京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18883号 / 医博第3994号 / 新制||医||1009(附属図書館) / 31834 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 野田 亮, 教授 藤田 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

Stroke is the world's leading cause of adult disability, caused by lack of oxygen and nutrients to the brain due to a blood clot in a major artery. This leads to ischemic damage of neuronal cells that leads to paralysis, motor, and speech deficits. While most stroke therapies aim at removing or reducing the blood clots in the brain, few treatments target cell damage. Neuroglobin (NGB) is a protein in the brain that is able to aid in neuroprotection following oxidative stress. Hypoxia-Inducible Factor-1 (HIF-1) is a transcription factor that serves as a marker for cell recovery after hypoxia or low oxygen levels. Exosomes are microscopic extracellular vesicles that can help deliver proteins across the blood-brain barrier. This thesis focuses on finding a correlation between exosomal-delivered neuroglobin to ischemic cells and the regulation of HIF-1 in order to develop an innovative treatment using exosomes. The specific aims of this thesis are as follows: Aim 1: Package NGB in exosomes of healthy cell The XPAK-NGB plasmid will be used to transfect NGB DNA into wild-type human embryonic kidney (HEK-293 cell line) cells. Exosomes will be harvested from the spent media. The exosomes will be analyzed to ensure that the protein is packaged inside the exosomes. Aim 2: Determine the limit of hypoxic conditions and effects of NGB on damaged cells A literature review will be performed to determine the ideal concentration of H2O2 for the survival of neuronal cells. This will include the composition of hypoxia as well as the length of time that cells can be exposed to and remain viable. Aim 3: Correlate NGB concentration and HIF-1 concentration Another literature review will determine the specific markers of NGB and HIF-1.

No description available.

Die ständige Verfügbarkeit von molekularem Sauerstoff (O2) ist ein elementarer Bestandteil multizellulärer Lebensformen. Zur Aufrechterhaltung der Homöostase sind diese auf die Bildung des Energiesubstrates ATP durch oxidative Phosphorylierung angewiesen. Aus diesem Grunde mußten höhere Organismen während der Evolution komplexe Systeme entwickeln, die die Aufnahme und Verteilung von O2 in jede Zelle sicherstellen, sowie eine Adaptation in Phasen der Hypoxie erlauben. Mit der Identifikation des Transkriptionsfaktors "Hypoxia-inducible Factor-1" (HIF-1, 1995) wurde ein entscheidender Regulator der hypoxischen Adaptation gefunden. Unter anderem werden Prozesse wie die Erythropoiese, die Angiogenese, die Modulation des Gefäßtonus, des Glukosetransportes und der Glykolyse wesentlich durch HIF reguliert. HIF ist ein Heterodimer bestehend aus zwei Untereinheiten; einer konstitutiven beta- und einer regulativen alpha-Untereinheit. Letztere zeigt ein inverses Expressionsmuster zur perizellulären O2-Konzentration. Unter normoxischen Bedingungen ist HIFalpha instabil und wird mit einer Halbwertzeit von nur wenigen Minuten degradiert. Erst unter Hypoxie wird HIFalpha stabilisiert und ist transkriptionell aktiv. Es konnten bisher zwei funktionell relevante O2-abhängige alpha-Untereinheiten identifiziert werden: HIF-1 und HIF-2alpha. Die Bedeutung dieser beiden Systeme, der unterliegenden Regulationsmechanismen sowie die Relevanz dieses Systems in vivo waren weitgehend ungeklärt und sind wesentlicher Teil der hier zusammengefaßten Arbeiten. In den vorgelegten Studien ist es gelungen, die Expression und Regulation der beiden unterschiedlichen HIFalpha Isoformen sowohl in der Zellkultur, als auch in gesunden Geweben zu charakterisieren. In Zellkulturen zeigte sich ein sehr ähnliches Regulationsmuster hinsichtlich der O2-abhängigen Degradation, bzw. dem Induktionsverhalten unter Hypoxie, sowie der chemisch/pharmakologischen Modulation, so dass offensichtlich beide Isoformen über den gleichen O2-Sensing- und