A study has been made of the effect of cell population density and virus-transformation on the transport of K+, inorganic phosphate (Pi) and 2-deoxyglucose in cultured mouse fibroblasts. Normal, untransformed 3T3 show a marked reduction (3-5 fold) in the influx of K+, Pi and 2-deoxyglucose with increasing; cell population density. The reduce-in transport begins at low cell densities and procedes the cessation of cell growth. In contrast, virus-transformed 3T3 cells do not exhibit similar density-dependent reductions in transport. In these cells the influx of K+ and 2-deoxyglucos is independent of cell density. The influx of Pi does decrease with increasing cell density but to a lesser extent and at much higher population densities than in cells. At low cell densities the influx of Pi and 2-deoxglucose in 3T3 cells is only slightly lower than the influx into transformed cells. For K+ the influx in 3T3 cells is slightly higher than in the transformed cells. At higher cell densities the transformed cells exhibit the higher rates of uptake of all three substrates due to the density-dependent transport reduction in the 3T3 cells. Virus-transformation per se does not, therefore, lead to any great increase in transport capacity. In all cases the density-dependent transport reductions are attributable to a decreased Vmax with no change in the Km of the system. The transport sites of normal and virus-transformed cells appear to be qualitatively similar since no significant differences were found for transport Km's between the cell lines. The density-dependent reduction of the K+ influx in 3T3 cells is due to a decrease in "Na-pump" activity with no change in the passive permeability of the cell membrane to K. This reduced "Na-pump" activity is accompanied by a decrease in [K+]i and an increase in [Na+]i. The [K+]i and [Na+]i of virus-transformed cells are not markedly affected by change in cell density. The [K+]i of transformed cells is slightly higher than the [K+]i of untransformed cells at low cell density and almost 2-fold greater than the [K+]i of high density untransformed cells. The cardiac glycoside, ouabain, inhibits Na-K exchange in both untransformed and transformed cells. In the transformed cells the drug also produces a secondary effect ie. a stimulation of K-K exchange. Evidence is presented which suggests that this difference is related known changes in the lipid properties of virus-transformed cell membranes. The inward transport of Pi occurs predominantly via a carrier-mediated, Na-dependent process. A small Na-independent influx of Pi is also present. Preliminary evidence suggests that the outward movement of Pi is also Na-dependent. A model for Na-coupled Pi transport is presented and discussed briefly. The density-dependent reduction of Pi transport in 3T3 cells is due to a decreased Na-dependent influx with no change in the Na-independent component. The addition of fresh complete medium to quiescent 3T3 cells causes a rapid increase in the influx of Pi and 2-deoxyglucose. Fresh medium without scrum does not increase transport. For bott substrates the increased transport results from a higher Vmax with no alteration in Km. Under normal culture conditions this scrum-stimulation of transport is independent of protein synthesis. However, an additional increase in Pi transport occurs when fresh complete medium is added to serum-starved 3T3 cells. This second increase is inhibited by cyclohoximide indicating a requirement for protein synthesis. A model is presented which attempts to explain the effects of serum growth factors in terms of their action on transport systems.

