter les CTC.Nous avons concentré notre étude sur les cellules ayant un phénotype hybride, entre épithélial et mésenchymal, qui pourraient correspondre à des CTC de plus fort potentiel métastatique compte tenu du rôle joué par la transition épithelio-mésenchymateuse dans ce processus. Nous avons tout d’abord isolé, par immunofluorescence et cytométrie en flux, une lignée cellulaire de cancer (A549, le carcinome de poumon humain) co-exprimant la E- et la N-cadhérine, de sorte qu’elle puisse être utilisée comme modèle de CTC dans le développement de nouvelles techniques de détection. Nous avons en particulier adapté le système de microscopie de fluorescence et d’analyse d'images PathfinderTM à haut-débit de la société Imstar S.A. pour identifier efficacement quelques milliers de cellules A549 mélangées à du sang de patient, après une étape de filtration par la taille. Afin d’améliorer l’identification des cellules hybrides, nous avons évalué la technique de transfert de Förster résolue en temps qui pourrait révéler avec un excellent rapport signal/bruit la présence à la membrane cellulaire d’agrégats compacts de N- et E-cadhérines. Enfin, afin d’augmenter le nombre de biomarqueurs simultanément détectés par immunofluorescence nous avons contribué à la mise au point de nanocristaux semi-conducteurs fluorescents conjugués avec un anticorps dirigé contre une protéine d'intérêt. Au final, nos résultats fournissent un ensemble de technologies qui pourront être utilisées pour améliorer la détection et la caractérisation des CTC. / Circulating tumor cells (CTCs) are rare cells (one in millions of normal cells) in blood circulatory system playing a key role in the process of metastasis, which is responsible for the majority of death of patients with cancer. Detecting cancer at early stage can give patients higher chances of survival. The aim of this work is to develop a set of technologies capable of characterizing and detecting the CTCs. We restricted our study to CTCs with hybrid phenotype, between epithelial and mesenchymal, that could correspond to circulating cells with the highest metastatic potential, considering the relation of the Epithelial to Mesenchymal Transition to cancer. Using immunofluorescence and flow cytometry, we first isolated a cancer cell line (A549, human lung carcinoma) co-expressing E- and N-cadherin, which is further used as a CTC model in the development of new detection techniques. In particular, we showed that the high throughput automated fluorescence microscope and image processing Imstar S.A. PathfinderTM system can recover efficiently a few thousands of A549 cells spiked in a blood sample, after an initial size-filtering step. We also used time-gated Fluorescence Resonant Energy Transfer to investigate the presence of E- and N-cadherin clusters at the cell membrane that could enhance the detection sensitivity of hybrid phenotype. Finally, in view of increasing the number of simultaneous biomarkers detection by immunofluorescence we contributed to the development of fluorescent semiconductor nanocrystals conjugated with antibody directed against the protein of interest. Altogether, our results provide a set of technologies that can be used to improve the detection and characterization of CTCs.

