Angiogenesis is known to be induced and maintained in tumors by the constant expression of the hypoxia inducible factor 1 alpha (HIF-1α) and human vascular endothelial growth factor (VEGF). In fact, tumor recurrence, aggressive metastatic legions and patient mortality rates are known to be positively correlated with overexpression of these two proteins. The HIF-1α and VEGF promoters contain a polypurine/polypyrimidine (pPu/pPy) tract, which are known to play critical roles in their transcriptional regulation, and are structurally dynamic where they can undergo a conformational transition between B-DNA, single stranded DNA and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We hypothesize that the i-motif and G-quadruplex structures can form within the pPu/pPy tracts of the HIF-1α and VEGF proximal promoters, which play important roles in the transcriptional regulation of these genes by acting as scaffolds for alternative transcription factor binding sites. The purpose of this dissertation was to elucidate the transcriptional regulation of the HIF-1α and VEGF genes through the atypical DNA structures that form within the pPu/pPy tracts of their proximal promoters. We investigated the interaction of the C-rich and guanine-rich (G-rich) strands of both of these tracts with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of HIF-1α and VEGF. In this dissertation, we demonstrate that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the HIF-1α and VEGF promoters. Specifically, the small interfering RNA-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, and the opposite effect was seen when the transcription factors were overexpressed, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within pPu/pPy tracts will provide new insight into mechanisms of transcriptional regulation of the HIF-1α and VEGF gene.

No description available.

Le pancréas est une glande mixte à composantes endocrine et exocrine. Le tissu endocrine, essentiellement composé de cellules bêta productrices d’insuline, joue un rôle prépondérant dans le maintient de l’homéostasie glucidique. La perte qualitative ou quantitative des cellules bêta conduit au développement de pathologies caractérisées par une hyperglycémie chronique et connues sous le nom de diabète. Le développement de stratégies thérapeutiques innovantes, thérapie cellulaire ou médecine régénérative, pour guérir le diabète repose sur une connaissance précise des mécanismes développementaux impliqués dans la formation des cellules bêta. Ainsi, au delà de l’intérêt cognitif, il est primordial de comprendre au mieux les évènements cellulaires et moléculaires qui régissent l’organogénèse pancréatique pour offrir des thérapies alternatives. Le développement embryonnaire s’effectue dans un environnement où la pression partielle en oxygène (pO2) est faible. Par ailleurs, une étude menée au sein du laboratoire a montré que la pO2 influence la différenciation des cellules bêta pancréatique in vitro. En effet, lorsque des pancréas embryonnaires sont cultivés sur filtre en hypoxie (pO2=3%), le développement des cellules bêta est drastiquement diminué comparativement à une condition de 21% d’O2. Le facteur de transcription HIF-1 (Hypoxia Inducible Factor-1), composé d’une sous-unité alpha sensible au niveau d’oxygène et d’une sous-unité bêta constitutivement présente, permet à la cellule de s’adapter à un environnement pauvre en O2, notamment en favorisant la formation de nouveaux vaisseaux sanguins au cours d’un processus appelé angiogénèse. L’objectif de ma thèse était d’étudier le rôle de HIF-1alpha au cours du développement embryonnaire du pancréas in vivo. Pour cela, nous avons utilisé des lignées murines génétiquement modifiées permettant de stabiliser constitutivement la protéine HIF-1alpha dans l’épithélium pancréatique. En utilisant ce modèle murin, nous avons montré que la différenciation endocrine et le développement des cellules bêta est altéré dans les pancréas mutants comparativement aux contrôles. Par ailleurs, en utilisant une approche pharmacologique in vitro conduisant à l’ablation des cellules endothéliales du pancréas, nous avons pu restaurer une différenciation endocrine comparable aux contrôles. Ce travail a permis d’éclaircir le rôle de HIF-1 et de la vascularisation au cours du développement embryonnaire du pancréas. Nos résultats indiquent que ces paramètres doivent être pris en compte pour améliorer les protocoles actuels permettant de générer des cellules bêta in vitro. / The pancreas is an endoderm-derived organ which is composed by both an exocrine and an endocrine compartment. Within the endocrine tissu, insulin-producing beta-cells are essential for the regulation of glucose homeostasis. The loss of beta-cells can lead to pathologies such as diabetes. Currently, people suffuring from diabetes can be treated but not permanently cured. The development of innovating therapeutical approaches, like cellular therapy or regenerative medecine, relies on the precise knowledge of the mechanisms regulating the ontogenesis of pancreatic beta-cells. Different studies have linked proper embryonic development and low-oxygen tension (pO2). Specifically, when embryonic pancreases are cultured in vitro under a hypoxic condition (pO2=3%), the beta-cells development is impaired compared to a normoxic condition (pO2=21%). Different pathways are involved in the cell adaptation to hypoxia, such as the ubiquitous Hypoxia Inducible Factor 1-alpha (HIF-1alpha). The aim of my PhD project was to elucidate the role of HIF-1alpha during pancreatic development in vivo. To do so, we used genetically modified mice allowing the constitutive stabilization of HIF-1alpha in pancreatic epithelial cells. We have shown that HIF-1alpha stabilization leads to a reduction of endocrine differentiation and beta-cells development. Moreover, using a pharmacological approach in vitro consisting in deleting endothelial cells, we rescued the endocrine differentiation in the mutant pancreases. In conclusion, my data demonstrated the negative influence of both HIF-1 and endothelial cells on endocrine differentiation processes.