Transduktionsapparat reguliert werden. An Geweben von gesunden Ratten führten wir eine systematische Analyse der Expression und Regulation der beiden HIFalpha Isoformen durch. Nur unter systemischer Hypoxie konnten deutliche Signale für beide Isoformen gesehen werden. Interessanterweise zeigte sich, daß beide nur von spezifischen Zellpopulationen exprimiert werden. In vivo lassen sich also klare Unterschiede im Expressionsmuster der beiden Systeme feststellen. Über die unterschiedlichen zellulären Funktionen und different exprimierten Zielgene vermuten wir einen funktionell relevanten Unterschied. Mit der Identifikation des "von Hippel Lindau" Tumor Suppressor Gens als der bindende Anteil der E3 Ubiquitin Ligase, die für die HIFalpha Destruktion verantwortlich ist, konnte ein wichtiger Beitrag zu der späteren Klärung des O2-Sensing-Mechanismus geleistet werden. Diese Befunde wurden initial anhand von Zellkultur-Linien erhoben, liessen sich aber auf Nierenzellkarzinome aus einer klinischen Sammlung übertragen. Letzterer Befund ist daher für das Verständnis der Rolle von HIF für die Tumorbiologie, eventuell aber auch für die Entwicklung therapeutischer Ansätze von Bedeutung. / The permanent availability of molecular oxygen (O2) is an elemental need of multicellular life. For the maintenance of hemeostasis these are dependent on generation of the energy substrate ATP by oxidative phoshorylation. For this reason higher organisms had to develop complex systems during evolution that ensure the uptake and distribution of O2 into each cell, as well as permit adaptation to phases of hypoxia. With the identification of the transcription factor "Hypoxia-inducible Factor-1" (HIF-1, 1995) a master regulator of hypoxic adaptation has been found. Amongst others processes like erythropoiesis, angiogenesis, modulation of vascular tone, glucose transport and glycolysis are largely regulated by HIF. HIF is a heterodimer consisting of two subunits, a constitutive beta and a regulative alpha subunit. The latter shows an inverse relationship to the pericellular O2 concentration. HIFalpha is instable under normoxic conditions and degrades with a half life of only a few minutes. Under hypoxia the HIFalpha subunits are stabilised and are transcriptionally active. To date two functionally relevant O2-dependent alpha subunits have been identified: HIF-1 and HIF-2alpha. The importance of these two systems, the underlying regulatory mechanisms, as well as the relevance of this system in vivo were largely unknown and are a major part of the summarised studies. The presented work succeeded in characterising the expression and regulation of both HIFalpha isoforms in cell culture as well as healthy tissues. In tissue culture a very similar pattern of regulation was seen for oxygen dependent degradation, induction under hypoxia and chemical/pharmacological modulation, indicating that both subunits are regulated by the same O2-sensing and transduction apparatus. We undertook a systematic analysis of expression and regulation of both HIFalpha subunits in tissues of healthy rats. Signals for HIFalpha could only be seen under systemic hypoxia. Interestingly, both subunits were expressed by specific and different cell populations. Therefore, clear differences can be seen in expression pattern of both systems in vivo. We suspect that these differences will be functionally relevant through differing cellular functions and gene expression profile. With the identification of the "von Hippel Lindau" tumor suppressor gene as the binding part of the E3 ubiquitin ligase, which is responsible for HIF degradation, an important contribution to the clarification of the oxygen sensing mechanism was provided. Initially this data was generated in tissue culture lines, but could also be confirmed in renal cell carcinomas of a clinical collection. The latter finding is of importance for the understanding of the role of HIF in tumor biology, possibly also for the development of therapeutic strategies.

