Le métabolisme des cellules souches embryonnaires est étroitement régulé par un équilibre entre<p>auto-renouvellement et différenciation. Ces cellules peuvent proliférer en culture de manière prolongée,<p>tout en restant indifférenciées, mais cela peut engendrer l’apparition d’anomalies karyotypiques, qui<p>accentuent la prolifération de ces cellules au détriment de leur potentiel de différenciation. Ce<p>phénomène d’adaptation à leurs conditions de culture et leurs caractéristiques communes avec les<p>cellules cancéreuses sont deux preuves majeures de la nature tumorigène de ces cellules souches.<p>Le but de ce travail a été d’élucider les voies de signalisation qui contrôlent le métabolisme des<p>cellules souches embryonnaires humaines, et plus particulièrement, leur différenciation. De plus, nous<p>avons souhaité étudier le caractère tumorigène de ces cellules et pour cela, nous avons travaillé en<p>comparant différentes lignées de cellules souches embryonnaires avec une lignée de carcinome<p>embryonnaire humain, leurs correspondantes cancéreuses. Nous avons choisi d’étudier la voie des<p>dérivés de l’inositol, dont la composante PI3K/AKT avait déjà été auparavant associée avec le<p>métabolisme des cellules souches embryonnaires murines, tout comme la composante des inositols<p>phosphates solubles qui avait été impliquée dans le contrôle de l’auto-renouvellement de ces cellules.<p>Nous avons tout d’abord étudié la distribution des inositols phosphates dans ces différents<p>modèles par HPLC et observé que leur profil différait entre les cellules souches embryonnaires normales<p>et cancéreuses, principalement au niveau de la quantité d’InsP5. L’analyse du profil des InsPs en cours de<p>différenciation spontanée a permis par la suite de mettre en évidence des modulations de ces différents<p>métabolites, avec notamment une diminution reproductible d’InsP5. Nous basant sur l’hypothèse que ces<p>modulations devaient se refléter au niveau de l’expression des enzymes régulant la production des<p>inositol phosphates, nous avons entamé une analyse par qPCR des kinases et phosphatases principales<p>impliquées dans cette voie. Nous avons effectivement pu mettre en évidence une augmentation<p>significative de deux isoformes de la triphosphate 3-kinase, ITPKB et ITPKA, en cours de différenciation<p>spontanée. Ces deux enzymes sont également davantage exprimées dans les cellules souches cancéreuses.<p>Ces augmentations d’expression ont ensuite pu être confirmées au niveau protéique pour ITPKB et au<p>niveau de leur activité enzymatique. Par ailleurs, au cours de ce travail, nous avons également mis au<p>point un modèle de différenciation dirigée de nos cellules souches embryonnaires vers des cellules<p>souches mésenchymateuses, que nous avons entièrement caractérisées et dont nous avons pu mettre en<p>évidence les propriétés immunomodulatrices. Des résultats préliminaires sont venus confirmer, dans ce<p>modèle, l’augmentation d’ITPKB préalablement observée en cours de différenciation spontanée.<p>Nous avons également montré que l’expression du gène suppresseur de tumeur PTEN était<p>augmentée en cours de différenciation spontanée des cellules souches embryonnaires humaines. A l’aide<p>de la technique d’immunofluorescence, nous avons mis en évidence une localisation subcellulaire<p>différente de PTEN dans nos cellules souches embryonnaires normales par rapport à leur<p>correspondantes cancéreuses ainsi qu’une plus faible expression de PTEN dans ces dernières, confirmant<p>ainsi nos résultats obtenus précédemment par qPCR. PTEN est le principal inhibiteur de la voie<p>PI3K/AKT dont nous avons également disséqué l’expression des effecteurs majeurs par qPCR. Nous<p>avons notamment mis en évidence une augmentation de l’expression des unités régulatrices p85α et<p>catalytique p110β en cours de différenciation. Ces mêmes enzymes étaient également surexprimées dans<p>les cellules de carcinome embryonnaire préalablement différenciées à l’aide d’acide rétinoïque.<p>En conclusion, l’ensemble de ces résultats met en évidence une modulation des voies de<p>signalisation dérivées de l’inositol en cours de différenciation spontanée et dirigée des cellules souches<p>embryonnaires, suggérant leur implication dans le contrôle de la différenciation de ces cellules. Nous<p>avons également observé des différences entre les cellules souches embryonnaires normales et<p>cancéreuses, dont l’étude devra être approfondie afin d’évaluer l’impact de ces divergences sur le risque<p>de transformation cancéreuse des cellules souches embryonnaires humaines. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

