Le syndrome d’apnées obstructives du sommeil (SAOS) est un problème de santé publique majeur qui est considéré comme un facteur indépendant de risque de survenue d’un infarctus du myocarde (IM). Les altérations cardiovasculaires associées au SAOS sont principalement dues à l’hypoxie intermittente (HI) chronique. En particulier, l’HI induit l’activation du facteur de transcription hypoxia-inducible factor-1 (HIF-1), susceptible d’être impliqué dans la vulnérabilité accrue du myocarde à l’ischémie-reperfusion. L’objectif de cette thèse était d’étudier le rôle de HIF-1 dans les mécanismes induits par l’HI et impliqués dans l’augmentation de la taille de l’infarctus suite à une ischémie-reperfusion. Ces travaux ont mis en évidence deux nouveaux effets délétères de l’HI, à savoir l’induction d’un stress du réticulum endoplasmique (RE) et d’altérations mitochondriales. A travers, l’inhibition génétique et/ou pharmacologique de HIF-1, nous avons montré que HIF-1 apparaît comme un acteur primordial dans l’ensemble des mécanismes délétères de l’HI, incluant ceux découverts lors de cette thèse. De plus, HIF-1 joue un rôle majeur dans l’augmentation de la taille de l’IM induite par l’HI chronique. Parallèlement, son activation myocardique est corrélée à l’index d’apnées-hypopnées chez des patients apnéiques atteints d’une maladie coronarienne (comparativement aux non-apnéiques). Par conséquent, l’activation de HIF-1 pourrait être utilisée comme marqueur diagnostic du SAOS chez les patients à risque cardiovasculaire. HIF-1 pourrait également représenter une cible pour le développement de nouvelles thérapies complémentaires ou substitutives aux traitements actuels. / Obstructive sleep apnea syndrome (OSAS) is a major public health problem that is considered an independent risk factor for the occurrence of myocardial infarction (MI). The cardiovascular alterations associated with OSA are mainly due to the chronic intermittent hypoxia (IH). In particular, activation by IH, the hypoxia-inducible factor-1 (HIF-1) transcription factor likely contributes to enhance myocardial vulnerability to ischemia-reperfusion injury. The aim of this thesis was to study the role of HIF-1 in the mechanisms involved in the increase in MI induced by chronic IH. This work has highlighted two new deleterious consequences of IH exposure, namely endoplasmic reticulum (ER) stress and mitochondrial alterations. Through genetic and/or pharmacological inhibition of HIF-1, we have shown that HIF-1 appears to be a primordial actor in all the deleterious mechanisms of IH, including those discovered during this thesis. HIF-1 also appears to play a major role in the IH-induced increase in MI size. In parallel, its myocardial activation is correlated with the apnea-hypopnea index in apnoeic, compared to non-apnoeic, patients with coronary heart disease. Therefore, HIF-1 activation could serve as a diagnostic marker of OSA in patients with cardiovascular risk. HIF 1 could also be a target for new therapeutic approaches, in complement or replacement of standard treatments.

PURPOSE: The anti-tumor activity of glucose analogs 2-deoxy-glucose (2-DG) and D-allose was investigated alone or in combination with p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 or platinum analogs as a strategy to pharmacologically target glycolytic tumor phenotypes. METHODS: Hypoxia inducible factor-1 alpha (HIF-1alpha) protein accumulation in pancreatic cell lines treated with SB202190 alone and in combination with glucose analogs was analyzed by Western blot. HIF-1alpha transcriptional activity was measured in MIA PaCa-2 cells stably transfected with a hypoxia response element luciferase reporter following treatment with glucose analogs alone, and in combination with SB202190. Induction of cleaved poly(ADP-ribose) polymerase (PARP) was measured by Western blot in the MIA PaCa-2 cells. In vitro anti-proliferative activity of 2-DG and D-allose alone, or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines), or with SB202190 were investigated using the MTT assay. RESULTS: SB202190 decreased HIF-1alpha protein accumulation and transcriptional activity. 2-DG demonstrated greater anti-proliferative activity than D-allose. Pre-treatment with SB202190 enhanced activity of both 2-DG and D-allose in MIA PaCa-2, BxPC-3, ASPC-1, and SK-OV-3 cells. The combination of D-allose and platinum agents was additive to moderately synergistic in all but the OVCAR-3 and HEY cells. SB202190 pre-treatment further enhanced activity of D-allose and 2-DG with platinum agents in most cell lines investigated. CONCLUSIONS: SB202190 induced sensitization of tumor cells to 2-DG and D-allose may be partially mediated by inhibition of HIF-1alpha activity. Combining glucose analogs and p38 MAPK inhibitors with chemotherapy may be an effective approach to target glycolytic tumor phenotypes.

