Hypoxic chemotransmission in the rat carotid body (CB) is mediated in part by ATP acting on P2X urinoceptors. Here we use RT-PCR, cloning, and sequencing techniques to show P2X2 and P2X3 receptor expression in petrosal neurons (PN). Confocal immunofluorescence indicated co-localization of P2X2 and P2X3 protein in many petrosal neurons and CB afferent terminals in situ. Petrosal and nodose neurons share a similar developmental origin (i.e. the ectodermal placodes) and GDNF is a required survival factor for both the neuronal groups. However, in the present study the profiles of P2X receptor subunit expression in the petrosal and nodose neurons differed from each other. The PNs showed a slight increase in the expression of P2X2 and P2X3 mRNA by P-21, whereas in nodose neurons the corresponding expression levels peaked by P-9, but fell significantly by P-21. However, in both cases the expression levels for the P2X2 mRNA were higher than those of P2X3 mRNA and the expression profile for both the P2X subunits developed along a parallel time course. Previous developmental studies have indicated that expression levels of various autoreceptors on type I cells (e.g. DA D2-receptors, A2a and A2b adenosine receptors) change during the first week of postnatal life, as the animal breathes normoxic air. In the present study, the possibility that oxygen tension might regulate P2X receptor expression on type I cells was examined. RT-PCR was employed to compare expression levels of the P2X subunits in isolated type I cells grown under different 0₂ tensions. Compared to nonnoxic (20% 02) cell clusters grown under chronic hypoxia ( 6% O₂). Immunofluorescence studies cells grown under CHox. Thus, ATP and P2X2/P2X3 purinoceptors play an important role under normal conditions and following exposure to chronic hypoxia. / Thesis / Master of Science (MSc)

Acute Lung Injury, characterised clinically as the Acute Respiratory Distress Syndrome is a catastrophic response to a range of pulmonary and non-pulmonary insults. Despite much work the key mechanisms involved in generating the exaggerated immune response that results in lung injury are not completely understood. Hypoxia-inducible factor-1 has been shown to be a key transcription factor in the myeloid cell response to inflammatory signals. The aims of this thesis were to develop a model of acute lung injury and to study the role of Hif-1 in the generation of lung injury in this model. A model of direct pulmonary injury as a result of intratracheal instillation of endotoxin is described. Using this model the role of myeloid cell Hif-1α was characterised using a myeloid cell specific conditional knockout system. The injury in Hif-1α deficient mice was quantitatively similar to the injury seen in wild type animals over a range of time points. However, the quality of the injury, assessed by a measure of nitric oxide mediated damage was reduced. The in vivo data were supported by in vitro studies using a murine macrophage cell line which showed that manipulation of the cellular oxygen tension in the presence of endotoxin alters the ability of the cell to generate nitric oxide. Furthermore, pharmacological manipulation of cellular Hif-1 levels by Dimethyloxallyl Glycine (DMOG) in the macrophage cell increased the generation of nitric oxide in response to endotoxin by altering the expression of a number of the isoforms of Nitric Oxide Synthase. In a final set of experiments the response to intratracheal endotoxin was modulated in mice by the concurrent administration of DMOG. As expected the qualitative response to endotoxin was similar but the NO mediated damage was enhanced in the animals administered DMOG. Manipulation of Hif-1 may have a role in the therapy of lung injury by altering the characteristics of the response.

