O prognóstico de pacientes portadores de sarcomas de partes moles (SPM) avançados é reservado. Aumentar o benefício de quimioterapia é necessário, sendo, uma das estratégias, quimioterapia em dose densa, a qual demonstrou benefício em câncer de mama. Por outro lado, a busca de um marcador prognóstico é importante para uma melhor seleção de pacientes que se beneficiariam de protocolo de tratamento mais intensivo. A ezrina é uma proteína que liga o citoesqueleto celular a proteínas de membrana, está associada a invasão celular e metástase e sua hiperexpressão tem sido associada a um pior prognóstico em sarcomas de partes moles. O objetivo deste estudo foi avaliar o papel de quimioterapia com dose densa em pacientes portadores de SPM de alto grau, avançados. O desfecho primário foi taxa de resposta e os secundários foram sobrevida global (SG), sobrevida livre de progressão (SLP), perfil de toxicidade, qualidade de vida e controle de dor. Avaliou-se também a expressão de ezrina por imunohistoquímica como marcador de prognóstico, com o intuito de estratificação da população que poderia se beneficiar mais desta abordagem intensificada. Neste estudo de fase II prospectivo, vinte e um pacientes foram incluídos. A idade mediana foi 37 anos (23-60) e extremidades inferiores foram o sítio primário mais comum. Sarcoma sinovial, leiomiossarcoma e sarcoma sem outras especificações foram as histologias mais frequentes. O protocolo consistiu de seis ciclos seqüenciais de doxorubicina 30mg/m2 D1-3 e ifosfamida 2,5g/m2 D1-5 a cada 14 e 21 dias, respectivamente, seguidos por sete dias de suporte hematopoiético. As intensidades de dose medianas de doxorrubicina e ifosfamida foram, respectivamente, 42mg/m2/semana e 3,63g/m2/semana (93% e 87% do planejado, respectivamente) e 15 pacientes (71%) receberam todo o tratamento. Toxicidades graus 3 e 4 foram observadas em 19 pacientes e em 77/105 ciclos, neutropenia febril em 6 ciclos (5 pacientes) e reduções da fração de ejeção de ventrículo esquerdo de pelo menos 10% em três pacientes. Não houve toxicidade renal provavelmente pela adminsitração da ifosfamida em duas horas. A resposta foi avaliada pelos critérios de RECIST, com três respostas parciais, totalizando uma taxa de resposta de 14%. Seis respostas deveriam ser observadas para que o estudo completasse a inclusão de todos os pacientes programados. Como não se atingiu a taxa de resposta prevista, o protocolo foi fechado. Três mortes precoces foram observadas com suspeita de toxicidade. Após seguimento mediano de 11 meses, a SLP e a SG medianas foram 8,1 e 20,1 meses respectivamente. Pacientes com sarcoma sinovial e idade inferior a 45 anos apresentaram maior sobrevida na análise univariada. A expresão de ezrina foi positiva em 10 pacientes (47%) e houve tendência a uma correlação direta entre sua expressão e sobrevida mais longa (p=0,1191). Todos os pacientes com histologia sinovial foram positivos para ezrina (teste de Fischer, p= 0,0325). Este esquema de quimioterapia sequencial com dose densa de doxorubicina e ifosfamida foi tóxico, a taxa de resposta foi baixa em um grupo de pacientes com doença avançada e não pode ser empregado na prática clínica diária fora de protocolo de pesquisa / Advanced soft tissue sarcoma (STS) patients have a dismal prognosis. Efforts to increase benefit from chemotherapy are needed and dose-dense chemotherapy could be an option, since this approach has demonstrated survival benefit in breast cancer. On the other hand, the identification of a prognostic marker is essential to stratify which patients could benefit most from intensified strategies. Ezrin is a member of the ERM (ezrin, radixin, moesin) cytoeskeleton-associated protein family associated with invasion and metastasis, and has been pointed as important prognostic marker in sarcomas. The objective of this study was to explore the role of dose-dense doxorubicin- and ifosfamide-based chemotherapy in advanced high grade STS patients. Primary endpoint was response rate and secondary endpoints were overall survival (OS), progression free survival (PFS), toxicity profile, quality of life and pain control evaluation. Tumor ezrin immunoreactivity was an exploratory endpoint as a predictor of response to chemotherapy and as a prognostic factor in this population, trying to find which patients could benefit most from this intensified strategy. This prospective, single arm, phase II study included 21 advanced STS patients. Median age was 37 years (23-60y) and lower limbs were the most frequent primary site. Synovial, leiomyo and unclassified sarcoma were the most common histologies. Protocol consisted of 6 cycles of sequential dose-dense doxorubicin 30 mg/m2 D1-3 and