L’Ins(1,3,4,5)P4 produit par l’Ins(1,4,5)P3 3-kinase de type B (Itpkb) est nécessaire au développement des thymocytes et lymphocytes T murins. Trois hypothèses sont admises quant à la fonction physiologique et au mécanisme d’action de cet inositol phosphorylé :la première postule que l’Ins(1,3,4,5)P4 module la réponse calcique intracellulaire ;la seconde, que cet inositolphosphate est un intermédiaire métabolique dans la synthèse d’inositols plus hautement phosphorylés ;la dernière, que l’Ins(1,3,4,5)P4 module la localisation subcellulaire et la fonction de protéines capables de la reconnaître par des domaines spécifiques de liaison. Afin d’investiguer cette dernière hypothèse, nous avons analysé la physiologie des lymphocytes B invalidés pour Itpkb et avons généré et analysé des souris transgéniques d’addition pour Rasa3, récepteur potentiel à l’Ins(1,3,4,5)P4.<p>Les lymphocytes B déficients en Itpkb présentent un défaut de survie car ils ne peuvent activer correctement les protéines kinases Erk1/2 suite à la stimulation du BCR de surface. Cela conduit à la surexpression anormale de la protéine pro-apoptotique Bim. La diminution de l’expression de Bim est suffisante dans ce modèle pour restaurer une fonction normale des lymphocytes B. In vitro, Nous avons montré que l’Ins(1,3,4,5)P4 est nécessaire à la translocation de Rasa3, protéine favorisant l’inactivation de la voie de Ras, de la membrane vers le cytoplasme. L’étude de lymphocytes invalidés pour Itpkb dans un modèle de BCR transgénique semble montrer que des anomalies de réponse calcique ne participent pas au phénotype.<p>En conclusion, nos résultats indiquent qu’une des voies de signalisation préférentielle de l’Ins(1,3,4,5)P4 passe par la modulation de la localisation subcellulaire de protéines possédant un domaine d’affinité pour l’Ins(1,3,4,5)P4 telle que Rasa3.<p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

A leishmaniose é uma doença parasitária de ampla distribuição, para a qual se dispõe de um limitado arsenal terapêutico. Trabalhos recentes mostraram que tamoxifeno é eficaz no tratamento de leishmaniose experimental. Nesse trabalho, avaliamos a terapia combinada de tamoxifeno com os fármacos utilizados atualmente no tratamento desta enfermidade. A interação entre os fármacos mostrou-se aditiva, tanto in vitro como in vivo. Em paralelo, analisamos os efeitos de tamoxifeno na biossíntese de esfingolipídios em Leishmania, sendo identificada a redução da síntese de fosfatidilinositol e inositolfosforil ceramida (IPC) e acúmulo de ceramida acilada. A redução na biossíntese de IPC não pode ser atribuída a redução no transporte de inositol, mas provavelmente está relacionada à inibição da enzima IPC sintase. Estes resultados indicam novas estratégias para superar as deficiências encontradas no tratamento de leishmaniose utilizando tamoxifeno, um fármaco clinicamente bem conhecido que exerce ações em múltiplos alvos em Leishmania. / Leishmaniasis is a parasitic disease with wide distribution and limited treatment. Recent reports demonstrate that tamoxifen is an effective drug for experimental leishmaniasis treatment. In this work, we evaluated the combined therapy of tamoxifen with current drugs used in leishmaniasis chemotherapy. The drug interaction was additive both, in vitro and in vivo. We also evaluated tamoxifen effect on in Leishmania sphingolipids biosynthesis. We found a reduction in phosphatidylinositol and inositol phosphorylceramide (IPC) synthesis and an accumulation of acilceramide. The reduction in IPC biosynthesis could not be assigned to the reduction observed in inositol transport, but probably is related to IPC synthase inhibition. These results show new strategies to circumvent shortcomings of leishmaniasis treatment using tamoxifen, a multitarget drug in Leishmania and widely used in the chemotherapy of breast cancer.