Introduction:Un des problèmes du cancer du Pancréas (CP) est le temps de latence entre la suspicion du CP et la mise en place des traitements. Les méthodes de biopsie liquide pourraient accélérer la mise en évidence d’éléments tumoraux et le diagnostic.Objectif :L’objectif principal de l’étude était de comparer la performance diagnostique de plusieurs techniques de biopsie liquide chez des patients atteint d’un CP résécable d’emblé. L’objectif secondaire était la corrélation avec le taux de récidive post-opératoire.Méthodes:Tout d'abord, nous avons testé 2 méthodes d'enrichissement CTC pour estimer la sensibilité de la détection CTC avec des expériences de cell-spiking de deux lignées de cellules tumorales pancréatiques dans des échantillons de sang de 24 volontaires sains en utilisant la méthode en gradient de densité OncoQuick® et la méthode de sélection négative RosetteSep™. De plus, les mutations KRAS ont été quantifiées dans l'ADN génomique de cellules purifiées par digital droplet PCR (dd-PCR) avec des amorces spécifiques des allèles.Nous avons conçu un essai clinique prospectif (NCT03032913) visant à détecter les cellules tumorales circulantes (CTC), l’ADN tumoral circulant (ADNct) et les onco-exosomes chez les patients atteint de CP et chez les patients d’un groupe témoin. Pour les CTCs : enrichissement et détection de CTCs par la méthode CellSearch©, méthode d’enrichissement de CTCs RosetteSep® et OncoQuick® puis quantification de l’ADN tumoral par dd-PCR. Les exosomes ont été isolés puis caractérisés avec le taux d’expression de Glypican-1. Tous les patients de l’étude ont eu un prélèvement de sang périphérique, les patients du groupe CP ont eu un prélèvement de sang portal peropératoire.Résultats:La sensibilité analytique était de 100 % pour OncoQuick®, quelle que soit la lignée cellulaire, et se situait entre 70 et 100 % pour RosetteSep™. Le taux moyen de récupération des cellules était de 56±23% pour OncoQuick® contre 39±27% pour RosetteSep™ (p<0,001). Les cellules tumorales de la population de cellules sanguines enrichies ont été détectées par dd-PCR après enrichissement par RosetteSep™ et OncoQuick® La détection des allèles K-RAS mutants par ddPCR après enrichissement de RosetteSepTM était 3 à 4 fois plus sensible qu'après OncoQuick®. Ainsi, RosetteSep™ est plus fiable en termes d'efficacité de récupération et de détection des mutants KRAS que OncoQuick®.De février à novembre 2017, 22 patients atteints de CP résécable et 28 patients témoins ont été inclus. Tous les patients ont été détectés positifs par au moins une méthode. Les CTCs ont été détectées chez 9 patients avec la méthode cellsearch (70% dans le sang portal exclusif) et 13 avec la méthode Rosettesep (60%). Les onco-exosomes ont été détecté chez 14 patients sur 22. L’ADNct n’a été détecté que chez deux patients métastatiques. La détection combinée des CTCs et des onco-exosomes était significativement corrélée à la survie sans récidive.Conclusion:Cette étude suggère que la biopsie liquide combinée peut être un outil prometteur à fois diagnostique et pronostique dans le CP à un stade précoce. / Introduction:One of the problems of pancreatic ductal adenocarcinoma (PC) is the latency time between the suspicion of PC and the initiation of treatments, especially neo-adjuvants that require histological evidence. Liquid biopsy methods could be a companion test for diagnosis.Objective :The main objective of the study was to compare the diagnostic performance of several liquid biopsy techniques in patients with resectable pancreatic without neo-adjuvant therapy cancer. The secondary objective was the correlation between the quantification of liquid biopsy parameters and clinic-pathologic features.Methods:First, we tested 2 CTC enrichment methods to estimate the sensitivity of CTC detection with cell spiking experiments of two pancreatic tumour cell lines in blood samples from 24 healthy volunteers using the onco-specific density gradient OncoQuick® and the negative selection enrichment method RosetteSep™. Additionally, KRAS mutations were quantified in genomic DNA of purified cells by digital droplet Q-PCR (dd-PCR) with allele specific primers.We designed a prospective clinical trial (PANC-CTC# NCT03032913) to detect circulating tumour cells (CTC), circulating tumour DNA (ADNct) and onco-exosomes in patients with pancreatic cancer and in patients in a control group using different methods. For CTCs, it was the enrichment and detection of CTCs by the CellSearch© method (reference method), the RosetteSep® and OncoQuick® CTC enrichment method and the quantification of tumor DNA by dd-PCR. Exosomes were isolated and characterized with the expression rate of Glypican-1. All patients in the study had a peripheral blood sample, patients in the PDAC group had a portal blood sample during surgery.Results:Analytical sensitivity was 100% for OncoQuick®, regardless of the cell line, and ranged between 70 and 100% for RosetteSep™. Mean recovery rate of cells was 56±23% for OncoQuick® versus 39±27% for RosetteSep™ (p<0.001). Molecular detection of mutant K-RAS alleles by ddPCR after RosetteSepTM enrichment was 3- to 4-fold more sensitive than after OncoQuick®. Thus, RosetteSep™ is more reliable in terms of recovery efficiency and KRAS mutant detection than OncoQuick®.From February to November 2017, 22 patients with resectable pancreatic cancer and 28 control patients were included. All patients were positive by at least one method. CTCs were detected in 9 patients with the cellsearch method (70% in the exclusive portal blood) and 13 with the Rosettesep method (59%), Onco-exosomes were detected in 14 out of 22(64%) patients in peripheral and/or portal blood. DNAct was detected in only two metastatic patients. The combined detection of CTCs with cellsearch and onco-exosomes was significantly correlated with progression free survival and overall survival when CTC cluster were found.Conclusion: This study suggests that combined liquid biopsy can be a promising tool for both diagnosis and prognosis in early pancreatic cancer.

