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.

No description available.

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.

Chlamydien durch die Modulation spezifischer Wirtszellproteine verschiedene Wege der Apoptose verhindern können. Mcl-1 und cIAP-2 erwiesen sich als bedeutende Faktoren, die durch die Infektion hochreguliert und absolut notwendig für die Inhibierung der Apoptose durch Chlamydien waren. Hochregulation der Mcl-1 Expression führte zu einem Block im apoptotischen Weg oberhalb der Mitochondrien. cIAP-2 zusammen mit anderen Inhibitor of Apoptosis Proteins (IAP) verhinderten die Aktivierung von Caspase-3, denfinalen Schritt in der apoptotischen Kaskade. Weiterhin wurde beobachtet, dass die Aktivierung des MAPKinase-Signalweges durch die Infektion wichtig war für die Hochregulierung von Mcl-1 und cIAP-2. Ein Hochdurchsatz-Screen wurde durchgeführt, um andere Wirtszellfaktoren, die für die Apoptoseinhibierung durch die Chlamydien verantwortlich sind, zu identifizieren. Neben Mcl-1 waren die identifizierten Faktoren hauptsächlich Mitglieder des MAPKinase-Signalweges. Dabei wurde deren Rolle für die Apoptoseresistenz bestätigt. Eine weiterführende Analyse der im Screen ermittelten Faktoren identifizierte eine Funktion von HIF-1a bei der Modulation der Expression anti-apoptotischer Faktoren während der Infektion. Es wurde beobachtet, dass HIF-1a stabilisiert und zum Nukleus transloziert wird. Es ist bekannt, dass HIF-1a HIF-1a im Nukleus binden kann, um den funktionalen Transkriptionsfaktor HIF zu bilden. Dieser reguliert die Expression verschiedener Überlebensfaktoren, unter anderem Mcl-1. HIF-1a Knockdown inhibierte die Chlamydien-induzierte Hochregulation von Mcl-1 mRNA-Expression. / chlamydial infection blocked the apoptotic pathway at multiple levels by modulation of specific host cell proteins. Mcl-1 and cIAP-2 were two most prominent factors that were up-regulated during the infection, and absolutely required for apoptosis inhibition. Increased expression of Mcl-1 led to a block in the apoptotic pathway upstream of the mitochondria. cIAP-2, together with other inhibitor of apoptosis proteins (IAPs), blocked the activation of caspase-3 at the final step of the apoptosis cascade. Further, it was observed that the activation of the MAPK pathways during infection was needed for the up-regulation of Mcl-1 and cIAP-2. A high throughput RNAi screen was performed to identify other host factors required for the apoptosis resistance during the infection. Besides Mcl-1, the targets from the screen prominently included members of the MAPK pathways, confirming their role in the apoptosis resistance. Pathway analysis of the targets identified the role of HIF-1a in modulating the expression of the anti-apoptotic factors during infection. It was observed that during infection, HIF-1a gets stabilized and translocates to the nucleus. It is known that HIF-1a can bind to HIF-1a in the nucleus to form the functional transcription factor HIF, which can regulate the expression of survival factors like Mcl-1. This was seen to be the case, because knock down of HIF-1a abrogated the infection induced up-regulation of Mcl-1 at the mRNA levels.

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-1α 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-1α 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-1α 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-1α 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-1α 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-1α induction linked to the expression of pro- inflammatory proteins and TB, indicating the share and/or co-segregation of the genes involved in AIR.

