Poly(ADP-ribose)polymerase-1 and -2 (PARP1/2) are nuclear proteins involved in DNA repair. Tumors with defects in homologous recombination, including BRCA1- and BRCA2-deficient cancers, have been shown to be sensitive to PARP inhibition. The Weissleder group has synthesized fluorescent and radioactive derivatives of the PARP1/2 inhibitor AZD2281. We hypothesized that fluorescent and radioactive AZD2281-based imaging agents would quantify PARP1/2 expression in vitro and in vivo. To test this hypothesis, a panel of pancreatic ductal adenocarcinoma and ovarian carcinoma cell lines were characterized by immunocytochemistry for PARP1/2 expression. AZD2281-derived fluorescence signal correlated with anti-PARP antibody fluorescence signal strength in vitro. Four cell lines representing a range of PARP1/2 expression levels were then xenografted into Nu/Nu mice. Mice bearing four tumor types each were imaged with AZD2281-derived imaging agents, sacrificed, and their tumors excised for stand-alone imaging and Western blot. AZD2281-derived signal correlated with tumor PARP1/2 expression determined by Western blot, indicating that PARP1/2 expression level is a determinant of fluorescent signal strength and SUVs of AZD2281-derived agents in vivo. These data indicate that AZD2281-derived agents are useful tools for quantifying intracellular PARP1/2 both in vitro and in vivo, which could one day enable prospective identification of tumors likely to respond to PARP inhibitors.

The aim of this thesis was to develop and validate prognostic and predictive biomarkers in order to better identify among patients with metastatic colorectal cancer those at high-risk of early death or progression. The interest in developing such biomarkers is that their subsequent use in clinical practice would avoid exposing a patient for months to the toxic side effects of ineffective and expensive treatments, and thus to limit the financial impact of these treatments on our healthcare systems.The projects carried out in the framework of this thesis have shown that:The biomarker WB-MATV (metabolically active tumor volume of the whole body) measured before the start of the last line treatment has a high prognostic value, higher than the general clinical parameters commonly used. This biomarker was then validated in first line treatment and was shown to have a high prognostic value, also higher than the general clinical parameters.The biomarker cfDNA (circulating DNA) also representing the tumor load was then investigated to assess its value added to the previously validated WB-MATV. We showed that the presence of high levels of cfDNA before starting the last-line treatment is significantly associated with poor prognosis and that these two biomarkers are prognostically complementary, each providing an added value.The biomarker of early metabolic response to last line treatment has a high negative predictive value (95%). This biomarker was then validated as a predictive biomarker independent of WB-MATV and clinical factors in first-line treatment setting.In conclusion, the results of this thesis strongly support the clinical use of these prognostic and predictive biomarkers in patients with metastatic colorectal cancer. Allowing a more accurate stratification of patients, the use of the combination of these biomarkers should become an essential tool to help oncologists in tailoring therapeutic strategies according to the patients’ individual risk. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18866号 / 医博第3977号 / 新制||医||1008(附属図書館) / 31817 / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸井 雅和, 教授 平岡 眞寛, 教授 三森 経世 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20252号 / 医博第4211号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 伊達 洋至, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21294号 / 医博第4383号 / 新制||医||1030(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 佐藤 俊哉, 教授 今中 雄一, 教授 増永 慎一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23811号 / 医博第4857号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小林 恭, 教授 万代 昌紀, 教授 佐藤 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Die Kombination von Positronen-Emissions-Tomographie (PET) und Röntgen-Computertomographie (CT) in Form moderner PET/CT-Geräte ermöglicht die Nutzung der CT-Information zur Korrektur der Photonenschwächung in der PET. Allerdings können Bewegungen, die zum Beispiel durch die Atmung hervorgerufen werden können, zu einer fehlerhaften Schwächungskorrektur führen. Die Einführung von zeitlich aufgelöster Bildgebung für beide Modalitäten (4D-PET/4D-CT) ermöglicht nicht nur die Auflösung von periodischen Bewegungen, sondern auch die Reduktion dieser Fehler in der Schwächungskorrektur. Dazu werden die einzelnen Datensätze des 4D-PET, die jeweils einer bestimmten Bewegungsphase entsprechen, mit dem entsprechenden CT-Datensatz dieser Atemphase schwächungskorrigiert. In der vorliegenden Arbeit wurde diese phasenkorrelierte Schwächungskorrektur des 4D-PET mit dem 4D-CT am Universitästsklinikum Dresden installierten PET/CT ermöglicht und anhand von Phantomexperimenten mit anderen Schwächungskorrekturmethoden für 4D-PET verglichen. Dazu musste zunächst die Aufnahme von 4D-CT an dem verwendeten PET/CT ermöglicht und dessen Synchronität mit dem 4D-PET hergestellt werden. Außerdem wurde ein vorhandenes Atemphantom so modifiziert, dass es typische Bewegungen von Bronchialkarzinomen in zwei Dimensionen und mit zwei möglichen Atemmustern simuliert. Die