Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

Circulating DNA is fragments of DNA which can be found in the blood of healthy as well as diseased individuals. Higher levels of these nucleic acid molecules can be found in diseased and pregnant individuals in contrast to healthy controls. The origin of circulating DNA has not been elucidated, but release of DNA after apoptosis or necrosis or active release by living cells has been hypothesized. It was concluded in this study that apoptosis or necrosis may only be a minor source of circulating DNA and that release of DNA by living cells might play a major role in the origin, while disturbance of the equilibrium between release by living cells and clearance mechanisms may cause the rise in the levels of circulating DNA observed in different conditions. Before circulating DNA can be analyzed, it has to be isolated from the blood. A number of different preanalytical conditions can have an impact on the quantity and quality of circulating DNA that can be isolated. Furthermore, the choice of isolation and quantification method may also influence the results obtained. Quantitative analysis of circulating DNA was done by real-time PCR analysis of the &Globin gene and the DNA levels obtained for healthy controls and cancer patients correlated with levels reported in the literature. Characterization of total circulating DNA may be beneficial in diagnosis and prognosis and may also contribute to determining the source and function of circulating DNA. In order for characterization to take place a method to clone total circulating DNA was developed and standardized and thirty-five clones were obtained and analyzed. It was found that the sequences contain a large amount of Alu repeats and the significance of this has not been determined yet. This is a first step towards future studies. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Kateřina Totzauerová Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Free circulating nucleic acids in blood plasma Outline: The aim of this thesis was to compare various commercially available DNA extraction kits and choose the universally optimal method for routine isolation of free circulating DNA from blood plasma samples according to their optical characteristics, the qPCR results and yield of short fragments. Methods: Four commercially available methods of extraction have been chosen to compare: QIAmp DNA Blood Mini Kit, QIAmp DSP Virus Spin Kit (both Qiagen), NucleoSpin Plasma XS (Macherey-Nagel) and Agencourt Genefind v2 (Beckman Coulter). DNA was isolated from aliquots of a pooled blood plasma of healthy individuals. Plasmatic DNA was quantified after extraction by spectrophotometry, fluorimetry and quantitative polymerase chain reaction. Fragmentation analysis on selected samples by capillary electrophoresis was also realized. Results: The best yield and purity provided the method from Qiagen QIAmp DSP Virus Spin Kit. Average value of the concentration determined qPCR was 49.95 ± 23.57 ng/mL and it gave the highest values of the fluorescence of...

O tratamento padrão para pacientes com câncer de reto localmente avançado consiste no uso de quimioradioterapia neoadjuvante (QRTn), seguida por cirurgia. Uma fração significativa dos pacientes responde completamente ao tratamento e no momento da reavaliação não apresenta evidência clínica nem radiológica de doença. Uma abordagem alternativa, Watch and Wait, propõe não operar imediatamente esses pacientes e submetê-los a um protocolo de observação frequente, a fim de evitar as morbidades associadas à cirurgia. No entanto, a avaliação da resposta ao tratamento ainda é um desafio, devido à subjetividade da avaliação clínica e a ausência de exames radiológicos suficientemente sensíveis e específicos para garantir a ausência de células tumorais residuais ou capazes de detectar a recorrência precoce da doença. DNA circulante contendo alterações genéticas específicas do tumor (ctDNA) pode ser encontrado na fração livre de células do sangue e tem sido utilizado para monitorar a dinâmica tumoral em tumores sólidos. Avanços recentes das tecnologias de sequenciamento permitem a identificação eficiente e rápida e a um custo relativamente baixo de alterações genéticas em tumores individuais, superando o problema imposto pela ausência de alterações genéticas recorrentes nesses tumores. Essas alterações podem ser utilizadas como biomarcadores personalizados para monitorar a resposta ao tratamento, detectar doença residual e a recidiva precoce do tumor. O objetivo deste trabalho foi identificar e estudar biomarcadores personalizados em pacientes com câncer de reto localmente avançado tratados com QRTn e avaliar a capacidade desses biomarcadores para monitorar a dinâmica tumoral, e auxiliar na definição da conduta cirúrgica e na detecção da recidiva precoce da doença. Biópsias de seis pacientes com