Made available in DSpace on 2016-01-26T12:51:46Z (GMT). No. of bitstreams: 1 marinagobbemoschetta_dissert.pdf: 2656918 bytes, checksum: d63acce15a1feb7170b4c9270fd920d9 (MD5) Previous issue date: 2013-09-16 / Fundação de Amparo a Pesquisa do Estado de São Paulo / Introduction: Mammary tumors are the most common type of tumor in dogs, with approximately half of these tumors are malignant. Hypoxia, characterized by oxygen levels below normal, is a known adverse factor to cancer treatment. The transcription factor HIF-1&#945; is the central regulator of pathophysiological response of mammalian cells to low oxygen levels, able to activate transcription of the vascular endothelial growth factor (VEGF), which in turn promotes angiogenesis through its ability to stimulate growth, migration and invasion of endothelial cells, contributing to tumor growth. Objectives: To evaluate the serum concentration and the gene expression of VEGF and HIF-1&#945; linking them with clinicopathological parameters and survival of dogs with mammary tumors in order to infer the possible prognostic value of these factors. Material and Methods: We collected tumor fragments of 30 bitches with mammary tumors to verify protein expression of VEGF and HIF-1&#945; by immunohistochemistry and gene expression by RT-PCR. To determine the serum concentration of VEGF and HIF-1&#945; by ELISA (Enzyme-linked immunosorbent), serum was collected from 50 bitches control (healthy) and 30 bitches with mammary neoplasia (study group). The results were statistically related to clinicopathological features. Results: The comparison between immunohistochemical staining of the two proteins analyzed showed increased intensity of immunostaining of VEGF (p=0.03). By ELISA, we observed relationship between high serum levels of VEGF and abundant vascularization (p=0.02), metastasis (p=0.003), death rate (p=0.007) and low survival (p<0.0001). In addition, increased serum levels of VEGF in the study group compared to the control group (p=0.03). In contrast, the percentage of serum HIF-1&#945; bitches with mammary neoplasia was 20% lower than the control group of female dogs (p=0.0006). However, bitches with a history of recurrent tumor showed a 15% increase in the percentage of serum HIF-1&#945; (p=0.03). Regarding gene expression, there was a relationship between increased gene expression of VEGFA and tumor abundant vascularization (p=0.02), bitches with a history of recurrence (p=0.01) and death (p=0.02). Moreover, we observed a statistically significant increase in gene expression of HIF-1A with moderate vascularization (p=0.01) and bitches that remained alive during the follow-up period (p=0.003). Conclusions: Our results demonstrate a correlation between VEGF and features of poor prognosis, suggesting that this factor plays an important role in tumor progression and can be used as a potential prognostic marker in clinical practice and is useful in predicting tumor progression in dogs with mammary neoplasia. / Introdução: As neoplasias mamárias são o tipo mais comum de tumor na espécie canina, sendo aproximadamente metade desses tumores de caráter maligno. A hipóxia, caraterizada por níveis de oxigênio abaixo do normal, é um conhecido fator adverso ao tratamento do câncer. O fator de transcrição HIF -1&#945; é o regulador central da resposta fisiopatológica das células de mamíferos para baixos níveis de oxigênio, capaz de ativar a transcrição do fator de crescimento endotelial vascular (VEGF), que por sua vez, promove a angiogênese através da sua capacidade de estimular o crescimento, migração e invasão de células endoteliais, contribuindo para o