Dissertationen zum Thema „Hypoxia regulators“

Les facteurs de transcription Twist1 et Twist2 (famille Twist) jouent un rôle majeur dans le développement embryonnaire et dans la progression tumorale. Leur potentiel oncogénique dérive directement de la combinaison de leurs nombreuses activités développementales. Les gènes Twist peuvent notamment, en induisant la transition épithélio-mésenchymateuse (EMT), promouvoir l’invasion des cellules cancéreuses et participer de ce fait aux processus métastatique. De plus, en bloquant l’activité des voies de signalisation Rb et p53, ils peuvent inhiber les deux principaux programmes de sauvegarde cellulaire que sont l’apoptose et la senescence. Enfin, ils sont également impliqués dans la résistance des cellules cancéreuses aux agents chimio-thérapeutiques. En plus de ces nombreuses activités, nos données préliminaires nous ont amené à considérer un rôle de Twist dans la réponse au stress. Les cellules cancéreuses doivent croitre dans un environnement en perpétuel changement qui génère de nombreux types de stress. Seules les cellules capables de s’adapter, peuvent survivre et acquérir de nouvelles capacités les rendant plus agressives. La résistance au stress fait donc partie intégrante de la progression tumorale. Nos travaux révèlent que Twist en induisant une résistance au stress, plus particulièrement métabolique, est un acteur essentiel de l’acquisition d’u phénotype agressif des cellules cancéreuses. Dans une première étude, nous avons montré que Twist module le stress oxydatif, une condition très fréquemment retrouvée dans les tumeurs. Ainsi, nos résultats indiquent que l’expression de Twist provoque une réduction du taux d’espèces réactives de l’oxygène (ROS) intracellulaire. Cette activité a pour conséquence directe d’induire une résistance accrue à l’apoptose déclenchée par divers traitements. Nous avons par la suite caractérisé cette activité et mis en évidence un programme génétique contrôlé par Twist impliquant divers facteurs possédant des propriétés anti-oxydantes. Dans un second temps, nous nous sommes intéressés à un autre type de stress métabolique, l’hypoxie. L’hypoxie définie par un taux insuffisant d’oxygène, est retrouvée dans la plupart des tumeurs solides du fait de l’absence ou de l’anomalité de la vascularisation. L’hypoxie mène à la stabilisation d’un facteur de transcription, HIF1α. Cette protéine est essentielle à l’adaptation hypoxique et contrôle l’expression de nombreux gènes impliqués dans le métabolisme du glucose, le transport de l’oxygène, l’angiogenèse ou l’apoptose. Dans les premiers temps d’hypoxie, l’effet d’adaptation induit par HIF1α est bénéfique pour les cellules. Cependant, si l’absence d’oxygène se prolonge, HIF1α, peut pousser les cellules vers la mort. Nos travaux démontrent que Twist est capable de rendre les cellules résistantes à une hypoxie prolongée. De plus, cette activité de protection contre le stress hypoxique agit via un effet paracrine. Enfin, nos données suggèrent que cet effet est médié par une interaction directe entre les protéines Twist et HIF1α. Au final, cette étude indique que l’expression de Twist dans les cellules cancéreuses, en conférant une résistance accrue à l’environnement hypoxique, joue un rôle essentiel dans l’adaptation au stress et à l’acquisition de nouveaux phénotypes agressifs. En résumé, L’objectif principal de ma thèse était de mettre en évidence de nouvelles propriétés cellulaires des oncogènes de la famille Twist. Nos résultats démontrent que Twist par ses capacités à contrôler le stress métabolique, permet à la cellule cancéreuse de mieux s’adapter et donc survivre dans un environnement en constante évolution. Nos travaux renforcent donc la notion de l’importance de ces facteurs dans la progression tumorale
Twist1 and Twist2 are related transcription factors that play major roles both during embryonic development and in several pathologies, including cancer. Twists' oncogenic potential arises from a combination of their multiple functions in development. Notably, both Twist induce epithelial-to mesenchymal transition, thus promoting tumour invasiveness and possibly conferring to cells self-renewal properties. Furthermore, through disruption of both Rb- and p53-driven pathways, Twist override two major oncogene-induced fail-safe programs, namely senescence and apoptosis, thereby promoting malignant conversion. Twist has also been reported to participate in acquisition of drug resistance and in promotion of neo-angiogenesis.Current knowledge of pleiotropic activities of Twist prompted us to postulate that these factors may be major regulators of stress response. Cancer cells survive and grow within a continuously changing environment that creates multiple stresses to which they must adapt in order to survive and strive. Such adaptations often give rise to the acquisition of an aggressive phenotype. Consistent with this hypothesis, we recently unveiled new activities of Twist proteins that are related to stress response. We have shown that Twist regulates response to oxidative stress, a condition exacerbated in cancer by stimuli such as inflammation, increased cellular metabolism and changes in tumour oxygenation. Our work has contributed to the understanding of molecular mechanisms through which Twist diminishes cellular ROS and thus participates in the escape from apoptosis and senescence. In the first part of my thesis, I worked on the antioxidant activity of Twist and described its molecular mechanisms.The second part of my work addressed the impact of Twist proteins on cellular response to hypoxia that is insufficient oxygen supply, frequently found in solid tumours. Cellular response to hypoxic stress relies on stabilization and activation of HIF1α, a key transcriptional mediator of the hypoxic response, regulating numerous genes involved in glucose metabolism, oxygen transport, angiogenesis, cell growth and apoptosis. HIF1α is beneficial for cancer cells in response to short hypoxic episodes, however its sustained activation in case of prolonged hypoxia may push cancer cells towards apoptosis. In this context, we have shown that Twist protects cancer cells from hypoxia-induced apoptosis. We have discovered HIF1α and Twist physically interact, suggesting a possible mechanistic basis for Twist's protective effect. These results led us to postulate that Twist plays a role in cellular response to hypoxia and thus participates in cancer cell adaptation and acquisition of aggressive phenotypes triggered by lack of oxygen.Our results reinforce the notion that Twist factors are major cellular stress modulators that might be important for adaptation of cancer cells to changing conditions in the process of tumour progression

Title from PDF of title page (University of Missouri--Columbia, viewed on March 8, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Michael J. Petris. Vita. Includes bibliographical references.

Abstract Breast cancer is the most common cancer affecting the female population of the Western world. It is a heterogeneous disease entity that encompasses tumors with remarkably different forms of behaviour, and it is therefore vital to distinguish patients with good and poor prognoses. The classical prognostic and predictive factors for breast cancer serve as tools for clinical oncologists when planning treatment, but the growing awareness of breast cancer biology is bringing about a need for novel prognostic and predictive biomarkers. This thesis examines the prognostic significance of hypoxia response and cell cycle regulators in ductal breast cancer and in triple-negative breast cancer (negative for hormone receptors and human epidermal growth factor receptor 2), concluding that PHD2 and PHD3 are associated with a good prognosis, while the role of PHD1 is controversial, as it is associated with proliferation in ductal breast cancer but with node-negative status in triple-negative breast cancer. In our experiments HIF-1α redeemed its role as a marker of an adverse prognosis, whereas the role of HIF-2α appeared to be the opposite. Our data suggest that PHDs can have other targets than the HIF-αs, and that triple-negative breast tumors express more HIF-1α and less HIF-2α and PHD3 than those with a good prognosis. Furthermore, we identified cyclin D1 as a biomarker with independent prognostic significance in ductal breast cancer, being associated with good prognostic factors and a better outcome, whereas the opposite was seen in triple-negative breast cancer. CDK4 was associated with high proliferation in triple-negative breast cancer. In addition, high levels of p16 correlated with increased survival in breast cancer patients independently of receptor status
Tiivistelmä Rintasyöpä on naisten yleisin syöpä läntisessä maailmassa. Rintasyöpä on heterogeeninen tautiryhmä, jossa kasvaimet vaihtelevat biologiselta käyttäytymiseltään huomattavasti. Tästä syystä on tärkeää erottaa hyvä- ja huonoennusteiset potilaat. Syöpälääkärit käyttävät klassisia ennustetekijöitä hoitopäätöksiä tehdessään, mutta lisääntynyt tieto rintasyövän biologiasta on saanut aikaan tarpeen löytää uusia ennustetekijöitä. Tässä väitöskirjatyössä tutkimme hypoksiavasteen ja solusyklin säätelijöiden ennusteellisuutta duktaalisessa rintasyövässä sekä kolmoisnegatiivisessa (ei ilmennä hormonireseptoreita eikä epidermaalikasvutekijäreseptoria) rintasyövässä. PHD2 ja PHD3:n vahva ilmentyminen liittyi parempaan ennusteeseen, mutta PHD1:n esiintymisen vaikutus oli ristiriitainen. PHD1:n ilmentyminen liittyi lisääntyneeseen solujakautumiseen duktaalisessa rintasyövässä, mutta kolmoisnegatiivisessa rintasyövässä sen esiintyminen liittyi vähentyneeseen imusolmukemetastasointiin. Tutkimuksessamme HIF-1α osoittautui huonon ennusteen merkiksi. Sitä vastoin HIF-2α:n ilmentymisen vaikutus näytti liittyvän parempaan ennusteeseen. Tuloksemme osoittavat, että PHD-entsyymeillä on mahdollisesti muitakin kohteita kuin HIF-α:t. Osoitimme myös, että HIF-1α:n ilmentyminen on yleisempää ja HIF-2α:n sekä PHD3:n ilmentyminen vähäisempää kolmoisnegatiivisessa kuin duktaalisessa rintasyövässä. Lisäksi totesimme, että sykliini D1 on itsenäinen ennustetekijä liittyen parempaan ennusteeseen duktaalisessa rintasyövässä. Huomioitavaa on kuitenkin, että kolmoisnegatiivisessa rintasyövän alaryhmässä sykliini D1:n esiintyminen oli huonon ennusteen merkki. CDK4 osoittautui voimakkaan proliferaation merkiksi kolmoisnegatiivisessa rintasyövässä. Lisäksi osoitimme, että p16:n ilmentyminen liittyy parempaan ennusteeseen sekä duktaalisessa rintasyövässä että kolmoisnegatiivisessa rintasyövässä