No description available.

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.

<p>One of today’s most widely spread health problems is urinary incontinence, affecting 60-80% of the US population from age 15 and up. Treatment based on the possibility to implant a scaffold seeded with the patients’ own urine-derived stem cells, hUSC, to regenerate the damaged muscle tissue, would prove effective. A main challenge in regenerating new tissue from cell-seeded scaffolds is the limited cell survival due to insufficient oxygen diffusion to the center of the scaffold. Ways of enhancing cell survival, and thereby, proliferation and differentiation, is by hypoxic preconditioning of the cells or implantation in an oxygen-release material. Hypoxic preconditioning has shown to enhance proliferation as well as the expression of vascular endothelial growth factor, VEGF, in for example human bone marrow derived stem cells, hBMSC. VEGF is involved in the establishment of vasculature structures and an upregulation of its expression may therefore help promote quicker angeogenisis, increasing the oxygen supply and the cell survival. Oxygen-release materials have shown to enhance cell survival and growth both <em>in vitro</em> and <em>in vivo</em>.<em></em></p><p>This study aims to investigate the effect of hypoxia on hUSC, during 9 days of hypoxic culturing (2.0% ± 0.1% O₂) with and without oxygen-release material (PLGA 75:25 with 5 w% CPO) <em>in vitro</em>. hBMSC, and human smooth muscle cells, hSMC, have been used as control groups. Cell proliferation, morphology, differentiation, production of VEGF, and expression of hypoxia inducible factor HIF-1α have been studied.</p><p>According to the results, combining hypoxic preconditioning of hUSC with implantation in oxygen-release material could be an effective way to regenerate muscular tissue. Hypoxic preconditioning enhanced cell proliferation, production of VEGF, and HIF-1α expression. The increase of VEGF and HIF-1α would promote vascularization when implanted. The oxygen-release material showed possible promotion of cell differentiation, which would augment the hUSCs’ myogenic differentiation, while supplying oxygen until the tissue’s vascular structure has been established.</p>

One of today’s most widely spread health problems is urinary incontinence, affecting 60-80% of the US population from age 15 and up. Treatment based on the possibility to implant a scaffold seeded with the patients’ own urine-derived stem cells, hUSC, to regenerate the damaged muscle tissue, would prove effective. A main challenge in regenerating new tissue from cell-seeded scaffolds is the limited cell survival due to insufficient oxygen diffusion to the center of the scaffold. Ways of enhancing cell survival, and thereby, proliferation and differentiation, is by hypoxic preconditioning of the cells or implantation in an oxygen-release material. Hypoxic preconditioning has shown to enhance proliferation as well as the expression of vascular endothelial growth factor, VEGF, in for example human bone marrow derived stem cells, hBMSC. VEGF is involved in the establishment of vasculature structures and an upregulation of its expression may therefore help promote quicker angeogenisis, increasing the oxygen supply and the cell survival. Oxygen-release materials have shown to enhance cell survival and growth both in vitro and in vivo. This study aims to investigate the effect of hypoxia on hUSC, during 9 days of hypoxic culturing (2.0% ± 0.1% O2) with and without oxygen-release material (PLGA 75:25 with 5 w% CPO) in vitro. hBMSC, and human smooth muscle cells, hSMC, have been used as control groups. Cell proliferation, morphology, differentiation, production of VEGF, and expression of hypoxia inducible factor HIF-1α have been studied. According to the results, combining hypoxic preconditioning of hUSC with implantation in oxygen-release material could be an effective way to regenerate muscular tissue. Hypoxic preconditioning enhanced cell proliferation, production of VEGF, and HIF-1α expression. The increase of VEGF and HIF-1α would promote vascularization when implanted. The oxygen-release material showed possible promotion of cell differentiation, which would augment the hUSCs’ myogenic differentiation, while supplying oxygen until the tissue’s vascular structure has been established.