Posttranslational modifications are of great importance in the mediation of transcriptional effects, necessary for signalling in cancer. A characteristic example of such modifications is acetylation of the p53 tumour suppressor, a transcription factor involved in several crucial cellular functions including cell-cycle arrest and apoptosis. p53 is stabilised under hypoxic and DNA damaging-conditions. However, only in the latter scenario is p53 fully capable of inducing the expression of its proapoptotic targets through acetylation. The hypoxia inducible factor 1 (HIF-1) transcription factor is stabilised at low oxygen levels to mediate a cellular adaptive response under these conditions, promoting cell survival. As these two opposing transcription factors share a common transcriptional regulator, p300/CBP, this study focused on deciphering the p300/CBP complex components under differential stress to determine its composition required for cellular responses elicited in response to DNA damage or hypoxia, in an effort to investigate a possible link between differential posttranslational modifications and the resulting cell fate. Hence, the aim of this study was to investigate the roles of p300/CBP components in dictating transcriptional regulation of both HIF-1 and p53 in hypoxic conditions. To carry out this study, the proapoptotic BID gene was the system used, as its promoter contains a p53 response element and a HIF-1 response element (HRE). The p300/CBP associated factors PCAF and Strap were appointed as potent candidates for posttranslational modifications under differential conditions, as they are stress-responsive cofactors. Under DNA damage, PCAF acetylates p53 at K320 and Strap augments p300 binding to p53, both of which amplify the p53 response. Evidence from this study demonstrates that under hypoxia-mimicking conditions PCAF-mediated p53 acetylation at K320 is reduced to a greater extent compared to p300/CBP acetylation at K382. The limited amounts of acetylated p53 at K320 are preferentially recruited to the promoter of the cell cycle arrest p21WAF-1/CIP-1 gene that appears to be unaffected by hypoxia, but fail to be recruited to the BID promoter, rendering p53 incapable of upregulating proapoptotic BID in hypoxic conditions. In addition, under the same conditions, PCAF was found to acetylate, and direct HIF-1 to a particular subset of its targets, leading to alterations in the net physiological effect. Moreover, the intrinsic acetyl transferase activity of PCAF was shown to increase the stability of HIF-1. An additional role was attributed to PCAF in relation to apoptosis, albeit from another angle. BID protein translocation to the cytoplasm in hypoxic conditions was facilitated by ectopically expressed PCAF.Strap was found to be preferentially recruited to the HRE of the BID promoter in hypoxic conditions, and to exert a transrepression effect that appeared to be p53-dependent. Strap also interacted with specific PCAF isoforms depending on the type of cellular stress. Contrary to PCAF, ectopically expressed Strap did not have any effect on BID subcellular distribution. This study has provided additional insight in the mechanisms by which cofactors are involved in cell fate, either by affecting activity and stability of HIF-1 and p53, or having a direct effect on Bcl-2 member subcellular distribution.