adenocarcinoma de reto distal (cT2- 3N0-1M0), foram coletadas prospectivamente pré-tratamento. O DNA genômico extraído a partir das biópsias foi usado para construir bibliotecas tipo mate-pair para o sequenciamento do genoma completo, utilizando a plataforma SOLiD. Rearranjos inter e intracromossômicos foram identificados utilizando programas computacionais desenvolvidos pelo nosso grupo de pesquisa e em seguida foram validados utilizando PCR e sequenciamento Sanger. Foram validadas, pelo menos, três variações estruturais para cada paciente. Amostras de plasma foram coletadas no momento do diagnóstico, depois da QRTn e durante o seguimento. DNA circulante total foi extraído a partir das amostras de plasma e ensaios personalizados foram desenvolvidos para monitorar a presença de variações estruturais através de PCR Digital. ctDNA foi detectado em todas amostras de plasma pré-tratamento de pacientes com tumores T3. A detecção desses biomarcadores apresentou boa correlação com a resposta ao tratamento, no entanto, esta abordagem não foi sensível o suficiente para detectar doença residual. Para dois pacientes que desenvolveram doença metastática foi verificado um aumento nos níveis de ctDNA com pelo menos 36 semanas antes do diagnóstico clínico de doença metastática, sendo possível correlacionar os níveis de ctDNA detectados em coletas subsequentes com a resposta ao tratamento sistêmico de segunda linha. Este estudo, embora de caráter exploratório, gerou dados relevantes e suficientes para justificar a realização de estudos adicionais para avaliar a aplicação dos biomarcadores personalizados na definição da conduta cirúrgica e no acompanhamento de pacientes com câncer de reto tratados com QRTn. / The standard treatment for patients with locally advanced rectal cancer comprises in neoadjuvant chemo radiotherapy (nCRT), followed by surgery. A significant fraction of these patients show complete response to the treatment and at the time of reassessment, there are no clinical and nor radiological evidence of residual tumor. An alternative approach, Watch and Wait, proposes not to immediately operate these patients, but to submit them to a protocol of frequent observation in order to avoid the morbidities associated with radical surgery. However, assessment of treatment response remains a significant challenge due to the subjectivity of the clinical examination and to the lack of sufficiently sensitive tools to ensure the absence of tumor cells or to detect early disease recurrence. Circulating DNA carrying tumor-specific genetic alterations (circulating tumor DNA - ctDNA) can be found in the cell-free fraction of the blood and has been successfully used to monitor the tumor dynamics in solid tumors. Recent advances in sequencing technologies have enabled the rapid and cost effective identification of genetic alterations in individual tumors, overcoming the problem imposed by the absence of recurrent genetic alterations in these tumors. These alterations can be used as personalized biomarkers to monitor treatment response, detect residual disease and early tumor recurrence. The purpose of this work was to identify and validate the use of personalized biomarkers for patients with locally advanced rectal cancer treated with nCRT and to evaluate the ability of these biomarkers to monitor the tumor dynamics, to define surgical approach and to detect early recurrence of the disease. Pre-treatment biopsies from 6 patients with cT2-3N0-1M0 distal rectal adenocarcinoma were prospectively collected. Genomic DNA extracted from the biopsies was used to construct mate-pair libraries for whole genome sequencing using SOLiD platform. Inter and intrachromosomal rearrangements were identified using an in-house bioinformatics pipeline and validated using PCR amplification and Sanger sequencing. At least three structural variations were validated for each patient. Plasma samples were collect at diagnosis, after nCRT and follow-up. Circulating DNA was obtained from the plasma samples and personalized assays were designed to monitor the presence of structural variations using Droplet Digital PCR. ctDNA was detected in all pre-treatment plasma samples for patients with T3 tumors. The detection of these biomarkers showed a good correlation with the treatment response, nonetheless, the approach was not sensitive enough to detect residual disease. In two patients who developed metastatic disease, an increase in ctDNA levels was observed at least 36 weeks before clinical detection of metastatic disease, and it was possible to correlate the level of ctDNA in subsequent plasma samples with response to the second-line treatment. This study, although exploratory, generated relevant and sufficient data to support additional studies to evaluate the use of personalized biomarkers in the surgical management and follow-up of rectal cancer patients treated with nCRT.