Les cancers du foie et du pancréas sont classés parmi les cancers les plus fréquents et agressifs à travers le monde et présentent une résistance à la chimiothérapie. L'efficacité des médicaments anticancéreux est affectée par les activités des enzymes métaboliques, transporteurs membranaires et par l’environnement tumoral. Le but de notre thèse est 1) de développer un modèle cellulaire hépatique et caractériser les mécanismes sous-jacents de la modulation de l’expression et de la fonctionnalité des transporteurs membranaires et des enzymes clés qui régissent le métabolisme des médicaments et 2) d’évaluer in vitro, dans différents modèles cellulaires (hépatique et pancréatique) en 2D et 3D, l’effet apoptotique de médicaments anticancéreux associés à des polyphénols en vue d’optimiser leur activité. Dans une première partie, nous avons mis en place une nouvelle lignée cellulaire hépatique humaine dérivée des HepG2, stable, exprimant suffisamment et significativement les enzymes CYP450 et les transporteurs hépatiques (MRP2, MDR1 et OATP1B1). Ce modèle pourrait être un outil de choix pour des études précliniques de métabolisme et de prédiction d’hépatotoxicité. Dans une deuxième partie, nous avons pu voir que les cellules pancréatiques et hépatiques dans un environnement 3D sont plus prédictives d’une tumeur in vivo et peuvent être un modèle de choix pour des études pharmacologiques de criblage de nouveaux médicaments anticancéreux ou des stratégies de combinaisons de molécules (avec des PP). Ainsi, nous avons montré que la quercétine, dans les cellules 3D, était capable d’augmenter l’activité de la gemcitabine et de la doxorubicine, en augmentant le taux des cellules mortes jusqu’à 60 %, par modulation des protéines MDR1 et par diminution significative du facteur HIF-1 alpha dans les cellules cancéreuses. En conclusion, les polyphénols peuvent être des molécules d’intérêt en combinaison avec des médicaments anticancéreux pour diminuer la résistance à ces traitements et servir d’outil pharmacologique pour mieux comprendre les mécanismes de résistance des cellules tumorales. / Liver and pancreatic cancers are among the most common and aggressive cancers worldwide that are resistant to chemotherapy. The efficacy of anticancer drugs is affected by the activities of metabolic enzymes, transporters and the tumor environment. The aim of my thesis was based on to main objectives: 1) developement of a hepatic cellular model and characterize the underlying mechanisms of modulation of the expression and functionality of transporters and key enzymes involved in the regulation of drug metabolism 2) study the effect of new strategies in vitro by combining anti-cancer drugs with polyphenols in these processes in order to optimize their activities on different cellular models (hepatic and pancreatic) in 2D and 3D cultures. Our results showed that we have developed a new human hepatic cell line derived from HepG2 cells. The novel cell line is a good in vitro model with a capacity of predicting hepatotoxicity of novel drugs with significant differences for chromosomes 5, 17 and 20 and high expression level of CYP450 and transporters (MRP2, MDR1 and OATP1B1). Secondly, our results indicate that the combination of anticancer drugs and polyphenols increased the rate of apoptosis in cancer cells by up regulation of the expression levels of cleaved caspase-3 and the regulator of apoptosis p53. Moreover, our results demonstrated that polyphenols inhibit the efflux activity of MDR1. In addition, our results indicate that the combination of anti-cancer drugs and quercetin down regulated the expression of HIF-1α and increased the expression levels of the cleaved caspase-3 and p53 on human pancreatic and liver cell line cultured in 3D culture. In conclusion, polyphenols may be promising agents for novel combination therapy since they potentialize the cytotoxic activity of anticancer drugs to eradicate cancer and therefore the cellular resistance.

No description available.