Made available in DSpace on 2016-06-02T20:19:13Z (GMT). No. of bitstreams: 1 2912.pdf: 5230025 bytes, checksum: e6829f4e9fed0f66d6055d44a7e1c4f0 (MD5) Previous issue date: 2010-03-26 / Universidade Federal de Sao Carlos / Skin flaps are widely used in plastic surgery, mainly in reconstruction surgeries (transference of skin graft, pre-made tissues). After the surgery one of the major complication is ischemia, which may cause necrosis of the flap. Several features have been studied with the aim of increasing the viability flaps. Among these features, the low laser therapy is an alternative treatment, since it can promote an increase in microcirculation and vascular neoformation. However, there are discrepancies in the literature of the parameters employed in the use of laser, especially the fluence used in treatment . The aim of this study was to investigate the effects of 2 different laser wavelengths (660nm e 780nm) at 30 and 40J/cm2, on the viability of skin flap in rats evaluated by the paper template, vessels blood counting, activity of matrix metalloproteinase-2 (MMP-2), evaluation of plasma levels of NO, and expression hypoxiainducible Factor 1_. Sixty male animals Wistar were used in this study and they were distributed into the following groups (n=12 each group): control group, group irradiated with 660nm, at 30J/cm2; group irradiated with 660nm, at 40J/cm2 group irradiated with 780nm, at 30J/cm2, and group irradiated with 780nm, at 40J/cm2. The skin flap was performed on the back of all animals studied, with a plastic sheet interposed between the flap and the donor site. The animals received laser irradiation immediately after surgery and within 4 days, using the technical point of contact, on 24 points on the skin surface and around it. On the seventh postoperative day was evaluated the percentage of necrotic area and collected samples of tissue for histological analysis, zymography and protein expression by Western blotting, animals were euthanized by exsanguination, and blood were evaluated plasma levels of NO. The data obtained from the evaluation by the method of the paper template showed no increase in the viability of skin flaps after laser treatment. For biochemical analysis we found that the laser modulates the activity of MMP-2 and expression of HIF-1_ and induced an increase in the number of vessels especially in the groups irradiated with 40J/cm2 Measurement plasma level of NO did not differ between groups. Molecular analysis showed that the application of laser parameters used here, although it stimulated angiogenesis by modulating HIF-1_ and activity of MMP-2 was not able to improve the viability of skin flaps. Thus, we conclude that to find the beneficial effects of laser therapy we need to understand their mechanisms of action and know the best parameters to use. / Os retalhos cutâneos são amplamente utilizados na cirurgia plástica, principalmente na reconstrutiva. Após o procedimento operatório, uma das principais complicações é a isquemia, podendo ocasionar a necrose do retalho. Vários recursos têm sido estudados com o intuito de aumentar a viabilidade desses retalhos. Dentre esses recursos, o laser de baixa intensidade é uma alternativa de tratamento, uma vez que pode promover um aumento da microcirculação e da neoformação vascular. Entretanto, existem discrepâncias na literatura em relação aos parâmetros empregados no uso do laser de baixa intensidade, principalmente das fluências utilizadas nos tratamentos. Este estudo teve como objetivo verificar o efeito de 2 comprimentos de onda diferentes (660nm e 780nm), com fluências de 30 e 40J/cm2, na viabilidade do retalho cutâneo randômico em ratos, avaliados pelo método do gabarito de papel, contagem dos vasos sanguíneos, atividade da metaloproteinase de matriz -2 (MMP-2), avaliação dos níveis plasmáticos de NO, e expressão do fator induzível por hipóxia (HIF-1_). Sessenta ratos da linhagem Wistar foram usados nesse estudo, sendo distribuídos em 5 grupos (n=12): grupo controle, grupo irradiado com 660nm a 30J/cm2; grupo irradiado com 660nm a 40J/cm2, grupo irradiado com 780nm a 30J/cm2, e grupo irradiado com 780nm a 40J/cm2. O retalho cutâneo foi realizado no dorso dos animais com dimensões de 10 X 4cm e uma barreira plástica foi interposta entre o retalho e o leito doador. Os animais receberam irradiação laser imediatamente após a cirurgia e nos 4 dias seguintes, utilizando-se a técnica pontual em contato em 24 pontos distribuídos sobre e ao redor do retalho. No sétimo dia pósoperatório foi avaliado a porcentagem de área de necrose e coletado amostras de tecido para análise histológica, zimografica e de expressão protéica por western blotting, os animais foram eutanaziados por exsanguinação, e no sangue foram avaliados os níveis plasmáticos de NO. Os dados obtidos a partir da avaliação pelo método de gabarito de papel mostraram que não houve aumento na viabilidade dos retalhos cutâneos após tratamento com laser. Pelas análises bioquímicas observamos que o laser modulou a atividade de MMP-2 e a expressão de HIF-1&#61537;, bem como induziu o aumento no número de vasos especialmente nos grupos irradiados com 40J/cm2 independente do comprimento de onda utilizado. A medida do NO circulante não apresentou diferença entre os grupos. A análise molecular mostrou que a aplicação do laser com os parâmetros aqui utilizados, embora tenha estimulado a angiogênese por modular HIF-1&#61537;&#61472;e a atividade de MMP-2 não foi capaz de melhorar a viabilidade dos retalhos cutâneos. Assim, concluímos que para encontrarmos os efeitos benéficos e seguros da laserterapia precisamos compreender seus mecanismos de ação e conhecer os melhores parâmetros de utilização.

