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Traumatic brain injury, hereon referred to as TBI, can be simply defined as a disruption to normal brain function as a result of an outside force to the head. TBI contributes to one third of all injury related deaths in the United States, and treatment strategies for TBI are supportive. Although primary and secondary mechanisms of injury have been clearly identified, the heterogeneous and intertwined pathophysiology of TBI is not fully understood. Primary injury results from the impact itself and causes immediate damage. However, secondary mechanisms of injury in TBI, such as oxidative stress and inflammation, are points at which intervention may reduce neuropathology. Trials taking advantage of the antioxidant and anti-inflammatory properties of several agents have had little clinical success, while the use of targeted therapeutics in TBI is relatively unexplored. Evidence suggests that reactive oxygen species (ROS) propagate blood-brain barrier (BBB) hyperpermeability and exacerbate inflammation following TBI. In the studies presented herein, we tested the hypothesis that targeted detoxification of ROS may improve the pathological outcomes using the controlled cortical impact mouse model of TBI. Following TBI, endothelial activation results in a time dependent increase in vascular expression of ICAM-1, an endothelial activation and cell adhesion molecule, as was observed by immunohistochemistry and immunofluorescence staining of isolated cortical microvessels. We conjugated catalase, an antioxidant enzyme, to anti-ICAM-1 antibodies and administered the conjugate intravenously to 8-week-old C57BL/6J mice at 30 minutes after moderate controlled cortical impact TBI. Results indicate that catalase targeted to ICAM-1 reduces markers of oxidative stress including levels of hydrogen peroxide and 3-nitrotyrosine detected in the cortex ipsilateral to the area of injury. Anti-ICAM-1/catalase also preserved BBB permeability based on two assays of barrier permeability to the plasma protein fibrinogen and small fluorescent tracer sodium fluorescein. Following TBI, mice receiving the conjugate exhibited attenuated neuropathological indices for astrocyte and microglia activation as well as cortical neuronal loss compared to controls. For each of these endpoints, anti-ICAM-1/catalase was found to be more effective than anti-ICAM-1 antibodies or catalase administered alone. An extensive study of microglia by two-photon microscopy of ex vivo brain segments from CX3CR1-GFP mice revealed that anti-ICAM-1/catalase prevented the transition of microglia to an activated phenotype after TBI. Finally, anti-ICAM-1/catalase offered functional improvement in Rotarod and elevated zero maze performance compared to controls at acute and chronic time points, respectively. Collectively, these findings demonstrate the use of a targeted antioxidant enzyme to interfere with oxidative stress mechanisms acutely in TBI. The results demonstrate histological and functional benefit of anti-ICAM-1/catalase administration and provide a proof-of-concept approach to improve acute TBI management that may also be applicable to other neuroinflammatory conditions. / Biomedical Sciences

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24549号 / 薬科博第166号 / 新制||薬科||18(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

L’étude des mécanismes aboutissant à la tumorigénèse a permis de révéler, dans beaucoup de cancers, une amplification ou une mutation de divers oncogènes, avec pour conséquence des capacités de prolifération et de survie accrues pour la cellule tumorale. L’identification des protéines kinases comme étant des éléments centraux de ces processus en ont fait des cibles thérapeutiques prometteuses. Plusieurs inhibiteurs ciblant de façon plus ou moins spécifique les tyrosines kinases oncogéniques ont ainsi été développés. Parmi eux, l’imatinib mesylate (Gleevec®, Novartis) a constitué la première chimiothérapie ciblée. Il correspond aujourd’hui au traitement de première intention contre la LMC. Cependant, malgré