Abstract The carbonic anhydrase (CA) gene family contains eleven active members, the basic physiological functions of which are linked to the interconversion of carbon dioxide and bicarbonate (CO2 + H2O ⇔ H+ + HCO3⇔ H2CO3-). They participate in a variety of physiological processes that involve pH regulation, CO2 and HCO3- transport and water and electrolyte balance, and some new functions have also been suggested recently. A novel tumour-associated antigen, MN, containing a CA-domain and named MN/CA IX, has been found to promote cell proliferation when transfected into NIH3T3 cells and has also been shown to be a potential biomarker for neoplasia in the uterine cervix. The present study examines the expression of MN/CA IX in the normal alimentary tract by immunohistochemistry and compares it with the expression of cytoplasmic CA I, CA II, apical plasma membrane associated CA IV and secretory CA VI. The distribution of mitochondrial CA V is examined by immunohistochemistry and Western blotting. The value of MN/CA IX as a potential biomarker of gastrointestinal tumours is assessed in a series of colorectal and hepatobiliary neoplasms. A positive immunoreaction for MN/CA IX was detected in the basolateral plasma membrane of the gastric, intestinal and biliary epithelium, but was confined to the proliferating cryptal enterocytes in the human gut, suggesting a role in cellular proliferation. In colorectal tumours, MN/CA IX immunoreaction was also located in the proliferative zone, indicating that it could be a useful marker of cellular proliferation. In the case of hepatobiliary tumours a positive signal was mainly associated with tumours of biliary epithelial parentage. These results demonstrate that MN/CA IX has a unique expression pattern in the alimentary tract relative to other CAs. Its localization and enzymatic properties suggest that it may have a dual function in the gastrointestinal epithelium. Through its CA activity it could participate in the regulation of carbon dioxide/bicarbonate homeostasis, while its localization to the basolateral surfaces of proliferating cryptal enterocytes suggests that it may serve as a ligand or receptor for one or more other proteins that regulate intercellular communication and/or cell proliferation. MN/CA IX may also serve as a new biomarker of gastrointestinal tumours.

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Les travaux de la thèse portent sur l'implication de la voie de signalisation Akt (sérine/thréonine kinase Akt) et mTOR (mammalian target of rapamycin) dans la physiopathologie de l'hypertension artérielle pulmonaire (HTAP) expérimentale. L'HTAP résulte d'une prolifération exagérée des cellules constitutives des vaisseaux pulmonaires, principalement les cellules musculaires lisses artérielles pulmonaires (CML-AP). De nombreux effecteurs biologiques et physiques préalablement identifiés agissent sur les CML-AP et participent à l'hyperplasie de celles-ci. Nous montrons que ces nombreux effecteurs convergent vers une voie de signalisation intracellulaire commune, la voie Akt/mTOR, qui de fait représente une cible thérapeutique pour le traitement de l'HTAP, et pourrait conditionner l'hyperplasie des CML-AP. mTOR est présent dans la cellule sous forme de deux complexes, mTORC1 et mTORC2, qui phosphorylent des substrats variés contrôlant la prolifération cellulaire. Les effecteurs de mTORC1 incluent les S6 kinases (S6K1 and S6K2) et les "eIF4E-binding proteins" (4EBP) alors que mTORC2 active la sérine/thréonine kinase Akt et parmi les kinases sous-jacentes, la kinase GSK3. La première étude est consacrée à l'évaluation des effets des inhibiteurs de protéases du VIH (ritonavir, amprenavir, nelfinavir) sur la progression de HTAP expérimentale, induite par la monocrotaline ou l'hypoxie. Nous montrons que ces deux formes d'HTAP sont associées à une activation de la voie Akt/mTOR dans les artères pulmonaires. Les traitements respectifs par les trois inhibiteurs des protéases du VIH durant 3 semaines induisent une réversibilité de l'HTAP, de l'hypertrophie ventriculaire droite et du remodelage des vaisseaux pulmonaires, de même qu'une inhibition de la phosphorylation d'Akt, de S6K et de GSK3. La prolifération des CML-AP induite par le PDGF ou le SVF 5%, associée à une augmentation de p-Akt et p-GSK3, est également bloquée par les inhibiteurs des protéases, de façon similaire et non additive à celle d'inhibiteurs spécifiques de la PI3 kinase et de GSK3. La conclusion est que ces traitements antirétroviraux inihibent la progression de l'HTAP en inhibant la voir Akt/mTOR dans les CML-AP. Cette proposition permettrait d'expliquer l'effet suspecté en clinique des traitements antirétroviraux sur l'HTAP compliquant l'infection par le VIH.Dans