Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The aggressiveness and therapeutic recalcitrance of this malignancy has been attributed to multiple factors including the influence of an active desmoplastic stroma. How the stromal microenvironment of PDAC contributes to the fatal nature of this disease is not well defined. In the analysis of clinical specimens, we observed diverse expression of the hypoxic marker carbonic anhydrase IX and the lactate transporter MCT4 in the stromal compartment. These stromal features were associated with the epithelial to mesenchymal phenotype in PDAC tumor cells, and with shorter patient survival. Cultured cancer- associated fibroblasts (CAFs) derived from primary PDAC exhibited a high basal level of hypoxia inducible factor 1a (HIF1 alpha) that was both required and sufficient to modulate the expression of MCT4. This event was associated with increased transcription and protein synthesis of HIF1a in CAFs relative to PDAC cell lines, while surprisingly the protein turnover rate was equivalent. CAFs utilized glucose predominantly for glycolytic intermediates, whereas glutamine was the preferred metabolite for the TCA cycle. Unlike PDAC cell lines, CAFs were resistant to glucose withdrawal but sensitive to glutamine depletion. Consistent with the lack of reliance on glucose, CAFs could survive the acute depletion of MCT4. In co-culture and xenograft studies CAFs stimulated the invasive potential and metastatic spread of PDAC cell lines through a mechanism dependent on HIF1a and MCT4. Together, these data indicate that stromal metabolic features influence PDAC tumor cells to promote invasiveness and metastatic potential and associate with poor outcome in patients with PDAC.

Solid tumors are commonly subject to hypoxia. Hypoxic cancer cells have undesirable properties such as a high tendency to metastasize and resistance to chemotherapy and radiotherapy. Hypoxia-inducible factors (HIFs) respond to the changes in oxygen levels, orchestrating the transcription of many proteins that are vital for the survival of hypoxic cancer cells. With their parent drug SN-38 as an inhibitor of both topoisomerase 1 and HIF-1, hypoxia-activated SN-38s may have a dual inhibitory effect on hypoxic tumor cells due to hypoxia-targeting and HIF-1 inhibition. To develop hypoxia-activated prodrugs of SN-38; 2-, 3-, and 4-nitrobenzyl SN-38s have been synthesized with good yields (78%, 67% and 68%, respectively). Topoisomerase 1 inhibitory assay on 2- and 4-nitrobenzyl SN-38s and cell viability assay on 2-, 3- and 4-nitrobenzyl SN-38s have been performed. All three derivatives showed less toxicity on K562 cells, which meets the principle of prodrug design. Cyclic voltammetry results suggest that the reduction potentials of these three derivatives may be not high enough for these compounds to be activated. The manner of reduction of three nitrobenzyl SN-38s is quasi-reversible under the testing condition, not against the proposed mechanism of activation. Two new derivatives of SN-38 have been designed to elevate reduction potential and further reduce toxicity. They are to be synthesized and tested for future work. / October 2016

No description available.

MicroRNAs (miRNAs) are short, non-coding RNA sequences which regulate gene expression. Regulation is mediated primarily through binding to complementary sites in their un-translated regions which leads to mRNA degradation or translational repression. Hypoxia is a known feature of many tumours, and increased hypoxia is associated with poor prognosis. Hypoxia leads to the up-regulation of many genes involved in a variety of functions including angiogenesis, a shift to glycolytic metabolism, and cell proliferation. This is mediated by the heterodimeric transcription factor HIF (hypoxia inducible factor), which is stabilised in hypoxia. In normoxia, the von-Hippel Lindau protein (VHL) targets HIF for degradation. Mutation in the VHL gene, as is frequently seen in clear cell renal cell cancer (CCRCC), results in constitutive over-expression of HIF and its gene targets, leading to a pro-angiogenic and pro-tumourigenic state. This thesis examined the expression of hypoxia-regulated miRNAs in cancer. The principal aims were to determine gene targets of miR-210, and to explore the effects of its over-expression and knock-down, both in vitro and in vivo. The expression of hypoxia-regulated miRNAs was examined in clinical renal tumour samples with matched normal tissue controls, and correlated with VHL mutation status. It was found that miR-210 targeted the iron sulphur cluster homologue (ISCU) gene, and was responsible for much of its down-regulation in hypoxia. Knock-down of ISCU had consequences on cell metabolism, in particular involving mitochondrial function and iron metabolism. miR-210 was found to be highly over-expressed in clear cell renal tumours (CCRCC), with greater expression seen in tumours with VHL mutations. miR-210 over-expression was also observed in papillary renal tumours, but to a lesser extent than in CCRCCs. miR-210 expression appeared to be correlated with reduced stage and grade, and improved survival. ISCU protein expression in CCRCCs was determined by immunohistochemistry, which showed that its expression correlated negatively with miR-210 expression, suggesting a functional role of miR-210 in vivo.