crescimento tumoral. Objetivos: Avaliar a concentração sérica e a expressão gênica do VEGF e do HIF-1&#945; relacionando-os com os parâmetros clínico-patológicos e a sobrevida de cadelas com neoplasia mamária a fim de inferir o possível valor prognóstico desses fatores. Material e Métodos: Foram coletados fragmentos tumorais de 30 cadelas com neoplasia mamária para verificar a expressão protéica do VEGF e do HIF-1&#945; por imuno-histoquímica e a expressão gênica por PCR em Tempo Real. Para determinar a concentração sérica do VEGF e do HIF-1&#945; pelo método de ELISA (Enzyme-linked immunosorbent), foram coletados soro sanguíneo de 50 cadelas controle (saudáveis) e 30 cadelas com neoplasia mamária (grupo de estudo). Os resultados encontrados foram estatisticamente relacionados às características clínico-patológicas. Resultados: A comparação entre a marcação imuno-histoquímica das duas proteínas analisadas demonstrou aumento da intensidade da imunomarcação do VEGF (p=0,03). Por meio da técnica de ELISA, foi possível observar relação entre altos níveis séricos de VEGF com vascularização abundante (p=0,02), metástase (p=0,003), óbito (p=0,007) e baixa taxa de sobrevida (p<0,0001). Além disso, houve aumento dos níveis séricos de VEGF no grupo de estudo quando comparados ao grupo controle (p=0,03). Ao contrário, o percentual sérico de HIF-1&#945; das cadelas com neoplasia mamária foi 20% menor do que das cadelas do grupo controle (p=0,0006). No entanto, cadelas com histórico de recidiva tumoral demonstraram aumento de 15% no percentual sérico de HIF-1&#945; (p=0,03). Quanto à expressão gênica, houve relação entre o aumento da expressão gênica do VEGFA e tumores com vascularização abundante (p=0,02), cadelas com histórico de recidiva tumoral (p=0,01) e óbito (p=0,02). Por outro lado, foi observado aumento estatisticamente significante da expressão gênica do HIF-1A com vascularização moderada (p=0,01) e cadelas que continuaram vivas durante o período de acompanhamento (p=0,003). Conclusões: Nossos resultados demostram correlação entre o VEGF e as características de pior prognóstico, sugerindo que este fator desempenha um importante papel na progressão tumoral, podendo ser utilizado como um potencial marcador prognóstico na rotina clínica, sendo útil na predição da progressão tumoral em cadelas com neoplasia mamária.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / O carcinoma epidermoide de esôfago (CEE) representa 90% dos casos de câncer de esôfago no Brasil. O CEE tem detecção tardia, um comportamento extremamente agressivo e baixa sobrevida, sendo, portanto, um alvo interessante para o estudo dos mecanismos envolvidos em sua carcinogênese, a fim de se identificar possíveis alvos terapêuticos ou marcadores moleculares que ajudem na prática clínica. Mudanças no metabolismo energético da célula tumoral parecem ter papel de destaque na transformação maligna. Sabe-se que células tumorais consomem glicose avidamente produzindo ácido lático, mesmo em condições de normóxia. Dentre os fatores que podem contribuir para o estímulo da glicólise em células tumorais destacam-se as alterações em enzimas da via glicolítica tais como: as piruvato-cinases M1 e M2 (PKM1 e PKM2), a hexocinase II (HKII), isofoma 1 do transportador de glicose, GLUT-1, e o fator de transcrição induzido por hipóxia (HIF1&#945;), responsável pela transcrição das proteínas citadas. O objetivo do estudo é avaliar a relação entre a expressão de HIF1&#945;, HK2, PKM2, PKM1 e GLUT-1 e dados clínico-patológicos no CEE. Para tal, foram avaliados tumores conservados em parafina de 44 pacientes com CEE matriculados no INCA e no Hospital das