ifosfamide 2.5 g/m2 D1-5 every 14 and 21 days, respectively, followed by seven days of hematopoietic support. The median doxorubicin and ifosfamide dose-intensities were, respectively, 42 mg/m2/week and 3.63 g/m2/week (93% and 87% of planned, respectively) and 15 patients (71%) received all cycles. Grade 3/4 toxicities occurred in 19 patients and 77/105 cycles, febrile neutropenia in 5 patients (six cycles) and three LVEF drops of at least 10%, one symptomatic. No renal toxicity was observed what could occurred due to the two-hour-schedule of ifosfamide. Responses were evaluated by RECIST criteria and three patients presented partial response (response rate of 14%). Six responses were necessary to the inclusion of the target population, however, this was not observed and the study was closed. Three deaths were probably related to toxicity. After a median follow-up was 11 months, PFS and OS were 8.1 months and 20.1 months, respectively. Patients with synovial sarcoma and those younger than 45y presented better survival at univariate analysis. Ezrin expression was positive in 10 patients (47%) and a trend was observed for a correlation between positive ezrin expression and longer survival (p= 0.1191). There was a statistically significant correlation between positive ezrin expression and synovial hystology (Fishers exact test, p= 0.0325). This sequential dosedense doxorubicin/ifosfamide-based chemotherapy protocol was toxic, response rate was low in advanced STS patients and can not be considered for routine practice outside clinical trials

<p>Histopathology is the clinical standard for tissue diagnosis. However, histopathology has several limitations including that it requires tissue processing, which can take 30 minutes or more, and requires a highly trained pathologist to diagnose the tissue. Additionally, the diagnosis is qualitative, and the lack of quantitation leads to possible observer-specific diagnosis. Taken together, it is difficult to diagnose tissue at the point of care using histopathology.</p><p>Several clinical situations could benefit from more rapid and automated histological processing, which could reduce the time and the number of steps required between obtaining a fresh tissue specimen and rendering a diagnosis. For example, there is need for rapid detection of residual cancer on the surface of tumor resection specimens during excisional surgeries, which is known as intraoperative tumor margin assessment. Additionally, rapid assessment of biopsy specimens at the point-of-care could enable clinicians to confirm that a suspicious lesion is successfully sampled, thus preventing an unnecessary repeat biopsy procedure. Rapid and low cost histological processing could also be potentially useful in settings lacking the human resources and equipment necessary to perform standard histologic assessment. Lastly, automated interpretation of tissue samples could potentially reduce inter-observer error, particularly in the diagnosis of borderline lesions. </p><p>To address these needs, high quality microscopic images of the tissue must be obtained in rapid timeframes, in order for a pathologic assessment to be useful for guiding the intervention. Optical microscopy is a powerful technique to obtain high-resolution images of tissue morphology in real-time at the point of care, without the need for tissue processing. In particular, a number of groups have combined fluorescence microscopy with vital fluorescent stains to visualize micro-anatomical features of thick (i.e. unsectioned or unprocessed) tissue. However, robust methods for segmentation and quantitative analysis of heterogeneous images are essential to enable automated diagnosis. Thus, the goal of this work was to obtain high resolution imaging of tissue morphology through employing fluorescence microscopy and vital fluorescent stains and to develop a quantitative strategy to segment and quantify tissue features in heterogeneous images, such as nuclei and the surrounding stroma, which will enable automated diagnosis of thick tissues.