Nous présentons deux dispositifs microfluidiques, aussi appelés Laboratoires-sur-Puces, fondés sur deux technologies : la microfluidique et les billes magnétiques. Un premier laboratoire-sur-puce est dédié à la recherche de cellules tumorales circulantes. Les billes magnétiques sont auto-organisées sur un dépôt magnétique de ferrofluide obtenu par tamponnage moléculaire et les cellules sont séparées sur la base de protéines de membranes à leur surface. Les expériences menées établissent les performances de rendement et de pureté de la séparation cellulaire. On démontre que les cellules capturées peuvent être remises en culture. Comme validation clinique, ce système a été utilisé pour le typage de cellules tumorales circulantes lymphoïdes issues de leucémies et de lymphomes à l'aide de microscopie confocale à haute résolution. Un deuxième laboratoire-sur-puce a été utilisé pour la recherche de bactéries infectieuses. Un module de concentration d'ADN a été mis en place à l'aide de billes magnétiques organisées en réseau très dense de billes. L'ADN est retenu par sa charge sur les billes à faible pH et élué par augmentation de pH. La preuve de concept du système est apportée.

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-05-02T18:31:29Z No. of bitstreams: 1 Arquivototal.pdf: 790723 bytes, checksum: 7e439258b05733c014dcd3e7de230c1f (MD5) / Made available in DSpace on 2018-05-02T18:31:29Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 790723 bytes, checksum: 7e439258b05733c014dcd3e7de230c1f (MD5) Previous issue date: 2017-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In this present work, principles and theorems associated to integers are returned, as well as eigenvalues and eigenvectors problems, highlighting a Hermitian matrix. Then it is emphasized to the Circulating Matrices, through which it is found the association to two well-defined polynomials: the representative and the characteristic. Later a brief account about the history of polynomial equations is made, drafting the Cardano-Tartaglia Formulas associated to them. Afterwards a unification is made in the resolution process of the polynomial equations of smaller degrees than the equal to 4, by means of the circulating matrices. The work is completed by proving a Fermat theorem for n = 3, using the Cardano-Tartaglia Formulas. / No presente trabalho, princípios e teoremas associados aos números inteiros são retomados, bem como problemas de autovalores e autovetores, sendo ressaltada a matriz Hermitiana. Em seguida é dado ênfase às Matrizes Circulantes, através das quais verifica-se a associação a dois polinômios bem definidos: o representante e o característico. Posteriormente realiza-se um breve relato acerca da história das equações polinomiais, destacandose as Fórmulas de Cardano-Tartaglia associadas às mesmas. Logo após é feita uma unificação no processo de resolução das equações polinomiais de graus menores do que o igual a 4, por meio das matrizes circulantes. O trabalho é finalizado, sendo provado o Teorema de Fermat para n = 3, recorrendo-se às Fórmulas de Cardano-Tartaglia.