1 |
Intérêt diagnostique de la biopsie liquide dans la prise en charge de l'adénocarcinome canalaire du pancréas à un stade précoce / Diagnostic interest of liquid biopsy in the management of early stage pancreatic ductal adenocarcinomaBuscail, Etienne 14 June 2019 (has links)
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.
|
2 |
Thermodynamic Characterization of Linker Histone Binding Interactions with ds-DNAMachha, Venkata Ramana 17 May 2014 (has links)
Linker histones (H1) are the basic proteins in higher eukaryotes that are responsible for the final condensation of chromatin. H1 also plays an important role in regulating gene expression. H1 has been described as a transcriptional repressor as it limits the access of transcriptional factors to DNA. Linker histone binds to DNA that enters or exits the nucleosome. Several crystal structures have been published for the nucleosome (histone core/DNA complex), and the interactions of the core histone proteins with DNA are well understood. In contrast the location of the linker histone and its interactions with ds-DNA are poorly understood. In this study we have used isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and CD spectropolarimetry to determine the thermodynamic signatures and structural changes that accompany H1 binding to ds-DNA. The thermodynamic parameters for the binding of intact linker histones (H1.1, H1.4, and H10) to highly polymerized calf-thymus DNA and to short double stranded DNA oligomers have been determined. We have also determined the thermodynamics for binding of H10 C-terminal tail (H10-C) and globular domain (H10-G) to calf-thymus DNA. The real surprise in the energetics is that the enthalpy change for formation of the H1/DNA complex is very unfavorable and that H1/DNA complex formation is driven by very large positive changes in entropy. The binding site sizes for H1.1, H1.4, and H10 were determined to be 36bp, 32bp, and 36bp respectively. CD results indicate that CT-DNA is restructured upon complexation with either the full length H1 protein (H10) or its C-terminal domain (H10-C). In contrast, the structure of H10 is largely unchanged in the DNA complex. Temperature dependence of enthalpy change, osmotic stress and ionic strength dependence of Ka were tested using ITC. These results indicate that the entropy driven H1/DNA complexes are a result primarily from the expulsion of bound water molecules from the binding interface. This study provides new insights into the binding of linker Histone H1 to DNA. A better understanding of the functional properties of H1 and its interactions with DNA could provide new insights in understanding the role H1 in DNA condensation and transcriptional regulation.
|
3 |
Sequencing and functional analysis of cT-DNAs in Nicotiana / Séquence et analyse fonctionnelle des ADN-T dans NicotianaChen, Ke 26 February 2016 (has links)
La bactérie Agrobacterium tumefaciens est bien connue pour son utilisation en génie génétique végétale où elle sert comme vecteur de gènes. A l’origine, cette bactérie ainsi que l’espèce voisine Agrobacterium rhizogenes sont des bactéries phytopathogènes qui induisent respectivement des tumeurs et des racines anormales sur des plantes sensibles telles que la vigne ou des arbres fruitiers. L’action pathogène résulte d’un transfert horizontal de gènes de la bactérie vers l’hôte végétal, à partir d’un plasmide, le pTi (plasmide inducteur de tumeurs) ou pRi (plasmide inducteur de racines). Mon travail de thèse concerne deux aspects particuliers de cette bactérie.1. Sa capacité à transformer durablement des espèces végétales dans la nature, donnant ainsi naissance à des plantes naturellement transformées, notamment dans le genre Nicotiana. Nous avons pu montrer par séquençage à haut débit du génome de N. tomentosiformis et par l’analyse d’autres séquences complètes de Nicotianées publiées récemment l’existence inattendue de 5 séquences venant d’Agrobacterium (cT-DNAs) avec une taille total de 65 kb, dont certaines portent des gènes intacts. Nous avons montré que deux de ces gènes (TB-mas2’ de N. tabacum et TE-6b de N. otophora) ont une activité biologique. Une étude comparative approfondie a permis de mieux comprendre l’évolution de ces cT-DNAs (Chen et al., 2014). Le gène mas2’ est bien connu, il code pour une enzyme qui catalyse la synthèse du désoxyfructosyl-glutamine (DFG) dans des tumeurs ou racines induites par Agrobacterium. Des résultats récents dans notre groupe portant sur le gène TB-mas2’ montrent que ce gène est exprimé de façon très active dans plusieurs cultivars de N. tabacum, et y donne naissance à l’apparition de quantités mesurables de DFG. Ce travail est présenté