L'hypoxie intermittente (HI), induite par les apnées du sommeil, conduit à des altérations de la sensibilité à l'insuline et de l'homéostasie glucidique mais les mécanismes impliqués restent mal connus. L'objectif de ce travail était d'étudier les effets et les mécanismes sous jacents d'une exposition chronique à l'HI sur l'homéostasie glucidique. L'HI induit une résistance à l'insuline à la fois systémique et tissulaire, ainsi qu'une amélioration de la tolérance au glucose associée à une activation de l'AMPK musculaire. L'HI cause également des altérations du foie et du tissu adipeux associées à un changement du pattern d'expression des gènes dans ces tissus et à un risque accru de développement de pathologies vasculaires comme l'athérosclérose. Enfin, la délétion de PHD1, une des protéines régulatrices de HIF-1, entraîne une résistance à l'insuline associée une stéatose hépatique, faisant de HIF-1 une cible potentielle impliquée dans les altérations metaboliques induites par l'HI. / Intermittent hypoxia (IH), induced by sleep apnea, leads to alterations in insulin sensitivity and glucose homeostasis but the mechanisms involved remains poorly understood. The objective of this work was to study the effects and the underlying mechanisms of chronic exposure to IH on glucose homeostasis. IH induces both systemic and tissue-specific insulin resistance , as well as improved glucose tolerance associated with an activation of muscle AMPK. IH also causes a change in the pattern of gene expression in liver and adipose tissue and an increased risk of vascular pathologies such as atherosclerosis development. Finally, the deletion of PHD1, a regulatory protein of HIF-1, leads to insulin resistance associated with hepatic steatosis, making HIF-1 a possible target involved in the metabolic changes induced by IH.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-09T12:38:26Z No. of bitstreams: 2 Dissertação - Maisa Ribeiro - 2015.pdf: 1621645 bytes, checksum: 2b3a1e65f1e53b4a1264cf3febb2c630 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-09T12:41:15Z (GMT) No. of bitstreams: 2 Dissertação - Maisa Ribeiro - 2015.pdf: 1621645 bytes, checksum: 2b3a1e65f1e53b4a1264cf3febb2c630 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-09T12:41:15Z (GMT). No. of bitstreams: 2 Dissertação - Maisa Ribeiro - 2015.pdf: 1621645 bytes, checksum: 2b3a1e65f1e53b4a1264cf3febb2c630 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / In the lesions carcinomatous, low oxygen tension plays a crucial step in the self-renewal, metastatic potential, and therapy resistance of cancers. To adapt to the hypoxic microenvironment, neoplastic cells activate hypoxia-induced factor-1 alpha (HIF-1 alpha), which may mediates invasion and metastasis. In addition, the human THY-1 (CD90) cell surface protein mediates cell adhesion expressed in stem cells, and seens to drive tumor development in some malignant tumors. The present study investigates HIF-1 alpha (n=98) and CD90 (n=97) expression in oral squamous cell carcinoma (OSCC) and metastatic lymph nodes (n=24), the intratumoral region and the invasive front, by immunohistochemistry. Furthermore, clinicopathological data revised from the medical records. In superficial OSCCs, most tumor cells overexpressed HIF-1 alpha, whereas was restricted in the intratumoral region in invasive conventional SCCs. Interestingly, metastatic lymph nodes (91.7%, p=0.001), and intratumoral regions of its corresponding primary tumors (83.3%, p<0.001) were invaded by HIF-1 alpha-positive neoplastic cells. Overall survival was poor in patients with nodal involvement. CD90 was expressed mostly in microvessels and granulocyte cells similar to mast cells. These cells expressed CD90 mostly in the peritumoral region of invasive SCC (p<0.001). Microvessels CD90 positive were higher in the intratumoral region (p=0.032). Interesting, mast cell and microvessels positively correlated in OSCC (p=0.006; r²=0.077). In conclusion, hypoxic environment may facilitate regional metastasis and serve as a potential diagnostic and prognostic marker in OSCC primary tumors. Microvessels CD90 positive seems to promote tumor growth except in BSCC. Mast cell may occur via CD90 for tumor progression. / Nas lesões carcinomatosas a baixa tensão de oxigênio desempenha um passo crucial para a auto-renovação, potencial metastático, e resistência à terapia no câncer. Para se adaptar ao ambiente hipóxico, células neoplásicas ativam o fator induzido por hipóxia-1 alfa (HIF-1 alfa), que pode facilitar a invasão e metástase. Além disso, o THY-1 (CD90) humano, uma proteína de superfície celular expressa em células estaminais, medeia a adesão celular, e parece promover o desenvolvimento em alguns tumores malignos. O presente estudo analisou a expressão das proteínas HIF-1 alfa (n = 98) e CD90 (n = 97) no carcinoma espinocelular de boca (CEC de boca) e linfonodos metastáticos (n=24), na região intratumoral e no fronte de invasão, por meio de imunoistoquímica. Além disso os dados clinicopatológicos foram revisados a partir dos prontuários médicos e a sobrevida foi analisada. No CEC microinvasivo, a maioria das células tumorais apresentaram superexpressão do HIF-1 alfa, enquanto que no CEC invasivo a superexpressão foi restrita na região intratumoral. Verificou-se que em linfonodos metastáticos (91,7%, p = 0,001), e regiões intratumorais dos seus tumores primários correspondentes (83,3%, p <0,001) houve forte expressão do HIF-1 alfa em células neoplásicas. A sobrevida global foi pior em pacientes com metástase regional. A proteína CD90 foi expressa principalmente em microvasos e células de granulócitos semelhantes aos mastócitos. Estas células expressaram CD90 principalmente na região fronte de invasão do CEC invasivo (p<0,001). A média de microvasos CD90 positivo foi maior na região intratumoral (p=0,032). Interessantemente, mastócitos e microvasos foram positivamente correlacionados no CEC de boca (p=0,006; r²=0,077). Em conclusão, o ambiente hipóxico pode facilitar metástases regionais e funcionar como um potencial marcador de diagnóstico e prognóstico em tumores primários do CEC de boca. Os microvasos CD90 positivo parecem promover o crescimento do tumor, exceto no carcinoma escamoso basalóide (CEB). Os mastócitos ativados via CD90 podem contribuir com a progressão do tumor.