</p><p>To achieve these goals, three specific aims were proposed. The first aim was to develop an image processing method that can differentiate nuclei from background tissue heterogeneity and enable automated diagnosis of thick tissue at the point of care. A computational technique called sparse component analysis (SCA) was adapted to isolate features of interest, such as nuclei, from the background. SCA has been used previously in the image processing community for image compression, enhancement, and restoration, but has never been applied to separate distinct tissue types in a heterogeneous image. In combination with a high resolution fluorescence microendoscope (HRME) and a contrast agent acriflavine, the utility of this technique was demonstrated through imaging preclinical sarcoma tumor margins. Acriflavine localizes to the nuclei of cells where it reversibly associates with RNA and DNA. Additionally, acriflavine shows some affinity for collagen and muscle. SCA was adapted to isolate acriflavine positive features or APFs (which correspond to RNA and DNA) from background tissue heterogeneity. The circle transform (CT) was applied to the SCA output to quantify the size and density of overlapping APFs. The sensitivity of the SCA+CT approach to variations in APF size, density and background heterogeneity was demonstrated through simulations. Specifically, SCA+CT achieved the lowest errors for higher contrast ratios and larger APF sizes. When applied to tissue images of excised sarcoma margins, SCA+CT correctly isolated APFs and showed consistently increased density in tumor and tumor + muscle images compared to images containing muscle. Next, variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82% and 75%. The utility of this approach was further tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78% and 82%. The results indicate that SCA+CT can accurately delineate APFs in heterogeneous tissue, which is essential to enable automated and rapid surveillance of tissue pathology. </p><p>Two primary challenges were identified in the work in aim 1. First, while SCA can be used to isolate features, such as APFs, from heterogeneous images, its performance is limited by the contrast between APFs and the background. Second, while it is feasible to create mosaics by scanning a sarcoma tumor bed in a mouse, which is on the order of 3-7 mm in any one dimension, it is not feasible to evaluate an entire human surgical margin. Thus, improvements to the microscopic imaging system were made to (1) improve image contrast through rejecting out-of-focus background fluorescence and to (2) increase the field of view (FOV) while maintaining the sub-cellular resolution needed for delineation of nuclei. To address these challenges, a technique called structured illumination microscopy (SIM) was employed in which the entire FOV is illuminated with a defined spatial pattern rather than scanning a focal spot, such as in confocal microscopy. </p><p>Thus, the second aim was to improve image contrast and increase the FOV through employing wide-field, non-contact structured illumination microscopy and optimize the segmentation algorithm for new imaging modality. Both image contrast and FOV were increased through the development of a wide-field fluorescence SIM system. Clear improvement in image contrast was seen in structured illumination images compared to uniform illumination images. Additionally, the FOV is over 13X larger than the fluorescence microendoscope used in aim 1. Initial segmentation results of SIM images revealed that SCA is unable to segment large numbers of APFs in the tumor images. Because the FOV of the SIM system is over 13X larger than the FOV of the fluorescence microendoscope, dense collections of APFs commonly seen in tumor images could no longer be sparsely represented, and the fundamental sparsity assumption associated with SCA was no longer met. Thus, an algorithm called maximally stable extremal regions (MSER) was investigated as an alternative approach for APF segmentation in SIM images. MSER was able to accurately segment large numbers of APFs in SIM images of tumor tissue. In addition to optimizing MSER for SIM image segmentation, an optimal frequency of the illumination pattern used in SIM was carefully selected because the image signal to noise ratio (SNR) is dependent on the grid frequency. A grid frequency of 31.7 mm-1 led to the highest SNR and lowest percent error associated with MSER segmentation. </p><p>Once MSER was optimized for SIM image segmentation and the optimal grid frequency was selected, a quantitative model was developed to diagnose mouse sarcoma tumor margins that were imaged ex vivo with SIM. Tumor margins were stained with acridine orange (AO) in aim 2 because AO was found to stain the sarcoma tissue more brightly than acriflavine. Both acriflavine and AO are intravital dyes, which have been shown to stain nuclei, skeletal muscle, and collagenous stroma. A tissue-type classification model was developed to differentiate localized regions (75x75 µm) of tumor from skeletal muscle and adipose tissue based on the MSER segmentation output. Specifically, a logistic regression model was used to classify each localized region. The logistic regression model yielded an output in terms of probability (0-100%) that tumor was located within