sa très grande efficacité, il est apparu que certains mécanismes de résistances pouvaient être mis en place pour diminuer son effet pro-apoptotique. Le travail de cette thèse a consisté à mieux comprendre les mécanismes d’apoptose induits par les inhibiteurs de tyrosine kinases (ITK), et à rechercher quelles voies alternatives de survie devront être bloquées pour leur assurer une meilleure efficacité. Trois modèles ont été utilisés : la leucémie myéloïde chronique (LMC), les leucémies aiguës myéloïdes (LAM) et les glioblastomes (GBM). La LMC a été utilisé comme modèle et la démarche utilisé pour essayer d’augmenter l’efficacité des ITK, a été transposée aux modèles des LAM et des GBM. L’ensemble des résultats obtenus a démontré qu’une meilleure compréhension de la réponse apoptotique et des mécanismes de résistance permettait l’identification de nouvelles cibles thérapeutiques. Nous avons pu observer que, tout en favorisant la diminution des doses de molécules administrées, l’inhibition simultanée de plusieurs cibles apportait plusieurs bénéfices. Elle permet d’augmenter l’action pro-apoptotique des ITK, de contrer certains mécanismes de résistances, d’atteindre la cellule souche cancéreuse résistance et par conséquent de cibler simultanément des populations a plusieurs de stades de différenciation. / Protein kinases have been identified as playing fundamental roles in cancer development, suggesting that they could represent a promising therapeutic target. Several kinase inhibitors have been developed and the most successful of them, by far, is Gleevec® (imatinib, STI57; Novartis), a BCR-ABL inhibitor. It is currently used as the treatment of reference for chronic myeloid leukemia. However, despite a huge efficiency, some resistance mechanisms could be used to decrease its pro-apopototic effect. The global aim of my PhD was to understand the apoptotic mechanisms induced by tyrosine kinase inhibitors (TKI) to identify new potential therapeutic targets. I work on three different tumors: Chronic Myeloid Leukemia (CML), Acute Myeloid Leukemia (AML) and Glioblastomas (GBM). CML has been used as a model and the approach followed to increase TKI efficiency has been transposed to AML and GBM models. Altogether, our results showed that a better understanding of apoptotic response and resistance mechanisms could lead to the identification of new therapeutic targets. We observed that combination therapy brings several benefits. It allows to increase the TKI-induced apoptotic response, to counter some resistance mechanism, to reach the resistant cancer stem cells, and thus, to target simultaneously several populations in the tumour.

Os tumores de musculatura liso do útero se desenvolvem a partir do miométrio e podem apresentar carcterísticas clínicas malignas e benignas. Dentre eles, o leiomiossarcoma (LMS) é o tumor maligno mais comum, com altas taxas de metástase e recidiva, mesmo sendo diagnosticado em estágios iniciais. Já os leiomiomas (LM) são os tumores benignos mais frequentes em mulheres em idade reprodutiva. Ambos possuem mesma diferenciação celular, porém com comportamentos clínico e biológico bastante distintos, e até o momento não se dispõe de tratamento específico ou curativo. Nesse contexto, a busca por novos alvos moleculares pode contribuir não só para um melhor entendimento dessas neoplasias, como também para a descoberta de novas terapias. Em estudo prévio foi observada a expressão aumentada de FOXO3a nos sarcomas uterinos, em comparação aos LMs e ao miométrio adjacente (MM). Além disso, sua expressão foi crescente de acordo com o potencial de malignidade do tumor. Assim, o objetivo do presente estudo foi avaliar in vitro o efeito de terapia alvo específica para FOXO3a em células de LM e LMS. Para isto, linhagens celulares de MM (ATCC PCS-460-011), LM (THESCs - CRL-4003) e LMS (SK-UT-1 - HTB-114) foram caracterizadas quanto à expressão basal de FOXO3a (gene e proteína) e submetidas a tratamento com Genisteína e Metformina ou inativação do gene por siRNA. Os efeitos dos tratamentos foram avaliados por PCR em tempo real, Western