Many fish face low oxygen concentrations (hypoxia) in their natural environments, and they respond to hypoxia through a variety of behavioral, physiological, and cellular mechanisms. Some of these responses involve changes in gene expression. In mammals, the hypoxia inducible factor (HIF) family of transcription factors are the “master regulators” of gene expression during hypoxia, but the study of HIF in fish has been hampered by the lack of reagents to detect this protein in non-mammalian vertebrates. The goals of this thesis are to affinity purify antibodies against HIF from the killifish Fundulus heteroclitus and use them to recover and quantify HIF from killifish cells and tissues. The purified, validated antibodies represent a critical reagent for future studies of the role of HIF in the molecular response of this and other fish to fluctuations in oxygen in their natural environments.

Background: The impairment of articular cartilage caused by trauma or degenerative pathology is one of the most challenging issues in clinical Orthopedics because of the limited intrinsic regenerative capability of this tissue. Hypoxia is a major stimulus to initiate gene programs in regulating chondrogenic lineage cell functions during cartilage development and regeneration. Hypoxia-inducible factor-1α (HIF-1α), the key transcription factor to sense oxygen fluctuations of cells, is abundantly expressed in the cartilage and considered as a potential therapeutic target for cartilage tissue homeostasis or repair. However, the molecular mechanisms and therapeutic efficacy of targeting the HIF-1α pathway remain to be well defined. / Methods: Osteochondral defect mouse model was generated to examine the hypoxia states during articular cartilage repair with the Hypoxyprobe. Specific HIF-1α deletion in the repairing tissue was established to determine its regulatory role during cartilage restoration. Deferoxamine (DFO), stabilizing HIF-1α from proteolysis by inhibiting the prolyl hydroxylases (PHDs), was investigated systemically on the function of chondroprogenitors and mesenchymal stem cells (MSCs) in vitro. Alcian blue staining determined the proteoglycan synthesis. HIF components, chondrogenic related genes and proteins were examined by quantitative PCR, western blotting and immunohistochemistry, respectively. The proliferation, differentiation and migration assays were performed to determine the influence of DFO onchondroprogenitors and MSCs. The recruitment or engraftment of MSCs in the injured site was traced by transplantation of GFP-labeled MSCs adjacent to the defect region, and examined by immunofluorescence staining. DFO incorporated in a 3D alginate-gelfoam scaffold was analyzed for its therapeutic effects on the articular cartilage regeneration. At 6 and 12 weeks following surgery, the cartilage tissue repair was scored and the expression of proliferating cell nuclear antigen (PCNA), Sox9 and collagen typeⅡ(Col2) was examined by immunohistochemistry. / Results: Hypoxia states and the expression of HIF-1α in the repair tissue were ubiquitously existed in the osteochondral defect model. DFO significantly upregulated HIF-1α expression and nuclear localization, and increased the levels of PHDs. DFO increased chondroprogenitor cell proliferation as visualized by colony forming unit assay, which was in accord with the