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-10-01T21:50:08Z No. of bitstreams: 1 DANIEL DE JESÚS LIMONTA VELÁZQUEZ.pdf: 919728 bytes, checksum: e0d58f37f2cdfe18820bd7ba77a8415c (MD5) / Made available in DSpace on 2013-10-01T21:50:08Z (GMT). No. of bitstreams: 1 DANIEL DE JESÚS LIMONTA VELÁZQUEZ.pdf: 919728 bytes, checksum: e0d58f37f2cdfe18820bd7ba77a8415c (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Dengue é a doença viral transmitida por mosquitos de maior importância mundial e é, causada por qualquer um dos quatro sorotipos do vírus (DENV-1,-2,-3,-4). É uma virose frequente em áreas tropicais e subtropicais e atualmente o Brasil notifica um número crescente de casos. Durante mais de 30 anos a dengue foi classificada em febre da dengue e febre hemorrágica da dengue. Porém, a OMS no ano 2009 propôs uma nova classificação: dengue com e sem sinais de alarme (DSSA e DCSA) e dengue grave (DG) no intuito de facilitar tanto o diagnóstico como o manejo do tratamento. A patogenia da dengue é multifatorial na qual se aceitam contribuições de fatores virais e do hospedeiro. Estudos sobre mecanismos da imunopatologia da dengue descreveram associações entre fenômenos descritos como “tempestade” de citocinas, hiperativação de células imunes, anticorpos e células T de reação cruzada entre diferentes sorotipos virais. Além disso, estudos vêm demonstrando a contribuição da apoptose na imunopatologia da dengue. Neste contexto, o objetivo do trabalho da tese foi avaliar o envolvimento de proteínas circulantes relacionadas a apoptose na patogênese da infecção pelos DENV. Para isso, 106 amostras foram obtidas de pacientes com suspeita clínica de infecção por DENV durante a epidemia de 2010 nos estados brasileiros de Mato Grosso do Sul e Rio de Janeiro. Para confirmação dos casos, cinco diferentes técnicas foram utilizadas: ELISA de captura de IgM, IgG anti-dengue, detecção da proteína viral NS1, isolamento viral e RT-PCR. Nesta casuística, 72 casos de dengue foram confirmados. A detecção de IgM anti-dengue confirmou um maior número de casos suspeitos. Os sorotipos encontrados foram DENV-1 e DENV-2. Baseado na nova classificação da OMS, tivemos 35 indivíduos com DSSA, 17 pacientes com DCSA e 15 indivíduos com DG. Contudo, a classificação tradicional da dengue diagnosticou apenas 5 casos com febre hemorrágica da dengue. Nos 67 pacientes da dengue foram dosados níveis plasmáticos de moléculas pró-apoptóticas (TNF-α, TRAIL, FasL) e anti-apoptótica (Survivina). Níveis aumentados de TNF-α, FasL e TRAIL foram encontrados nos pacientes com a forma DSSA quando comparados aos controles e pacientes graves (DG). Nossos resultados não indicaram correlações importantes entre os níveis de TNF-α e parâmetros associados à gravidade, mas, observamos correlações diretas entre FasL ou TRAIL com plaquetas e TRAIL com hematócrito. Todavia, o único caso fatal apresentou o maior nível plasmático de FasL. De forma interessante, TRAIL se correlacionou inversamente com a contagem dos linfócitos. Em relação à Survivina, os resultados indicaram que o grupo DCSA apresentou níveis diminuídos em comparação aos controles e pacientes graves e de forma interessante, Survivina se correlacionou diretamente com a contagem de leucócitos. Assim, o TRAIL seria indicador de bom prognóstico da doença, e TRAIL poderia estar envolvido com a maior susceptibilidade dos linfócitos à morte. Por outro lado, Survivina poderia estar participando da proliferação e/ou migração dos leucócitos. Até o presente, este é o primeiro trabalho abordando mediadores circulantes de apoptose segundo a nova classificação clínica da dengue. Mais pesquisas precisam ser realizadas para discernir a complexa interação entre moléculas pró e