Clínicas de Porto Alegre. Além disso, foram coletadas amostras de biópsia de esôfago em 67 pacientes sem doença esofágica, que foram submetidos à endoscopia no Hospital Universitário Pedro Ernesto (HUPE). A expressão das proteínas foi avaliada nos tecidos por imuno-histoquímica, enquanto que a expressão do mRNA de GLUT-1 também foi avaliada nas amostras controle. Foi observado que as amostras controle expressam HK2, PKM1, PKM2, HIF1&#945; nas camadas do epitélio esofágico. Já GLUT-1 e Ki-67 são vistos apenas na camada basal. Além disso, a expressão do mRNA de GLUT-1 não teve correlação com fatores etiológicos da doença. Em CEE a expressão de HK2, PKM2 e GLUT-1 foi vista em todos os tumores, já a expressão de HIF1&#945; e PKM1 foi variável. Além disso, observou-se que maior expressão de HIF-1&#945; apresenta correlação com invasão linfonodal e diferenciação, enquanto que a expressão de HK2 tem relação com sobrevida e PKM1 com diferenciação. As correlações clínicas encontradas sugerem que alterações no metabolismo energético é um alvo de estudo interessante para desenvolvimento de marcadores moleculares que auxiliem a prática clínica. / The esophageal squamous cell carcinoma (ESCC) represents 90% of cases of esophageal cancer in Brazil. The ESCC has late diagnosis, highly aggressive behavior and poor survival. ESCC is an interesting target to the study of mechanism involved in its carcinogenesis, in order to identify potential drug targets or biomarkers to help clinical practice. Changes in tumor cell energy metabolism appear to have a prominent role in malignant transformation. Tumor cells consume glucose avidly and produce lactic acid, even under normoxia. Among the factors that may contribute to the stimulation of glycolysis in tumor cells, there are changes in the glycolytic pathway enzymes such as: pyruvate kinase M1 and M2 (PKM2 and PKM1), hexokinase II (HKII), glucose transporter isoform 1, GLUT-1, and transcription factor induced by hypoxia (HIF1&#945;), responsible for the transcription of proteins cited. The goal of the study is to evaluate the relationship between the expression of HIF1&#945;, HK2, PKM2, PKM1 and GLUT-1 and clinicopathological data in ESCC. Biopsy of the esophagus in patients without esophageal disease were collected, who underwent endoscopy at University Hospital Pedro Ernesto (HUPE). Tissue samples were collected from 44 patients with a histologically confirmed diagnosis of ESCC recruted from Hospital Universitário Pedro Ernesto (HUPE-UERJ), and Instituto Nacional de Câncer (INCA). Tissue samples from healthy individuals submitted to endoscopic routine examination, not related to cancer or esophageal disorders, at HUPE-UERJ were also included in this study. The expression of proteins in tissues was evaluated by immunohistochemistry, while mRNA expression of GLUT-1 was also evaluated in the control samples. It was observed that the control samples express HK2, PKM1, PKM2, HIF1&#945; layers of the esophageal epithelium. GLUT-1 and Ki-67 are seen only in the basal layer. Furthermore, expression of GLUT-1 mRNA did not correlate with disease etiological factors. In ESCC expression of HK2, PKM2 and GLUT-1 was seen in all tumors, and the expression of HIF1&#945; and PKM1 was variable. We found that increased expression of HIF-1&#945; correlates with lymph node invasion and differentiation, whereas the expression of HK2 is related to survival, and differentiation with PKM1. The clinical correlations found suggest that alterations in energy metabolism are an interesting subject of study for development of biomarkers that help clinical practice.

No description available.