la seconde étude, nous montrons que les CML-AP en culture prélevées à partir de rats ayant développé une HTAP induite par la monocrotaline proliférent de façon exagérée en comparaison avec les cellules de rats témoins. Ce phénotype prolifératif est observé en présence de nombreux facteurs mitogènes parmi lesquels le SVF 5%, le PDGF, la sérotonine ou 5-HT, l'IGF1 ou l'IL1-beta, et est associé à une activation des substrats de mTORC1 et mTORC2. Le traitement in vitro par la rapamycine des CML-AP de rats avec HTAP établie permet d'inhiber la prolifération de ces cellules et de bloquer à la fois mTORC1 et mTORC2. De même, le traitement par la rapamycine de rats porteurs de l'HTAP préétablie pendant une semaine permet de normaliser la prolifération des CML-AP in vitro et in vivo et d'inhiber mTORC1 et mTORC2, effets non observés par l'Imatinib ou la Fluoxetine. De plus, le traitement des rats par la rapamycine prévient ou corrige l'HTAP induite par la monocrotaline de façon plus importante que l'Imatinib ou la Fluoxétine.Ces résultats indiquent donc que l'activation de la voie Akt/mTOR, très étroitement associée au développement de l'HTAP expérimentale, pourrait expliquer le phénotype prolifératif anormal des CML-AP inhérent à la pathologie, et ainsi représenter une cible thérapeutique de choix pour le traitement de l'HTAP humaine. / The major objectives of research described in this thesis is focused on the cell signaling pathway of Akt (serine/threonine kinase Akt) and mTOR (mammalian target of rapamycin) in the patho-physiology of experimental pulmonary arterial hypertension (PAH). PAH occurs as a result ofhyperplasia of the components of pulmonary vessels, principally the pulmonary arterial smooth muscle cells (PA-SMCs). Numerous previously identified biological and physical effectors act on the PA-SMCs and participate in PA-SMC hyperplasia. Here we show studied that these different effectors converge into a common intracellular signaling pathway, Akt/mTOR signaling pathway, which represents actually a therapeutic target for PAH treatment, and could be involved in the hyperplasia of PA-SMCs. In cells mTOR, is presented in the form of two complexes, mTORC1 and mTORC2, which phosphorylate various substrates controlling the cellular proliferation. The effectors of mTORC1 include the S6 kinases (S6K1 and S6K2) and eIF4E-binding proteins (4EBP), meanwhile mTORC2 activates the serine/threonine kinase Akt and the underlying kinases, e.g. GSK3 kinase.The first study is devoted to evaluate the effects of the protease inhibitors of HIV (ritonavir, amprenavir, nelfinavir) on experimental PAH development induced by monocrotaline or hypoxia. We studied that the two forms of PAH are associated with an activation of Akt/mTOR signaling pathway in pulmonary arteries. The treatment by the three protease inhibitors of HIV during 3 weeks causes reversibility in experimental PAH with decreased right ventricular hypertrophy and pulmonary vascular remodeling as well as inhibition of phosphorylation of Akt, S6K and GSK3. The proliferation of PA-SMCs stimulated by PDGF or FCS 5%, which is associated with an increased p-Akt and p-GSK3, is also blocked by the proteases inhibitors, in a similar and non additive way like the specific inhibitors of PI3 kinase and GSK3. We conclude that the antiretroviral treatments significantly inhibits PAH development by inhibiting Akt/mTOR signaling pathway in PA-SMCs. This proposition allows explaining the effect of antiretroviral treatments of PAH accompanied with HIV in patients.In the second study, we studied that the cultured PA-SMCs extracted from the rats with monocrotaline induced-PAH(MCT-PAH) proliferates faster as compared to control. This proliferative phenotype is observed in the presence of different mitogenic factors including FCS 5%, PDGF, 5-HT, IGF1 or IL-1β, and is associated with an activation of the substrates of mTORC1 and mTORC2. Treatment with rapamycin in the PA-SMCs extracted from the rats with PAH in vitro inhibits the proliferation and also blocks the activation of mTORC1 and mTORC2. The treatment by rapamycin in the rats with PAH during one week allows normalizing the proliferation of PA-SMCs in vitro and inhibiting the activation of mTORC1 and mTORC2 in vivo. These effects were not observed when treated with imatinib or fluoxetine. Moreover, treatment with rapamycin prevents or reverse MCT induced PAH more significantly than that by imatinib or fluoxetine.These results indicate that the activation of Akt/mTOR signaling pathway isclosely related to experimental PAH development, which can explain the abnormal proliferative phenotype of PA-SMCs involved in the patho-physiology of PAH, and represent a therapeutic target for the treatment of PAH in human.