phasenkorrelierte Schwächungskorrektur führte zu einer quantitativ korrekten Wiederherstellung des Aktivitätsvolumens, der darin enthaltenen Aktivität sowie der Bewegungsamplitude und stellt somit die beste der hier verglichenen 4D-PET-Schwächungskorrekturmethoden dar. Diese Ergebnisse lassen vermuten, dass die phasenkorrelierte Schwächungskorrektur auch bei klinischer Anwendung eine signifikante Verbesserung in oben genannten Punkten darstellt. Dies sollte in Zukunft an Patientendaten überprüft werden. / The combination of Positron Emission Tomography (PET) and Computed Tomography (CT) in one device allows the use of CT-information for attenuation correction in PET. Though motion, for example induced by respiration, can cause inaccurate attenuation correction. The implementation of time-resolved imaging methods for both modalities (4D-PET/4D-CT) enables not only the resolution of motion but also the reduction of artifacts caused by attenuation correction. Therefore, the single datasets of the 4D-PET that are related to a individual respiratory phase, are attenuation corrected with the corresponding dataset of the 4D-CT. This phase correlated attenuation correction of the 4D-PET with the 4D-CT was implemented at the PET/CT installed at the Universitätsklinikum Dresden. For that purpose the acquisition of 4D-CT was implemented at the PET/CT and its synchronisation with the 4D-PET was verified. Furthermore the new attenuation correction method was compared with other attenuation correction methods by performing phantom experiments. Therefore an exisisting respiratory phantom had to be modified to perform typical lung tumor motion in two dimensions with two possible patterns of respiration. The phase correlated attenuation correction leads to a quantitatively correct restauration of the activity volume, its total activity and its motion amplitude. Compared with other correction methods, the phase correlated attenuation correction shows the best results in all examined criteria. This findings suggest that the clinical application of the phase correlated attenuation correction will also lead to a significant improvement in all mentioned points. This has to be verified by analyzing patient data.

As doenças cardiovasculares são a principal causa de mortalidade no mundo. A aterosclerose é a base fisiopatológica dessas doenças, sendo definida como um processo crônico-inflamatório multifatorial, resultando da interação de diferentes células como linfócitos, macrófagos, células endoteliais e células musculares lisas na parede arterial. A lipoproteína de baixa densidade eletronegativa [LDL(-)], uma subfração modificada da LDL nativa, desempenha um papel-chave na aterosclerose, uma vez que as modificações sofridas por esta partícula são capazes de induzir o acúmulo de ésteres de colesterol em macrófagos e a subsequente formação de células espumosas. O sistema imunológico é crucial no processo aterogênico e estratégias terapêuticas direcionadas à imunoregulação deste processo têm sido utilizadas como novas alternativas tanto na prevenção do desenvolvimento quanto da progressão desta doença. Dentre essas estratégias, destaca-se o uso de fragmentos de anticorpos como o scFv (do inglês, single chain fragment variable), que podem ainda estar conjugados a nanopartículas com o intuito de aumentar sua eficiência de ação no organismo. Diante do papel da LDL(-) na aterosclerose, este projeto objetivou avaliar os efeitos in vitro e in vivo de um sistema nanoestruturado contendo fragmentos scFv anti-LDL(-) derivatizados na superfície de nanocápsulas sobre macrófagos murinos e humanos primários e em camundongos knockout para o gene do receptor da LDL (Ldlr-/-) no desenvolvimento e na progressão dessa doença. Demonstrou-se que o tratamento de macrófagos com a formulação scFv anti-LDL(-)-MCMN-Zn diminuiu de forma significativa a captação de LDL(-), assim como a expressão de IL-1β (mRNA e proteína) e MCP-1 (mRNA). Foi demonstrada a internalização da nanoformulação pelos macrófagos via diferentes mecanismos de endocitose, demonstrando seu potencial uso como carreador de fármacos. In vivo, a nanoformulação diminuiu de forma significativa a área da lesão aterosclerótica em camundongos Ldlr-/- submetidos à avaliação pela técnica de tomografia por emissão de pósitrons (do inglês, PET), utilizando o radiotraçador 18F-FDG (18F-desoxiglicose), associada à tomografia computadorizada (CT) com agente de contraste iodado, além da análise morfométrica das lesões no arco aórtico. O conjunto dos resultados obtidos evidenciou a ação ateroprotetora da formulação scFv anti-LDL(-)-MCMN-Zn, reforçando seu potencial como estratégia terapêutica na aterosclerose. / Cardiovascular diseases are the leading cause of mortality worldwide. Atherosclerosis is the pathophysiological basis of these diseases, defined as a chronic inflammatory multifactorial process, resulting from the interaction of several cells such as lymphocytes macrophages, endothelial cells and smooth muscle cells within the arterial wall. The electronegative low-density lipoprotein [LDL(-)], a modified subfraction of native LDL, plays a key role in atherosclerosis, since its modifications are capable of inducing the accumulation of cholesteryl esters in macrophages and the subsequent foam cells formation. The immune system is crucial in atherogenic process and therapeutic strategies directed to the immunoregulation of this process have been used as a new alternative in the prevention of the