This thesis summarizes a research programme on the role of hypoxia-inducible factor (HIF) and its 2alpha subunit in systemic human physiology. Experiments were performed to assess the role of HIF in the regulation of skeletal muscle metabolism, cardiac anatomy, function and energy metabolism, and in cardiopulmonary physiology. Patients with different genetic mutations affecting the HIF pathway were recruited for each main study. Chapter 1 presents an overview of human physiological responses to hypoxia in a historical perspective, with particular attention to the areas of human physiology that are relevant for the studies presented in the experimental chapters. Chapter 1 also presents a summary of the HIF pathway and the novel findings presented in this thesis. Chapter 2 illustrates the methods used to perform the experiments. Chapter 3 investigates skeletal muscle metabolism, cardiac anatomy, function and energy metabolism in patients with Chuvash polycythaemia, who have mildly elevated levels of HIF, associated with a mutation in von Hippel-Lindau gene, at whole body level. Chapter 3 shows major abnormalities associated with HIF pathway alterations in skeletal muscle energy metabolism, especially in conditions of metabolic stress such as during exercise and digestion of a meal. Chapter 4 shows that patients with Chuvash polycythaemia also have small hearts and reduced cardiac energy levels. Chapter 5 explores cardiopulmonary abnormalities in patients with gain-of-function mutations specifically in HIF-2alpha subunit; these patients are polycythaemic like patients with Chuvash polycythaemia. Observed abnormalities include pulmonary hypertension, elevated heart rate, cardiac output, ventilation, and the increment in pulmonary blood pressure in response to moderate hypoxia. Chapter 6 presents results from experiments in patients with classic von Hippel- Lindau disease, who are not usually polycythaemic. However, some degree of haploinsufficiency was observed in their neutrophils, suggesting a pseudo-hypoxic phenotype. Chapter 6 shows that von Hippel-Lindau disease is not associated with major cardiopulmonary abnormalities. Overall, the research reported in this thesis presents original experimental evidence for the effects of alterations in the HIF pathway on human physiology.

The principal substrate used by the normal adult human heart is free fatty acids, the remainder being, predominantly,carbohydrate. During failure, the heart becomes less reliant on fatty acid metabolism, possibly as a result of tissue hypoxia. Therefore, understanding hypoxic adaptation may explain the metabolic changes that occur during the development of heart failure.As peroxisome proliferator activated receptor alpha (PPARα) modulates cardiac fatty acid metabolism, the work in this thesis focused on the role of PPARα in cardiac metabolic adaptation to chronic hypoxia. It was found that isolated hearts from chronically hypoxic (11% O₂ for 3 weeks)mice were more glycolytic, had reduced PPARα expression and decreased fatty acid metabolism,but had normal function, determined using in vivocine-MRI. ³¹P MRS of isolated perfused mouse hearts showed a drop in PCr with hypoxia, but ΔG_ATP was not altered, indicating that metabolic reprogramming was sufficient to maintain ATP production and contractile function. Increased or decreasedPPARα expression, using a high fat diet or PPARα null mice, respectively, prevented metabolic adaptation to hypoxia and caused cardiac dysfunction. Hypoxia with high fat feeding was particularly deleterious, reducing ejection fraction by 9%,possibly due to increased mitochondrial uncoupling. PPARβ/δ and γ were not involved in the adaptation to hypoxia, and none were modified by PPARα stimulation or ablation. Cardiac VEGF and PDK1, prominent hypoxia-inducible factor (HIF) targets, were increased by hypoxia, indicating that HIF may have been involved in metabolic adaption. However, high fat feeding prevented VEGF accumulation during hypoxia, suggesting that impaired HIF signalling may have contributed to the maladaptive response to hypoxia. In order to determine the relationship between HIF and PPARα, HIFwas stabilised pharmacologically using FG2216/BIC in HL-1 cardiomyocytes, to show decreased PPARα expression and caused similar metabolic changes to those seen in the in vivo hypoxic heart. In conclusion, this study demonstrated that HIF downregulation of PPARα is crucial for metabolic adaptation and maintenance of cardiac function during chronic hypoxia. Similar metabolic changes that occur in end-stage heart failure may also be a response to increasing hypoxia.