Alterações no mecanismo de transporte de oxigênio (O2) frequentes em inflamações, infecções, tumores, transplantes e isquemia, levam a hipóxia tecidual. Espécies reativas do O2 são produzidas e citocinas inflamatórias são liberadas engatilhando uma série de eventos, os quais são amplificados após a restituição do fluxo sanguíneo resultando em inflamação sistêmica. No presente estudo, caracterizamos a regulação da Resposta Inflamatória Aguda (AIR) após indução de isquemia e reperfusão intestinal (I/Ri) e a participação do HIF-1&alpha; neste fenótipo. Camundongos selecionados para alta (AIRmax) e baixa (AIRmin) AIR foram submetidos a I/Ri e avaliados em diferentes períodos de reperfusão (0, 1, 4 e 24h). Nossos resultados demonstraram maior sensibilidade da linhagem AIRmax frente a I/Ri, confirmada por: 1) maior mobilização de neutrófilos para circulação periférica; 2) maior adesão celular e aumento da migração granulocítica no intestino e pulmão; 3) aumento da expressão de genes de citocinas e daqueles expressos em hipóxia (Tnfa, Il1, Il6 e Hif1a); 4) Translocação Bacteriana (TB); 5) maior expressão pulmonar da proteína HIF-1&alpha; e de proteínas envolvidas em processos inflamatórios tais como S100A9, Anexina 1, Profilina 1, Tropomiosina. Por outro lado, a linhagem AIRmin foi considerada pouco responsiva aos efeitos da I/Ri. Diante do exposto, nós concluímos que a sensibilidade dos camundongos AIRmax à injuria após indução de IRi está associada ao agravamento da inflamação sistemica, a qual foi determinada pela indução de HIF-1&alpha; atrelada à expressão de proteínas pró- inflamatórias e TB, indicando o compartilhamento ou a co- segregação entre os genes envolvidos na AIR e na hipóxia. / Changes in oxygen transport mechanism (O2) frequent in inflammation, infection, tumors, transplantation and ischemia, lead to tissue hypoxia. Reactive species of O2 are produced and inflammatory cytokines are released triggering a series of events, which are amplified after blood flow refund resulting in systemic inflammation. In the present study, we characterized the regulation of Acute Inflammatory Response (AIR) after intestinal ischemia and reperfusion (I/Ri) induction and the involvement of HIF-1&alpha; in this phenotype. Mice selected for high (AIRmax) and low (AIRmin) AIR were subjected to I/Ri and evaluated in different periods of reperfusion (0, 1, 4 and 24h). Our results show sensitivity of AIRmax front line I/Ri, confirmed by: 1) higher neutrophils mobilization to peripheral circulation; 2) increase in cell adhesion and granulocyte migration in lung and intestine; 3) higher expression of cytokine genes and those expressed in hypoxia (TNFa, IL-1, IL-6 and HIF1a); 4) Bacterial Translocation (BT), 5) increase in HIF-1&alpha; pulmonary protein expression and those involved in inflammatory processes such as S100A9, Annexin 1, profilin 1 Tropomyosin. On the other hand, the AIRmin line was considered unresponsive to effects of I/Ri. We concluded that the I/Ri sensitivity of the AIRmax mice were associated with worsening of systemic inflammation, which was determined by HIF-1&alpha; induction linked to the expression of pro- inflammatory proteins and TB, indicating the share and/or co-segregation of the genes involved in AIR.