each 75x75 µm region. The model performance was tested using a receiver operator characteristic (ROC) curve analysis that revealed 77% sensitivity and 81% specificity. For margin classification, the whole margin image was divided into localized regions and this tissue-type classification model was applied. In a subset of 6 margins (3 negative, 3 positive), it was shown that with a tumor probability threshold of 50%, 8% of all regions from negative margins exceeded this threshold, while over 17% of all regions exceeded the threshold in the positive margins. Thus, 8% of regions in negative margins were considered false positives. These false positive regions are likely due to the high density of APFs present in normal tissues, which clearly demonstrates a challenge in implementing this automatic algorithm based on AO staining alone. </p><p>Thus, the third aim was to improve the specificity of the diagnostic model through leveraging other sources of contrast. Modifications were made to the SIM system to enable fluorescence imaging at a variety of wavelengths. Specifically, the SIM system was modified to enabling imaging of red fluorescent protein (RFP) expressing sarcomas, which were used to delineate the location of tumor cells within each image. Initial analysis of AO stained panels confirmed that there was room for improvement in tumor detection, particularly in regards to false positive regions that were negative for RFP. One approach for improving the specificity of the diagnostic model was to investigate using a fluorophore that was more specific to staining tumor. Specifically, tetracycline was selected because it appeared to specifically stain freshly excised tumor tissue in a matter of minutes, and was non-toxic and stable in solution. Results indicated that tetracycline staining has promise for increasing the specificity of tumor detection in SIM images of a preclinical sarcoma model and further investigation is warranted. </p><p>In conclusion, this work presents the development of a combination of tools that is capable of automated segmentation and quantification of micro-anatomical images of thick tissue. When compared to the fluorescence microendoscope, wide-field multispectral fluorescence SIM imaging provided improved image contrast, a larger FOV with comparable resolution, and the ability to image a variety of fluorophores. MSER was an appropriate and rapid approach to segment dense collections of APFs from wide-field SIM images. Variables that reflect the morphology of the tissue, such as the density, size, and shape of nuclei and nucleoli, can be used to automatically diagnose SIM images. The clinical utility of SIM imaging and MSER segmentation to detect microscopic residual disease has been demonstrated by imaging excised preclinical sarcoma margins. Ultimately, this work demonstrates that fluorescence imaging of tissue micro-anatomy combined with a specialized algorithm for delineation and quantification of features is a means for rapid, non-destructive and automated detection of microscopic disease, which could improve cancer management in a variety of clinical scenarios.</p> / Dissertation

Herpesviral infections are currently incurable and are associated with severe human diseases, such as cancer. Kaposi’s Sarcoma-associated Herpesvirus (KSHV), like all herpesviruses, undergoes a long-term, latent infection where few viral products are made as a mechanism to evade the host immune system. Recently, the KSHV latent genome was shown to have bivalent histone marks thought to keep the virus poised for replication. However, it is unclear how the virus prevents spurious leaky transcription from this primed state. The 3' untranslated region (3'-UTR) of transcripts is a common site of gene expression regulation, however less than half of the KSHV 3'-UTRs have been mapped and few studies have interrogated their role during infection. The work presented here is the first large-scale map and analysis of the KSHV 3'-UTRs. Four methods were used to identify the 3'-UTRs expressed by the ~85 KSHV genes, including prediction algorithms, 3'-RACE, DNA tiling array, and next generation deep sequencing analysis. The role of each KSHV 3'-UTR in gene expression was then examined using luciferase reporter assays and showed a surprising prevalence of negative regulation conveyed during latent infection. Sequential deletions across numerous 3'-UTRs indicated RNA structure is likely involved in this regulation. In addition, several KSHV 3'-UTRs conveyed an increase in translation during lytic infection through enhanced recognition by the cap-dependent translation initiation machinery activated via the MNK1 kinase. A second mechanism