Intracellular redox potential (IRP) is a measure of how oxidising or reducing the environment is within a cell. It is a function of numerous factors including redox couples, antioxidant enzymes and reactive oxygen species. Disruption of the tightly regulated redox status has been linked to the initiation and progression of cancer. However, there is very limited knowledge about the quantitative nature of the redox potential and pH gradients that exist in cancer tumour models. Multicellular tumour spheroids (MTS) are three-dimensional cell cultures that possess their own microenvironments, similar to those found in tumours. From the necrotic core to the outer proliferating layer there exist gradients of oxygen, lactate, pH and drug penetration. Tumours also have inadequate vasculature resulting in a state of hypoxia. Hypoxia is a key player in metabolic dysregulation but can also provide cells with resistance against cancer treatments, particularly chemotherapy and radiation therapy. The primary hypoxia regulators are HIFs (Hypoxia Inducible Factors) which under low O2 conditions bind a hypoxia response element, inhibiting oxidative phosphorylation and upregulating glycolysis which has two significant implications: the first is an increase in levels of NADPH/NADH, the main electron donors found in cells which impacts the redox state, whilst the second is a decrease in intracellular pH (pHi) because of increased lactate production. Thus, redox state and intracellular pHi can be used as indicators of metabolic changes within 3D cultures and provide insight into cellular response to therapy. Surface-Enhanced Raman Spectroscopy (SERS) provides a real-time, high resolution method of measuring pHi and IRP in cell culture. It allows for quick and potentially portable analysis of MTS, providing a new platform for monitoring response to drugs and therapy in an unobtrusive manner. Redox and pH-active probes functionalised to Au nanoshells were readily taken up by prostate cancer cell lines and predominantly found to localise in the cytosol. These probes were characterised by density functional theory and spectroelectrochemistry, and their in vitro behaviour modelled by the chemical induction of oxidative and reductive stress. Next, targeting nanosensors to different zones of the MTS allowed for spatial quantification of redox state and pHi throughout the structure and the ability to map the effects of drug treatments on MTS redox biology. The magnitude of the potential gradient can be quantified as free energy (ΔG) and used as a measurement of MTS viability. Treatment of PC3 MTS with staurosporine, an apoptosis inducer, was accompanied by a decrease in free energy gradients over time, whereas treatment of MTS with cisplatin, a drug to which they are resistant, showed an increase in viability indicating a compensatory mechanism and hence resistance. Finally, using this technique the effects of ionising radiation on IRP and pHi in the tumour model was explored. Following exposure to a range of doses of x-ray radiation, as well as single and multi-fractionated regimes, IRP and pHi were measured and MTS viability assessed. Increased radiation dosage diminished the potential gradient across the MTS and decreased viability. Similarly, fractionation of a single large dose was found to enhance MTS death. This novel SERS approach therefore has the potential to not only be used as a mode of drug screening and tool for drug development, but also for pre-clinical characterisation of tumours enabling clinicians to optimise radiation regimes in a patient-specific manner.

Transcriptional control plays a major role in regulating hypoxic responses in plants. However, the transcriptional regulatory networks associated with hypoxia remain to be constructed. By transcriptomic analysis I show here that a novel systemic transcriptional reprogramming, which is mediated via the interplay of hormones, facilitates the survival of plants under flooding. A feasible strategy for identifying downstream targets of transcription factors (TFs) was developed. The downstream pathways of a hypoxia-responsive TF, WRKY22, were constructed. The results also show that AtERF73/HRE1 (Arabidopsis thaliana Ethylene Response Factor 73/Hypoxia Responsive ERF 1) modulate ethylene-dependent and -independent responses during hypoxia. Transcriptomic analysis of Arabidopsis in both root and shoot tissues during flooding of roots indicates the existence of a systemic communication through transcriptional reprogramming. By functional classification of affected genes, a comprehensive managing program of carbohydrate metabolism was observed. Through transcriptional profiling in ethylene and abscisic acid (ABA) signaling mutants, ein2-5 and abi4-1, an alteration of long-distance hypoxic regulation was uncovered in ein2-5 and abi4-1. Moreover, genes involved in ABA biosynthesis were also found to be differentially regulated between shoots and roots. Many members of the WRKY TF family were highly induced by hypoxia. One of the early-induced WRKYs, WRKY22, which has the highest induced level, was chosen for identifying its downstream targets. Anoxic tolerance was affected in WRKY22 overexpressing (WRKY22-OX) and knock-out (wrky22-ko) lines. Comparison of differential gene expression profiles between the wild-type and WRKY22-OX and between the wild-type and wrky22-ko lines by microarray analysis identified novel hypoxia-responsive genes as WRKY22 targets. Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (ChIP-chip) and ChIP followed by quantitative PCR (ChIP-qPCR) were utilized to analyze in vivo interactions. To study the role of ethylene during hypoxia, I characterized an AP2/ERF (APETALA2/ethylene response factor) AtERF73/HRE1 that is specifically induced during hypoxia. I showed that the expression of AtERF73/HRE1 can be induced by exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene. Its hypoxic induction was reduced but not completely abolished in ethylene-insensitive mutants and in the presence of inhibitors of ethylene biosynthesis and responses. Increased ethylene sensitivity and exaggerated triple responses were observed in HRE1-RNAi knock-down lines. By comparing expression differences between the wild-type and HRE1-RNAi lines, I found that hypoxic induction of glycolytic and fermentative genes was reduced by the HRE1-RNAi knock-down mutations, whereas induction of a number of peroxidase and cytochrome P450 genes was increased. Collectively, these results show that AtERF73/HRE1 is involved in modulating ethylene responses under both normoxia and hypoxia.

In response to low oxygen (hypoxia), cells have evolved sophisticated gene expression programmes for survival and adaption. How the chromatin state coordinates these changes remains largely unknown. Global histone methylation changes occur in response to hypoxia, however, temporal dynamics of histone methylation changes and how they correlate with hypoxia induced gene transcription changes is ill defined. The Jumonji C (Jmjc) histone demethylases are oxygen dependent enzymes and represent a potential link between chromatin structure and oxygen sensing. Many of these enzymes are differentially expressed in hypoxia and some have been found to influence the hypoxic response. Here, the JmjC histone demethylase, KDM2B, is found to be induced at the mRNA level but not at the protein level in response to hypoxia. KDM2B was also found to regulate the transcriptional response hypoxia, in a cell type dependent manner, through control of Hypoxia Inducible Factor (HIF) subunits, HIF 1 and 2α. These findings highlight complex HIF-KDM2B crosstalk involved in the cells response to low oxygen. Additionally, it was found that various histone methylation marks are induced in the early response to hypoxia prior to hypoxia induced gene transcription changes. This demonstrates that chromatin structural marks responds rapidly to changes in oxygen availability. Furthermore the methylation landscape of 2 two active transcription histone methylation marks, H3K4me3 and H3K36me3, were mapped by ChIP sequencing in the acute response to hypoxia. This analyses found specific changes in histone methylation, which correlate with the core gene transcription changes in hypoxia, pointing towards a mechanism by which rapid chromatin changes programs the cell for hypoxic transcription. Finally, KDM5A was identified to, at least in part, regulate early hypoxia H3K4me3 changes and changes in gene expression of a subset of hypoxia responsive genes. Findings described herein provide evidence for the role of chromatin structure dynamics, mediated by chromatin modifying enzymes, in regulating the hypoxic response. Specifically, early histone methylation changes elicited in acute hypoxia may help establish a chromatin landscape for the cell to transcriptionally respond, which is essential for survival and adaptation to hypoxia. Insights into chromatin dynamics in the response to hypoxia and the role played by JmjC histone demethylases in regulating the hypoxic response has the potential for new drug discovery in diseases such as cancer, were hypoxia, epigenetics and JmjC enzymes are often implicated in disease progression.

Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2alpha gene (EPAS1) in normal cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2alpha gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumours and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2alpha pathway in the hypoxic tumour microenvironment, which is necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumour growth in vivo. In addition, restoring HIF-2alpha expression in DNMT3a-reintroduced cancer cells restores full tumorigenic potential, including the capacity to traverse the hypoxic barrier. These data support a tumour-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2alpha oxygen-sensing pathway and the cellular response to hypoxia.

The hypoxia signalling pathway controls the adaptation of cells to decreased oxygen availability. When oxygen becomes limiting, the central transcription factor of the pathway, hypoxia-inducible factor (HIF), is activated and induces the expression of a set of genes, which cause a glycolytic switch, enhance cell survival and induce angiogenesis. While those are necessary physiological adaptive processes, they are also hallmarks of cancer and hypoxia signalling is often found activated in cancer. However, current knowledge about the regulation of the hypoxia signalling pathway is not able to satisfactorily explain the deregulation found in cancer. Therefore, the aim of this work was to discover new regulatory mechanisms that could be responsible for sustained hypoxia signalling and therefore might represent future therapeutic targets. We focused on how post-translational modifications affected the properties of the central proteins of the pathway. Using standard biochemical approaches and fluorescence lifetime imaging, we found that the ubiquitin specific protease 29 (USP29) is a deubiquitinase for HIF-1α. Moreover, USP29 is the first ubiquitin protease towards HIF-2α. USP29 de-conjugates poly-ubiquitin from HIF-α and prevents its proteasomal degradation, leading to its stabilisation even in normoxic conditions. Importantly, this action of USP29, while dependent on its catalytic activity, was not dependent on the classical prolyl-hydroxylation and pVHL-mediated ubiquitination, and therefore we propose that USP29 is a non-canonical HIF activator. As hardly any reports about USP29 were available, we next aimed to characterise this maternally imprinted protein. We found that USP29 itself was subject to poly-ubiquitin-mediated destabilisation, but was able to remove the ubiquitination itself and therefore auto-stabilise in a catalytic activity-dependent way. Furthermore, we demonstrated that USP29 formed homodimers in living cells. Finally, we found that USP29 mRNA levels correlated with disease progression and severity in prostate cancer and suggest that USP29 might become an attractive target for therapeutic targeting of hypoxia signalling in the future.

Cancer develops in many organs and tissues in the body through genetic and environmental modifications to acquire the hallmarks of cancer. The hallmarks of cancer allow the cells to become malignant and progress to a tumorigenic state. It has previously been shown in various carcinomas that HIF-2a, a key component in hypoxia adaptation, has a role in autonomous growth, the first hallmark of cancer. Ovarian cancer is the most lethal of the gynecological malignancies and accounts for 3% of new cases in women annually but is the fifth most common cause of death due to cancer. Here, it is shown in two ovarian carcinoma cell lines that HIF-2a is involved in in vitro and in vivo growth. It is also shown that the effect of HIF-2a is due to its role in autonomous growth and not vascularization with the use of in vitro spheroids. From recent findings in the laboratory the oxygen-stimulated translation initiation complex was discovered and HIF-2a is one of its components. In the absence of HIF-2a there is a downregulation in translation in hypoxia in ovarian carcinoma. This is also seen in a HIF-2a translational target, IGF1R and its downstream signaling pathway, which may be involved in autonomous growth as well as other hallmarks of cancer. Taken together, the data in this thesis presents the importance of HIF-2a in autonomous growth and cancer progression in ovarian carcinoma, as well as verifying its role in translation.

Limited oxygen availability (hypoxia) influences cell migration and invasion, but the underlying mechanisms are poorly understood. Much of the cellular response to hypoxia is regulated by a family of Hypoxia Inducible Factor (HIF) prolyl hydroxylases (PHD1-3), each of which is thought to regulate specific pathways.Their activity is dependent on the availability of oxygen and alpha-ketoglutarate but despite intensive studies their activity in vivo and their substrates are poorly defined. In this study we performed a quantitative proteomic screen to identify new substrates of PHDs. Co-immunoprecipitations using FLAG-tagged PHDs were performed under hypoxia to trap the enzyme-substrate interactions, and binding partners were identified by mass spectrometry. Actin was identified to interact with PHD3 specifically under hypoxia. Subsequently two defined prolyl residues in beta-actin were shown to be hydroxylated. Hypoxia-induced rearrangement of the actin cytoskeleton was shown to be dependent on PHD3 activity as a knockdown of PHD3 was sufficient to increase the intracellular G- to F-actin ratio. An increase in cell migration and invasion was also found to be dependent on PHD3 activity. Mutation of both hydroxylated prolyl residues led to a similar phenotype regarding actin rearrangement and cell migration. Using constantly active HIF-mutants, we could show that these PHD3-dependent pathways are independent of HIF. All together, this study shows a pro-invasive pathway linking HIF-independent oxygen-sensing pathways and actin signalling. However, the mechanism of how hypoxia-induced actin rearrangement leads to increased migration and invasion remains to be elucidated.