Objetivos: Determinar a expressão imunohistoquímica do fator indutor de hipóxia 1-alfa (HIF-1-alfa) e suas variáveis associadas em pacientes com câncer de mama localmente avançado. Pacientes e método: Vinte e sete mulheres foram biopsiadas para diagnóstico histopatológico do carcinoma mamário e submetidas a tratamento quimioterápico pré-cirúrgico. Analisou-se a associação do HIF-1-alfa com idade, tamanho tumoral, grau histológico, estadio clínico, status hormonal e axilar, resposta clínica e patológica após tratamento quimioterápico, expressão do receptor de estrogênio, progesterona e cerbB2. Resultados: A expressão de HIF-1-alfa foi presente em 66,7% das pacientes. O único fator associado à sua presença foi o status axilar positivo (p=0,02), tendo permanecido durante a análise univariada. As demais variáveis não apresentaram associação estatisticamente significante. Conclusão: Existe uma associação estatisticamente significante entre o acometimento linfonodal e a presença de HIF-1-alfa em pacientes com câncer de mama localmente avançado. / Objectives: To assess the expression of HIF-1 and its associated variables with locally advanced breast cancer (LABC) patients. Methods: Twenty-seven women were submitted to incisional biopsy for histopathological diagnosis of breast carcinoma and undertaken to neoadjuvant chemotherapy (NACT). It was studied the association of HIF-1 with age, tumoral size, histological grade, clinical stage, hormonal and axillary status, clinical and pathological response after NACT, expression of estrogen and progesterone receptors, as well as the presence of cerbB2 antigen. Results: HIF-1-alpha expression was found in 66.7% of patients. Only axillary status was the associated factor with its presence (p=0.02), and remained after univariate analysis. The others did not present any significant statistically difference. Conclusion: There is a significant statistically association between axillary status and HIF-1-alpha expression in LABC patients.

Les traitements des cancers des VADS reposent sur la chirurgie, la radiothérapie, et la chimiothérapie. Malgré ces traitements, la survie globale des patients à 5 ans est de l’ordre de 50%. Les causes d’échec thérapeutique sont dues au profil de résistance des tumeurs. Le ciblage de l’axe EGFR/mTOR/HIF-1 par une combinaison de rapalogues et d’anti-EGFR a montré son efficacité sur certaines tumeurs solides. L’objet de ce travail de thèse a été de caractériser l’impact d’une combinaison de drogues à faibles doses sur des lignées cellulaires des VADS au moyen d’une approche pharmacologique et moléculaires. Nos résultats montrent que la combinaison de la rapamycine (5nM) au cetuximab (2,5μg/ml) diminue la survie clonogénique des cellules et permet une inhibition du facteur de transcription HIF-1α. Cette combinaison de drogue améliore l’efficacité de la radiothérapie. En revanche, l’induction de HIF-2a induite par le traitement provoque la résistance des cellules aux traitements par, et une rechute rapide des tumeurs in vivo. L’inhibition de HIF-2 permet une inhibition de la survie cellulaire d’environ 100% dans un modèle résistant. / Management of HNSCC relies on surgery, radiotherapy, and chemotherapy. Despite these treatments, the 5 years overall survival of patient is lower than 50%. Main causes of therapeutic failure are due to the profile of resistance of tumors. The efficacy of a combination rapalogues and anti-EGFR therapies in targeting the EGFR/mTOR/HIF-1 axis in solid tumors was shown previously. In this PhD work, we have evaluated the impact of a low-dose drug combination on head and neck cancer cells lines with a pharmacological and molecular approach. We show that the combination of rapamycine (5nM) and cetuximab (2,5μg/ml) efficiently inhibits the HIF-1 transcription factor and impairs cell clonogenic survival. The efficacy of radiation therapy is improved by this drug combination. However, cell resistance to the treatment is acquired via the induction of HIF-2 in our resistant model cell line. This induction is associated with more tumor relapse in tumors mice xenograft. The inhibition of HIF-2 achieves a dramatic drop of cell clonogenic survival to < 1%.