of KSHV gene regulation was identified through motifs encoded in the K7 3'-UTR. This work indicated that a previously characterized RNA element and a novel putative hairpin are both partially responsible for negative regulation conveyed by the K7 3'-UTR. We hypothesize that these structural motifs control expression of the K7 transcript by altering its sub-cellular location and/or via RNA stability. This work represents a broad 3'-UTR study that mapped the KSHV 3'-UTRs and is the first large-scale functional analysis of 3'-UTRs from a large genome virus. We have implicated post-transcriptional mechanisms, along with known transcriptional regulation, in viral evasion of the immune response during latency and the escape of viral-mediated host shutoff. These results identify new potential targets for therapeutic intervention of KSHV-associated disease. / text

Les sarcomes sont des tumeurs malignes rares, représentant 1% de tous les cancers mais regroupant un large spectre d’entités histopathologiques distinctes. Du fait de leur rareté et de leur difficulté diagnostique, l’épidémiologie des sarcomes reste mal connue. Nous avons décrit l’incidence et la répartition géographique des sarcomes en région Rhône-Alpes grâce à la constitution d’une cohorte prospective et exhaustive de tous les cas incidents en 2005 et 2006, après relecture centralisée systématique des diagnostics par un pathologiste référent. Nous rapportons : - un taux d’incidence brut de 6,2 cas/100 000/an, supérieur à celui publié dans la littérature et confirmé sur les deux années de recueil de données ; - une répartition géographique et une épidémiologie spécifiques selon les sous-types histologiques ; - des discordances de diagnostic portant sur 45% des cas dont 19% de discordances majeures. Nos résultats sont différents des données publiées portant sur des séries rétrospectives ou incomplètes. Ils contribuent à une meilleure connaissance de l’épidémiologie des sarcomes. Ils permettent de poser des hypothèses étiopathogéniques dans la répartition géographique observée notamment pour les tumeurs stromales gastro intestinales et les liposarcomes / Sarcoma is a rare malignant tumour accounting for 1% of all cancers but comprising a wide range of distinct histopathological types. Given the rarity of cases and the difficulty of diagnosis, the epidemiology of sarcomas is still unclear. We have examined the incidence and the geographical distribution of sarcomas based on the results of a prospective cohort study of all incident cases diagnosed in 2005 and 2006 in the Rhône-Alpes region and after centralized and systematic review of diagnosis by a referent pathologist. We report: - a crude incidence rate of 6.2 cases/100 000/year confirmed on these two years of data collection, higher than the one published in the literature ; - a geographical distribution and epidemiology characteristic of each different histological sub-type ; - diagnostic discordances in 45% of cases, of which 19% were major discordances. Our findings, which differ from data published in retrospective or incomplete series, contribute to a better knowledge of the epidemiology of sarcoma. They make it possible to formulate etiopathogenic hypotheses regarding the geographical distribution of the disease, especially for gastrointestinal stromal tumours and liposarcomas

<p>Soft tissue sarcomas (STSs) are a heterogeneous group of mesenchymal tumors comprised of >70 subtypes. An important question is how the cell of origin and the pathways to tumor development shape the broad array of STS subtypes. By forcing identical tumor-promoting mutations to different cell types in Genetically Engineered Mouse Models (GEMMs) of STS, I have a unique model system to investigate this question. In the process of performing these experiments I observed that genetic mutations are necessary, but not sufficient for rapid sarcoma formation. However, tissue injury dramatically accelerates sarcoma formation in our GEMM of STS. For my thesis, I have worked to understand how cell of origin affects sarcoma subtype and how the microenvironment in our models promotes transformation. I have observed that cell of origin plays an important, but not the only, role in defining STS subtype. Additionally, I have concluded that the microenvironment, and specifically the HGF/c-MET signaling pathway play a crucial role in promoting sarcoma development after acute tissue injury.</p> / Dissertation