development as well as the progression of this disease. Among these strategies, it is the use of antibody fragments such as scFv (single chain fragment variable), which may be also conjugated to nanoparticles in order to increase their efficiency in the body. Given the role of LDL(-) in atherosclerosis, the aim of this project was to evaluate the in vitro and in vivo effects of a nanostructured system containing scFv anti-LDL(-) fragments derivatized on the surface of nanocapsules on murine and human primary macrophages and in the development and progression of the disease in LDL receptor knockout mice (Ldlr-/-). It was demonstrated that the treatment of macrophages with scFv anti-LDL(-)-MCMN-Zn formulation significantly decreases the uptake of LDL(-) and the expression IL-1β (mRNA and protein) and MCP-1 (mRNA). Moreover, the internalization of the nanoformulation by macrophages through different endocytosis mechanisms was shown, demonstrating its potential use as a nanocarrier. In vivo, the nanoformulation decreased the area of atherosclerotic lesions in Ldlr-/- mice evaluated by positron emission tomography with 18F-FDG associated with computed tomography with iodinated contrast agent (PET/CT), besides the lesion morphometric analysis at the aortic arch Thus, these data provide evidence of the atheroprotection action of the ateroprotection action of the scFv anti-LDL(-)-MCMN-Zn formulation, suggesting its promising use as a therapeutic strategy for atherosclerosis.

O sistema microPET/CT é um importante equipamento utilizado nas pesquisas de imagem diagnóstica em pequenos animais. O radiofármaco mais usado nesta tecnologia é o fluordeoxiglicose marcado com flúor-18. Este estudo tem como objetivo efetuar o controle radiológico no laboratório de pesquisa microPET/CT do Centro de Radiofarmácia do IPEN-CNEN/SP, de forma a satisfazer tanto as normas nacionais como as recomendações internacionais. O laboratório está classificado pela equipe de radioproteção da instalação como área supervisionada, nas quais embora não seja obrigatória a adoção de medidas específicas de proteção e segurança, devem ser submetidas reavaliações regulares das condições do ambiente de trabalho. Visando assegurar a proteção radiológica dos trabalhadores diretamente envolvidos no manuseio do equipamento, realizou-se o monitoramento do local de trabalho e a avaliação do controle de dose individual. Inicialmente foi feito o monitoramento pré-operacional, isto é, o levantamento radiométrico no laboratório. Além disso, mediu-se nível de radiação externa nas instalações do laboratório e suas adjacências, por meio da colocação de nove dosímetros termoluminescentes (TL) de CaSO4:Dy, em locais previamente selecionados. Os indivíduos ocupacionalmente expostos foram avaliados mensalmente por meio do uso de dosímetros TL posicionados no tórax e por medidas de corpo inteiro, tomadas a cada seis meses. O período do estudo foi de dois anos, com início em abril de 2014. Para o controle do microPET/CT realizou-se testes de desempenho de acordo com o protocolo padrão do equipamento e em conformidade com a norma desenvolvida pela força tarefa para estudos com PET em animais Animal PET Standard Task Force. O presente estudo permitiu demonstrar que os níveis de radiação das áreas (estimativas de dose ambiente e dose efetiva), assim como a blindagem do equipamento estão adequados de acordo com os limites da exposição ocupacional. Ressalta-se a importância de se seguir rigorosamente os princípios de radioproteção, já que se trata de pesquisas com fontes radioativas não seladas. / Micro PET/CT scanner is an essential tool generally used for small animal molecular imaging. Fluorine-18-labeled fluorodeoxyglucose is the most widely used radioisotope in this technique. The present study aimed to evaluate the radiation levels in a micro PET/CT research laboratory of the Radiopharmacy Center at IPEN-CNEN / SP, in order to accomplish both national standards and international recommendations. The radioprotection team has classified the laboratory as supervised area; even this laboratory does not require the adoption of specific measures for protection and safety, should be done regular re-evaluation of the conditions of occupational exposures. Workplace monitoring and individual control assessment were carried out to ensure the radiological protection of all workers directly involved in handling the scanner. Initially, there was conducted a radiometric survey, as well as measurements of the external radiation level in the workplace and its surroundings. To achieve this goal, there were placed nine thermoluminescent dosimeters of CaSO4:Dy in preselected locations. Monthly evaluations of the occupationally exposed individuals were carried out through the use of TL dosimeters, ported in the workers´ chest. Moreover, whole body measurements were performed every six months. The study period was about two-years which started in April 2014. All tests to evaluate micro PET/CT performance were based on the standard protocol of the equipment in accordance with the standard developed by the Animal PET Standard Task Force. Present study\'s results demonstrated that the ambient radiation levels (ambient and effective estimated radiation dose), as well as the effective shielding equipment are both adequate. This study emphasizes that it is essential to strictly follow the principles of radioprotection in workplace, whenever researches involve radioactive unsealed sources.