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.

Iron is an essential nutrient for many biochemical functions within the body. However, its flexible redox potential is a "double edged" sword that makes it both essential for life but also renders it potentially toxic. As iron excretion is limited, iron absorption is tightly regulated. Systemic iron homeostasis is controlled through the hepatic hormone hepcidin. Iron and other stimuli control hepcidin transcription. When produced hepcidin circulates through out the body and binds to the cellular iron exporter ferroportin (FPN1) expressed on duodenal enterocytes, macrophages, and hepatocytes causing FPN1's subsequent internalization and degradation. Ergo hepcidin function to control the efflux of iron from duodenal enterocytes, hepatocytes, and macrophages in to the blood stream. Cellular iron metabolism is controlled through iron regulatory proteins 1 and 2 (IRP1 and IRP2). IRPs repress or stabilize translation of mRNAs, encoding proteins of iron uptake, utilization, and storage, which contain iron responsive elements (IRESs) within their untranslated regions.This work examines in vivo regulation of hypoxia inducible factor 2 alpha (HIF2α) by IRP1. HIF2α is a transcription factor involved in the transcription of many genes involved in the body's response to hypoxic stress including, most notable, erythropoietin the hormone responsible for erythropoiesis. In chapter II it is hypothesized that translational de-repression of HIF2α mRNA by the absence of IRP1 leads to polycythemia through the induction of erythropoietin. Here it is demonstrated that IRP1 but not IRP2 is involved in the translational de-repression of HIF2α which subsequently leads to an accumulation of HIF2α protein in the kidneys of IRP1-/- mice. It is further demonstrated that the accumulation of HIF2α protein in the kidneys leads to an increase serum erythropoietin and subsequent polycythemia in IRP1-/- mice. In chapter III it is hypothesized that IRP1-/- mice will demonstrate a metabolic phenotype. Here it is demonstrated that IRP1-/- mice exhibit hypoglycaemia. Through the global knock out of IRP1 couple with the hepatocyte specific deletion of HIF2α we observe a partial rescue of the hypoglycaemic phenotype seen in IRP1-/- mice.
Le fer est un nutriment essentiel à de nombreuses fonctions biochimiques du corps. Cependant, son potentiel redox flexible est une épée à double tranchant qui rend le fer à la fois essentiel à la vie mais aussi potentiellement toxique. Alors que l'excrétion du fer est limitée, son absorption est amplement régulée. L'homéostasie systémique du fer est contrôlée par l'hormone hépatique hepcidine. Le fer et d'autres stimuli contrôlent la transcription de l'hepcidine. Une fois produite, l'hepcidine circule dans l'organisme et se fixe à la ferroportine, exporteur cellulaire du fer, qui est exprimé à la surface des entérocytes duodénaux, des macrophages et des hépatocytes causant son internalisation et ensuite sa dégradation. En conséquence, l'hepcidine fonctionne pour contrôler l'efflux du fer de ces cellules dans la circulation sanguine. Le métabolisme cellulaire du fer est contrôlé par les protéines de régulation du fer 1 et 2 (IRP1 et IRP2). Les IRP répriment ou stabilisent la traduction d'ARNm codant les protéines responsables de la capture, l'utilisation, et le stockage du fer, lesquels contiennent des éléments de réponse au fer (IRE) dans leurs régions non traduites. Ce travail examine la régulation in vivo du facteur inductible de l'hypoxie 2 alpha (HIF2α) par IRP1. HIF2a est un facteur de transcription impliqué dans la transcription de nombreux gènes incluant, le plus notable, l'érythropoïétine l'hormone responsable de l'érythropoïèse. Dans le chapitre II, l'hypothèse est que la dérépression traductionnelle de l'ARNm de HIF2α par l'absence de IRP1 conduit à la polyglobulie à travers l'induction de l'érythropoïétine. Ici il est démontré que IRP1, et non IRP2, est impliqué dans la dérépression traductionnelle de HIF2α qui mène à l'accumulation de la protéine HIF2α dans les reins des souris IRP1-/-, aboutissant à l'augmentation de l'érythropoïétine dans le sérum et conséquemment à la polyglobulie dans ces souris. Dans le chapitre III, L'hypothèse est que les souris IRP1-/- exhibent des anomalies métaboliques. Ici nous établissons que les souris IRP1-/- présentent un phénotype d'hypoglycémie. Il est ensuite observé que ce phénotype est partiellement secouru à travers l'élimination globale de IRP1 couplée à la délétion spécifique de HIF2α dans les hépatocytes.

Polychlorinated biphenyls (PCBs) are synthetic organic chemicals that persist in the environment and are known to be carcinogenic to humans. Virtually all of the deleterious effects of PCB 126, the most potent dioxin-like PCB, are mediated by the aryl hydrocarbon receptor (AhR). By means of the common cofactor ARNT, the AhR signaling pathway can crosstalk with the hypoxia signaling pathway. Regulated by hypoxia-inducible factors (HIFs), the hypoxia pathway mediates responses to environments of reduced oxygen availability (hypoxia). This dissertation specifically examines the crosstalk and interference between these two pathways in the context of PCB 126 exposure. The results of this dissertation show that the antagonistic relationship between the AhR and hypoxia signaling pathways affects the function and responses of both AhR and HIF-1Α. We provide substantial evidence that ARNT is indeed a crucial factor in both the AhR and HIF-1Α signaling pathways. Furthermore, this dissertation examines regulatory mechanisms involved in AhR-mediated gene expression and identifies epigenetic regulation as a critical factor in AhR target gene expression. In summary, this dissertation helped to improve the understanding of mechanisms of PCB 126 toxicity. Understanding the detrimental biological effects of these ubiquitous environmental pollutants might ultimately have significant implications for human health.

Rheumatoid arthritis (RA) is a chronic inflammatory disease with a significant impact on patients' quality of life. One of the well-described features in RA is hypoxia, together with increased infiltration of macrophages into the inflamed joints. Members of the Hypoxia-inducible factor (HIF) family play key roles in activating the transcription of hypoxia regulated genes. Regulation of HIFs, including the enzymes which regulate their stabilisation and transactivation, namely prolyl hydroxylase domain (PHD) enzymes and factor inhibiting HIF-1 (FIH-1), were the main focus of this thesis. This work aimed to shed more light on the influence of monocyte-to-macrophage differentiation on the Hypoxia/HIF axis as well as how PHDs are being regulated during the differentiation process and thus influence the HIF pathway. For this purpose I have used macrophages derived from a monocytic cell line namely THP-1, or from freshly isolated monocytes, derived from peripheral blood mononuclear cells. Monocytes were differentiated to either the classical (M1) or the alternative (M2) macrophages using granulocyte-macrophage colony stimulating factor or macrophage-colony stimulating factor respectively. THP-1 monocytes were differentiated to a M2-like phenotype using phorbol 12-myristate 13-acetate. Possible differences between the two macrophage phenotypes with regard to the Hypoxia/HIF pathway were also investigated in this study. One of the major findings was that macrophage differentiation leads to stabilisation of HIF-1α isoform even in normoxic (20 % O2) conditions. In particular, in PMA-treated THP-1 cells, the normoxic HIF-1α stabilisation was partly due to an increased HIF α gene transcription. Moreover, PHD-2 expression and enzymatic activity were also affected during the differentiation process, and were linked to reduced HIF hydroxylation and increased HIFα protein accumulation. However, expression of downstream HIF dependent angiogenic genes was not affected during the differentiation process, suggesting an additional level of control, mediated possibly by transcriptional inactivation of HIF via asparagine hydroxylation by FIH-1, or inhibition of the HIF α nuclear translocation machinery. Using the M1 and M2 polarised macrophages, there were no significant differences observed between the two phenotypes with regard to the HIF α isoform expression. In both phenotypes downstream gene expression was observed for Bcl2/adenovirus E1B 19d-interactin protein (BNIP3) and ephrinA3 (EPHRINA3), although in the M2 phenotype expression of both genes was significantly greater. Exposure of macrophages to the hypoxia mimetic dimethyloxaloyglycine (DMOG) stabilised both HIF α protein isoforms with no siginificant differences in expression levels, and led to transcription of the HIF α target genes BNIP3 and EphrinA3. Expresion of PHD-2 and PHD-3 were also increased in response to DMOG but more in the M2 macrophages. The effect of HIF stabilisation by DMOG was also studied in two different in vivo mouse models of arthritis; collagen induced arthritis (CIA) and antigen induced arthritis (AIA). HIF activation by DMOG was observed by imaging analysis, using a previously described transgenic mouse oxygen-dependent degradation domain (ODD)-luciferase reporter mice. It was demonstrated that DMOG activates the HIF pathway in vivo, and appears to exert a protective role in the setting of arthritis (reduced paw/knee swelling), possibly by promoting the expansion of the anti-inflammatory M2 macrophage population. Furthermore the effect of DMOG in anaemia of inflammation was also observed in the CIA mouse model. Arthritic mice showed decreased haematocrit levels, which were corrected back to normal levels after DMOG treatment. In conclusion my data contributes to a better understanding of the Hypoxia/HIF pathway in RA, and also suggest an additional possibly protective role of HIF possibly by shifting macrophages towards a less inflammatory phenotype, and also inhibiting anaemia of inflammation. These findings also set the basis for further research that will allow the development of new therapeutic strategies for the treatment of RA.

There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O2 tension. In high O2 tension (normoxia) the PHDs hydroxylate HIFα subunits on 2 conserved proline residues inducing binding of the von-Hippel-Lindau (VHL) tumour suppressor, the recognition component of a multi-protein ubiquitin-ligase complex, initiating HIFα ubiquitylation and degradation by the 26S proteasome. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIFα or as a multi-protein complex. In this thesis, data are presented that shows that the tumour suppressor protein LIMD1 acts as a molecular scaffold simultaneously binding the PHDs and pVHL into a normoxic protein complex (normoxiplex), increasing their physical proximity in order to enable efficient and rapid sequential modifications and thus degradation of HIF1α. Data are presented which indicates that increased LIMD1 expression down regulates HIF transcriptional activity, by promoting HIF1α degradation via the oxygen dependent degradation domain in a manner dependent on hydroxylase and 26S proteasome activities. However, degradation of this domain is not wholly dependent on the well characterised proline residues subject to hydroxylation, suggesting that LIMD1 may alter proline hydroxylation specificity or modulate HIF via a different mechanism. Furthermore, endogenous depletion of LIMD1 results in the converse, leading to HIF1α stabilisation and accumulation, enhancing HIF transcriptional activity. Moreover, Limd1-/- MEFs show increased HIF transcriptional activity. One mechanism by which this is achieved involves the binding of PHD2 within the N-terminal portion of LIMD1 while allowing concurrent binding of VHL to the C-terminal zinc-finger LIM domains. However, the LIMD1 mediated mechanism regulating HIF1α independently of proline residues 402 and 564 is still unclear. Finally, data are presented that show that the LIMD1 family member proteins Ajuba and WTIP all bind specifically to VHL but differentially to PHDs 1, 2 and 3 and thus these three LIM domain containing proteins represent a new group of hypoxic regulators.