Inositolpyrophosphate (PP-InsP) sind eine Gruppe sekundärer Signalmoleküle, die in einer Vielzahl zellulärer Prozesse, von Phosphathomeostase über Insulinsignalisierung bis Apoptose eine Rolle spielen. Die Art und Weise, wie PP-InsPs ihre Funktion ausführen, noch weitgehend unbekannt. Deshalb wurden zwei neue analytische Methoden basierend auf Kernspinresonanzspektroskopie und Flüssigchromatographie mit Massenspektrometrie-Kopplung (LCMS) entwickelt. Um die limitierende Sensitivität der Kernresonanzspektroskopie zu umgehen, wurde die Synthese von kernspinresonanzaktivem, 13C-markiertem Inositol optimiert. Des Weiteren wurde eine chemoenzymatische Synthese für alle Säugetier-PP-InsP-Isomere entwickelt, die auf der skalierbaren Ausfällung mittels Mg2+ Ionen basiert. Menschliche Zellen wurden mit 13C-Inositol isotopenmarkiert und in den Spektren der Zellextrakte wurde, basierend auf den PP-InsP-Standards, Fingerabdrucksignale identifiziert mit denen die Konzentrationen der dazugehörigen Moleküle bestimmt werden konnte. Die LCMS basierte Methode wurde auf dem Prinzip der Umsetzung von hochgeladenen Inositolpyrophosphaten zu ihren korrespondieren Methylestern mittels Trimethylsilyldiazomethan geplant. Die ungeladenen, permethylierten PP-InsPs wären geeignet für LC-Auftrennungen und MS-Messungen und sollten eine von Kernspinresonanzspektroskopie nicht erreichbare Sensitivität ermöglichen. Die Methode wurde mittels Inositolhexakisphosphat (InsP6), einem einfacheren PP-InsP-Analog, etabliert und methyliertes InsP6 konnte in Mengen von 10 femtomol detektiert werden. Die Adaption der Methode für die PP-InsPs gestaltete sich jedoch herausfordernd, da der Analyt während der Reaktion zersetzt wurde. Ein Wechsel zu Diazomethan als Methylierungsagens zeigte vielversprechende Resultate. / Inositol pyrophosphates (PP-InsPs) are a well conserved group of second messengers that are involved in a plethora of cellular processes including phosphate homeostasis, insulin signaling, and apoptosis. Despite much effort, it is still mostly unknown how PP-InsPs exert their diverse functions. In order to decipher the mechanisms, researchers have relied either on metabolic labeling with radioactive inositol or on electrophoretic separation on polyacrylamide gels but these methods either lack ease of use or sensitivity. Therefore, two new analytical tools, based on nuclear magnetic resonance (NMR) spectroscopy, and liquid chromatography coupled mass spectrometry (LCMS), were developed. To overcome the limited sensitivity provided by NMR spectroscopy, a high yielding synthesis of NMR-active 13C-labeled inositol was designed and optimized. Furthermore, a chemoenzymatic synthesis of all mammalian PP-InsPs isomers was developed that relied on a scalable purification strategy utilizing precipitation with Mg2+ ions. Human cells were metabolically labeled with 13C-inositol and the prepared PP-InsPs were used as standards to identify peaks in the NMRspectra. These fingerprint signals enabled the quantification of the corresponding molecules. The LCMS-based method was based on the derivatization of the highly charged inositol pyrophosphates to their corresponding methyl esters by trimethylsilyldiazomethane. The permethylated InsPs and PP-InsPs were suitable for LC separation and MS measurement, and provide a sensitivity unmatched by NMR spectroscopy. The method was established using inositol hexakisphosphate, a simpler analog of PP-InsPs, and methylated InsP6 could be detected at quantities as low as 10 femtomole. However, the adaptation of the derivatization for PP-InsPs proved challenging as the reaction caused degradation of the analyte but strategies to circumvent the decay by changing the derivatization agent to diazomethane were promising.