anti-apoptóticas e suas implicações na gravidade da dengue. / Dengue é a doença viral transmitida por mosquitos de maior importância mundial e é, causada por qualquer um dos quatro sorotipos do vírus (DENV-1,-2,-3,-4). É uma virose frequente em áreas tropicais e subtropicais e atualmente o Brasil notifica um número crescente de casos. Durante mais de 30 anos a dengue foi classificada em febre da dengue e febre hemorrágica da dengue. Porém, a OMS no ano 2009 propôs uma nova classificação: dengue com e sem sinais de alarme (DSSA e DCSA) e dengue grave (DG) no intuito de facilitar tanto o diagnóstico como o manejo do tratamento. A patogenia da dengue é multifatorial na qual se aceitam contribuições de fatores virais e do hospedeiro. Estudos sobre mecanismos da imunopatologia da dengue descreveram associações entre fenômenos descritos como “tempestade” de citocinas, hiperativação de células imunes, anticorpos e células T de reação cruzada entre diferentes sorotipos virais. Além disso, estudos vêm demonstrando a contribuição da apoptose na imunopatologia da dengue. Neste contexto, o objetivo do trabalho da tese foi avaliar o envolvimento de proteínas circulantes relacionadas a apoptose na patogênese da infecção pelos DENV. Para isso, 106 amostras foram obtidas de pacientes com suspeita clínica de infecção por DENV durante a epidemia de 2010 nos estados brasileiros de Mato Grosso do Sul e Rio de Janeiro. Para confirmação dos casos, cinco diferentes técnicas foram utilizadas: ELISA de captura de IgM, IgG anti-dengue, detecção da proteína viral NS1, isolamento viral e RT-PCR. Nesta casuística, 72 casos de dengue foram confirmados. A detecção de IgM anti-dengue confirmou um maior número de casos suspeitos. Os sorotipos encontrados foram DENV-1 e DENV-2. Baseado na nova classificação da OMS, tivemos 35 indivíduos com DSSA, 17 pacientes com DCSA e 15 indivíduos com DG. Contudo, a classificação tradicional da dengue diagnosticou apenas 5 casos com febre hemorrágica da dengue. Nos 67 pacientes da dengue foram dosados níveis plasmáticos de moléculas pró-apoptóticas (TNF-α, TRAIL, FasL) e anti-apoptótica (Survivina). Níveis aumentados de TNF-α, FasL e TRAIL foram encontrados nos pacientes com a forma DSSA quando comparados aos controles e pacientes graves (DG). Nossos resultados não indicaram correlações importantes entre os níveis de TNF-α e parâmetros associados à gravidade, mas, observamos correlações diretas entre FasL ou TRAIL com plaquetas e TRAIL com hematócrito. Todavia, o único caso fatal apresentou o maior nível plasmático de FasL. De forma interessante, TRAIL se correlacionou inversamente com a contagem dos linfócitos. Em relação à Survivina, os resultados indicaram que o grupo DCSA apresentou níveis diminuídos em comparação aos controles e pacientes graves e de forma interessante, Survivina se correlacionou diretamente com a contagem de leucócitos. Assim, o TRAIL seria indicador de bom prognóstico da doença, e TRAIL poderia estar envolvido com a maior susceptibilidade dos linfócitos à morte. Por outro lado, Survivina poderia estar participando da proliferação e/ou migração dos leucócitos. Até o presente, este é o primeiro trabalho abordando mediadores circulantes de apoptose segundo a nova classificação clínica da dengue. Mais pesquisas precisam ser realizadas para discernir a complexa interação entre moléculas pró e anti-apoptóticas e suas implicações na gravidade da dengue.