L’hypertension artérielle pulmonaire (HTAP) est une maladie grave caractérisée par une obstruction progressive des artères pulmonaires de petit calibre, conduisant à une augmentation des résistances vasculaires pulmonaires et, à terme, à une défaillance cardiaque droite et au décès du patient. La vasoconstriction, le remodelage vasculaire et la dysfonction endothéliale pulmonaire sont autant de facteurs qui contribuent au développement et à la progression de la maladie. Plusieurs arguments sont également en faveur d’une hypothèse de désordres immunologiques, voire autoimmuns, dans la physiopathologie de l’HTAP. Malgré ces données, le lien entre endothélium pulmonaire et système immunitaire dans cette maladie restent peu connus. Ce travail de thèse a donc eu pour objectif d’étudier et mieux comprendre la nature et les conséquences d’une communication aberrante entre cellules endothéliales pulmonaires et système immunitaire dans la pathogénèse de l‘HTAP, afin d’identifier de nouvelles cibles thérapeutiques. Pour cela, nous avons analysé le rôle de la dysfonction endothéliale dans la régulation de deux processus de la dysfonction immunitaire : l’autoimmunité pour la réponse adaptative d’une part, et la sécrétion de cytokines en ce qui concerne la réponse innée d’autre part. A travers ce travail de thèse, nous avons mis en évidence l’existence d’une communication aberrante entre endothélium pulmonaire et système immunitaire dans l’HTAP et montré que l‘endothélium pulmonaire jouait un rôle primordial dans le contrôle des réponses adaptatives, en régulant la fonction des lymphocytes T régulateurs via la leptine, et dans la participation active à la réponse innée, en acquérant un phénotype pro-inflammatoire. Cette meilleure compréhension du rôle de la dysfonction endothéliale dans la dérégulation du système immunitaire présente dans l’HTAP pourrait aider au développement de nouvelles stratégies thérapeutiques dans cette maladie
Pulmonary arterial hypertension (PAH) is a severe disease characterized by a progressive obstruction of small pulmonary arteries, leading to an increase in pulmonary vascular resistance and ultimately right heart failure and death. Vasoconstriction, vascular remodeling and pulmonary endothelial dysfunction contribute to the disease development and progression. Increasing evidence are also suggesting the importance of immune disorders, such as autoimmunity, in PAH pathophysiology. Despite these data, the link between pulmonary endothelium and immune system is still unclear. The objective of this work was to investigate and elucidate the nature and the consequences of an aberrant communication between pulmonary endothelial cells and immune system in PAH pathogenesis, in order to identify new therapeutical targets. Therefore, we analyzed the role of endothelial dysfunction in the control of two types of altered immune responses: autoimmunity for the adaptive response and cytokine secretion for the innate response. In this work, we highlighted the existence of an aberrant communication between pulmonary endothelium and immune system in PAH and showed that pulmonary endothelium played a key role in the control of adaptive responses, by regulating regulatory T lymphocyte function in a leptin-dependent manner, and by actively participating to the innate responses through a pro-inflammatory phenotype. A better understanding of the role of endothelial dysfunction in PAH immune system dysregulation may help to the development of new therapeutical strategies for this disease

Sterol regulatory element binding proteins (SREBPs) are transcription factors that regulate the expression of genes involved in fatty acid and cholesterol biosynthesis. It has been established that SREBPs are regulated downstream of the PI3-Kinase/Akt/ mTORC1 signalling axis, a pathway that is frequently hyper-activated in cancer. SREBP target genes are upregulated in some forms of human cancer and a role for lipid metabolism in tumourigenesis has been suggested. Glioblastoma multiforme (GBM) is a cancer type that is associated with hyperactivation of the PI3-kinase/Akt signalling pathway and frequently displays poorly oxygenated (hypoxic) regions. SREBP1 has been implicated in the tumourigenic potential of this cancer type. However, the exact role of SREBPs in tumourigenesis is not known. In oder to investigate the SREBP-transcriptional response in cancer cells, a gene expression microarry analysis was carried out. It was found that SREBPs regulate genes involved in a variety of cellular processes including lipid metabolism, cell cycle regulation, redox regulation and cellular stress response. In addition, the role of SREBPs in lipid metabolism in hypoxia was investigated. It was found that hypoxia leads to distinct changes in the expression of different SREBP isoforms and their target genes and is associated with a decrease in pyruvate-dependent lipid synthesis and increased lipid storage. SREBPs are regulated downstream of the Akt/mTORC1 pathway, although the exact mechanism of this regulation remains to be elucidated. Possible mechanisms by which Akt and mTORC1 regulate SREBPs were investigated. It was found that inhibition of mTORC1 differentially affects the expression of individual SREBP isoforms. The results described in this thesis also show that mTORC1 modulates the transcriptional activity of mature SREBP and may regulate its stability in a GSK3-independent manner.

Cortisol is a glucocorticoid produced by the zona fasciculata (ZF) of the adrenal cortex. Traditionally, cortisol production and release was seen as being regulated strictly by adrenocorticotropic hormone (ACTH). While this is true of baseline cortisol levels and in response to acute mental stress, the picture is somewhat more complicated in other situations.Interleukin-6 (IL-6) contributes to the maintenance of cortisol levels in situations of prolonged immune or inflammatory stress. AMP activated protein kinase (AMPK) was investigated as a possible mediator of the action of IL-6 or as an independent actor in raising cortisol levels in response to hypoxemic or hypoglycemic stress.5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside (AICAR) was used to activate AMPK. Bovine ZF tissue fragments were exposed to AICAR alone and together with a known AMPK inhibitor, compound C. Protein or mRNA was then extracted from these tissue fragments. As an indicator of overall steroidogenic activity, these extracts were tested using RT-PCR and western blot assays for relative protein and mRNA levels of steroidogenic acute regulatory (StAR) protein, steroidogenic factor-1 (SF-1), and dosage sensitive sex reversal adrenal hypoplasia congenita gene on the X chromosome, gene 1 (DAX-1). Also a reporter gene assay was performed on H295R cells with a transfected StAR promoter.In bovine ZF tissue fragments, AICAR caused a significant increase of StAR protein and mRNA and SF-1 protein with a decrease of DAX-1 protein in a dose and time dependant manner. DAX-1 mRNA was shown to decrease in response to AICAR administration in a dose dependant manner. AICAR induced increases in StAR protein and SF-1 protein, and the attendant decrease in DAX-1 protein were all shown to be reduced by administration of compound C. This demonstrated that in this situation AICAR is acting through AMPK. When IL-6 was given with compound C the levels of StAR, SF-1, and DAX-1 were significantly reduced from samples treated with IL-6 alone. AICAR exposure also increased StAR promoter activity in a dose and time dependant manner. This AMPK induced increase in steroidogenic activity provides a possible mechanism for increased cortisol during hypoxia and hypoglycemia, and a possible mediator for IL-6 in the ZF.

In addition to host defense, alternative functions of immune cells are emerging. Immune cells are crucial during healing of injured tissue, in formation of new blood vessels, angiogenesis, and also in maintaining the balance in inflammation having immune regulating functions. Over the last decade a higher degree of heterogeneity and plasticity of immune cells have been reported and immune cells develop different characteristics in different situations in vivo. This thesis investigates roles for immune cells in situations of muscle hypoxia and reduced blood perfusion, wound healing in skin and at sites of transplantation of allogeneic islets of Langerhans and on top of this, ways to steer immune cell function for future therapeutic purposes. A specific neutrophil subset (CD49d+VEGFR1+CXCR4high) was found to be recruited to VEGF-A released at hypoxia and these neutrophils were crucial for functional angiogenesis. In muscle with restricted blood flow macrophages were detected in perivascular positions and started to express aSMA and PDGFR1b and were found to directly assist in blood flow regulation by iNOS-dependent NO production. This essential function in muscle regain of function could be boosted by plasmid overexpression of CXCL12 where the effect of these macrophages chaperoning the vasculature was amplified improving limb blood flow regulation. The effect on macrophages accelerating tissue regeneration being amplified by CXCL12 was tested in a model of cutaneous wound healing where the administration of CXCL12 was optimized for high bioavailability. In the skin, CXCL12-treatment induced accumulation of TGFb-expressing macrophages close to the wound driving the healing process, and subsequently the wounds healed with an efficiency never reported before. In the last study means to circumvent systemic immune suppressive therapy required in allogeneic transplantation was investigated. Allogeneic islets of Langerhans transplanted to muscle were immediately destroyed by the host immune system. Co-transplanting islets and CCL22-encoding plasmids we could curb this fast rejection for 10 days by accumulating CD4+CD25+FoxP3+ regulatory T lymphocytes at the site for transplantation preventing islet grafts from being attacked by the host cytotoxic T lymphocytes. In summary this thesis outlines distinct immune cell subsets being essential for regain of tissue function in hypoxia, ischemia and post injury and ways to amplify specific immune cell functions in these situations that are feasible for clinical use.

A hipóxia ocorre quando a demanda de oxigênio molecular necessário para gerar ATP é insuficiente. Os genes ativados por hipóxia compreendem o gene Hif-1a (Hipóxia-fator induzível 1a), Vegf-a (fator de crescimento endotelial vascular a), Arnt e Vhl (von Hippel-Lindau). Neste estudo foram utilizadas linhagens de camundongos geneticamente selecionados para alta (AIRmax) ou baixa (AIRmin) resposta inflamatória aguda (AIR). Foram realizados testes biológicos para caracterizar as reações inflamatórias produzidas por Biogel e TPA, bem como o tipo PAH cancerígeno. Testamos a expressão de mRNA de genes de hipóxia e caracterização de polimorfismo da região codificadora do Hif-1a no cromossomo 12. Camundongos AIRmax demonstraram uma maior reação inflamatória que os AIRmin para biogel e TPA enquanto o inverso foi observado com o DMBA. Os conjuntos de dados de fenótipos, expressão gênica e polimorfismo candidatam a região do cromossomo 12, que contém, entre outros, o gene Hif-1a, como participante da regulação da AIR.
Hypoxia occurs when the demand for molecular oxygen necessary to generate ATP is insufficient. Genes activated by hypoxia comprise the Hif-1a gene (Hypoxia-Inducible Factor 1a), Vegf-a (Vascular Endothelial Growth Factor a), Arnt and Vhl (von Hippel-Lindau). In this study we used lines of mice genetically selected for high (AIRmax) or low (AIRmin) acute inflammatory response (AIR). We conducted biological tests to characterize the inflammatory reactions produced by Biogel and TPA, and the type PAH carcinogen. We tested the mRNA expression of genes of hypoxia and characterization of polymorphism of the coding region of Hif-1a gene on chromosome 12. We found that the mice AIRmax had greater intensity of the inflammatory reaction that AIRmin to biogel and TPA while the reverse was observed with the DMBA. The data sets of phenotypes, gene expression and polymorphism applying the region of chromosome 12 that contains, among others, the gene Hif-1a, as part of the regulation of AIR.