No description available.

Traumatic brain injury (TBI) is becoming a national epidemic, as it accounts for 1.5 million cases each year. This disorder affects primarily the young population and elderly. Currently, there is no treatment for TBI, which means that ~2% of the U.S. population is currently living with prolonged neurological damage and dysfunction. Recently, there have been many studies showing that TBI negatively impacts mitochondrial function. It has been proposed that in order to save the cell from destruction mitochondrial function must be preserved. The ketogenic diet, originally designed to mimic fasting physiology, is effective in treating epilepsy. Therefore, we have used fasting as a post injury treatment and attempted to elucidate its underlying mechanism. 24 hours of fasting after a moderate TBI increased tissue sparing, cognitive recovery, improved mitochondrial function, and decreased mitochondrial biomarkers of injury. Fasting results in hypoglycemia, the production of ketones, and the upregulation of free fatty acids (FFA). As such, we investigated the neuroprotective effect of hypoglycemia in the absence of fasting through insulin administration. Insulin administration was not neuroprotective and increased mortality in some treatment groups. However, ketone administration resulted in increased tissue sparing. Also, reduced reactive oxygen species (ROS) production, increased the efficiency of NADH utilization, and increased respiratory function. FFAs and uncoupling proteins (UCP) have been implicated in an endogenously regulated anti-ROS mechanism. FFAs of various chain lengths and saturation were screened for their ability to activate UCP mediated mitochondrial respiration and attenuate ROS production. We also measured FFA levels in serum, brain, and CSF after a 24 hour fast. We also used UCP2 transgenic overexpressing and knockout mice in our CCI injury model, which showed UCP2 overexpression increased tissue sparing, however UCP2 deficient mice did not show a decrease in tissue sparing, compared with their wild type littermates. Together our results indicate that post injury initiated fasting is neuroprotective and that this treatment is able to preserve mitochondrial function. Our work also indicates ketones and UCPs may be working together to preserve mitochondrial and cellular function in a concerted mechanism, and that this cooperative system is the underlying mechanism of fasting induced neuroprotection.

Myeloid-derived suppressor cells (MDSC) are a major component of the immune suppressive network that develops during cancer. MDSC down-regulate immune surveillance and antitumor immunity and facilitate tumor growth. The ability of MDSC to suppress T cell responses has been documented; however the mechanisms regulating this suppression remain to be understood. This work proposes a biological dichotomy of MDSC regulated by the tumor microenvironment. In peripheral lymphoid organs MDSC cause T-cell non-responsiveness that is antigen-specific. These MDSC have increased expression of NOX2, enabling them to produce large amounts of reactive oxygen species. Since the transcription factor STAT3 is substantially activated in MDSC, its potential role in upregulation of NOX2 expression was investigated. Over-expression of a constitutively active form of STAT3 increases expression of NOX2 subunits, whereas attenuation of STAT3 activity leads to decreased expression of NOX2. The significance of NOX2 in ROS generation is demonstrated in mice devoid of NOX2 function; NOX2- deficient MDSC are unable to inhibit antigen-induced activation of T cells. In contrast, MDSC within the tumor microenvironment have a diminished potential to generate ROS but acquire expression of arginase and inducible nitric oxide synthase, enzymes plicated in T cell non-responsiveness. Upregulation of these enzymes results in MDSC ability to inhibit lymphocyte response in absence of antigen presentation. The tumor microenvironment also promotes the differentiation of MDSC to tumor associated macrophages. Hypoxia is an exclusive feature to the tumor microenvironment and we investigated its involvement in the properties of MDSC at the tumor site. Exposure of spleen MDSC to hypoxia converts MDSC to non-specific suppressors and induces a preferential differentiation to macrophages. Stabilization of HIF-1!, a transcription factor activated by hypoxia, induces similar changes in MDCS as hypoxic exposure. Finally, ablation of HIF-1! prevents MDSC from acquiring factors that enable the suppression of T cells in absence of antigen. These findings help to expand our understanding of the biology of MDSC and suggest a regulatory pathway of myeloid cell function exclusive to the tumor microenvironment. They may also open new opportunities for therapeutic regulation as we now should take into consideration how systemic location affects the function of MDSC.

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.