Le syndrome d’apnées obstructives du sommeil (SAOS) est associé à une forte morbi-mortalité cardiovasculaire. L’hypoxie intermittente (HI), conséquence majeure des apnées, est à l’origine d’un stress oxydant, d’une activation de HIF-1 (hypoxia inducible factor 1) et d’une expression d’endothéline (ET-1), tous impliqués dans les complications cardiovasculaires liées à l’HI.Dans un premier temps, nous avons démontré que l’HI augmentait l’incidence des arythmies ventriculaires létales associées à l’ischémie myocardique. Parmi les mécanismes potentiels impliqués, l’analyse spectrale de la variabilité de la fréquence cardiaque et de la pression artérielle et le dosage des catécholamines ont mis en évidence une activation sympathique chez les animaux exposés à l’HI. L’HI est également à l’origine d’altérations de la repolarisation ventriculaire (allongement du QTc et du Tpeak-Tend) et d’une dispersion du gradient transmural (allongement de la durée du potentiel d’action endocardique) associées à une augmentation de l’expression de canaux calciques de type LTCC et TRPC dans l’endocarde.Dans la seconde partie de ce travail de thèse, nous nous sommes intéressés aux perturbations de l’homéostasie du zinc en réponse au stress oxydant causé par l’ischémie-reperfusion (IR) ou par l’HI et aux propriétés cardioprotectives de la supplémentation en zinc dans ce contexte. Nous avons montré que l’IR et l’HI induisaient une diminution des concentrations de zinc myocardiques et plasmatiques, respectivement. Nous avons mis en évidence les effets bénéfiques de la supplémentation en zinc vis-à-vis des arythmies ventriculaires et des altérations myocardiques induites par l’IR. L’administration de zinc lors de la reperfusion a également permis d’abolir l’augmentation de la taille d’infarctus induite par l’exposition chronique à l’HI.Finalement, nous avons étudié les effets de la déplétion en zinc sur des cellules endothéliales à l’aide d’un chélateur spécifique du zinc, le TPEN. Nous avons observé que l’exposition des cellules au TPEN entraînait une translocation nucléaire de HIF-1α et une augmentation de la sécrétion d’ET-1 avec, comme conséquence, une augmentation de la capacité migratoire des cellules endothéliales. Ainsi, une déplétion en zinc semble conduire à une activation de l’axe HIF-1-ET-1 connu pour ses effets délétères lors de l’HI.En résumé, l’exposition chronique à l’HI exacerbe les arythmies et augmente la taille d’infarctus lors de l’IR. L’activation sympathique, le stress oxydant et l’altération de l’homéostasie du zinc pourraient être impliqués. L’utilisation d’outils pharmacologiques permettrait de confirmer leur rôle et potentiellement de prévenir les altérations cardiovasculaires liées à l’HI et au SAOS. / Obstructive sleep apnea (OSA) is associated with increased cardiovascular morbidity and mortality. Intermittent hypoxia (IH), one of the major consequences of apneas, leads to oxidative stress, activation of HIF-1 (hypoxia inducible factor 1) and endothelin (ET-1) expression, all known to play an important role in the cardiovascular consequences of OSA.First, we have demonstrated that IH increases the incidence of ischemia-related lethal ventricular arrhythmias. Among the potential mechanisms involved, spectral analysis of heart rate and blood pressure variability and catecholamine assay, showed a sympathetic activation in animals exposed to IH. IH was also responsible for alterations in ventricular repolarisation (increased QTc and Tpeak-Tend intervals) and dispersion of the transmural gradient (increased endocardial action potential duration). These alterations were associated with increased expression of endocardial LTCC and TRPC calcium channels.The second part of the thesis aimed at investigating zinc homeostasis in response to the oxidative stress induced by ischemia-reperfusion (IR) or IH as well as the beneficial effects of zinc supplementation in this context. We observed that IR and IH induced a decrease in myocardial and plasma zinc concentrations, respectively. We also highlighted the protective effects of zinc supplementation during reperfusion against the ventricular arrhythmias and myocardial dysfunction induced by IR. Zinc administration during reperfusion also abolished the increase in infarct size induced by chronic IH exposure.Finally, we investigated the effects of zinc depletion in endothelial cells exposed to TPEN, a specific zinc chelator. We observed that TPEN induced a nuclear translocation of HIF-1α and an increase in ET-1 secretion with a resulting increase in endothelial cell migration. Thus, zinc depletion appears to promote activation of the HIF-1-ET-1 axis, known for its deleterious effects upon IH.In summary, chronic IH exposure enhances ventricular arrhythmias and increases infarct size upon myocardial I/R. Sympathetic activation, oxidative stress and alterations of zinc homeostasis appear to be contributing factors. Pharmacological targeting of these alterations should be performed in order to confirm their role as well as to potentially prevent the deleterious cardiovascular consequences of IH and OSA.