Os sistemas de sinalização de dois componentes são sistemas prevalentes em bactérias, permitindo a adaptação a diferentes condições ambientais. O sistema de dois componentes classicamente possui uma proteína histidina quinase, o primeiro componente, capaz de reconhecer o estímulo ambiental e fosforilar o regulador de resposta, o segundo componente. Pseudomonas aeruginosa é uma proteobactéria ubíqua, capaz de infectar hospedeiros filogeneticamente distintos. Esse patógeno oportunista apresenta um dos maiores conjuntos de sistemas de dois componentes em bactérias, que permite que ela sobreviva numa grande gama de ambientes, incluindo humanos. P. aeruginosa UCBPP-PA14 apresenta pelo menos 64 histidina quinases e 76 reguladores de resposta codificados em seu genoma. Diversos sistemas de dois componentes já foram correlacionados com a virulência, sendo o sistema GacSA o exemplo melhor caracterizado. Há poucos estudos sistemáticos sobre o envolvimendo dos reguladores de resposta na virulência de P. aeruginosa e os sinais que induzem a ativação dos reguladores de resposta precisam ser encontrados. Para identificar novos reguladores de resposta envolvidos na patogenicidade, infecções in vitro em macrófagos e in vivo em Drosophila melanogaster foram realizadas neste trabalho. Os macrófagos foram infectados com cada mutante dos reguladores de resposta ou com a linhagem selvagem, e a produção da citocina pró-inflamatória TNF-α e o clearance bacteriano foram determinados. Alternativamente, as moscas foram infectadas utilizando-se a estratégia de feeding e a sobrevivência foi verificada. Utilizando-se essas abordagens, a identificação de diversos reguladores de resposta com papel na virulência foi alcançada, além de se corfirmar o papel de reguladores de resposta já estudados. Um dos novos genes envolvidos em virulência, PA14_26570 (nomeado neste trabalho de atvR), codifica um regulador de resposta atípico com substituição no aspartato fosforilável para glutamato, o que usualmente induz um estado sempre ativo. Um mutante não polar em atvR foi construído e macrófagos infectados com a linhagem ΔatvR confirmaram um maior clearance bacteriano e maior produção de TNF-α em comparação aos macrófagos infectados com a linhagem selvagem. Para comprovar a participação de AtvR durante a patogênese, um modelo de pneumonia aguda em camundongos foi utilizado. Camundongos infectados com a linhagem ΔatvR apresentaram uma maior sobrevivência em comparação aos camundongos infectados com a linhagem selvagem. Além disso, os camundongos infectados com ΔatvR apresentaram menor carga bacteriana, aumento no recrutamento de neutrófilos ativados e aumento na produção de citocinas pró-inflamatórias (TNF-α e IFN-γ). Utilizando-se uma abordagem transcritômica (RNA-Seq), foi determindo diversos genes são regulados positivamente na linhagem superexpressando AtvR em relação à linhagem controle. Dentre esses, os clusters de respiração anaeróbia nar, nir, nor e nos estão incluídos. Esse resultado foi confirmado por qRT-PCR e análises fenotípicas, em que a linhagem ΔatvR apresentou menor crescimento e expressão da nitrato redutase durante condições de hipóxia em comparação à linhagem selvagem. Em suma, neste trabalho foi demonstrado que diversos reguladores de resposta são importantes para a virulência de P. aeruginosa em macrófagos in vitro e in vivo em Drosophila, além de caracterizar o regulador de resposta atípico AtvR, que regula a respiração anaeróbica por desnitrificação, permitindo que P. aeruginosa possa infectar e colonizar o hospedeiro com maior eficiência.
Two-component systems are widespread in bacteria, allowing the adaptation to environmental changes. A two-component system is classically composed by a sensor kinase that phosphorylates a cognate response regulator. Pseudomonas aeruginosa is a ubiquitous proteobacterium able to cause disease in several hosts. This opportunistic pathogen presents one of the largest sets of two-component systems known in bacteria, which certainly contributes to its ability to thrive in a wide range of environmental settings, including humans. P. aeruginosa UCBPP-PA14 genome codes for at least 64 sensor kinases and 76 response regulators. Some response regulators are already known to be related to virulence, with the GacSA system as the best characterized. There are no systematic studies about the involvement of P. aeruginosa response regulators in virulence. Moreover, the input signal that triggers the response regulator activation is yet to be uncovered for most systems. To find new response regulators involved in virulence, in vitro infections werecarried out using macrophages. Briefly, the macrophages were infected with each response regulator mutant or the wild-type strain, the pro-inflammatory cytokine production (TNF-α) and the bacterial clearance were evaluated. Using this approach, we identified several response regulators involved in virulence, and we also confirmed the involvement of known response regulators in this process. One of the novel virulence-related response regulators, PA14_26570 (named here as AtvR), is an atypical response regulator with a substitution in the phosphorylable aspartate to glutamate, that usually leads to an always-on state. A non-polar mutant was constructed, and macrophage infection with ΔatvR confirmed an increased bacterial clearance as well as a higher TNF-α production as compared to the wild-type strain. To ascertain the role of AtvR during the pathogenic process, an acute pneumonia model was used. Mice infected with ΔatvR showed an increased survival as compared to mice infected with the wildtype strain. In addition, ΔatvR infected mice showed reduced bacterial burden, increased neutrophil recruitment and activation, as well as increased pro-inflammatory cytokine production (TNF-α and IFN-γ). Also, using a transcriptomic approach (RNASeq), we showed that several genes were upregulated in the strain overexpressing AtvR. These genes include the anaerobic respiration clusters nar, nir, nor and nos. This result was confirmed by qRT-PCR and phenotypic analysis, in which ΔatvR showed reduced growth and nitrate reductase expression during hypoxic conditions as compared to the wild-type strain. In conclusion, we have demonstrated that several response regulators are important for P. aeruginosa virulence in vitro. In addition, we further characterized the atypical response regulator AtvR, which regulates anaerobic respiration via denitrification, allowing this bacterium to infect and colonize the host more efficiently.