Tumors need the development of new vessels from the pre-existing vasculature to bring nutrients and oxygen to the whole tumor mass. The tumor vascular network is known to be poorly functional due to architectural and functional abnormalities. The end result is an inadequate and heterogeneous tumor perfusion leading to the development of tumor hypoxia. From a therapeutic perspective, hypoxia is a source of radioresistance and the dysfunctional perfusion hampers drug delivery. Historically, tumor hypoxia refers to chronic hypoxia (or diffusion-limited hypoxia) that results from the increasing distance between O2-consuming cells and blood vessels due to the high metabolic rate of tumor cells. Many studies have demonstrated the impact of chronic hypoxia on the clonal selection of tumor cells resistant to conventional anti-cancer therapies. Growing evidence for the existence of another form of hypoxia caused by heterogeneities in tumor perfusion, namely acute or perfusion-limited hypoxia, plead however for a non-genetic source of phenotype conversion reaching not only tumor cells but also the tumor vasculature and in particular endothelial cells. In the cardiovascular field, the cyclic exposure to different pO2 levels is known to precondition cardiac myocytes to resist more prolonged ischemic insults. We hypothesized that this concept of myocardium preconditionning to promote the resistance vs pro-apoptotic stresses could be translated in tumors. Indeed, intermittent hypoxia in tumors is nothing else than cyclic changes in pO2 and radio- and chemotherapy can be viewed as pro-apoptotic stresses that the tumor can face. In particular, in the case of the tumor vasculature, the resistance could be a capacity to re-initiate angiogenesis after treatment. Radioresistance would be further potentiated since low pO2 is per se associated to reversibility of the damages. Also, since intermittent hypoxia is thought to be due in part to fluctuations in tumor blood flow (TBF), access of chemotherapy to the tumor could also further participate to chemoresistance. To address the above hypotheses, we first aimed to explore the extent and the origin of TBF fluctuations in tumor mouse models and to determine whether therapeutic modulation of such potential TBF heterogeneities could improve the efficacy of chemotherapy. We then more directly examined whether and how intermittent hypoxia could influence endothelial cell survival and modulate resistance to radiotherapy. We also took advantage of this study to dissect the molecular mechanisms driving the phenotype conversion of endothelial cells exposed to intermittent hypoxia. Finally, because VEGF plays a major role in hypoxia-mediated angiogenesis but also regulates major pro-survival pathways in endothelial cells, we evaluated the potential role of caveolin as a new therapeutic target to tackle EC resistance. Caveolin is, indeed, a key structural protein recently documented to interact with many downstream targets of VEGF. 1. To explore the extent and the origin of TBF fluctuations in tumor mouse models and to determine whether therapeutic modulation of such potential TBF heterogeneities could improve chemotherapy. We focused this part of the work on the vascular tone modulator endothelin-1. Indeed, this peptide is over-expressed in many mouse and human tumors where it is documented to act as a mitogenic factor in both para- and autocrine manners. Endothelin-1 is also a potent vasoconstrictor acting through the ETA receptors located on VSMCs. In our lab, we previously showed that over-expression of endothelin-1 in tumors accounted for the development of a myogenic tone within the tumor vasculature. We have now documented that an ETA receptor antagonist induces the relaxation of microdissected tumor arterioles and selectively and quantitatively increases tumor blood flow in experimental tumor models. We also combined dye staining of functional vessels, fluorescent microsphere-based mapping, and magnetic resonance imaging to identify heterogeneities in tumor blood flow and to examine the reversibility of such phenomena. We showed that administration of an ETA receptor antagonist reduces the extent of underperfused tumor areas, proving the key role of vessel tone variations in tumor blood flow heterogeneity. We also provided evidence that ETA antagonist could improve the access of cyclophosphamide to the tumor compartment and thereby induces a significant tumor growth delay. 2. To examine whether and how intermittent hypoxia could influence endothelial cell survival and modulate resistance to radiotherapy. To dissect the mechanisms driving the phenotype conversion of endothelial cells exposed to intermittent hypoxia. This second part of our work, is a comprehensive investigation of the consequences of intermittent hypoxia, as caused by TBF heterogeneities, on the endothelial cell phenotype. First, we postulated that intermittent hypoxia (IH) favors endothelial cell (EC) survival, thereby extending the concept of hypoxia-driven resistance to the tumor vasculature. We showed that exposing EC to cycles of hypoxia/re-oxygenation reduces radiation-induced cell death and promotes angiogenesis. In contrast, prolonged hypoxia failed to achieve such protection and even appeared deleterious. We also observed that although HIF-1£ is completely degraded during each re-oxygenation, its abundance is paradoxically found higher at each new hypoxic challenge. Moreover, the use of siRNA targeting HIF-1£ pointed out that HIF-1ƓÑ accumulation account for the increased resistance of EC to radiotherapy. Finally, we extended this concept in vivo by forcing IH in tumor-bearing mice