O sistema microPET/CT é um importante equipamento utilizado nas pesquisas de imagem diagnóstica em pequenos animais. O radiofármaco mais usado nesta tecnologia é o fluordeoxiglicose marcado com flúor-18. Este estudo tem como objetivo efetuar o controle radiológico no laboratório de pesquisa microPET/CT do Centro de Radiofarmácia do IPEN-CNEN/SP, de forma a satisfazer tanto as normas nacionais como as recomendações internacionais. O laboratório está classificado pela equipe de radioproteção da instalação como área supervisionada, nas quais embora não seja obrigatória a adoção de medidas específicas de proteção e segurança, devem ser submetidas reavaliações regulares das condições do ambiente de trabalho. Visando assegurar a proteção radiológica dos trabalhadores diretamente envolvidos no manuseio do equipamento, realizou-se o monitoramento do local de trabalho e a avaliação do controle de dose individual. Inicialmente foi feito o monitoramento pré-operacional, isto é, o levantamento radiométrico no laboratório. Além disso, mediu-se nível de radiação externa nas instalações do laboratório e suas adjacências, por meio da colocação de nove dosímetros termoluminescentes (TL) de CaSO4:Dy, em locais previamente selecionados. Os indivíduos ocupacionalmente expostos foram avaliados mensalmente por meio do uso de dosímetros TL posicionados no tórax e por medidas de corpo inteiro, tomadas a cada seis meses. O período do estudo foi de dois anos, com início em abril de 2014. Para o controle do microPET/CT realizou-se testes de desempenho de acordo com o protocolo padrão do equipamento e em conformidade com a norma desenvolvida pela força tarefa para estudos com PET em animais Animal PET Standard Task Force. O presente estudo permitiu demonstrar que os níveis de radiação das áreas (estimativas de dose ambiente e dose efetiva), assim como a blindagem do equipamento estão adequados de acordo com os limites da exposição ocupacional. Ressalta-se a importância de se seguir rigorosamente os princípios de radioproteção, já que se trata de pesquisas com fontes radioativas não seladas. / Micro PET/CT scanner is an essential tool generally used for small animal molecular imaging. Fluorine-18-labeled fluorodeoxyglucose is the most widely used radioisotope in this technique. The present study aimed to evaluate the radiation levels in a micro PET/CT research laboratory of the Radiopharmacy Center at IPEN-CNEN / SP, in order to accomplish both national standards and international recommendations. The radioprotection team has classified the laboratory as supervised area; even this laboratory does not require the adoption of specific measures for protection and safety, should be done regular re-evaluation of the conditions of occupational exposures. Workplace monitoring and individual control assessment were carried out to ensure the radiological protection of all workers directly involved in handling the scanner. Initially, there was conducted a radiometric survey, as well as measurements of the external radiation level in the workplace and its surroundings. To achieve this goal, there were placed nine thermoluminescent dosimeters of CaSO4:Dy in preselected locations. Monthly evaluations of the occupationally exposed individuals were carried out through the use of TL dosimeters, ported in the workers´ chest. Moreover, whole body measurements were performed every six months. The study period was about two-years which started in April 2014. All tests to evaluate micro PET/CT performance were based on the standard protocol of the equipment in accordance with the standard developed by the Animal PET Standard Task Force. Present study\'s results demonstrated that the ambient radiation levels (ambient and effective estimated radiation dose), as well as the effective shielding equipment are both adequate. This study emphasizes that it is essential to strictly follow the principles of radioprotection in workplace, whenever researches involve radioactive unsealed sources.