Inflammation has modulatory effects on the brain, particularly during development. These plastic changes can hold severe functional consequences. Perinatal hypoxia-ischemia (HI)-induced inflammation can result in cerebral palsy and cognitive impairment. In an attempt to reduce inflammation in the brain, we assessed the probiotic Bifidobacterium (B.) infantis as an HI intervention, using a rat model. Rat pups, developmentally equivalent to preterm infants, were exposed to chronic hypoxia from postnatal (PND) 3 –PND 10. Inflammation was assessed through hippocampal concentrations of the cytokine interleukin-6 (IL-6). Tissue was collected from pups on PND 10 and analyzed via enzyme-linked immunosorbent assay (ELISA). Results showed lower IL-6 concentrations in hypoxic groups , regardless of B. infantis administration. Qualitative observations suggested poor gut health in association with hypoxia and probiotic exposure. These preliminary findings support the chronic hypoxia exposure model of HI and suggest the association with IL-6 and HI events is less straightforward than expected. / October 2016

This study determines effects of oxygen levels on morphology and VEGF expression of developing chicken lungs following incubation in normoxia (21% O2), hypoxia (15% O2) or hyperoxia (30% O2), until developmental days 16 or 18. Lung morphology was assessed using light microscopy, while VEGF expression was determined with ELISA. In hypoxia, the proportion of parabronchial tissue and parabronchi including lumina increased from day 16 to 18 (61 to 68% and 74.2 to 82.2%, respectively). Non-parabronchial tissue was higher in hypoxia than in hyperoxia on day 16 (26 to 20%). However, by day 18, there were no differences between groups. VEGF expression was 33% higher in hypoxia than in hyperoxia on day 16 (736 vs. 492 pg/ml). On day 18, VEGF expression was 43% higher in hyperoxia than in normoxia (673 to 381pg/ml), and remained elevated by 40% in hypoxia over normoxia (631 pg/ml). VEGF may be a mechanism by which parabronchial tissue is stimulated from day 16 to 18 following exposure to chronic hypoxia.