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

Submitted by Maicon Juliano Schmidt (maicons) on 2015-04-15T20:36:34Z No. of bitstreams: 1 Fabiano Anderson Pedroso.pdf: 4283933 bytes, checksum: 9e8c395fe9319e367efba6745553280e (MD5) / Made available in DSpace on 2015-04-15T20:36:34Z (GMT). No. of bitstreams: 1 Fabiano Anderson Pedroso.pdf: 4283933 bytes, checksum: 9e8c395fe9319e367efba6745553280e (MD5) Previous issue date: 2013-01-31 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / PROSUP - Programa de Suporte à Pós-Gradução de Instituições de Ensino Particulares / A combustão em Leito Fluidizado (LF) é caracterizada por sua capacidade de redução das emissões de poluentes em relação aos métodos tradicionais de queima do combustível pulverizado. No Brasil, há um potencial de geração de energia em LF pela combustão do carvão mineral, dada a quantidade de reservas disponíveis, das quais mais de 99% se concentram nos estados do Rio Grande do Sul e de Santa Catarina. Dentre as tecnologias disponíveis para combustão e gaseificação em LF, destaca-se a de Leito Fluidizado Circulante (LFC), devido ao potencial de uso em gaseificação integrada a um ciclo combinado de conversão de energia (IGCC). No projeto, dimensionamento e operação de LFs, o entendimento do escoamento multifásico gás-sólido é de suma importância. As leis de escalonamento de Glicksman fornecem as regras necessárias para construção de leitos em escala com similaridade fluidodinâmica, permitindo reproduzir em escala piloto ou mesmo de laboratório a fluidodinâmica de um leito em escala industrial. Aliado a isso, a Dinâmica dos Fluidos Computacional (CFD) vem se estabelecendo como uma poderosa ferramenta para a simulação dos processos em LFC. Portanto, o objetivo deste trabalho é desenvolver um modelo computacional para a simulação da fluidodinâmica de LFCs utilizando o código livre MFIX e aplicar esse modelo para validação das leis de escala através da modelagem numérica de um LFC em escala real com validação experimental e um leito em escala reduzida de acordo com o conjunto simplificado. Para isso, foram desenvolvidos um leito em completa correspondência com o conjunto simplificado, seis leitos escalonados com alteração de parâmetros operacionais e um leito escalonado pelo conjunto completo das leis de escalonamento de Glicksman. O modelo computacional é baseado na abordagem Euler-Granular, em que as fases gás e sólido são consideradas como meios contínuos interpenetrantes. A fase sólida é modelada como um fluido cujo tensor tensão é construído de modo a descrever o escoamento da fase particulada conforme a teoria cinética dos escoamentos granulares (KTGF). O modelo físico aproximado para validação da simulação para a escala real foi o Terceiro Desafio promovido em parceria pela NETL e PSRI. Entre as escalas, foram comparados os perfis horizontal e vertical de fração volumétrica de gás; perfis horizontais de velocidade vertical adimensional dos sólidos e fluxo mássico adimensional de sólidos; perfil vertical de perda de carga adimensional e a evolução temporal da fração volumétrica de gás média. No presente estudo, os resultados permitiram verificar que, na modelagem numérica de uma escala reduzida a partir do conjunto simplificado das leis de escalonamento de Glicksman, a média do Erro Relativo Médio (ERM) ponderado sobre todos os perfis analisados apresentou um valor de 14,2% em relação a escala real, aceitável para esse tipo de sistema. Também se verificou que a diminuição do diâmetro das partículas em não conformidade com as leis de escala, em alguns dos perfis analisados, implicou em uma redução do ERM em comparação com aquele obtido pelos resultados do conjunto simplificado, devido a maior aproximação do diâmetro da partícula do valor determinado pelo conjunto completo. Em relação à comparação dos resultados obtidos pelo conjunto simplificado e completo das leis de escalonamento de Glicksman, confirma-se o esperado - uma maior