Blot, imunocitoquímica, ensaios de proliferação, migração e apoptose. Nossos resultados mostraram que todos os tratamentos realizados interferiram na capacidade de proliferação e migração das células, com maior inibição após as 48 horas nos LMS e 72h nos LM. O efeito obtido na transfecção com siRNA apresentou maior eficiência após 48 h da transfecção nos LMS e 72h nos LM. Os efeitos da inibição de FOXO3a foram maiores na proliferação e migração dos LM, porém os resultados não foram estatisticamente significativos. Dentre as substâncias testadas, a Metformina apresentou maior efeito sobre a proliferação, migração e viabilidade das linhagens celulares. A Genisteína também apresentou efeito inibitório nas células, porém o controle com veículo também apresentou o mesmo efeito citotóxico. De modo geral, os efeitos obtidos com os fármacos, foram tempo e concentração dependentes. Em conjunto, nossos resultados sugerem um relevante do FOXO3a nos tumores de musculatura lisa uterinos, além de apresentá-lo como potencial alvo para terapia específica / Smooth muscle tumors of the uterus develop from the myometrium and may present benign and malignant clinical features. Among them, leiomyosarcoma (LMS) is the most frequent malignant tumor, with high rates of metastasis and relapse, even when diagnosed in early stages. On the other hand, leiomyomas (LM) are the most frequent benign tumors in women of reproductive age. Both have the same cellular differentiation, but with very different clinical and biological behaviors, and so far no specific or curative treatment is available. In this context, the search for new molecular targets can contribute not only for a better understanding of these neoplasms, but also for the discovery of new therapies. In a previous study, increased expression of FOXO3a in uterine sarcomas was observed, compared to LMs and adjacent myometrium (MM). In addition, its expression was increasing according to the malignancy potential of the tumor. Thus, the aim of the present study was to evaluate in vitro, the effect of specific targeted therapy for FOXO3a on LM and LMS cells. For this, MM (ATCC PCS-460-011), LM (THESCs-CRL-4003) and LMS (SK-UT-1-HTB-114) cell lines were characterized for basal expression of FOXO3a (gene and protein) and subsequently submitted to treatment with metformin and genistein, or silencing of FOXO3a by siRNA. The effects of the treatments were evaluated by real-time PCR, Western Blot, immunocytochemistry, proliferation, migration and apoptosis assays. Our results showed that all treatments interfered in the proliferation and migration capacity of the cells, with greater inhibition after 48 hours for LMS and 72 hours LM. The effect obtained in the transfection with siRNA showed higher efficiency after 48 hours of transfection in LMS and 72 hours in LM. The effects of inhibition of FOXO3a were greater in the proliferation and migration of the LM, but the results were not statistically significant. Among the substances tested, Metformin had a greater effect on proliferation, migration and viability of the cell lines. Genistein also had an inhibitory effect on the cells, but the control with the vehicle also presented the same cytotoxic effect. In general, the effects obtained with the drugs were time and concentration dependent. Together, our results suggest a relevant role of FOXO3a in uterine smooth muscle tumors, in addition to presenting it as a potential target for specific therapy

Les mastocytes (MCs) sont issus des cellules hématopoïétiques multipotentes non engagées, CD34+, et jouent un rôle important dans l’initiation de la réponse immunitaire innée et adaptative, ainsi que dans les réactions allergiques IgE-dépendantes. Les mastocytoses sont des néoplasies myéloïdes caractérisées par une accumulation anormale et l'activation fréquente de mastocytes dans divers organes. Les organes généralement atteints sont la moelle osseuse, la peau, le foie et le tractus gastro-intestinal. Elles sont caractérisées dans l’immense majorité des cas par la présence de mutations acquises de la structure du