La radiothérapie thoracique peut induire le développement de pneumopathies aiguës et de fibroses. La dysfonction du système vasculaire participe au développement de lésions radiques. Dans l'intestin, un KO endothélial de PAI-1 protège les souris de la fibrose radique. Le premier objectif de ce projet est d'explorer le rôle de PAI-1 dans l'apparition de la fibrose radique pulmonaire. L'irradiation thoracique de souris à 17 Gy altère sévèrement le parenchyme pulmonaire et l'analyse histologique révèle que l'invalidation de PAI-1 aggrave les lésions à 2 et 13 semaines. Cette invalidation ne protège donc pas les animaux des dommages radiques pulmonaires. L'organisation en parallèle du poumon permet d'envisager une tolérance à de fortes doses par fraction sur des petits volumes. Des irradiations en conditions stéréotaxiques ont donc été réalisées chez la souris. Les analyses histologiques montrent une déstructuration alvéolaire et un fort infiltrat inflammatoire au niveau de la zone cible. Un œdème est observable dans l'ensemble du poumon ipsilatéral deux semaines après irradiation. Le poumon ipsilatéral est également affecté par des altérations de structure, tel un épaississement des septa alvéolaires. Ces bouleversements se traduisent également au niveau transcriptomique. A la vue de l'ensemble de ces altérations, un test à l'effort a été réalisé pour évaluer l'impact potentiel sur la fonction pulmonaire. Les résultats mettent en évidence une diminution des performances des animaux. Les analyses sont à approfondir mais elles démontrent l'importance de s'intéresser aux tissus sains situés hors du volume cible mais recevant des fractions variables de la dose délivrée. / Radiation-induced endothelial dysfunction is known to participate to the development of normal tissue damage. PAI- is implicated in the phenotypic changes of irradiated endothelial cells and KOendo mice are protected from radiation damage to the gut. Whole thorax of PAI-1 KOendo and floxed mice were exposed to 17 Gy. Histological analyzes showed that PAI-1 KOendo induces a worsening of injuries at 2 and 13 weeks. Consequently, contrary to the gut no protection from radiation-induced lung damage is observed in PAI-1 KOendo mice. Our second aim was to study the effects of a single high dose stereotactic irradiation on pulmonary tissues. Histological analyzes and scanner imaging show important injuries on the targeted volume. An ipsilateral edema can also be observed 2 weeks after irradiation. Ipsilateral lung is moreover importantly damaged. A thickening of alveolar septa is notably observable. A transcriptomic analysis show important similarities between tissues from the ipsilateral lung and the focal lesion. As really highly damages have been observed in both scanner and histological analyzes, we decided to perform forced physical activity test on treadmill. A drastic decrease of maximal distance traveled has been observed from two weeks. These experiments highlighted a deficiency in respiratory function and all of these results show the importance of non-targeted irradiated pulmonary volume in the development of radiation-induced fibrosis. Effect of an endothelium-specific deletion of HIF-1α has been investigated in this model of stereotactic irradiation. Only few differences have been observed between KOendo and control mice. Experiments are still ongoing.