The basis of this investigation is to characterize growth property differences in normal versus virally transformed cells. Using a temperature-sensitive mutant of Rous sarcoma virus, the cells' transformation state is regulated by the growth temperature; at 33°C the cells are transformed, while at 39°C the cells have normal characteristics. The morphology of NRK cells is elongated and fibroblastic; when transformed the cells are rounded. Normal cells grow to a monolayer and stop, while transformed cells grow to saturation densities greater than just a monolayer amount. Transformed cells can form foci when grown in mixture with normal cells. Normal cells must be in contact with the culture vessel in order to grow, but transformed cells lack anchorage dependence for growth.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several tumours and hyperproliferative disorders. Hypoxia plays an important role in KSHV lifecycle, as hypoxia-inducible factors (HIFs) are involved in the latent/lytic switch and affect other KSHV genes, and as KSHV infection can in turn enhance cellular levels of HIFs. Two KSHV-associated tumours tend to develop in settings of relative hypoxia; Kaposi's sarcoma (KS) often occurs in the lower extremities and primary effusion lymphoma (PEL) exists in pleural effusions. A better knowledge of the pathways that regulate KSHV infection in hypoxia is therefore essential for an improved understanding of viral infection and pathogenesis. MicroRNAs (miRNAs) have been shown to play important roles in regulating the expression of genes in oncogenesis, and herpesviruses, including KSHV, encode for miRNAs. This thesis describes a multidisciplinary approach toward understanding the mechanisms behind the hypoxia-regulated miRNA-mRNA networks in the context of KSHV infection. The question of miRNA and mRNA regulation through hypoxia, KSHV or both is addressed in this thesis by deep sequencing and gene expression assays as well as various transfection and functional assays. In chronically infected cells compared to uninfected controls, it is demonstrated that the majority of cellular miRNAs whose expression is affected are substantially down-regulated. A third of this down-regulation can be attributed to a single genomic region, 14q32 cluster, where miRNAs are lowly expressed in infected cells. In hypoxia, hsa-miR-210 is the only miRNA to be consistently up-regulated in the KSHVinfected cell lines subjected to deep sequencing in this study. Computational approaches additionally allowed for the investigation of mRNA targets. Inversely correlated miRNAmRNA target pairs were identified and distributed into canonical pathways and biological networks. Taken together, these results suggest that miRNAs affected by hypoxic stress and/or viral infection are implicated in the pathogenesis of KSHV-related diseases. It is expected that the outcomes of these studies will change our understanding of how KSHV uses the host RNA silencing machinery to its advantage and how this intersects with the use of the cell's response to hypoxia.

Les sarcomes sont des cancers rares touchant toutes les zones du corps humain, caractérisés par une grande diversité de nature, de comportement clinique et de réponse aux thérapies existantes, certains étant de bon pronostic, d’autres très difficilement curables.Leur traitement de référence est la chirurgie ; la radiothérapie et les protocoles de chimiothérapie n’ayant que peu évolué lors des 30 dernières années.Récemment des caractéristiques génétiques leur étant propres ont été découvertes, prédictives de leur agressivité et contre lesquelles il est possible de diriger des drogues spécifiques pouvant améliorer le pronostic et diminuer les effets secondaires des thérapies conventionnelles.C’est sur l’inhibition d’Aurora Kinase A et B et CHK1 que s’est focalisé ce travail avec le test des effets de deux nouvelles drogues sur 9 types de cellules cancéreuses sarcomateuses avec des résultats très prometteurs, qu’il conviendra de conforter par d’autres expériences, notamment sur l’animal. / Sarcomas are rare cancers, which may arise in all parts of human body. They are characterized by great diversity in their nature, clinical behavior and response to existing therapeutics. Some are of good prognosis and others hard to cure.Their treatment essentially relies on surgery and radiotherapy or chemotherapy haven’t know major breakthrough over the last 3 decades.Recently new genetics abnormalities linked to sarcomas have been discovered. Their analysis can predict their aggressiveness and it is now possible to develop targeted therapies against them. This could help improving cancer prognosis and/or limiting conventional drugs adverse effects.Our work focused on Aurora Kinase A and B and CHK1 inhibition, testing the effects of 2 new drugs on 9 types of sarcoma cells with promising results, which we will confort by other experiments, including on the animal.