upregulation of cyclin D1. DFO significantly induced chondrogenic differentiation indexed by increased Col2 and Sox9 protein expression and elevated proteoglycan synthesis. With sustained upregulation of HIF-1α DFO was supposed to effectively promote chondrogenesis in mimic of hypoxic microenvironment. DFO also increased the migration of MSCs, and elevated the expression of tissue inhibitor metalloproteinase-3 (TIMP3) through transcriptional control by HIF-1α. Furthermore, DFO initiated MSCs membrane protrusion through regulating the expression and interaction of the key focal adhesion proteins vinculin and paxillin. In vivo study showed that DFO dramatically facilitated the recruitment and functional engraftment of MSCs to the lesion site compared with the controls. Alginate-gelfoam scaffold incorporated with DFO enhanced articular cartilage repair through increasing chondrogenic cell proliferation, differentiation and proteoglycan synthesis. The enhanced therapeutic effect of DFO on articular cartilage repair was eliminated following HIF-1α deletion in the repairing cells of the cartilage lesion. The results indicate that the positive effect of DFO on articular cartilage repair is at least partially mediated by HIF-1α. / Conclusion: HIF-1α is an essential mediator during articular cartilage repair. Activation of HIF-1α by PHD inhibitor DFO increases chondroprogenitor cell proliferation, differentiation and migration in vitro. DFO enhances articular cartilage repair through coordinating MSCs migration, chondrogenic differentiation and functional engraftment. The results provide proof of principle that targeting the HIF-1α pathway may serve as a novel approach for promoting articular cartilage regeneration. / 背景：关节软骨自愈能力非常有限，由创伤或退行性病变引起关节软骨损伤的治疗是骨科领域的一大难题。在软骨发育和再生过程中，低氧条件对启动基因表达及调控软骨系细胞功能至关重要。低氧诱导因子-1α（HIF-1α）作为关键的转录因子可感应细胞外氧含量变化，广泛存在于软骨组织中，并被认为对维持软骨组织内稳态及促进软骨修复有重要作用。然而，以HIF-1α 通路为靶点的小分子靶向药物的分子机制与治疗效果尚不明确。 / 方法：本课题系统性地研究了HIF 信号通路激活剂去铁胺（DFO）对软骨损伤的作用。我们构建了骨软骨缺损模型，应用缺氧探针检测了软骨缺损过程中修复组织的低氧状态，并特异性敲除软骨修复组织中HIF-1α 表达，研究其在软骨再生过程中的调节作用。我们用阿利新蓝染色检测软骨细胞蛋白多糖的合成及分泌。通过实时荧光定量聚合酶链式反应，免疫印迹以及免疫组化等方法检测了HIF 家族成员和软骨分化标志物的基因和蛋白含量变化。通过增殖及迁移实验检测了DFO 对软骨细胞或者骨髓间充质干细胞（MSC）功能的影响。另外，我们还将GFP 标记的MSC 注射到与小鼠软骨缺损区域相邻的软骨下骨中，观察其在软骨缺损模型中的募集及功能性植入。我们以藻酸盐和明胶海绵复合物为给药系统，包载DFO 并作用于关节软骨缺损部位。术后6 周及12 周取材，以番红O 染色检测DFO 对小鼠关节软骨缺损的修复效果，并通过免疫组化检测增殖细胞核抗原（PCNA），Sox9 以及Col2 等蛋白的表达。 / 结果：低氧状态和HIF-1α 在骨软骨缺损模型中的软骨缺损区域广泛存在和表达。DFO 显著提高了HIF-1α 蛋白表达及转运入核，增加了脯氨酸羟化酶（PHD）表达。在软骨祖细胞中，DFO 可提高其增殖、克隆能力，并增加周期蛋白D1的表达。同时，DFO 能明显促进软骨祖细胞分化，增加软骨分化标志物基因以及Sox9 和Col2 蛋白表达，提高蛋白多糖分泌。通过持续性激活HIF-1α，DFO可模仿低氧微环境来提高软骨细胞增殖、分化能力。分子机制研究发现，DFO激活HIF-1α 后，HIF-1α 作用在靶基因金属蛋白酶组织抑制剂-3 启动子上，增加其转录和蛋白表达，进而提高MSC 的迁移能力。另外，激活HIF-1α 蛋白可增加黏着斑蛋白，桩蛋白表达以及它们的相互作用，促进MSC 伪足延伸。体内实验中，通过追踪小鼠体内GFP 标记的MSC 发现， DFO 可在软骨损伤早期（7 天及14 天）提高受损部位MSC 募集数量，并促进其向软骨细胞谱系分化。通过增加软骨系细胞增殖、分化、蛋白多糖合成，包载DFO 的藻酸盐明胶海绵给药系统显著提高了软骨缺损组织的修复效果。而在软骨修复组织中特异性敲除HIF-1α 蛋白后，明显降低了DFO 对软骨缺损的治疗效果，提示DFO对软骨修复的作用至少部分由HIF-1α 介导。 / 结论：HIF-1α 是关节软骨修复过程中的重要调控因子。PHD 抑制剂DFO 可以激活HIF-1α 表达，增加软骨祖细胞增殖、分化和迁移。DFO 通过调控MSC 募集、软骨细胞谱系分化以及功能性植入，明显改善关节软骨再生修复的效果。本研究为HIF-1α 信号通路作为一种新的治疗靶点促进关节软骨再生提供了重要证据。 / Shu, Yinglan. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 155-181). / Abstracts also in Chinese. / Title from PDF title page (viewed on 09, September, 2016). / Shu, Yinglan. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 19, 2010). Includes bibliographical references.