correspondência para o leito escalonado pelo conjunto completo, com destaque à correta previsão do perfil horizontal do fluxo mássico adimensional de sólidos, não previsto pelo conjunto simplificado. Dessa forma, considera-se que o conjunto simplificado das leis de escala de Glicksman, dentro de suas limitações intrínsecas, fornece uma boa aproximação para o escalonamento de LFCs através da simulação numérica Euler-Granular. / Fluidized bed (FB) combustion has as main feature its capacity to reduce the release of pollutants in relation to conventional burning methods of pulverized fuel. Brazil has a potential in energy generation with FB through the combustion of coal, given the number of available reservoirs, of which 99% are located in the Southern states of Rio Grande do Sul and Santa Catarina. Among the available technologies for combustion and gasification on FB, we can highlight that of Circulating Fluidized Beds (CFB), given its use in Integrated Gasification Combined Cycles (IGCC). In the project, design and operation of FBs, the understanding of the gas-solid multiphase flow is highly important. Glicksman’s scaling laws provide the guidance needed for building beds in scale with fluid dynamics similarity, allowing the reproduction in pilot or even laboratory level of the fluid dynamics of a bed in industrial level. Along with that, Computational Fluid Dynamics (CFD) has established itself as a powerful tool in the simulation of CFB processes. Therefore, the aim of this paper is to develop a computational model for the simulation of CFBs fluid dynamics, using the MFIX code and to apply this model to the validation of scaling rules through the numerical modeling of a CFB in real scale with experimental validation and a bed in reduced scale according to a reduced set. For that to happen, a bed in fully correspondence wtih the reduced set, six scaled beds with alterations in their operational parameters, and a bed scaled by the full-set of Glicksman’s scaling laws have been developed. The computational model is based on the Euler-Granular Approach, in which the solid and gas phases are considered as interpenetrating continua. The solid phase is modeled as a fluid whose tensors are built in order to describe the flow of the granular phase according to the kinetic theory of granular flows (KTGF). The approximate physical model for the validation of this simulation to real scale was the Third Challenge held by NETL and PSRI. A comparison was made among the scales, one of the horizontal and vertical profiles of gas volume fraction; horizontal of vertical dimensionless speed of solids and dimensionless mass flux of solids; vertical of dimensionless pressure drop and the temporal evolution of the average gas volume fraction. In this study, the results allowed to verify that, in the numeral modeling of a reduced scale based on the reduced set of Glicksman’s scaling laws, the average of Relative Error (RE) considered over all the analyzed profiles showed a 14.2% value in relation to the real scale, which is acceptable for this kind of system. It has also been verified that the reduction in diameter of particles which were not suitable with the scaling laws, in some of the analyzed profiles, resulted in a reduction of RE when compared to that obtained through the results of the reduced set, due to a larger approximation of the particles diameter to the value determined by the full-set. Regarding the comparison of the results obtained through the reduced and full-set of Glicksman’s scaling laws, the most expected was confirmed – a larger matching for the bed scaled through the full-set, highlighting the correct prediction of the horizontal profile of the dimensionless mass flux of solids, which was not predicted by the reduced set. Thus, the reduced set of Glicksman’s scaling laws provides, within its inherent limitations, a good approximation for the scaling of CFBs through the Euler-Granular numerical simulation.