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / O carcinoma epidermoide de esôfago (CEE) representa 90% dos casos de câncer de esôfago no Brasil. O CEE tem detecção tardia, um comportamento extremamente agressivo e baixa sobrevida, sendo, portanto, um alvo interessante para o estudo dos mecanismos envolvidos em sua carcinogênese, a fim de se identificar possíveis alvos terapêuticos ou marcadores moleculares que ajudem na prática clínica. Mudanças no metabolismo energético da célula tumoral parecem ter papel de destaque na transformação maligna. Sabe-se que células tumorais consomem glicose avidamente produzindo ácido lático, mesmo em condições de normóxia. Dentre os fatores que podem contribuir para o estímulo da glicólise em células tumorais destacam-se as alterações em enzimas da via glicolítica tais como: as piruvato-cinases M1 e M2 (PKM1 e PKM2), a hexocinase II (HKII), isofoma 1 do transportador de glicose, GLUT-1, e o fator de transcrição induzido por hipóxia (HIF1&#945;), responsável pela transcrição das proteínas citadas. O objetivo do estudo é avaliar a relação entre a expressão de HIF1&#945;, HK2, PKM2, PKM1 e GLUT-1 e dados clínico-patológicos no CEE. Para tal, foram avaliados tumores conservados em parafina de 44 pacientes com CEE matriculados no INCA e no Hospital das Clínicas de Porto Alegre. Além disso, foram coletadas amostras de biópsia de esôfago em 67 pacientes sem doença esofágica, que foram submetidos à endoscopia no Hospital Universitário Pedro Ernesto (HUPE). A expressão das proteínas foi avaliada nos tecidos por imuno-histoquímica, enquanto que a expressão do mRNA de GLUT-1 também foi avaliada nas amostras controle. Foi observado que as amostras controle expressam HK2, PKM1, PKM2, HIF1&#945; nas camadas do epitélio esofágico. Já GLUT-1 e Ki-67 são vistos apenas na camada basal. Além disso, a expressão do mRNA de GLUT-1 não teve correlação com fatores etiológicos da doença. Em CEE a expressão de HK2, PKM2 e GLUT-1 foi vista em todos os tumores, já a expressão de HIF1&#945; e PKM1 foi variável. Além disso, observou-se que maior expressão de HIF-1&#945; apresenta correlação com invasão linfonodal e diferenciação, enquanto que a expressão de HK2 tem relação com sobrevida e PKM1 com diferenciação. As correlações clínicas encontradas sugerem que alterações no metabolismo energético é um alvo de estudo interessante para desenvolvimento de marcadores moleculares que auxiliem a prática clínica. / The esophageal squamous cell carcinoma (ESCC) represents 90% of cases of esophageal cancer in Brazil. The ESCC has late diagnosis, highly aggressive behavior and poor survival. ESCC is an interesting target to the study of mechanism involved in its carcinogenesis, in order to identify potential drug targets or biomarkers to help clinical practice. Changes in tumor cell energy metabolism appear to have a prominent role in malignant transformation. Tumor cells consume glucose avidly and produce lactic acid, even under normoxia. Among the factors that may contribute to the stimulation of glycolysis in tumor cells, there are changes in the glycolytic pathway enzymes such as: pyruvate kinase M1 and M2 (PKM2 and PKM1), hexokinase II (HKII), glucose transporter isoform 1, GLUT-1, and transcription factor induced by hypoxia (HIF1&#945;), responsible for the transcription of proteins cited. The goal of the study is to evaluate the relationship between the expression of HIF1&#945;, HK2, PKM2, PKM1 and GLUT-1 and clinicopathological data in ESCC. Biopsy of the esophagus in patients without esophageal disease were collected, who underwent endoscopy at University Hospital Pedro Ernesto (HUPE). Tissue samples were collected from 44 patients with a histologically confirmed diagnosis of ESCC recruted from Hospital Universitário Pedro Ernesto (HUPE-UERJ), and Instituto Nacional de Câncer (INCA). Tissue samples from healthy individuals submitted to endoscopic routine examination, not related to cancer or esophageal disorders, at HUPE-UERJ were also included in this study. The expression of proteins in tissues was evaluated by immunohistochemistry, while mRNA expression of GLUT-1 was also evaluated in the control samples. It was observed that the control samples express HK2, PKM1, PKM2, HIF1&#945; layers of the esophageal epithelium. GLUT-1 and Ki-67 are seen only in the basal layer. Furthermore, expression of GLUT-1 mRNA did not correlate with disease etiological factors. In ESCC expression of HK2, PKM2 and GLUT-1 was seen in all tumors, and the expression of HIF1&#945; and PKM1 was variable. We found that increased expression of HIF-1&#945; correlates with lymph node invasion and differentiation, whereas the expression of HK2 is related to survival, and differentiation with PKM1. The clinical correlations found suggest that alterations in energy metabolism are an interesting subject of study for development of biomarkers that help clinical practice.