No description available.

Inositol phosphates (IPs) have important cellular functions from teleomere maintenance (IP7 and IP8) to Ca2+ signaling pathways (1, 4, 5-IP3). Yet there is no robust, quantitative method to separate all the inositol phosphate isomers from IP1 to IP8. Four findings contributed heavily towards the development of a robust, quantitative IP isomer separation and identification method on the EskpertTM MicroLC 200+ QTrap 6500 system with a SelexIonTM DMS attachment. 1) TCA from inositol phosphate algal extractions was removed by elution with 100 mM ammonium carbonate, ammonium formate, or ammonium bicarbonate or by immobilized metal affinity chromatography (Fe-NTA columns). 2) A 250 mM ammonium carbonate and 25% methanol gradient was run on a weak anion exchange column to separate all the inositol phosphates from each other (IP1 through IP8. 3) Using ten different inositol phosphate isomer standards and fluoro- IP3 as an internal standard for future quantitation and recovery studies, isomer separation was obtained using SelexIonTM DMS with a 2- propanol modifier. 4) Ion suppression of inositol phosphate signals caused by 250 mM ammonium carbonate can be alleviated with a post-column dilution. The final assembled EskpertTM MicroLC 200+ QTrap 6500 system with a SelexIonTM DMS attachment and post-column dilution method was able to separate out IP isomers from IP1 to IP6 and detect IP7 and IP8. Once further optimized using the full compensation voltage range and a more polar modifier such as methanol, this method will allow the lipid biosynthesis pathways of C. reinhardtii, a promising candidate for algal biofuels, to be better studied.

<p>The homeostasis of naïve T lymphocytes is maintained by several mechanisms involving basal TCR and cytokine signaling, and nutrient factors. One of the common net results of these input signals is the production and stabilization of anti-apoptotic Bcl-2 family members. A second result of these processes is the induction of autophagy, an intracellular, catabolic, lysosomal targeting pathway. Autophagy induction in most systems involves the class III phosphatidylinositol-3 kinase (PI3K), Vps34, to produce phosphatidylinositol-3-phosphate (PI(3)P). To test this in T lymphocytes, I generated mice specifically lacking Vps34 in T cells (Vps34f/fLck-cre mice). However, Vps34-deficient T lymphocytes have normal levels of basal autophagy, and upregulate autophagy normally in response to cytokine or nutrient withdrawal, or TCR stimulation. Therefore I conclude that Vps34 activity is not required for autophagy induction in T lymphocytes. T lymphocytes lacking Vps34 do have enhanced rates of apoptosis, but this is due to defects in intracellular trafficking, specifically of the Interleukin-7 receptor alpha subunit (IL-7R&#945;). Additionally, multivesicular body (MVB) maturation is impaired in T cells lacking Vps34 such that extracellular ligands are not efficiently targeted to the lysosome. </p><p>Autophagy induction in Vps34-deficient T lymphocytes is still sensitive to pan-PI3K inhibitors, such as wortmannin and 3-methyladenine (3MA). Therefore, I hypothesized that other classes of PI3K are necessary to induce autophagy in T lymphocytes through the production of PI(3)P. Autophagy induction is sensitive to specific class I PI3K (PI3KI) inhibitors, such as PIK75. Additionally, T cells lacking the p85 regulatory subunit of PI3KI also have severe defects in T cell receptor (TCR) mediated autophagy induction. PI3KI activity results in the production of PI(3,4,5)P3, though, and not PI(3)P. Because of this specificity, I hypothesize that additional inositol polyphosphatases (Inpp) are required for autophagy induction downstream of PI3KI activity. Indeed, utilizing both inhibitors of pharmacological inhibition and siRNA-mediated knockdown of two classes of phosphatidylinositol phosphatases, inositol polyphosphate-4-phosphatase (Inpp4) and SH2 containing inositol phosphatase (SHIP), had dramatic impacts on autophagy induction. Furthermore, exogenous addition of PI(3,4)P2, a hypothesized intermediate in this pathway, positively regulates autophagy induction and leads to enhanced progression of autophagy. These observations indicate that PI3KI activity, linked to Inpp activity, are necessary and positive regulators of autophagy through the production of PI(3)P.</p> / Dissertation