récepteur KIT (plus particulièrement KIT D816V) qui induisent l’activation constitutive de ce récepteur à activité tyrosine kinase intrinsèque. Le traitement actuel de ces pathologies est décevant car la mutation KIT D816V résiste à la plupart des inhibiteurs de tyrosine kinases (ITKs).Dès le début de ma thèse, nous avons pu établir, à partir de cellules souches hématopoïétiques de sang de cordon humain normal cultivées à long terme en présence de stem cell factor (SCF), une nouvelle lignée mastocytaire humaine stable, dénommée ROSA KIT WT, restant strictement dépendante pour sa survie et sa prolifération du SCF, exprimant le récepteur de haute affinité aux IgE (FcεRI), et présentant un récepteur KIT de structure normale. Cette lignée, facile à cultiver en grandes quantités, permet d’envisager l’étude approfondie des évènements intracellulaires menant à l’activation mastocytaire IgE-dépendante et la mise au point de tests de criblage à haut débit dans le domaine de la thérapeutique anti-allergique et/ou anti-inflammatoire.Par ailleurs, afin de pouvoir étudier le rôle transformant des mutants de KIT retrouvés au cours des mastocytoses, nous avons transfecté les cellules ROSA KIT WT par des vecteurs lentiviraux apportant une construction codant pour le KIT muté en D816V ou le KIT muté Delta 417-419 insY. Nous avons ainsi obtenu deux nouvelles lignées mastocytaires humaines SCF-indépendantes, ROSA KIT D816V et ROSA KIT Delta 417-419 insY, pour lesquelles nous avons montré qu’il existe une activation constitutive de KIT, mais aussi de STAT5 et d’AKT. Ces deux lignées de pourront être utilisées soit pour étudier l’impact des mutations de KIT sur la signalisation intracellulaire, soit pour le criblage molécules à activité antiproliférative potentielle dirigées soit contre KIT muté, soit contre l'une ou l’autre des molécules intracellulaires impliquées dans la transduction du signal KIT muté / Mast cells are cells with ubiquitous tissue distribution, derived from CD34 + multipotent hematopoietic cells. These cells play a fundamental role in the initiation of innate and adaptive immune response and in IgE-dependent allergic reactions or in various inflammatory reactions.Mastocytosis is defined as a myeloproliferative neoplastic disorder, caused by an abnormal accumulation of mast cells in one or more organ systems. Mastocytosis presents in cutaneous and systemic forms. In patients with systemic mastocytosis, the most frequent point mutation is KIT D816V, whereas in pediatric patients, where the disease is usually restricted to the skin, different KIT defects have been detected, mostly in the extracellular portion of KIT.In a primary culture of mast cells made from precursors of one cord blood, we have successfully isolated a new human mast cell line called ROSA KIT WT with a phenotype and reactivity comparable to those of normal mast cells. This cell line is dependent on SCF for growth and expresses the KIT receptor wild. It is easy to grow in large quantities, to freeze and to activate by IgE-anti IgE couple or a couple of allergen and corresponding specific IgE. This cell line can be used to study pathophysiologic mechanisms of allergy and to develop and use high-throughput screening tests of molecules in search of anti-allergic properties.In addition, we transfected these cells by lentiviral vectors providing constructs encoding the mutated KIT D816V or the mutated KIT Delta 417-419 insY, two KIT abnormalities encountered in the course of mastocytosis. This allowed us to establish two new cell lines independent of SCF for proliferation, ROSA KIT D816V and ROSA KIT Delta417-419 insY, which are particularly easy to grow in large quantities, and whose phenotype is similar to that of abnormal mast cells during mastocytosis. These two cell lines can be used for pathophysiologic studies on mastocytosis and for high throughput screening of molecules in search of antiproliferative effects specifically directed against the mutated KIT or against one or other of the intracellular molecules involved in signal transduction induced by mutant KIT.