Hintergrund: Endothelzellen spielen sowohl in Homöostase als auch in Stresssituationen eine wesentliche Rolle bei der Regulation und der Erhaltung der hämatopoetischen Stammzellen (HSC). Viele Zytokine und Wachstumsfaktoren werden für die natürliche HSC-Aktivität benötigt und von den Endothelzellen exprimiert. Neben der Unterstützung der hämatopoetischen Stammzellaktivität stellen Endothelzellen ein Gefäßversorgungsnetzwerk zur Verfügung, um eine ausreichende Sauerstoffversorgung zu gewährleisten. Fragestellung: In der hier vorgestellten Arbeit habe ich die Hypothese verfolgt, dass eine Veränderung der Sauerstoffsensorik in Endothelzellen eine Modifizierung der Aktivität hämatopoetischer Stammzellen ermöglicht. Material und Methoden: Trotz des Wissens um die Bedeutung von Sauerstoff für die Endothelzellen fehlt uns das Verständnis dafür, wie die Sauerstoffsensorik der Endothelzellen die lokalen Knochenmark-Nischenzellen und die HSCs reguliert. Um einen Einblick in diesen Mechanismus zu erhalten, haben wir ein Mausmodell mit einem endothelzellenspezifischen Knockout des zentralen Sauerstoffsensors PHD2 entwickelt. Mit diesem in vivo-Ansatz habe ich versucht, den Einfluss von Veränderungen der Hypoxie-Signalwegproteine in Endothelzellen auf HSCs und ihre Nische zu untersuchen. Ergebnisse: Ich konnte in der vorliegenden Arbeit zeigen, dass die Morphologie der sinusförmigen Endothelzellen des Knochenmarks nach Verlust von PHD2 verändert ist. Außerdem konnte ich eine ausgeprägte Gefäßvasodilatation, begleitet von reduzierten hypoxischen Bereichen im angrenzenden Knochenmark beobachten. Zudem stellte ich fest, dass die Inaktivierung von PHD2 in Endothelzellen zu einem Rückgang des Knochenvolumens und zu einer Verminderung der an das Endothel angrenzenden Perizyten führt. Auffallend ist, dass sich gravierende Unterschiede in den hämatopoetischen Zellen der Peripherie zeigten, insbesondere war ein deutlicher Anstieg der zirkulierenden Leukozyten bei den KO-Mäusen zu erkennen. Dieser Phänotyp ist mit einer Vermehrung der hämatopoetischen Stamm- und Vorläuferzellen in Knochenmark und Milz verbunden. Darüber hinaus konnte ich zeigen dass die B-Zelldifferenzierung in der Milz vollständig zum Erliegen kommt, was zu einem signifikanten Rückgang der B-Zellen in der Marginalzone führt. Um die Funktionalität von Endothelzellen mit deletiertem PHD2 zu beurteilen, habe ich die Mäuse mit einer nicht-tödlichen Dosis ionisierender Strahlung behandelt. Die Analyse der endothelialen Zellregeneration im KO-Knochenmark zeigte eine Verminderung der Gefäßneubildung ohne Einfluss auf das gesamte Gefäßlumen im Vergleich zu unbehandelten Wurfgeschwistern. Außerdem stellte ich fest, dass sich in den ersten 3 Wochen nach der Bestrahlung bei Mäusen mit fehlendem PHD2 auf der Endothelzellenoberfläche das RBC-Kompartiments schneller regeneriert. Ich konnte ausschließen, dass dieser Effekt auf eine erhöhte Produktion von RBCs zurückzuführen ist, was zu der Hypothese führte, dass eine Verminderung des endothelialen PHD2 entweder zu einem längerfristigen Überleben der RBCs oder zu einer beeinträchtigten RBC-Clearance führt. Zusätzlich habe ich einen Anstieg in der Anzahl der quieszenten hämatopoetischen Vorläuferzellen bei Mäusen mit endothelialer PHD2-Deletion beobachtet, was darauf hindeutet, dass das endotheliale PHD2 Downstream-Signaling den Zellzyklus von hämatopoetischen Vorläuferzellen bei myeloablativen Stress beeinflusst. Abschließend konnte ich zeigen, dass ein Großteil der beobachteten Phänotypen bei KO-Mäusen durch den nachgeschalteten Transkriptionsfaktor HIF-2α vermittelt wird. Die Verwendung meiner selbstgenerierten Doppel-Knockout-Mauslinie, bei der erstmalig gleichzeitig PHD2 und HIF-2 in den Endothelzellen deletiert sind, führte zu eine vollständige Umkehrung des hämatopoetischen Phänotyps der bei den PHD2-Knockout-Mäusen im Steady-State beobachtet wurde. Zusätzlich wird der zuvor beobachtete signifikante Anstieg der Lymphozyten und der Rückgang der Erythrozyten- und Thrombozytenzahl in den Doppel-Knockout-Mäusen genetisch wiederhergestellt. Gleichzeitig reduziert sich die Entwicklung der marginalen B-Zellen im stationären Zustand wieder auf das Wildtyp-Niveau. Des Weiteren habe ich nach der Bestrahlung von Mäusen mit ionisierender Strahlung keine signifikanten Unterschiede zwischen WT und KO in ihrer hämatopoetischen Zellregeneration mehr feststellen können. Schlussfolgerungen: Zusammengenommen konnte ich in meiner Dissertation neue Eigenschaften von Hypoxie-Pathway-Proteinen in Endothelzellen mit Einfluss auf hämatopoetische Stamm- und Vorläuferzellen sowie auf verschiedene Kompartimente ihrer Nische demonstrieren; sowohl im stationären Zustand als auch nach Belastung durch Bestrahlung. Abschließend unterstreicht meine Arbeit die entscheidende Bedeutung der Sauerstoffsensorik im Knochenmark und gibt neue Einblicke in das Zusammenspiel zwischen dem Knochenmark-Endothel und dem hämatopoetischen System.:1 Introduction 1.1 Oxygen is necessary for survival of multicellular organisms 1.2 Oxygen sensing is necessary for induction of rapid cellular response 1.3 Endothelial and hematopoietic tissues together deliver oxygen 1.4 Hematopoietic stem cells give rise to blood cells 1.5 Regulation of HSCs is dependent on cells of the hematopoietic niche 1.6 Types of HSC regulation 1.7 Cancer therapy affects all highly proliferating cells 1.8 Irradiation stress disrupts hematopoietic stem cell niche signaling 1.9 Hypoxia induced signaling during recovery after irradiation 2 Aims of the thesis Aim 1: Determination of the role of endothelial PHD2 on the signaling towards the hematopoietic stem cells and its niche. 3 Materials and methods 3.1 Mice. 3.2 Histology, immunohistochemistry and immunofluorescence staining 3.2.1 Tissue processing prior to staining: 3.2.2 Staining: 3.3 Endothelial cell sorting 3.4 Mature hematopoietic cell isolation 3.5 Expression analysis 3.6 Hypoxyprobe 3.7 Quantitative image analysis 3.8 Bone structure analysis 3.9 Blood analysis 3.10 FACS analysis 3.11 Cell cycle analysis 3.12 RBC transfusion 3.13 Statistics. 4 Results 4.1 Determination of the role of endothelial PHD2 on the signaling towards the hematopoietic stem cells and its niche 4.1.1 Validation of the Flk1:cre line endothelial cell targeting 4.1.2 Characterization of the endothelial cell morphology and vessel function upon PHD2 inactivation 4.1.3 Loss of endothelial PHD2 leads to a decrease in bone marrow niche 4.1.4 Loss of PHD2 in endothelial cells leads to alterations of hematopoietic cells in the periphery 4.1.5 Significant increase of white blood cells in the periphery upon loss of endothelial PHD2 4.1.6 Loss of endothelial PHD2 does not impact the hematopoietic stem cells in the bone marrow. 4.1.7 Loss of PHD2 leads to increase in hematopoietic stem cells in the spleen 4.1.8 Loss of endothelial PHD2 impacts lineage-committed progenitors in bone marrow and the spleen 4.1.9 Loss of PHD2 in endothelial cells leads to an increase in frequency and activity of myeloid progenitors in bone marrow and the spleen 4.1.10 Loss of endothelial PHD2 impacts lymphocyte progenitors in secondary lymphatic organs but not in the bone marrow 4.2 Defining the impact of radiation exposure on the recovery of PHD2-deficient endothelial cells and its hematopoietic compartment. 4.2.1 Characterization of the non-lethal ionizing radiation damage to bone marrow endothelial and hematopoietic recovery 4.2.2 Loss of PHD2 from endothelium impacts endothelial recovery after myeloablative assault 4.2.3 Loss of PHD2 in endothelial cells and its subsequent effect on hematopoietic recovery following ionizing radiation exposure 4.2.4 Mechanism of increased RBC numbers is not due to an increase in hematopoietic stem cell frequency or activity in bone marrow or the spleen, 2 weeks after irradiation 4.3 Characterization of the influence of the transcription factor HIF-2α in mice lacking EC PHD2 4.3.1 The molecular signal transduction upon PHD2 inactivation is mediated by the HIF-2 transcription factor. 4.3.2 Loss of PHD2 and HIF-2α from endothelial cells partially rescues alterations in bone marrow endothelial cell morphology 4.3.3 Simultaneous loss of PHD2 and HIF-2α in endothelial cells completely reverses the hematopoietic phenotype observed in KO mice. 4.3.4 HIF-2α transcription factor induce signaling on endothelial cells that leads to marginal zone B cell impairment 4.3.5 Loss of PHD2 and subsequent stabilization of the HIF-2αtranscription factor is responsible for the increased recovery of RBCs following ionizing radiation 5 Discussion 6 References 7 List of abbreviations 8 Abstract 9 Zussamenfassung 10 Acknowledgements 11 Deklarations
Background: Endothelial cells have an essential role in hematopoietic stem cell (HSC) regulation and maintenance during homeostasis and stress. Many cytokines and growth factors are required for normal HSC activity and are expressed by the endothelial cells. Along the support of hematopoietic stem cell activity, endothelial cells provide vessel delivery network to ensure proper oxygen delivery. Despite the importance of oxygen on endothelial cells, we lack the understanding of how oxygen sensing in endothelial cells regulates the local bone marrow niche cells and HSCs. Hypothesis: I have hypothesized that by modulating oxygen sensor in endothelial cells I will be able to modify the activity of hematopoietic stem cells Material and methods: To gain insight into this system, we developed a mouse model with an endothelial cell-specific knockout of the central oxygen sensor PHD2. Using this in vivo approach I sought to determine the impact of changes in hypoxia pathway proteins in endothelial cells on HSCs and their niche. Results: First, I revealed that the morphology of bone marrow sinusoidal endothelial cells is altered upon loss of PHD2. I observed prominent vessel vasodilation accompanied by reduced hypoxic areas in their adjacent marrow. Moreover, I determined that inactivation of PHD2 in endothelial cells led to a decrease in bone volume and pericytes adjacent to endothelium. Remarkably, I observed profound differences in the hematopoietic cells of the periphery. Specifically, I observed a profound increase in circulating leukocytes of KO mice. This phenotype was related to an increase in hematopoietic stem and progenitor cells in bone marrow and spleen. Moreover, I found a complete impairment of B cell differentiation in the spleen, which consequently led to a profound decrease in marginal zone B cells. To assess the functionality of endothelial cells lacking PHD2, I subjected the mice to a non-lethal dose of ionizing radiation. Analysis of endothelial cell recovery in KO bone marrow revealed a decrease in the formation of new vessels without an impact on the overall vascular lumen compared to WT littermates. Similarly, I found an enhanced recovery of the RBC compartment during the first 3 weeks after irradiation in mice lacking PHD2 on endothelial cells. I excluded the possibility that this effect was due to an increased RBC production, which led to hypothesis that inhibition of endothelial PHD2 results in prolonged RBC survival or impaired RBC clearance. Additionally, I observed an increase in quiescent hematopoietic progenitors cells in mice lacking PHD2 in endothelial cells that implies that endothelial PHD2 downstream signaling impact cycling of hematopoietic progenitors upon myeloablative stress. Finally, I demonstrated that a majority of the observed phenotypes in KO mice are mediated by the downstream HIF-2α transcription factor. Using my unique self-made double knockout mouse line simultaneously lacking PHD2 and HIF-2 in their endothelial cells, I was able to reveal a reversal of the hematopoietic phenotypes observed in the single PHD2 knockout mice during steady state. Additionally, the previously observed significant increase in lymphocyte and decrease in erythrocyte and thrombocyte numbers was genetically rescued in double knockout mice. Similarly, marginal zone B cell development returned to wild-type levels during steady state. Moreover, after subjecting mice to ionizing radiation I did not observe any significant differences between WT and KO in their hematopoietic cell recovery. Conclusions: Taken together, during my thesis I was able to demonstrate novel properties of hypoxia pathway proteins in endothelial cells having an impact on hematopoietic stem and progenitor cells as well as different compartments of their niche; both during steady state and radiation stress. In conclusion, my work underscores the critical importance of oxygen sensor signaling in the bone/bone marrow and provides new insight into the interplay between the bone marrow endothelium and the hematopoietic system.:1 Introduction 1.1 Oxygen is necessary for survival of multicellular organisms 1.2 Oxygen sensing is necessary for induction of rapid cellular response 1.3 Endothelial and hematopoietic tissues together deliver oxygen 1.4 Hematopoietic stem cells give rise to blood cells 1.5 Regulation of HSCs is dependent on cells of the hematopoietic niche 1.6 Types of HSC regulation 1.7 Cancer therapy affects all highly proliferating cells 1.8 Irradiation stress disrupts hematopoietic stem cell niche signaling 1.9 Hypoxia induced signaling during recovery after irradiation 2 Aims of the thesis Aim 1: Determination of the role of endothelial PHD2 on the signaling towards the hematopoietic stem cells and its niche. 3 Materials and methods 3.1 Mice. 3.2 Histology, immunohistochemistry and immunofluorescence staining 3.2.1 Tissue processing prior to staining: 3.2.2 Staining: 3.3 Endothelial cell sorting 3.4 Mature hematopoietic cell isolation 3.5 Expression analysis 3.6 Hypoxyprobe 3.7 Quantitative image analysis 3.8 Bone structure analysis 3.9 Blood analysis 3.10 FACS analysis 3.11 Cell cycle analysis 3.12 RBC transfusion 3.13 Statistics. 4 Results 4.1 Determination of the role of endothelial PHD2 on the signaling towards the hematopoietic stem cells and its niche 4.1.1 Validation of the Flk1:cre line endothelial cell targeting 4.1.2 Characterization of the endothelial cell morphology and vessel function upon PHD2 inactivation 4.1.3 Loss of endothelial PHD2 leads to a decrease in bone marrow niche 4.1.4 Loss of PHD2 in endothelial cells leads to alterations of hematopoietic cells in the periphery 4.1.5 Significant increase of white blood cells in the periphery upon loss of endothelial PHD2 4.1.6 Loss of endothelial PHD2 does not impact the hematopoietic stem cells in the bone marrow. 4.1.7 Loss of PHD2 leads to increase in hematopoietic stem cells in the spleen 4.1.8 Loss of endothelial PHD2 impacts lineage-committed progenitors in bone marrow and the spleen 4.1.9 Loss of PHD2 in endothelial cells leads to an increase in frequency and activity of myeloid progenitors in bone marrow and the spleen 4.1.10 Loss of endothelial PHD2 impacts lymphocyte progenitors in secondary lymphatic organs but not in the bone marrow 4.2 Defining the impact of radiation exposure on the recovery of PHD2-deficient endothelial cells and its hematopoietic compartment. 4.2.1 Characterization of the non-lethal ionizing radiation damage to bone marrow endothelial and hematopoietic recovery 4.2.2 Loss of PHD2 from endothelium impacts endothelial recovery after myeloablative assault 4.2.3 Loss of PHD2 in endothelial cells and its subsequent effect on hematopoietic recovery following ionizing radiation exposure 4.2.4 Mechanism of increased RBC numbers is not due to an increase in hematopoietic stem cell frequency or activity in bone marrow or the spleen, 2 weeks after irradiation 4.3 Characterization of the influence of the transcription factor HIF-2α in mice lacking EC PHD2 4.3.1 The molecular signal transduction upon PHD2 inactivation is mediated by the HIF-2 transcription factor. 4.3.2 Loss of PHD2 and HIF-2α from endothelial cells partially rescues alterations in bone marrow endothelial cell morphology 4.3.3 Simultaneous loss of PHD2 and HIF-2α in endothelial cells completely reverses the hematopoietic phenotype observed in KO mice. 4.3.4 HIF-2α transcription factor induce signaling on endothelial cells that leads to marginal zone B cell impairment 4.3.5 Loss of PHD2 and subsequent stabilization of the HIF-2αtranscription factor is responsible for the increased recovery of RBCs following ionizing radiation 5 Discussion 6 References 7 List of abbreviations 8 Abstract 9 Zussamenfassung 10 Acknowledgements 11 Deklarations

Zhang, Jian.
Thesis Ph.D. Chinese University of Hong Kong 2015.
Includes bibliographical references (leaves 154-173).
Abstracts also in Chinese.
Title from PDF title page (viewed on 10, November, 2016).