There is a need for informative biomarkers in localised urological cancers. At present, no method can accurately distinguish between indolent and aggressive prostate cancers, and men often require repeated biopsies. Patients with muscle invasive bladder cancer undergo neo-adjuvant chemotherapy (NAC) to improve survival. However many do not respond to NAC, delaying definitive treatment. Cell-free mutant DNA (mutDNA) analysis represents an opportunity for non-invasive monitoring of cancer through tumour genome analysis. MutDNA derived from plasma can monitor tumour burden. There is emerging evidence that mutDNA can identify mutations from multiple clones and is abundant in adjacent body fluids. This work explores the utility of plasma and urinary mutDNA in localised prostate and bladder cancers. This thesis describes the optimisation of urinary mutDNA analysis by assessing urinary DNA processing and extraction methods using healthy volunteer and bladder cancer patient urine samples. Primer panels were designed and validated to target frequently mutated regions in prostate and bladder cancers, as well as for analysis of patient-specific mutations. Sequencing-based methods and dPCR were employed to analyse clinical samples including plasma and urine, to detect and quantify mutDNA. Molecular and clinical data were integrated to explore potential areas of application of mutDNA analysis. For bladder cancer, mutDNA was analysed from liquid-biopsy samples including plasma, cell pellets from urine and urine supernatant from multiple time-points of 17 MIBC patients undergoing NAC. I showed that mutDNA was more frequently detected and was present at higher AFs in urine compared to plasma samples. Of potential clinical relevance, I showed that the presence of mutDNA after starting NAC was associated with disease recurrence. This original contribution to knowledge could offer patients an opportunity to expedite surgical resection in a timely manner, if corroborated in large-scale trials. For prostate cancer, a TP53 specific panel was applied to men with metastatic disease, to demonstrate that clones containing TP53 mutations, which are dominant in at the metastatic stage were present in historical prostatectomy samples taken when then patient was believed to have localised disease only. Furthermore, I showed that these TP53 mutations could be detected at the localised stage of disease. To investigate the ability of mutDNA detection private clonal mutations I developed a method for higher sensitivity analysis (MRD-Seq). This was applied to a clinical cohort of 2 men with multi-focal localised prostate cancer to demonstrate the though the overall levels of mutDNA is low, private clonal mutations may be detectable. Taken together, these original contributions to knowledge could allow for less invasive surveillance of men with low risk prostate cancer and warrants further investigation. In this thesis, I used a range of molecular methods were applied to small cohorts of clinical samples from patients with urological malignancies, in an exploratory analysis. The molecular data was analysed in conjunction with clinical information to draw hypotheses on the biology and natural history of these cancer, and to suggest possible utility of mutDNA analysis in their clinical management. Some of the findings suggest areas of potential utility, which merit further validation or investigation in larger cohorts or clinical studies.

A methodology for the thermodynamic-kinetic evaluation of circulating systems as TSA carbon capture processes is developed and used in the assessment of a novel CFB configuration against a benchmark (co-current riser). The novel CFB features a counter-current adsorber, a counter-current regenerator and a riser, the latter element playing a double role of solids conveyer and co-current adsorber. The advantages sought by using a counter-current adsorber are not only the more efficient gas-solid contact mode with respect co-current, but also a low pressure drop derived from operation at lower gas velocities and hydrostatic head partially supported on the contactor internals. Knowledge of the adsorption equilibrium alone is sufficient to realise the much higher sorbent circulation rates required by co-current configurations –compared to counter-current– to meet the stringent carbon capture specifications of 90% recovery and 95% purity. Higher solids circulation rates imply higher energy requirements for regeneration, and therefore research and development of co-current gas-solid contactors cannot be justified in terms of searching for energy-efficient post-combustion carbon capture processes. Parallel experimental investigation in the operation and fluid dynamics of cold model CFB rigs is carried out with the purposes of: 1) providing information that may impact the process performance and can be fed into the mathematical model used in the theoretical assessment for more realistic evaluation, and 2) determine gas and solids residence time distributions (RTDs), which are used for the estimation of axial dispersion and comparison with published results in similar systems. Gas RTD data is generated using a tracer pulse injection-detection technique, whereas RTD for the solid phase is studied using positron emission particle tracking (PEPT). The PEPT technique proved to be adequate for the identification of flow regimes in the novel design of the counter-current adsorber, featuring inclined orifice trays. At low gas velocities the particles flow straight down through the tray holes, whereas at higher velocities the particles flow down in zig-zag, increasing the residence time of the particles and reducing the particle axial dispersion, both beneficial in terms of separation efficiency.