Les tumeurs solides sont souvent confrontées à un environnement déficient en oxygène, dit hypoxique. Hypoxia-Inducible Factor 1 (HIF1) est le facteur de transcription clé de l’adaptation cellulaire à l’hypoxie, régulant de nombreux gènes impliqués dans l’angiogenèse, le métabolisme cellulaire ou la régulation du pH. Ma thèse s’articule en trois axes autour de HIF1 et de la reprogrammation métabolique hypoxique. J’ai d’abord étudié Factor-Inhibiting HIF1 (FIH), l’un des deux senseurs d’oxygène régulant HIF1. Nous avons montré que FIH est essentiel dans le développement tumoral en inhibant à la fois l’activité transcriptionnelle de HIF1 et la voie p53-p21. J’ai ensuite étudié le « shift » du métabolisme cellulaire vers la glycolyse induit par HIF1, générant une addiction pour le glucose. Nos travaux ont montré que paradoxalement, les cellules hypoxiques synthétisent du glycogène via HIF1 constituant ainsi une réserve de glucose intracellulaire. Le glycogène confère alors une résistance accrue des cellules tumorales suite à une carence en glucose. Enfin, j’ai pu montrer que l’AMPK, « gardien de la balance énergétique », n’est pas nécessaire au maintien d’un niveau viable d’ATP suite à l’inhibition de la glycolyse, via le blocage de l’export de lactate, mais exerce, un effet protecteur en absence de glucose. Cependant, l’inhibition conjointe du transporteur de lactate, MCT4, et de l’AMPK réduit fortement le développement tumoral dans un modèle de xénogreffes chez la souris, suggérant un rôle crucial de ces deux acteurs dans ce contexte. L’ensemble de ces travaux a permis d’identifier plusieurs cibles potentielles impliquées dans la plasticité métabolique en hypoxie. / Cells of solid tumors are often exposed to an environment deficient in oxygen, i.e. hypoxic. The Hypoxia-Inducible Factor-1 (HIF-1) is the major transcription factor involved in cellular adaptation to hypoxia. HIF-1 regulates a wide array of genes involved in angiogenesis, cellular metabolism or pH regulation. My thesis is organized into three axes around HIF-1 and metabolic reprogramming in hypoxia. I first studied Factor-Inhibiting HIF-1 (FIH), one of two oxygen sensors regulating HIF-1. We showed that FIH is essential for tumor development through inhibition of the HIF-1 transcriptional activity as well as through the suppression of the p53-p21 axis. I then studied the HIF-1-induced « shift » in cellular metabolism toward glycolysis, which generates a type of “glucose addiction”. We showed that paradoxically, tumor cells store glycogen in hypoxia through a HIF-1 dependant mechanism. Glycogen served as a reservoir of intracellular glucose, which allows hypoxic cells to survive periods of glucose starvation. Finally, I studied AMPK «the guardian of energy », and showed that surprisingly, this kinase is not necessary in maintaining a viable level of ATP when glycolysis is inhibited (by blockade of lactate export). However, as expected, AMPK protected cells during glucose starvation. Moreover, combined inhibition of the lactate transporter MCT4 and of AMPK reduced dramatically tumor development in a xenograft model, suggesting a crucial role for these two actors in the context of growth of tumor cells in a hostile environment. Taken together these results identified several potential drug targets involved in the metabolic plasticity of hypoxic cells.

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.

Le syndrome d’apnées obstructives du sommeil est un problème de santé publique affectant plus de 5 % de la population, se caractérisant par des obstructions répétées des voies aériennes durant la nuit. L’hypoxie intermittente (HI) qui en résulte, induit des complications cardiovasculaires (hypertension, athérosclérose, insuffisance cardiaque). Le but de cette thèse est d’explorer le remodelage cardiovasculaire induit par l’HI et de comprendre le rôle de HIF-1 et de l’endothéline (ET-1) dans ces modifications. Pour cela nous avons exposé à 14 jours d’HI des souris HIF1α+/- ou traitées avec un antagoniste des récepteurs à l’ET-1. Chez les souris contrôles, nous avons observé un épaississement de la paroi aortique, une inflammation systémique et locale dans l’aorte ainsi qu’une hypertrophie du ventricule droit qui sont absents chez les souris HIF1α+/- ou traitées au bosentan. La délétion de HIF1a prévient également l’augmentation de contractilité observée dans le ventricule gauche après HI. En conclusion HIF-1 et ET-1 semblent fortement impliqués dans le remodelage vasculaire et myocardique induit par l’HI. / Obstructive sleep apnea is a public health problem affecting more than 5% of the population, characterized by repeated airway obstructions during sleep. The resulting intermittent hypoxia (IH) induces cardiovascular complications (hypertension, atherosclerosis, heart failure). The aim of this thesis is to characterize the cardiovascular remodeling induced IH and understand the role of HIF-1 and endothelin (ET-1) in these alterations. For this, we exposed to IH for 14 days HIF1α+/- mice or mice treated with an ET-1 receptor antagonist. Control mice developed thickening of the aortic wall, systemic and local inflammation in the aorta and right ventricular hypertrophy that were absent in HIF1α+/- or bosentan-treated mice. HIF1a deletion also prevents the increase in left ventricular contractility observed after HI. In conclusion, HIF-1 and ET-1 appear to be strongly involved in the vascular and myocardial remodeling induced by HI.