sous forme d’un manuscrit à soumettre.2. Une deuxième partie de la Thèse concerne les propriétés du gène T-6b, qui fait partie de l’ADN transféré par A. vitis souche Tm4 et provoque une croissance anormale caractérisée par l’apparition d’énations, sans que l’on connaisse son mode d’action. Le gène 6b fait partie de la famille des gènes plast (pour plasticité phénotypique), avec des effets différents et souvent remarquables sur la croissance des plantes. Le gène T-6b a été mis sous contrôle d’un promoteur inductible par le dexaméthasone, et des plantes de tabac transformées par cette construction ont été étudiées en détail, à différents moments après son induction. Un grand nombre de changements a été décrit incluant des analyses anatomiques montrant des modifications encore jamais décrites chez les plantes, comme par exemple l’apparition de méristèmes foliaires ectopiques à la base de trichomes, ou l’apparition de systèmes vasculaires ectopiques parallèles au système vasculaire normal avec un développement régulier menant à des structures complexes ordonnées (Chen and Otten, 2015). Le gène TE-6b de N. otophora a été mis sous contrôle d’un promoteur fort constitutif et introduit dans des plantes de tabac, où il provoque des changements de croissance différents de ce qui a été observé pour le gène T-6b. Ces derniers résultats préliminaires sont présentés en complément des observations sur le gène T-6b. Ils indiquent que le transfert horizontal du gène TE-6b vers l’ancêtre de N. otophora aurait pu contribuer à une modification de la croissance et ainsi à la création d’une nouvelle espèce. / The bacterium Agrobacterium tumefaciens is well-known for its utilisation in plant genetic engineering where it serves as a gene vector. This bacterium and the related species Agrobacterium rhizogenes are phytopathogens that induce tumors and hairy roots respectively on susceptible plants like grapevine or fruit trees. Their phytopathogenicity is due to horizontal transfer of bacterial genes to the plant host, from a plasmid called the Ti (tumor-inducing) or Ri (root-inducing) plasmid. The subject of my Thesis concerns two particular aspects of this bacterium.1. Their capacity to stably transform several plant species in nature, thereby yielding naturally transformed plants, especially in the genus Nicotiana. We have shown by deep sequencing of the Nicotiana tomentosiformis genome and by analysis of other recently published Nicotiana sequences the presence of five different Agrobacterium-derived sequences (cT-DNAs), totalling 65 kb, some of which carry intact genes. We have shown that two of them (TB-mas2’ from N. tabacum and TE-6b from N. otophora) have biological activity. A detailed comparative study has allowed us to better understand the evolution of these cT-DNAs (Chen et al., 2014). The mas2’ gene is well-known, it codes for the synthesis of desoxyfructosyl-glutamine (DFG) in tumors or roots induced by Agrobacterium. Recent work in our group has shown that the TB-mas2’ gene is highly expressed in some N. tabacum cultivars and leads to the accumulation of detectable amounts of DFG. This work is presented as a manuscript to be submitted.2. A second part of the Thesis describes new properties of the T-6b gene, which is part of the DNA transferred by A. vitis strain Tm4 and leads to abnormal growth caracterized by the appearance of enations, so far the mode of action of this gene is unknown. The 6b gene is part of the so called plast family (for phenotypic plasticity), with different and often remarkable growth effects on plants. The T-6b gene wasearlier placed under control of a dexamethasone-inducible promoter, and tobacco plants transformed with this construct have now been studied in detail, at different times after the start of induction. A large number of changes was analyzed, both at the morphological and anatomical level, these include various unprecedented morphological changes, like for example the appearance of shoot primordia at the base of trichomes, or the appearance of ectopic vascular strands parallel to the normal strands with a regular development leading to complex but predictable structures (Chen and Otten, 2015). The TE-6b gene from N. otophora was placed under strong and constitutive promoter control and introduced into tobacco, where it was found to cause new types of morphological change, different from those observed for T-6b. The latter results are preliminary and will be presented as a complement to the work on T-6b. They indicate that the introduction of the TE-6b gene in the N. otophora ancestor could have caused a change in growth pattern, and might have favored the appearance of a new species.