L’analyse de l’ADN circulant dans le contexte de la tumorogenèse et comme outil diagnostic Le projet de thèse ici décrit est fondé sur la découverte remarquable qu'une quantité importante d’ADN circule dans le sang de patients atteints de cancer [1-5]. Le développement d'une technologie basée sur la détection de l'ADN circulant représente une avancée scientifique et médicale pour le diagnostic et le suivi dans la prise en charge thérapeutique des patients atteints de cancer. Malgré de nombreuses études menées au cours des dix dernières années [4,5] sur l'ADN circulant, les origines de la libération de l'ADN circulant dans les liquides biologiques sont hypothétiques et sa structure n'est pas élucidée. Ces données ne valident pas jusqu'à présent l'ADN circulant en tant que biomarqueur. Pour cette raison, les objectifs du groupe dirigé par Alain Thierry sont axés sur l'élucidation des formes structurelles de l'ADN circulant. Ainsi, en utilisant des souris nude xénogreffées avec des lignées cellulaires tumorales humaines de cancer colorectal ainsi que des échantillons sanguins cliniques provenant de patients atteints de cancer colorectal, l'équipe a montré que la concentration en ADN circulant était corrélée positivement avec la taille de la tumeur [6, 7] et ces résultats se sont révélés optimaux pour des tailles inférieures à 100 pb. Une discrimination significative entre les individus sains et les patients du cancer a été observée grâce à l'analyse de la fragmentation de l'ADN circulant. L'originalité de ces découvertes a donné naissance à la technologie Intplex récemment breveté par le CNRS [8]. L'objectif de la thèse est de valider la quantification et la fragmentation de l'ADN circulant comme un outil de diagnostic et de suivi de la maladie dans la prise en charge du cancer en analysant de près les facteurs qui peuvent influencer la quantification et la fragmentation de l'ADN circulant. Grâce au modèle animal développé par l’équipe et l’étroite collaboration avec les centres anti-cancéreux, différents paramètres seront analysés. Une partie de la thèse se concentrera sur la comparaison et la standardisation des résultats en fonction de nombreux facteurs spécifiques à la tumeur, comme son type, sa progression, sa différenciation et sa localisation tissulaire. Le travail de thèse portera également sur l'influence de facteurs individuels pouvant affecter la quantité et la fragmentation de l'ADN circulant: âge, sexe, antécédents médicaux, états physiologiques spécifiques, situations physiopathologiques. L'influence du traitement sera également explorée. Des études seront menées afin de standardiser l'analyse biologique: influence du rythme circadien, prise de nourriture .... Techniquement, la variation analytique et l'influence des facteurs pré-analytiques seront déterminées afin d’établir un guide de bonnes pratiques analytiques pour éliminer tout artefact susceptible d’affecter la quantité et l'intégrité de l'ADN circulant dans les échantillons. Ces deux paramètres seront testés dans une évaluation clinique prospective multicentrique sur une cohorte de 450 patients atteints de cancer colorectal. Ce travail garantit un impact considérable dans la littérature et dans la pratique clinique comme test non invasif de diagnostic et de suivi et comme un outil pour améliorer les connaissances de base sur l'ADN circulant et le cancer. / Analysis of circulating DNA circulating in the context of tumorigenesis and as a diagnostic tool The thesis project described here is based on the remarkable discovery that a significant amount of DNA circulates in blood of cancer patients [1-5]. The development of a technology based on the detection of circulating DNA represents a scientific and medical breakthrough for diagnosis and follow up in therapeutic care of cancer patients. Despite numerous studies conducted over the last decade [4,5] on circulating DNA, origins of release of circulating DNA in biological fluids are hypothetical and its structure is unclear. These data do not validate so far circulating DNA as a biomarker. For this reason, objectives of the group led by Alain Thierry are focused on elucidating structural forms of circulating DNA. Thus, using nude mice xenografted with human tumor cell lines of colorectal cancer as clinical samples from colorectal cancer patients, the team showed that the concentration was positively correlated with tumor size [6 , 7] and these results were optimal for sizes below 100 bp. A significative discrimination between healthy individuals and cancer patients was found by the analysis of circulating DNA fragmentation. The originality