Au cours des dernières années, une nouvelle classe de composés anticancéreux à base de ruthénium, appelés RDCs (Ruthenium-Derived Compounds), a été développé pour dépasser les limitations des agents chimiothérapiques contenants du platine. Contrairement à ces derniers, l’activité anticancéreuse des RDCs est en partie indépendante de l'interaction avec l'ADN. L’objectif principal de ma thèse a été ainsi de comprendre les mécanismes moléculaires quiinter viennent dans l’action anticancéreuse et antimétastatique des RDCs au-delà du dommage à l’ADN.J’ai démontré que le RDC11, contrairement au cisplatine, affecte les voies de signalisation de HIF-1 et mTOR, deux voies qui jouent un rôle clé dans le métabolisme cellulaire et qui sont souvent altérées dans les cellules cancéreuses.En parallèle, j’ai effectué un analyse structure/activité pour sélectionner des nouveaux RDCs ayant meilleures propriétés chimiques et pharmacologiques que le RDC11. Cette étude a permis d’identifier deux nouveaux RDCs qui réduisent la croissance tumorale in vivo avec un dosage beaucoup plus faible que le RDC11 et qui induisent la mort des cellules cancéreuses par une surproduction d'espèces réactives de l'oxygène et par l'activation de la caspase8. En conclusion, mes travaux ont conduit à l’identification de nouveaux mécanismes à la base de l’activité anticancéreuse du RDC11 qui pourraient expliquer certaines différences entre le mode d’action du RDC11 et du cisplatine. De plus, ils ont permis de sélectionner deux nouveaux RDCs plus efficaces que le RDC11. Ces résultat sont un impact important pour le développement de nouvelles thérapies anticancéreuses ou antimétastatiques. / In recent years, a new class of anticancer ruthenium-based drugs, called RDCs (Ruthenium-Derived Compounds), has been developed to overcome the limitations of classic platinum chemotherapeutics. Unlike the latter, the anticancer activity of RDCs is in part independent of DNA interaction. Therefore, the main objective of my thesis work was to elucidate the molecular mechanisms involved in RDCs anticancer and antimetastatic activity beyond DNA damage. I demonstrated that RDC11, unlike cisplatin, affects the HIF-1 and mTOR signaling pathways, two pathways that play a key role in cellular metabolism and that are frequently altered in cancer cells. In parallel, I performed a structure/activity analysis to select new RDCs endowed with better chemical and pharmacological properties than RDC11. This study allowed to identify two novel RDCs that reduce tumor growth in vivo at much lower doses than RDC11 and that induce cancer cell death by an overproduction of reactive oxygen species and activation of caspase 8. In conclusion, my work led to the identification of new mechanisms underlying the anticancer activity of RDC11 that could explain some of the differences between the mode of action of RDC11 and cisplatin. In addition, it allowed to select two novel RDCs which are more effective than RDC11. These results have a significant impact on the development of new anticancer or antimetastatic therapies.