|
4 |
Desenvolvimento de sensor nanoestruturado e biossensor de dsDNA determinação de substâncias de interesse biológico: nitrotirosina, ácido ascórbico e ácido úrico / Development of nanoestrutured sensor and dsDNA biosensor for determination of biological interest substances: nitrotyrosine, ascorbic acid and uric acidCosta, Erivaldo de Oliveira 13 December 2016 (has links)
In this work we developed an electrochemical sensor modified with multi-walled carbon nanotubes and 5-nitroindole (5-NI) for the simultaneous determination of ascorbic (AA) and uric (UA) acids in urine and serum samples and a biosensor based on dsDNA for determination of 3-nitro-L-tyrosine ethyl ester (3NO2TEE), as a biomarker of biological peroxynitration. The polymer of 5-NI was electrogenerated in situ on the carbon nanotubes deposited on glassy carbon electrode (GCE). Thereafter, the aromatic nitro group, present in the molecule, was reduced, generating a hydroxylamine/nitroso redox couple, then used for detection and quantification of the analytes in the biological samples. The electrochemical behavior of the modified electrodes were studied using cyclic voltammetry, differential pulse voltammetry and chronoamperometry, which were used for the detection of the analytes and to obtain the kinetic parameters and the analytical characterization of the platforms. The chronoamperometric studies were performed in order to obtain more information about the redox processes between AA and the sensor, since it proved to be an electrocatalytic process. Thus, by means of graphs and Cottrell equations, it was possible to obtain values for the diffusion coefficient (DAA) and the catalytic constant (kcat) for the AA electrocatalytic oxidation. The values for DAA and kcat, determined for AA were 4.2 x10-6 cm-2 s-1 and 1.1 x 106 M-1 s-1, respectively. The platform for the detection of AA e AU showed good sensitivity and stability in urine and serum samples, with LOD of 1.9 mol L-1 for AA and 2.1 mol L-1 for UA. The analytical performance obtained for the nanostructured platform GCE/MWCNT/poly-5-NID justifies its use as a sensor for the simultaneous determination of AA and UA. Studies of 3NO2TEE in protic media (pH 4.5 acetate buffer and pH 7.4 and 10.0, in phosphate buffer) and in aprotic media (DMF/TBAPF6, 0.1 mol L-1), using Pt and glassy carbon electrodes, were necessary before the construction of the CT-DNA biosensor. The spectrolectrochemistry of 3NO2TEE, held in aprotic media was useful for rationalizing the electrochemical behavior of 3NO2TEE and intermediates generated during the reduction. For the biosensor construction, the amount of dsDNA, the conditioning time for reduction of the analyte, the pH value, on GCE, were optimized. The developed biosensor was used to determine the concentration of 3NO2TEE and showed a linear range from 60 to 320 nmol L-1 and LOD and LOQ of 58 nmol L-1 and 200 nmol L-1, respectively. These results indicate that the prepared sensor and biosensor were effective for the detection of substances with biological interest. (P.S.: some expressions out of formatting) / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Neste trabalho foram desenvolvidos um sensor eletroquímico modificado com nanotubos de carbono de paredes múltiplas (MWCNT) e 5-nitroindol (5-NI) para determinação simultânea dos ácidos ascórbico (AA) e úrico (AU), em amostras de urina e soro, e um biossensor à base de dsDNA para determinação do éster etílico da 3-nitro-L-tirosina (3NO2TEE), como biomarcador de peroxinitração biológica. O trímero do 5-nitroindol foi eletrogerado in situ sobre os nanotubos de carbono depositados em eletrodo de carbono vítreo (ECV). Após esse processo, o grupo nitro aromático, presente na molécula foi reduzido gerando o par redox hidroxilamina/nitroso, utilizado para detecção e quantificação dos analitos presentes nas amostras biológicas. Os comportamentos eletroquímicos dos eletrodos modificados foram estudados, empregando as técnicas de voltametria cíclica, voltametria de pulso diferencial e cronoamperometria, as quais foram utilizadas para a detecção dos analitos e para obtenção dos parâmetros cinéticos e caracterização analítica da plataforma. Os estudos cronoamperométricos foram realizados com o objetivo de obter maiores informações dos processos redox entre AA e o sensor, uma vez que este demonstrou ser um processo eletrocatalítico. Assim, por meio de gráficos e equações de Cottrell, foi possível obter os valores para o coeficiente de difusão (DAA) e a constante catalítica (kcat) da reação para o AA. Os valores do DAA e de kcat, determinados para AA, foram de 4,2 x10-6 cm-2 s-1 e 1,1 x 106 M-1 s-1, respectivamente. O método desenvolvido de detecção de AA e AU apresentou boa sensibilidade e estabilidade em amostras de urina e soro, com limite de detecção (LD) de 1,9 mol L-1 para AA e 2,1 mol L-1 para AU. A partir do desempenho analítico obtido da plataforma nanoestruturada ECV/MWCNT/poli-5-NID, justifica-se a sua utilização deste como sensor para a determinação simultânea de AA e AU. Antes da construção do biossensor de DNA, para nitroaromáticos de importância biológica, foi necessária a realização dos estudos da 3NO2TEE, em meios prótico: tampão acetato pH 4,5 e tampão fosfato pH 7,4 e 10,0 e, em meio aprótico (DMF/ TBAPF6, 0,1 mol L-1), utilizando eletrodo de Pt e carbono vítreo, como eletrodos de trabalho. A espectroeletroquímica de 3NO2TEE, realizada em meio aprótico, foi útil para racionalizar o comportamento eletroquímico de 3NO2TEE e de seus intermediários gerados durante a redução. Para a construção do biossensor, sobre o eletrodo de carbono vítreo, foram otimizados: concentração de dsDNA, tempo de condicionamento para a redução do nitrocomposto, valor de pH. O biossensor ECV/dsDNA apresentou faixa linear de 60 - 320 nmol L-1 e LD e LQ (limite de quantificação) encontrados foram 58 nmol L-1 e 200 nmol L-1. Estes resultados indicaram que o sensor e o biossensor foram produzidos e aplicados de forma eficaz para detecção de substâncias de interesse biológico. (obs.: algumas expressões fora da formatação)
|
Page generated in 0.029 seconds