by Wang, Wei Vivian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 237-266). / ACKNOWLEDGEMENTS --- p.viii / SUMMARY --- p.ix / PUBLICATION --- p.xiv / STATEMENT OF ORIGINALITY --- p.xv / LIST OF ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1 --- INTRODUCTION --- p.3 / Chapter 1.1 --- Preamble --- p.3 / Chapter 1.2 --- Free oxygen radicals --- p.7 / Chapter 1.2.1 --- Free oxygen radicals and mechanism of radical damage / Chapter 1.2.1.1 --- What is a free radical? / Chapter 1.2.1.2 --- Mechanism of free radical damage / Chapter 1.2.2 --- Detection and characterisation of free radical species / Chapter 1.2.2.1 --- Direct methods / Chapter 1.2.2.1.1 --- Electron spin resonance (ESR) spectroscopy / Chapter 1.2.2.1.2 --- Chemiluminescence / Chapter 1.2.2.2 --- Indirect methods / Chapter 1.2.2.2.1 --- Lipid peroxidation / Chapter 1.2.2.2.2 --- Protein and DNA oxidation / Chapter 1.2.2.2.3 --- Purine and pyrimidine metabolites / Chapter 1.2.3 --- Free oxygen radicals and major disease / Chapter 1.2.4 --- Oxygen-derived free radicals and fetal hypoxia / Chapter 1.3 --- Acid-base status in cord blood --- p.41 / Chapter 1.3.1 --- Correlation between obstetric clinical events and cord blood acid-base / Chapter 1.3.2 --- Practical implications of cord blood acid-base studies / Chapter 1.4 --- Intrapartum cardiotocography (CTG) analysis --- p.58 / Chapter 1.4.1 --- Base line / Chapter 1.4.1.1 --- Baseline rate / Chapter 1.4.1.2 --- Baseline variability / Chapter 1.4.2 --- Accelerations and decelerations / Chapter 1.4.3 --- Fetal outcome of labour / Chapter 1.4.3.1 --- Fetal heart rate (FHR) changes during labour / Chapter 1.4.3.2 --- Acidaemia during labour / Chapter 1.4.4 --- Computerised analysis of cardiotocogram / Chapter 1.5 --- Intrapartum complications --- p.83 / Chapter 1.5.1 --- Meconium stained amniotic fluid / Chapter 1.5.2 --- Nuchal cord entanglement / Chapter 1.5.3 --- Prolonged 1st and 2nd stage of labour / Chapter 1.6 --- Objectives of project --- p.93 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.98 / Chapter 2.1 --- Materials --- p.98 / Chapter 2.1.1 --- Clinical materials / Chapter 2.1.2 --- Chemicals and reagents / Chapter 2.1.2.1 --- The measurement of malondialdehyde (MDA) / Chapter 2.1.2.2 --- The measurement of organic hydroperoxides (OHP) / Chapter 2.1.2.3 --- The measurement of purine and pyrimidine metabolites / Chapter 2.1.3 --- Equipment / Chapter 2.1.3.1 --- Fetal monitor / Chapter 2.1.3.2 --- Fetal heart rate analysis system / Chapter 2.1.3.3 --- Blood gas analyser / Chapter 2.1.3.4 --- UV-VIS Spectrophotometer / Chapter 2.1.3.5 --- Fluorescence Spectrophotometer / Chapter 2.1.3.6 --- High Performance Liquid Chromatography (HPLC) / Chapter 2.2 --- Investigation Methods --- p.105 / Chapter 2.2.1 --- Blood gas / Chapter 2.2.2 --- Lipid peroxidation in umbilical cord blood / Chapter 2.2.2.1 --- The measurement of MDA / Chapter 2.2.2.2 --- The measurement of OHP / Chapter 2.2.3 --- Purine and pyrimidine metabolites in umbilical cord blood / Chapter 2.2.4 --- Computer analysis of CTG / Chapter 2.2.4.1 --- Data and signal processing / Chapter 2.2.4.2 --- The algorithm / Chapter 2.3 --- Statistical analysis --- p.112 / Chapter CHAPTER 3 --- RESULTS --- p.116 / Chapter 3.1 --- Umbilical blood pH and gas measurements --- p.118 / Chapter 3.2 --- Lipid peroxidation in cord blood plasma --- p.121 / Chapter 3.2.1 --- Validation of assay / Chapter 3.2.1.1 --- Performance characteristics of the MDA assay / Chapter 3.2.1.2 --- Performance characteristics of the OHP assay / Chapter 3.2.2 --- "Inter-relationship among MDA, OHP and acid-base status" / Chapter 3.3 --- Nucleotide metabolites in cord blood plasma --- p.142 / Chapter 3.3.1 --- Calibration of assay / Chapter 3.3.2 --- Inter-relationship among nucleotides and acid-base status / Chapter 3.4 --- Analysis of FHR patterns --- p.150 / Chapter 3.4.1 --- Umbilical blood gas and CTG analysis / Chapter 3.4.2 --- Biochemical parameters and CTG analysis / Chapter 3.5 --- "Relations of umbilical arterial blood pH and gas, lipid peroxidation, purine or pyrimidine metabolites and FHR patterns with intrapartum complications" --- p.166 / Chapter 3.5.1 --- Meconium stained amniotic fluid / Chapter 3.5.1.1 --- Clinical features / Chapter 3.5.1.2 --- Relationship between meconium stained amniotic fluid and biochemical parameters / Chapter 3.5.1.3 --- Relationship between meconium stained amniotic fluid and FHR patterns / Chapter 3.5.2 --- Nuchal cord / Chapter 3.5.2.1 --- Clinical features / Chapter 3.5.2.2 --- Relationship between nuchal cord and biochemical parameters / Chapter 3.5.2.3 --- Relationship between nuchal cord and FHR patterns / Chapter 3.5.3 --- The length of second stage of labour / Chapter 3.5.3.1 --- Clinical features / Chapter 3.5.3.2 --- Relationship between the length of second stage and acidaemia or FHR patterns / Chapter 3.5.4 --- Apgar scores / Chapter 3.5.4.1 --- Clinical features / Chapter 3.5.4.2 --- Relationship between Apgar scores and biochemical parameters / Chapter 3.5.4.3 --- Relationship between Apgar scores and FHR patterns / Chapter 3.5.4.4 --- "Relationship between Apgar scores and nuchal cord, meconium or second stage of labour" / Chapter CHAPTER 4 --- DISCUSSION --- p.189 / Chapter 4.1 --- Blood pH and gas in fetal asphyxia --- p.189 / Chapter 4.2 --- Lipid peroxidation in cord blood at birth --- p.194 / Chapter 4.2.1 --- Method for measurement of the cord plasma MDA / Chapter 4.2.2 --- Method for measurement of the cord plasma OHP / Chapter 4.2.3 --- Relationship between the fetal asphyxia and lipid peroxidation in cord plasma / Chapter 4.3 --- Purine and pyrimidine metabolites in cord blood at birth --- p.203 / Chapter 4.3.1 --- Limitations imposed by the tcchniqucs used / Chapter 4.3.2 --- Relationship between the fetal asphyxia and purine and pyrimidine metabolites in cord plasma / Chapter 4.4 --- Computerised analysis of CTG --- p.210 / Chapter 4.4.1 --- CTG patterns and cord blood acid base balance / Chapter 4.4.2 --- CTG patterns and cord blood biochemical parameters / Chapter 4.5 --- "Intrapartum complications 2,9" / Chapter 4.5.1 --- Meconium stained amniotic fluid / Chapter 4.5.2 --- Nuchal cord / Chapter 4.5.3 --- The length of second stage / Chapter 4.5.4 --- Apgar scores / Chapter CHAPTER 5 --- CONCLUSION --- p.233 / REFERENCES --- p.237