Os tumores de musculatura liso do útero se desenvolvem a partir do miométrio e podem apresentar carcterísticas clínicas malignas e benignas. Dentre eles, o leiomiossarcoma (LMS) é o tumor maligno mais comum, com altas taxas de metástase e recidiva, mesmo sendo diagnosticado em estágios iniciais. Já os leiomiomas (LM) são os tumores benignos mais frequentes em mulheres em idade reprodutiva. Ambos possuem mesma diferenciação celular, porém com comportamentos clínico e biológico bastante distintos, e até o momento não se dispõe de tratamento específico ou curativo. Nesse contexto, a busca por novos alvos moleculares pode contribuir não só para um melhor entendimento dessas neoplasias, como também para a descoberta de novas terapias. Em estudo prévio foi observada a expressão aumentada de FOXO3a nos sarcomas uterinos, em comparação aos LMs e ao miométrio adjacente (MM). Além disso, sua expressão foi crescente de acordo com o potencial de malignidade do tumor. Assim, o objetivo do presente estudo foi avaliar in vitro o efeito de terapia alvo específica para FOXO3a em células de LM e LMS. Para isto, linhagens celulares de MM (ATCC PCS-460-011), LM (THESCs - CRL-4003) e LMS (SK-UT-1 - HTB-114) foram caracterizadas quanto à expressão basal de FOXO3a (gene e proteína) e submetidas a tratamento com Genisteína e Metformina ou inativação do gene por siRNA. Os efeitos dos tratamentos foram avaliados por PCR em tempo real, Western Blot, imunocitoquímica, ensaios de proliferação, migração e apoptose. Nossos resultados mostraram que todos os tratamentos realizados interferiram na capacidade de proliferação e migração das células, com maior inibição após as 48 horas nos LMS e 72h nos LM. O efeito obtido na transfecção com siRNA apresentou maior eficiência após 48 h da transfecção nos LMS e 72h nos LM. Os efeitos da inibição de FOXO3a foram maiores na proliferação e migração dos LM, porém os resultados não foram estatisticamente significativos. Dentre as substâncias testadas, a Metformina apresentou maior efeito sobre a proliferação, migração e viabilidade das linhagens celulares. A Genisteína também apresentou efeito inibitório nas células, porém o controle com veículo também apresentou o mesmo efeito citotóxico. De modo geral, os efeitos obtidos com os fármacos, foram tempo e concentração dependentes. Em conjunto, nossos resultados sugerem um relevante do FOXO3a nos tumores de musculatura lisa uterinos, além de apresentá-lo como potencial alvo para terapia específica / Smooth muscle tumors of the uterus develop from the myometrium and may present benign and malignant clinical features. Among them, leiomyosarcoma (LMS) is the most frequent malignant tumor, with high rates of metastasis and relapse, even when diagnosed in early stages. On the other hand, leiomyomas (LM) are the most frequent benign tumors in women of reproductive age. Both have the same cellular differentiation, but with very different clinical and biological behaviors, and so far no specific or curative treatment is available. In this context, the search for new molecular targets can contribute not only for a better understanding of these neoplasms, but also for the discovery of new therapies. In a previous study, increased expression of FOXO3a in uterine sarcomas was observed, compared to LMs and adjacent myometrium (MM). In addition, its expression was increasing according to the malignancy potential of the tumor. Thus, the aim of the present study was to evaluate in vitro, the effect of specific targeted therapy for FOXO3a on LM and LMS cells. For this, MM (ATCC PCS-460-011), LM (THESCs-CRL-4003) and LMS (SK-UT-1-HTB-114) cell lines were characterized for basal expression of FOXO3a (gene and protein) and subsequently submitted to treatment with metformin and genistein, or silencing of FOXO3a by siRNA. The effects of the treatments were evaluated by real-time PCR, Western Blot, immunocytochemistry, proliferation, migration and apoptosis assays. Our results showed that all treatments interfered in the proliferation and migration capacity of the cells, with greater inhibition after 48 hours for LMS and 72 hours LM. The effect obtained in the transfection with siRNA showed higher efficiency after 48 hours of transfection in LMS and 72 hours in LM. The effects of inhibition of FOXO3a were greater in the proliferation and migration of the LM, but the results were not statistically significant. Among the substances tested, Metformin had a greater effect on proliferation, migration and viability of the cell lines. Genistein also had an inhibitory effect on the cells, but the control with the vehicle also presented the same cytotoxic effect. In general, the effects obtained with the drugs were time and concentration dependent. Together, our results suggest a relevant role of FOXO3a in uterine smooth muscle tumors, in addition to presenting it as a potential target for specific therapy

O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico. / Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.