Persistent organic pollutants (POPs) and adipose tissue hypoxia have been shown to independently affect adipocyte functions. The goals of this study were to (1) determine the effect of PCB-77, PCB-153, and DDE on the differentiation of human preadipocytes, and (2) investigate the cross-talk between PCB-77 and hypoxia in differentiated human adipocytes. First, human preadipocytes were exposed to PCB-77, PCB-153, or DDE during the entire 14-day differentiation period. We found no effect of low POP levels on lipid accumulation. Second, differentiated human adipocytes were exposed to a combination of PCB-77 and hypoxia. We demonstrated gene-specific cross-talk between PCB-77 and hypoxia, showing an additive effect of PCB-77 on VEGF, MCP-1, and adiponectin, as well as an inhibition of PCB-77-induced expression of CYP1A1 by hypoxia. This work has expanded our understanding of the role of POPs and hypoxia in differentiated human adipocytes.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 174-182). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

This thesis is concerned with a very common disorder of iron homeostasis: iron deficiency. The specific focus is the manner in which iron deficiency influences physiological responses to hypoxia in humans. This work is predicated on observations made over many decades in vitro and in vivo, suggesting that variations in the bioavailability of iron have important consequences for certain biological processes known to depend on oxygen availability. Three separate but related studies together form the basis for this thesis. The first two, Study A and Study B, adopt a similar approach in recruiting healthy volunteers who differ according to iron status, yielding iron-deficient and iron-replete groups in both cases. In Study A, the behaviour of the pulmonary circulation is investigated during a sustained hypoxic exposure, before and after an intravenous infusion of iron. In Study B, skeletal muscle metabolism is explored, both at the level of high-energy phosphate metabolism and the integrated physiological responses to exercise on a cycle ergometer. In the third study, Study C, a different approach is taken, recruiting patients with chronic obstructive pulmonary disease (COPD), and exploring the prevalence and associations of iron deficiency in this condition. Chapters 2 and 3 describe experiments using sustained hypoxia in a normobaric chamber, during which the pulmonary circulation is assessed non-invasively using Doppler echocardiography. These reveal augmented hypoxic pulmonary vasoconstriction (HPV) in iron-deficient individuals, who also exhibit greater sensitivity to the effects of an infusion of intravenous iron. Additionally, the way in which certain circulating mediators important for iron haemostasis change over the course of these hypoxic exposures, and how iron status influences these responses, is explored. Chapter 4 reports the findings of experiments using 31P-magnetic resonance spectroscopy and cardiopulmonary exercise testing, which demonstrate abnormal whole-body metabolism in iron-deficient individuals during large muscle-mass exercise, despite the absence of a clear defect in mitochondrial oxidative phosphorylation. Intravenous iron is found to have significant effects to alter the lactate threshold in healthy individuals, but the effects are more striking in iron-deficient individuals. Collectively, these experiments imply that iron deficiency promotes a more glycolytic phenotype. Chapter 5 explores iron deficiency in COPD, a condition in which pulmonary vascular disease, hypoxia and skeletal muscle dysfunction coexist, and examines some of the difficulties in assessing iron status in the setting of a chronic inflammatory disorder. Iron deficiency is found to be common, and unexpectedly associated with significantly more severe hypoxaemia, in patients with COPD. Possible reasons for these findings, and their clinical implications, are considered. Chapter 6 provides a summary of the main conclusions to be drawn from the studies presented in this thesis.