Les métastases représentent l’étape ultime de la progression tumorale. Les traitements inhibiteurs de l’angiogenèse offrent de nouvelles perspectives thérapeutiques, notamment pour les stades invasifs, mais cependant ils n’apportent qu’une prolongation modeste de la survie des patients. La faible réponse de certains types de cancers aux traitements ciblant l’angiogenèse tumorale, comme le carcinome de la prostate chez l’homme ou du sein chez la femme, est due au développement de résistances tumorales d’une part, et à l’accélération du processus métastatique mise en évidence par certaines études précliniques et cliniques d’autre part. L’objectif de ce travail de thèse a été d’étudier le rôle d’une protéine anti-angiogénique endogène, la thrombospondine 1 (TSP1), dans l’invasion et la dissémination métastatique dans le carcinome de la prostate et du sein. Notre laboratoire a montré que la densité microvasculaire est inversement corrélée à l’expression de la TSP1 dans les stades précoces de ces carcinomes. Cependant, cette corrélation est perdue aux stades avancés où l’expression de la TSP1 devient associée à un mauvais pronostic. Les travaux de ma thèse ont permis de montrer que la TSP1 stimule un ensemble de processus intervenant dans la progression tumorale, dont la migration et l’invasion cellulaires, l’extravasation et la dissémination métastatique. Nous avons aussi pu mettre en évidence que l’hypoxie induite par l’inhibition de l’angiogenèse est un activateur majeur de l’invasion et de la formation de métastases, que cette hypoxie soit produite par des molécules endogènes comme la TSP1 ou par des inhibiteurs pharmacologiques de ce processus. De plus, nous démontrons que la TSP1 est un facteur de mauvais pronostic puisque son expression est corrélée avec l’augmentation de l’invasion et de la rechute des patients atteints de cancers de la prostate androgéno-résistants. En conclusion, nos résultats suggèrent que l’expression de la TSP1 pourrait être un facteur prédictif de l’invasion et de l’occurrence de métastases, et que le ciblage de la TSP1 présente un fort potentiel thérapeutique pour bloquer à terme ces processus. / Prognosis is poor once tumors developed metastasis, and overall survival has been only modestly improved by the development of drugs inhibiting angiogenesis. The poor response of certain types of cancer to therapies that target tumor angiogenesis, including prostate and breast carcinomas, is due to the development of an evasion and to the acceleration of the metastatic process noted in some preclinical and clinical studies. The objective of this thesis was to study the role of an endogenous anti-angiogenic factor, thrombospondin 1 (TSP1), in the process of invasion and metastasis of prostate and breast carcinomas. Our laboratory has previously shown that microvessel density is inversely correlated with the expression of TSP1 in primary stages of breast and prostate carcinomas. However, this correlation is lost in advanced cancers, where TSP1 expression becomes associated with poor prognosis. We show that TSP1 stimulates a set of processes involved in tumor progression, including cell migration and invasion, extravasation and metastatic dissemination. We also demonstrate that hypoxia induced by an inhibition of angiogenesis resulting from the activity of endogenous or pharmacological molecules is a major activator of invasion and metastasis. In addition, we demonstrate that TSP1 is a poor prognostic factor since its expression is correlated with increased invasion and relapse in patients with androgen-resistant prostate cancer. In conclusion, our results suggest that the expression of TSP1 could be a predictor of invasion and metastasis occurrence, and that targeting TSP1 could be a great therapeutic potential to block these processes.