of these discoveries gave rise to the Intplex technology recently patented by the CNRS [8]. The aim of the thesis is to validate quantification and fragmentation of circulating DNA as a diagnostic and follow-up test in the management of cancer by closely analyzing the factors that may influence quantification and fragmentation of circulating DNA. Thanks to mouse model developed by the team and close collaboration with clinical cancer centers, different parameters will be analyzed. One part of the thesis will be focused on comparison and standardization of the different results depending on many factors specific to the tumor, such as its type, its progression, its differentiation and its tissue localization. The thesis work will also focus on the influence of individual factors that may affect the quantity and the fragmentation of circulating DNA: age, sex, medical history, specific physiological states, pathophysiological situations. The influence of treatment will also be explored. Studies will be undertaken in order to standardize the biological analysis: influence of circadian rhythm, food intake.... Technically, the analytical variation and the influence of pre-analytical factors will be determined to establish a good practice guide to eliminate any artifacts altering the amount and integrity of circulating DNA in the samples. These two parameters will be tested in a prospective multicentric clinical evaluation on a cohort including 450 patients with colorectal cancer. This work warrants a significant impact in the literature and in cancer clinical practice as a non invasive diagnostic and follow-up test and as a tool to improve the basic knowledge on circulating DNA and cancer.

Survival rates in cancers have improved vastly over the years. However, some survival rates remain extremely low, as is the case for ovarian and lung cancer. The lack of robust and reliable biomarkers is strongly reflected in the absence of pre-screening programs, and as such, most patients in these cancer types are diagnosed only in advanced stages, leaving few treatment options. Moreover, relapse and resistance to therapies adds to the complexities of treating these diseases, even in the era of targeted drug development. Research has shown the presence of cancer material, in the form of circulating cancer cells (CTCs) and genomic material in the blood of patients, opening the possibility of 'liquid biopsies'. Liquid biopsies allow sampling of the disease to provide phenotypic and genomic data on the cancer in real-time and on a routine basis. Moreover, they overcome obstacles currently faced by conventional tissue biopsies. In this work we evaluate the use of a novel CTC imaging flow-cytometry platform, and report the ability to characterise and quantify these cells in blood samples. Moreover, we report significantly higher levels of CTCs in cancer patients compared to controls, and found them to be associated with a poorer prognosis. In particular, in lung cancer we observe these findings even in the early stages, suggesting a potential diagnostic use for this assay. We detect a similar trend in when analysing the ctDNA and suggest the possibility of using this technique with a prognostic value in the advanced setting. We also report on the analysis of existing microarray data by use of unique gene regulatory networks to identify biomarkers of interest. RAD51AP1 was identified by this process. Clinical validation revealed an over-expression of this gene in both tissue and blood of ovarian and lung cancers. Moreover, the gene over-expression was associated with a poor overall survival. Functional analysis in vitro revealed silencing RAD51AP1 suppressed tumour growth, in addition, various tumorigenic proteins were down-regulated, whilst apoptotic and immune genes were up-regulated. These results suggest a role for RAD51AP1 as a potential therapeutic target. In this study, we also demonstrate the ability to further exploit tumour genomic material in the blood by means of RNAseq, cancer panels, and CNI scoring to identify novel markers, that play an important role in disease genesis and evolution. RNAseq analysis identified XIST as a gene up-regulated in the blood and tissue of lung cancers. The ovarian cancer panels revealed 2 unique gene signatures in the ovarian cancer patients. With the CNI analyses also highlighting chromosomal aberrations from plasma analysis of cancer patients. Collectively, the use of all these techniques and exploitation of available blood based biomarkers could see significant improvements to survival rates in these, currently devastating diseases.