and found that it is associated with less radiation-induced apoptosis within both the vascular and the tumor cell compartments (vs normoxia or prolonged hypoxia). Next, we focused our work on the underlying mechanisms of EC phenotype conversion exposed to IH and particularly on potential actors that may favor HIF-1£ accumulation during IH. Prolylhydroxylases (PHD), MAPK and PI3K/Akt pathways as well as eNOS are known to regulate HIF-1£ abundance and transcriptional activity. We documented that PHD2 and PHD3 abundance are slightly decreased during IH, whereas prolonged hypoxia increases PHD3 expression in EC. We then showed that, ERK, Akt as well as eNOS were phosphorylated during reoxygenation periods of the IH protocol. We also used specific inhibitors of these cascades (i.e. PD98059, LY294002 and L-NAME, respectively), to evaluate their specific impact on HIF-1£ abundance and performed clonogenic assays to evaluate their consequences on EC survival. We showed that although, PD98059 and LY294002 sensitizes EC to pro-apoptotic stresses, only the PI3K/Akt inhibitor abrogates the HIF-1£ signal during IH. Conversely, L-NAME, a non-specific NOS-inhibitor, appears to potentiate the expression of HIF-1£ and to favor the EC survival. 3. To identify new therapeutic targets to prevent endothelial cell resistance by studying VEGF signaling, the major pro-survival and pro-angiogenic growth factor in endothelial cells. Because VEGF plays a central role in hypoxia-mediated angiogenesis and cell survival, the VEGF signaling cascade is a an obvious therapeutic target. To more specifically identify the pathways leading to cell survival and the resistance phenomena that we observed in response to intermittent hypoxia, a careful dissection of the downstream VEGF signaling cascades was performed. In this part of the work, we focused our attention on caveolin since it modulates the activity of eNOS, ERK and Akt that are major effectors acting downstream VEGF stimulation. We demonstrated the paradoxical role of caveolin-1 preventing signaling in basal conditions and ensuring the coupling between VEGFR2 and the downstream cascades upon VEGF stimulation. We used mice deficient for the caveolin-1 gene (Cav-/-) to examine the impact of caveolae suppression in a model of adaptive angiogenesis obtained after femoral artery resection. Evaluation of the ischemic tissue perfusion and histochemical analyses revealed that contrary to Cav+/+ mice, Cav-/- mice fails to recover a functional vasculature and actually loose part of the ligated limbs. We also isolated endothelial cells (ECs) from Cav-/- aorta and showed that on VEGF stimulation, endothelial tube formation is dramatically abrogated when compared with Cav +/+ ECs. The Ser1177 eNOS phosphorylation and Thr495 dephosphorylation but also the ERK phosphorylation were similarly altered in VEGF-treated Cav-/- ECs. Interestingly, caveolin transfection in Cav-/- ECs redirected the VEGFR-2 in caveolar membranes and restored the VEGF-induced ERK and eNOS activation. However, when high levels of recombinant caveolin are reached, VEGF exposure fails to activate ERK and eNOS. Altogether, these data identify caveolin as a new therapeutic target to alter VEGF signaling, in particular the cascades leading to angiogenesis and resistance to stresses.

The development of intratumoral hypoxia is associated with resistance to therapy in many forms of human cancer, and pre-exposure of tumor cells to hypoxia confers multidrug resistance. Research over the last several years has led to considerable advances in the understanding of the cellular response to oxygen deprivation, however the hypoxia-induced mechanisms that contribute to the chemoresistance phenotype are still not well understood. Recent studies have identified hypoxia-inducible factor 1 (HIF-1), a master transcriptional regulator of oxygen homeostasis, as an important mediator of hypoxia-induced chemoresistance in cancer cells. The research described in this thesis confirms these findings and demonstrates HIF-1 is required for hypoxia-induced resistance to doxorubicin and etoposide in human tumor cells. In addition, novel findings revealed that hypoxia-induced drug resistance occurred independently of changes in the apoptotic fraction and was associated with decreased drug-induced senescence. DNA damage measured at the single-cell level revealed that the increase in survival correlated well with a HIF-1-dependent decrease in etoposide-induced DNA strand breaks, providing direct evidence that exposure of tumor cells to hypoxia leads to protection against some forms of drug-induced DNA damage. Characterization of several classical mechanisms of drug resistance upstream of DNA damage identified multiple determinants of cellular resistance to anticancer agents. The relative contributions of each varied depending on the particular drug and cancer cell line studied. Together, the findings presented here support a model in which hypoxia-induced chemoresistance is a multifactorial phenomenon that is regulated, at least in part, through HIF-1-dependent mechanisms. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2008-08-29 12:35:36.219