Magister Philosophiae - MPhil / The Nuclear Non-Proliferation Treaty (NPT) was negotiated to stop the proliferation of nuclear weapons, resulting from the dangers associated with the use of these weapons well visible during 1945, in Hiroshima and Nagasaki and a nuclear arms race as seen during the Cuban Missile Crisis. During NPT Review Conferences, held every five years, the strength and integrity of this treaty is tested. Evident in NPT review conferences is the disagreement between nuclear weapon states and non-nuclear weapon states over the role and importance placed on nuclear weapons and the slow pace of nuclear disarmament. The NPT has been in force for over 40 years; however the threat of nuclear weapons still exists. It then becomes necessary to understand what role the NPT plays in the international system, which differs depending on the theoretical lens used to interpret the NPT. A realist perspective of the NPT reveals that this treaty is an instrument used by dominant states to safeguard and legitimise their hold over nuclear weapons, while denying other states access to these weapons, instead protecting their allies through extended nuclear deterrence. A liberal perspective of the NPT highlights the moral influence of this treaty as an instrument for the benefit of the greater good, to shield humanity from the dangers of a nuclear explosion by delegitimizing nuclear weapons, key to shaping the perceptions of the decision makers of states regarding state security and nuclear weapons particularly. A constructivist interpretation of the NPT argues that this treaty is a social construction by states to impose a measure of order in their relations. At particular times in history, the NPT moves between a realist and liberal interpretation based on critical events that inform its direction. Social agents (decision makers of the state) through their thinking and ideas construct and give meaning to “reality” which is constantly negotiated. With that in mind, no interpretation of the NPT is fixed and for that reason, a constructivist conclusion seems ultimately applicable, namely that the NPT is what states make of it.

To investigate the effects of proliferation on metabolism and cell product yields, proliferating and growth-limited EPOBF7 hybridoma cells have been compared as to their growth rates, energy demand, relative energy distributions, and monoclonal antibody (MAb) yield. Medium deprivation of leucine or serum was used to prevent growth. Energy consumption rates were determined in cell suspensions from rates of glucose consumption, lactate production, and oxygen utilization. In addition, the energy consumption of pathways critical to cell growth and survival were estimated from the relative decreases occurring in oxygen and glucose consumption upon pathway inhibition. The overall rate of energy consumption was significantly lower among growth-limited cultures. In addition, the distributions of oxidative and glycolytic energy among cellular synthetic pathways differed significantly between the culture conditions. Non-growing cultures also produced significantly lower antibody yields. Cell growth was also investigated using ³¹p nuclear magnetic resonance (NMR) spectroscopy of cells grown and maintained in bioreactor culture. Saturation transfer methods detected measurable transfer between inorganic phosphate (P(i)) and the gamma resonance of ATP. This transfer rate could be correlated with cellular growth rates within the reactor. Transfer of magnetization from the gamma resonance of ATP to P(i) was also detected, although the rate of this transfer did not appear to be related to the growth rate. The ratio of these transfer rates was consistently near 4. This information is believed to suggest the importance of other reactions through which ATP may donate its terminal phosphate. Cells in bioreactor culture were found to grow more slowly and produce lower levels of monoclonal antibody when compared to cells proliferating in suspension. such phenomena may be a function of diffusion limitations within the reactor such that the cells cannot obtain the nutrient supply required for optimal cell growth or product formation.

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