Hintergrund: Die Versorgung von Immunzellen mit Energie in Form von ATP ist Grundlage eines funktionstüchtigen Immunsystems. Diese wird durch die mitochondriale OXPHOS oder durch die zytosolische Glykolyse gewährleistet. Sauerstoff und Glukose stellen die Hauptsubstrate dieser Stoffwechselprozesse dar. Fragestellung: Unter pathologischen Bedingungen wie sie in Entzündungsgebieten herrschen, konnte ein relativer Sauerstoffmangel experimentell nachgewiesen werden. Ziel dieser Arbeit war es herauszufinden, in welcher Weise die Funktionen einer definierten Lymphozytenpopulation (CD4+) durch Sauerstoffmangel beeinflusst werden. Methoden: Nach Isolation von CD4+ Zellen aus peripherem Blut gesunder Spender, wurden definierte Zellmengen stimuliert und in einem mit einer Sauerstoffelektrode ausgestatteten Gefäß unter Luftabschluß inkubiert. Zu definierten Zeitpunkten wurden Proben zur ATP-Messung entnommen, sowie Protein- und RNA-Lysate hergestellt. Die Vitalität zu Anfang und zum Ende der Inkubation wurde mittels Propidium-Jodid-Färbung im FACS bestimmt. Aus gesammelten Überständen wurden mittels Multiplex-ELISA die Konzentrationen von IL-1beta, IL-2, IL-6, IL-8, IL-10, TNF-alpha und MCAF gemessen. Als Kontrollen dienten unter Normoxie inkubierte Aliquots der Zellsuspensionen. HIF-1alpha wurde mit Immunoblotting nachgewiesen. Transkriptionsänderungen von SOD1 und HK1 wurden durch SYBR-Green Real-Time-PCR quantifiziert. Ergebnisse: Stimulierte CD4+-Zellen von Normalspendern schütten unter dem Einfluss von Hypoxie vermehrt proinflammatorische und chemotaktisch wirksame Zytokine, sowie zur Differenzierung notwendige antiinflammatorische Zytokine aus. Die Verfügbarkeit von Glukose hat hierauf einen verstärkenden Effekt. Eine hypoxische Umgebung sorgt in Abhängigkeit von der Versorgung mit Glukose für eine Anpassung der zellulären Atmungsrate. Glukose ist für die Aufrechterhaltung eines konstanten ATP-Levels verantwortlich. Die glykolytische Energiegewinnung unter Hypoxie kompensiert den Ausfall der OXPHOS. Hypoxie führt bei stimulierten CD4+-Zellen bei freier Glukoseverfügbarkeit zu einer vermehrten Transkription des Hexokinase1-Gens. Glukosemangel bewirkt dagegen in hypoxischer Umgebung eine Transkriptionssteigerung des SOD1-Gens. / Background: The energy supply of immune cells in form of ATP is the cornerstone of a functional immune system. This supply is realized by either mitochondrial OXPHOS or cytosolic glycolysis. Oxygen and glucose present the main substrates in these metabolic processes. Objective: Relative shortness of oxygen could be determined experimentally under pathological conditions present in inflamed tissues. The aim of this study was to determine the extent of hypoxic influence on the cellular function of CD4+ lymphocytes. Methods: Human CD4+ cells were isolated from peripheral blood of healthy blood donors by MACS sorting. Following a defined protocol cells were stimulated and incubated in a sealed container with a Clark type electrode. Samples were taken for measurements of ATP content. RNA- and Protein lysates were made to quantify the transcription of SOD1 and HK1 by SYBR green RT-PCR and look for the presence of HIF-1alpha by immunoblot analysis respectively. Supernatants were used to measure the expression of IL-1beta, IL-2, IL-6, IL-8, IL-10, TNF-alpha and MCAF using a multiplex ELISA assay. Aliquots of cell supspensions incubated under normoxic conditions served as controls. Results / Conclusion: Under the influence of hypoxia stimulated CD4+ lymphocytes of healthy blood donors express proinflammatory and chemotactically active as well as anti-inflammatory cytokines important for cell differentiation. The availability of glucose leads to an increase of this effect. An hypoxic environment dependant on the availability of glucose leads to an adaptation of cellular respiration. Glucose deficiency provokes an increase in cellular oxygen utilization. The availability of glucose is responsible for a constant intracellular ATP level. This proves that in CD4+ lymphocytes glycolysis is capable of compensating for hypoxically impaired oxidative phosphorylation thus providing enough ATP to enable cellular function. Hypoxia under glucose provision leads to an increase in mRNA expression for HK1, a key enzyme of glycolysis. Lack of glucose under hypoxic conditions results in an increase in mRNA expression for SOD1. Glucose therefore serves in CD4+ cells as an agent of constant energy supply that leads to cell survival and an upkeep of a proinflammatory environment through cytokine expression.