碩士
國立臺灣大學
生理學研究所
101
Hypoxia has been intensively investigated over the past decades based on the observations that hypoxic tumors were more resistant to therapy and had a worse prognosis. Previously, our lab identified that N-myc downstream-regulated gene 1 (NDRG1) was strongly up-regulated under hypoxia and may play an important role in tumor adaptation to fluctuation of oxygen concentrations. However, the regulatory mechanism of NDRG1 under hypoxia remains elusive. Therefore, the purpose of this study is to identify the novel transcription factors that regulate NDRG1 upon changes in oxygen concentrations. First of all, bioinformatic tools, MatInspector and MatchTM 1.0, were used to search the DNA binding sites of transcription factors in the promoter of NDRG1. Based on the similarities and numbers of transcription factor binding sites existing in the NDRG1 promoter (-783 ~ +312 bp), aryl hydrocarbon receptor (AHR) was identified as the most potential candidate and herein chosen for further validation. Western blotting showed that nuclear AHR was up-regulated in the presence of cobalt and hypoxia. Luciferase reporter assays showed that binding site of AHR at -402 ~ -398 bp played a crucial role in regulating NDRG1 under hypoxia-mimicking conditions. Moreover, hypoxia-mimetic induction of NDRG1 by was attenuated by knockdown of AHR expression using short interfering RNA. In summary, these results showed for the first time that AHR positively regulates NDRG1 transcription through a putative AHR binding site in the promoter by hypoxia-mimetic signaling, which may lead to development of a specific therapeutic regime to prevent tumor malignancy under hypoxia.

博士
國立中興大學
分子生物學研究所
105
Purpose: Forkhead box C1 (FOXC1) belongs to the forkhead family of transcription factors which with helix-turn-helix forkhead-like DNA binding domain. More and more reports indicate that FOXC1 is involved in various tumor progressions. However, the mechanism of tumor hypoxia in FOXC1 regulation and its role on lung cancer progression remain unclear. Results: We find that the expression of FOXC1 was increased in hypoxic areas of lung cancer tissues from rodents or humans. Hypoxia activated FOXC1 transcription through binding of hypoxia-inducible factor-1α (HIF-1α) to the hypoxia-responsive element (HRE) in the FOXC1 promoter via chromatin immunoprecipitation assay. FOXC1 overexpression in lung cancer CL1-0 cells promoted proliferation, migration, invasion, angiogenesis, and epithelial–mesenchymal transition in vitro, whereas FOXC1 shRNA in lung cancer CL1-5 cells inhibited these effects. Moreover, we used specific HIF-1-mediated FOXC1 shRNAs in lung cancer xenograft models to knockdown the tumor hypoxia-induced FOXC1 expression and could suppress tumor growth and angiogenesis through immunofluorescence staining. Finally, systemic delivery of FOXC1 siRNA with 2’-O-methyl modification encapsulated in lipid nanoparticles from tail vein inhibited tumor growth and increased survival time in lung cancer xenograft mice. Conclusion: These results point out that FOXC1 is a novel hypoxia response gene and plays a critical transcriptional role in tumor microenvironment which promoted lung cancer progression. Systemic delivery FOXC1 siRNA to decrease FOXC1 expression may be an effective therapeutic development for lung cancer. Keywords: Tumor hypoxia, HIF-1α, Lung cancer, FOXC1

博士
國防醫學院
生命科學研究所
89
The function of the dorsomedial (DM), the rostral ventrolateral (RVLM) and the caudal ventrolateral medulla (CVLM) in the cardiovascular control is well documented. However, in pathophysiological conditions such as cerebral ischemia or hypoxia, only the RVLM has been well investigated. The DM and the CVLM, received much less attention. The aims of this dissertation were to investigate the functional changes and the mechanisms of changes of the DM, the RVLM and the CVLM in the cardiovascular control after different degrees of hypoxia in anesthetized cats. Hypoxia of various degrees (I - III) was produced by inhalation of 5% O2 and 95% N2 for different durations. The responses of systemic arterial pressure (SAP) and vertebral nerve activity (VNA) elicited by microinjection of glutamate (0.1M, 70nL) into the DM, the RVLM and the CVLM were used to assess the neuronal functions. It was found that hypoxia has differential effect on the Glu-induced SAP and VNA responses of the DM, the RVLM and the CVLM in intact cats. For the DM, the Glu-induced sympathoexcitatory responses were depressed after all degrees of hypoxia. For the RVLM, post-hypoxia depression was found only when a moderate degree of hypoxia (II and III) was reached. For the CVLM, the Glu-induced responses virtually remained unchanged after all degrees of hypoxia. It is also indicated that such depression was not due to the depletion of norepinephrine storage in postganglionic sympathetic terminals, since intravenous administration of tyramine after different degrees of hypoxia produced similar pressor responses in intact cats. Precollicular decerebration prevented the post-hypoxia depression in the RVLM, but for the DM it was effective only after hypoxia I. Baro- and chemo-receptor denervation effectively abolished the post-hypoxia depression of the DM and the RVLM. To further investigate the mechanism of post-hypoxia depression, various intensities of electrical stimulation (50 - 200 mA, 80 Hz, 0.5 msec, 10 sec) were applied to the DM and the RVLM before and after hypoxia I. The post-hypoxia I depression was observed only for SAP response induced by 50 mA stimulation in the DM. The post-hypoxia I depression were not observed both in the DM following a higher intensities of electrical stimulation and in the RVLM following any intensities of electrical stimulation. Intravenous phenylephrine- or nitroprusside-induced SAP responses were also not changed before and after hypoxia I. These results suggest that the nearby sympathetic neurons or their processes adjacent to the stimulating sites in the DM may also involve in such depression. After hypoxia I, the sympathoexcitatory responses induced by microinjection of NMDA and AMPA into the DM were also depressed. Pretreatment with various Glu receptor antagonists, kynurenic acid (KYN, 10mM) and DL-2-amino-5-phosphonopentanoic acid (AP5, 100mM) in the DM, the post-hypoxia I depression was completely abolished. However, pretreatment with 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 2mM) was not effective in preventing such depression. In summary, the DM apparently is more susceptible to hypoxia than the RVLM, while the CVLM is almost resistant to hypoxia under the condition of the present study. The peripheral baro- and chemo-receptors and the suprapontine structures evidently play an important role in post-hypoxia depression. In the DM, the post-hypoxia I depression may be mediated via AMPA and NMDA receptors, and particularly, the later was predominant.

碩士
中華醫事科技大學
生物科技研究所
98
Lung cancer is the major cause of malignancy-related deaths worldwide, and its incidence is rising in many countries. It is notorious for its low cure rate, and the treatment of lung cancer is usually compromised by metastasis. In the processes of tumor growth, a rapidly growing tumor can induce lack of oxygen and develop a hypoxia environment. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that regulates genes involved in angiogenesis and metastasis. Andrographolide (Andro) is isolated from a traditional Chinese herbal medicine, Andrographis paniculate, and has been reported to possess a variety of pharmacological activities. Our previous study has been demonstrated that Andro can inhibit human lung cancer A549 cell migration and invasion which was associated with the inhibition of intracellular protein kinase cascades. In this study, we investigate whether Andro is an inhibitor of HIF-1α and its regulatory proteins, mediating downstream invasive or angiogenic signals. Using Western blotting and RT-PCR assay, it was revealed that the decreased expression of HIF-1α and vascular endothelial growth factor (VEGF), and the increase expressions of hydroxyl-HIF-1α (Pro564) and prolyl hydroxylase PHD2 in the Andro-treated A549 cells. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1α inactivation, because that it DNA binding activity was suppressed by Andro. In conclusion, we have elucidated the HIF-1α-VEGF signals was regulated by Andro. These results highlight the potential therapeutic effects of Andro, which may be developed as a chemotheraptic or an anti-angiogenesis agent in the future.

碩士
中國醫藥大學
基礎醫學研究所碩士班
100
Myocardial infarction (MI) is the common cause of cardiomyocyte death. Even hypoxia alone is sufficient to induce apoptosis of cardiomyocytes. In hearts, autophagy play important roles in hypoxia-mediated cardioprotection or myocardial injury effects. To date, the hypoxia-inducible factor-1α (HIF-1α) transcriptional factor and the BH-3 only protein, Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3), are known to play fundamental roles in adaptive or death process in response to hypoxia. In addition, hypoxia induces insulin-like growth factor binding protein 3 (IGFBP-3) to block the IGF1R/PI3K/Akt survival pathway. Therefore, we aim to investigate the molecular mechanisms and the correlation of HIF-1α, BNIP3 and IGFBP-3 in hypoxia-induced cardiomyocytes injuries. In the present study, heart-derived H9c2 cells and neonatal rat ventricular myocytes (NRVMs) were incubated in normoxic (21% oxygen) or hypoxic (1% oxygen) conditions for up to 48 h. Results showed that hypoxia primarily highly increased HIF-1α expression, then activated downstream genes such as BNIP3 and IGFBP-3, and further triggered mitochondria-dependent apoptotic pathways. Moreover, IGF1R/PI3K/Akt signaling obviously attenuated by up-regulated expression of HIF-1α-dependent IGFBP-3 expression to enhance hypoxia-induced cell apoptosis. In addition, suppression of autophagy with 3-methyladenine (3MA) or siRNA of ATG5 or Beclin-1 significantly decreased the myocardial apoptosis under hypoxic conditions. The data also showed that the activation of autophagy during hypoxia was obviously induced by Forkhead box O3 (FoxO3a)-dependent BNIP3 expression. Importantly, knockdown of FoxO3a or BNIP3 significantly abrogated hypoxia-induced autophagy and mitochondria-dependent apoptosis effects. Taken together, our present results confirmed that autophagy is a pivotal regulator for hypoxia-induced cardiomyocyte apoptosis modulated by FoxO3a-dependent BNIP3 expression. Moreover, prolonged-hypoxia induced HIF-1α not only stimulated BNIP3 expression but also enhanced IGFBP-3 activation to inhibit IGF1R/PI3K/Akt survival pathway and mediate mitochondria-dependent cardiomyocyte apoptosis. We believe that HIF-1α and FoxO3a blockage are sufficient to annul the change of excessive hypoxia of hearts.