abstract: A tumor is a heterogeneous combination of proliferating tumor cells, infiltrating immune cells and stromal components along with a variety of associated host tissue cells, collectively termed the tumor microenvironment (TME). The constituents of the TME and their interaction with the host organ shape and define the properties of tumors and contribute towards the acquisition of hallmark traits such as hypoxia. Hypoxia imparts resistance to cancer from chemotherapy and radiotherapy due to the decreased production of reactive oxygen species and also promotes angiogenesis, malignant progression and metastasis. It also provides a powerful physiological stimulus that can be exploited as a tumor-specific condition, allowing for the rational design of anticancer hypoxia-activated pro-drugs (HAP). Accurate evaluation of tumor oxygenation in response to therapeutics interventions at various stages of growth should provide a better understanding of tumor response to therapy, potentially allowing therapy to be tailored to individual characteristics. The primary goal of this research was to investigate the utility of prospective identification of hypoxic tumors, by two different Magnetic Resonance Imaging (MRI) based oximetry approaches, in successful treatment with hypoxia activated therapy. In the present study, I report the utility of these two techniques 1) PISTOL (Proton Imaging of Siloxanes to map Tissue Oxygenation Levels) and 2) use of a hypoxia binding T1 contrast agent GdDO3NI in reporting the modulations of hypoxia pre and post hypoxia activated therapies in pre-clinical models of cancer. I have performed these studies in non-small cell lung cancer (NSCLC) and epidermoid carcinoma (NCI-H1975 and A431 cell lines, respectively) as well as in patient derived xenograft models of NSCLC. Both the oximetry techniques have the potential to differentiate between normoxic and hypoxic regions of the tumor and reveal both baseline heterogeneity and differential response to therapeutic intervention. The response of the tumor models to therapeutic interventions indicates that, in conjunction with pO2, other factors such as tumor perfusion (essential for delivering HAPs) and relative expression of nitroreductases (essential for activating HAPs) may play an important role. The long term goal of the proposed research is the clinical translation of both the MRI techniques and aiding the design and development of personalized therapy (e.g. patient stratification for novel hypoxia activated pro-drugs) particularly for cancer. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2017

The brain requires a continuous supply of oxygen and nutrients, and even a short period of reduced oxygen supply can cause severe and lifelong consequences for the affected individual. The unborn baby is fairly robust, but there are of course limits also for these individuals. The mostsensitive and most important organ is the brain. When the brain is deprivedof oxygen, a process can start that ultimately may lead to the death of braincells and irreparable brain damage. This process has two phases; one more orless immediate and one delayed. There is a window of time of up to 24 hourswhere action can be taken to prevent the delayed secondary damage. One recently clinically available technique is to reduce the metabolism and thereby stop the secondary damage in the brain by cooling the baby.It is important to be able to quickly diagnose hypoxic injuries and to followthe development of the processes in the brain. For this, the electroencephalogram (EEG) is an important tool. The EEG is a voltage signal that originates within the brain and that can be recorded easily andnon-invasively at bedside. The signals are, however, highly complex and require special competence to interpret, a competence that typically is not available at the intensive care unit, and particularly not continuously day and night. This thesis addresses the problem of automatic classification ofneonatal EEG and proposes methods that would be possible to use in bedside monitoring equipment for neonatal intensive care units.The thesis is a compilation of six papers. The first four deal with the segmentation of pathological signals (burst suppression) from post-asphyctic full term newborn babies. These studies investigate the use of various classification techniques, using both supervised and unsupervised learning.In paper V the scope is widened to include both classification of pathologicalactivity versus activity found in healthy babies as well as application of thesegmentation methods on the parts of the EEG signal that are found to be of the pathological type. The use of genetic algorithms for feature selection isalso investigated. In paper VI the segmentation methods are applied onsignals from pre-term babies to investigate the impact of a certain medication on the brain.The results of this thesis demonstrate ways to improve the monitoring of the brain during intensive care of newborn babies. Hopefully it will someday be implemented in monitoring equipment and help to prevent permanent brain damage in post asphyctic babies.

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Acute kidney injury (AKI) is associated with renal medullary hypoxia. The medullary thick ascending limb (mTAL) in the renal outer medulla is most susceptible to hypoxic injury, due to marginal O2 supply and high O2 consumption. The objectives of this study were to document the earliest effect of hypoxia (8% O2 for 2.5 hrs) on the mTAL function, and to identify strategies to protect the mTAL from hypoxia. The earliest effect of hypoxia is large water diuresis, due to a fall in the medullary osmolality and increase in vasopressinase. Desmopressin acetate (dDAVP), a synthetic vasopressin analogue resistant to vasopressinase that may also increase O2 delivery, prevented water diuresis. A low dose (0.8mg/kg) of furosemide may significantly reduce the mTAL work without a large excretion of essential electrolytes. Large water diuresis may be diagnostically valuable in detecting renal tissue hypoxia, and dDAVP and furosemide may prevent AKI in the clinical setting.