Les siRNA se sont des agents actifs et spécifiques couramment utilisés en thérapie génique. L’intérêt d’utiliser des nanoparticules en tant que vecteurs des siRNA est leur capacité à protéger ces derniers de la dégradation par les nucléases et également de leur permettre d’être délivrés au niveau de leur cible thérapeutique. Afin d’appuyer cette théorie, ce travail s’est concentré sur un modèle cellulaire de sarcome d’Ewing, dans le but de mettre au point un nouveau système galénique pour le transport de siRNA formé des nanoparticules bifonctionnelles de diamants fluorescentes recouvertes par des polymères cationiques. Ces nano-vecteurs sont capables d’induire efficacement l’inhibition de l’expression de l’oncogène EWS-Fli1 dans les cellules en culture s’ils transportent des siRNA dirigés contre ce gène. Par ailleurs, les nanodiamants, grâce à leurs propriétés de fluorescence stable et intense, ont constitué des outils de détection permettant de suivre leurs voies de pénétration, leur biodistribution cellulaire, ainsi que la cinétique de libération des siRNA dans le cytoplasme. Enfin, un modèle de nanodiamants fonctionnalisés par le polyéthylenimine a été choisi pour la poursuite des travaux biologiques en raison de son efficacité de vectorisation. / SiRNA are powerful and commonly used agent for the specific inhibition of gene expression. They need to be vectorized by nanoparticles to facilitate cell penetration and their protection from degradation in biological media. At first, cationic nanodiamonds coated with cationic polymers were developed and were able to adsorb siRNA on their surface. Using antisense siRNA against the oncogene EWS-Fli1, nanodiamonds allowed to efficiently induce the inhibition of expression of the oncogene EWS-FLI1 in cultured Ewing sarcoma cells. As a second goal of this study, the fluorescence of red color center created in the nanodiamonds was used to follow their pathways, their cellular biodistribution and the kinetics of release of siRNA into the cytoplasm. In conclusion, nanodiamonds functionalized by polyethylenimine showed a better transfection efficiency and were chosen for further biological studies.

Ce travail a permis de réaliser une évaluation complète de différentes séquences de siRNA dirigées contre EWS/Fli-1 dans le cadre du traitement des tumeurs de la famille du sarcome d’Ewing Un siRNA a été vectorisé de manière efficace par des nanoparticules polymères ciblées contre un marqueur membranaire spécifique des ESFT. Ces nanoparticules ont été caractérisées et semblent bien tolérées à la fois in vitro et in vivo. Leur évaluation a été réalisée sur des cellules humaines et après prise des tumeurs ce qui représente une avancée intéressante dans la lutte contre les ESFT. La mise au point d’un modèle fluorescent de sarcome d‘Ewing permettra de mieux caractériser leur effet sur les métastases, facteur essentiel dans la survie des patients. Enfin, il a été montré que les techniques d’imagerie in vivo permettaient de suivre le devenir in vivo des nanoparticules ce qui permettra de comprendre leur biodistribution et leur mode d’action. / This work has enabled a comprehensive evaluation of different sequences of siRNAs directed against EWS/Fli-1 in the treatment of tumors of the Ewing sarcoma family of tumors (ESFT). A siRNA was efficiently vectorized by polymeric nanoparticles targeted against a specific membrane marker of ESFT. These nanoparticles were characterized and appear to be well tolerated both in vitro and in vivo. Their evaluation was conducted on human cells and tumors which represents an interesting step forward in the fight against ESFT. The development of a fluorescent model of Ewing's sarcoma will better characterize their effect on metastasis, a key factor in patient survival. Finally, it was shown that in vivo imaging techniques allow to follow the fate of nanoparticles in vivo. That will allow understanding their biodistribution and their mode of action.