碩士
長庚大學
復健科學研究所
94
Background and purpose: Prolonged hypoxic acclimatization alters aerobic metabolism and may improve exercise performance. However, acute hypoxia results in oxidative stress and produces an increase in leukocyte reactive oxygen species (ROS) release, adherence in vessels and eventually damages underlying tissue. Platelet-neutrophil/eosinophil aggregation plays an essential role in allergic disease and inflammation. In addition, acute, vigorous exercise induces platelet-leukocyte aggregation and subsequently activates leukocyte to release ROS. Whether intermittent hypoxia (IH) influences exercise-induced oxidative stress and platelet-neutrophil/eosinophil aggregation is unclear. This study investigates different dosage of IH, 1h daily, 5 days a week for 8 weeks on the following issue: 1) exercise performance in heath young men; 2) platelet-neutrophil/eosinophil aggregation induced by exercise ex vivo; 3) inflammatory cytokine and antioxidant capacity. Methods: 21 health young men randomly received either 4500m (12% O2) or 3000m (15% O2) of IH 1h daily, 5 days a week for 8 weeks. Progressive graded exercise test acute hypoxia test were performed and acid-base equilibrium, cell count profile, platelet-neutrophil/eosinophil aggregation, cytokine and antioxidative capacity were determined at 0-, 4- and 8-wk. Results: 1) 12% and 15% IH significantly increase VO2max and anaerobic threshold. 2) Blood pH value is unchanged during 15% acute hypoxia, whereas pH raises during 12% acute hypoxia and is unchanged at 4-wk. 3) total leukocyte count is unchanged during 15% acute hypoxia, whereas total leukocyte count decreases during 12% acute hypoxia and is unchanged at 8-wk. 4) Platelet-eosinophil aggregation is unchanged during 15% hypoxia. However, platelet-eosinophil aggregation increases during 12% acute hypoxia and further increases after exercise at 4-wk. 15% hypoxia suppresses platelet-eosinophil aggregation induced by exercise at 4-wk. 12% hypoxia suppresses the aggregation at 8-wk. In addition, platelet-neutrophil aggregation is unchanged during acute hypoxia and after exercise. 5) After 8-wk of IH, exercise-induced IL-1β is suppressed and IL-6 as well as IL-10 increase. 6) 4 wk of 12% IH decreases Vitamin A, Vitamin E and total antioxidant capacity. Vitamin A and total antioxidant capacity recover to baseline at 8-wk. Vitamin E increases at 8-wk. Conclusion: 12% and 15% IH improve exercise performance. Although 15% and 12% IH of 8-wk suppress exercise-induced platelet-eosinophil aggregation, 12% IH of 4-wk enhances the aggregation. We suggest the extent of platelet-eosinophil aggregation is associated with inflammatory cytokines and antioxidative capacity. Accordingly, 15% IH is relatively safe prescription applied to exercise performance.

Indiana University-Purdue University Indianapolis (IUPUI)
Rationale: CD55 down-regulation on airway epithelium correlates with local complement activation observed in hypoxia-associated pulmonary diseases. Therefore, we hypothesized that induction of hypoxia inducible factor 1 alpha (HIF-1α) in hypoxic airway epithelium, mediates CD55 down-regulation. Methods: Chetomin and HIF-1α siRNA inhibited HIF-1α in hypoxic SAECs (1% O2), and mice lungs (10% O2). DMOG mediated HIF-1α stabilization in normoxic SAECs and mice lungs (21% O2). Transduction of SAECs with AdCA5 also stabilized HIF-1α. CD55 and CA9 transcripts were measured by RT-PCR. CD55 and HIF-1α protein expression was assessed by western blots. In vivo, immunohistochemistry (IHC) confirmed CD55 and HIF-1α expression. C3a and C5a levels in bronchoalveolar lavage fluid (BALF) were measured by ELISA. Results: HIF-1α was induced in 6 hour hypoxic SAECs (p<0.05), but CD55 transcripts were repressed (p<0.05). CD55 protein was down-regulated by 72 hours (p<0.05). CA9 transcripts were elevated by 48 -72 hours (p<0.05 and p<0.01, respectively). In vivo, CD55 transcripts and protein were down- regulated by 24 hours post-hypoxia (p<0.01) which corresponded to complement activation (p<0.05) in BALF. However, CA9 was increased (p<0.01). Chetomin (100nM) treatment in 6 hour hypoxic SAECs, recovered CD55 transcripts (p<0.01) and protein (p<0.05), but down-regulated CA9 (p<0.05). Similarly, in vivo chetomin (1mg/ml) treatment recovered CD55 protein (p<0.01) and down-regulated CA9 (p<0.01). Silencing HIF-1α (50nM) in hypoxic SAECs restored CD55 transcripts by 6 hours (p<0.05), and protein expression by 24 hours (p<0.05). However, CA9 was repressed (p<0.01). In vivo silencing of HIF-1α (50µg) restored CD55 protein expression (p<0.05) but down-regulated CA9 (p<0.05). Stabilizing HIF-1α in normoxic SAECs via DMOG (1µM), down-regulated CD55 transcripts and protein (p<0.01), but increased CA9 within 6-24 hours (p<0.05 and p<0.01, respectively). HIF-1α induction by DMOG (1mg/ml) in normoxic mice lungs down-regulated CD55 transcripts (p<0.01) and protein (p<0.01), but increased CA9 (p<0.05). Induction of HIF-1α in AdCA5 (50 PFUs/cell) transduced normoxic SAECs, resulted in CD55 protein down-regulation (p<0.05), but increased CA9 (p<0.001). Conclusions: HIF-1α down-regulates CD55 on airway epithelium. Targeting this mechanism may be a potential therapeutic intervention for attenuating complement activation in hypoxic pulmonary diseases.

碩士
國立清華大學
分子與細胞生物研究所
101
Methylation/demethylation of histone tail, one of the major post-translational modifications (PTMs) in chromatin, plays an important role in chromatin remodeling to regulate gene expression. Dysregulation of these processes links to carcinogenesis. KDM8 (Jmjd5), an H3K36me2 histone demethylase containing jmjC domain, is crucial to regulate embryonic development, osteoclastogenesis and cell proliferation. Along with its general over expression in breast cancer, KDM8 is suggested to play a role in carcinogenesis. Swiftly proliferating cancer cells that encounter intermittent hypoxia trigger the expression of a master regulator, Hypoxia-inducible factor (HIF). We have previously found that KDM8 interacted with HIF1-α and was up-regulated under hypoxia (by Dr. Hung-Jung Wang). Moreover, knockdown KDM8 inhibited breast cancer cell metastasis (by So-Fang Yang). In this study, we aim to address the mechanism in which KDM8 is involved in cell proliferation under hypoxia, as well as tumor metastasis. A significantly reduced level of cell proliferation was found in KDM8 knockdown cells, especially when these cells were cultured under hypoxia. Knockdown cells were arrested in both G1/S and G2/M phases. Several cell cycle transition regulatory proteins including cyclins, cyclin-dependent kinases (CDKs), and cyclin-CDK complex inhibitors were affected accordingly. Moreover, KDM8 knockdown cells significantly lost hypoxia-induced c-Myc and p53 transactivation activity. And several epithelial mesenchymal transition inducers which belong to downstream targets of canonical Wnt signaling exhibited a lower level of expression, suggesting a link to hypoxia-induced Wnt signaling pathway.

碩士
中臺科技大學
藥物科技研究所
104
Abstract Fenofibrate (FF) is a lipid-lowering agent that can suppress tumorigenesis in many cancers. In the present study, we demonstrated that treatment of human U87MG glioma cells with FF decreased the protein levels of HIF-1α under normoxia and hypoxia conditions. The suppression of HIF-1α was reversed by pretreatment with proteasome inhibitor. The reduction of HIF-1α level was associated with decreased expression levels of glucose transporter Glut-1, hexokinase-2 (HK-2) and lactate dehydrogenase A (LDHA). On the other hand, treatment with FF also decreased the expression of pyruvate dehydrogenase kinase-1 (PDK-1) and pyruvate dehydrogenase phosphorylation (p-PDH), which reactivated mitochondria flux of acetyl CoA and TCA cycle. However, FF decreased mitochondria membrane potential presumably by reactive oxygen species (ROS) induced mitochondrial damage. These results suggest that FF may not only suppress the Warburg effect and reactive mitochondria oxidative phosphorylation but also increase ROS accumulation which leads to mitochondrial damage and subsequent cell death. Key word: Fenofibrate (FF), Warburg effect , HIF-1α, energy metabolism, ROS

博士
國防醫學院
生命科學研究所
101
A shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis is the biochemical hallmark of malignant cancer cells. In the present study, we demonstrated that Nodal stimulated the expression of glycolytic enzymes and decreased reliance on mitochondrial oxidative phosphorylation in human glioma cancer cells. The shift in glucose metabolism was mediated by induction of the hypoxia-inducible factor (HIF). Nodal protein expression was shown to be correlated with expression levels of glucose transporter (Glut)-1, hexokinase (HK)-II, and pyruvate dehydrogenase kinase (PDK)-1, the phosphorylation level of pyruvate dehydrogenase (PDH), glucose uptake, and lactate accumulation in human glioma cells. These effects were inversely correlated with mitochondrial oxygen consumption and ATP production. Knockdown of Nodal expression with specific small-hairpin RNA reduced Glut-1, HK-II, and PDK-1 expressions and PDH phosphorylation. Nodal knockdown also reduced glucose uptake and lactate generation, which in turn increased the mitochondria membrane potential (Ψ), O2 utilization, and ATP synthesis. The ectopic expression of Nodal in low-expressing Nodal glioma cells resulted in opposite results compared to those of Nodal-knockdown glioma cells. Treatment of cells with recombinant Nodal increased HIF-1 expression, and this effect was regulated at the transcriptional level. Blockage of the Nodal receptor by a pharmacological inhibitor or Nodal knockdown in U87MG cells decreased HIF-1α expression. Furthermore, HIF-1α knockdown in U87MG cells decreased Glut-1, HK-II, and PDK-1 expressions and PDH phosphorylation, which were similar to results in Nodal knockdown cells. Taken together, these results suggest that Nodal affects energy metabolism through HIF-1α.

碩士
國立臺灣大學
動物學研究所
100
Hypoxia is known to regulate gene expression via hypoxia- inducible factors (HIFs) and plays a role in many developmental processes, including vasculogenesis, angiogenesis, heart and central nervous system development. The Hif1α knockout mice shows defective heart development, including cardia bifida and abnormal neural crest migration. Recently, we have observed heart left-right asymmetric defects and cardia bifida in zebrafish embryos deficient in lysophosphatidic acid (LPA) synthesizing enzyme autotoxin and on of its receptors, LPAR3. LPA signaling stimulates cell proliferation, cell migration, survival of many cell types, tumorigenesis, angiogenesis, and metastasis. More interestingly, hypoxia is known to enhance LPA- induced Hif1α expression. Therefore, I hypothesize that Hif1α may regulate heart development through the Atx-Lpar3 pathway. Thus, I tested the idea that LPA may interact with HIF signaling to mediate cardiogenesis. Using the MO knockdown approach, I demonstrate that Hif1α and Lpar3 reciprocally regulate each other’s gene expression and rescues respective cardiac defects. Also, I investigate the connection between Hif1α and Atx. These results suggest that Hif1α, Atx and Lpar3 work together to mediate cardiac development.