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Inositol phosphates (IPs) are important signalling molecules with various biological roles in a cell. One such role it is often associated with is DNA repair. The DNA repair process following DNA insult is considered crucial for the genomic integrity and stability. Failure to perform this task will result in mutations and possibly disease. Thus, it is important that we expand our knowledge on how these repair processes occur and identify the key factors involved in its regulation. The aim of this project was to investigate whether DNA repair was mediated by inositol diphosphates (IDPs). Using a family of yeast knockout mutants with modulated levels of IPs, it was found that IDPs are crucial in repair of DNA following insult with bleomycin and 5-fluorouracil. The observed sensitivity of the mutants was thought to be due to lack of functional repair protein, UDG-like or APE-like, in the absence of essential cofactor such as IDPs. Experiments conducted revealed that the hypersensitive kcs1Δ contain both the repair proteins required to process the DNA lesions. However, extreme extraction methods were required to access these proteins, suggesting that the proteins are mislocalised and unavailable to access the damage site and perform DNA repair. GFP-tagging the proteins Ung1, Apn1 and Rad52 in kcs1Δ proved to be of little use as it failed to show exact localisation, movement and functionality status of these proteins following bleomycin insult. The enzymes accountable for the dephosphorylation of the IDPs in vivo are the diphosphoinositol polyphosphate phosphohydrolases (DIPPs). Little is known regarding the Michaelis-Menten kinetics parameters for Ddp1p/DIPPs. In this study, using improved methods for the enzymatic synthesis and electrophoretic purification of 1-InsP7, 5-InsP7 and InsP8, the DIPP family has been kinetically characterised. Each DIPP was found to ii display similar Km values for every substrate tested (range: 35-148 nM). The rank order of Kcat values (1-InsP7 > 5-InsP7 = InsP8) was identical for each enzyme, although DIPP-1 activity was observed to be 10- to 60-fold more than DIPP-2α/β and DIPP-3α/β, irrespective of the substrate. This study reveals that Ddp1, the yeast DIPP, is capable of hydrolysing not only 5-InsP7 but also 1-InsP7 and InsP8 to a single product, InsP6. The HPLC data found InsP7 accumulation to be relatively little during InsP8 breakdown by DIPPs. Such low build-up was found to be due to rapid conversion of InsP7 to InsP6. Through this study it is also clear that InsP8 prefers to dephosphorylate through 1-InsP7. In contrary, metabolically and functionally significant steady-state route of InsP8 synthesis was observed to be via 5-InsP7. Oesophageal cancer is considered as one of the deadliest cancers worldwide because of its aggressive nature and low survival rate. Epidemiologic studies have shown that low-dose daily intake of aspirin can decrease the incidence of oesophageal cancer. The data presented in this study show the effects of a number of in-house synthesized novel aspirin analogues on oesophageal cancer cell lines, squamous cell carcinoma (SSC) and adenocarcinoma (ADC). The aspirin analogues, fumaryldiaspirin (PN517) and benzoylsalicylates (PN524, PN528 and PN529), were observed to be more potent against the oesophageal cell lines than aspirin itself. Both, quantitative and qualitative apoptosis experiments conducted revealed that these compounds largely induced apoptosis, although some necrosis was evident with PN528 and PN529. Failure to recover following the treatment with these analogues emphasized that these drugs are largely cytotoxic in nature. The SSC cells (oe21) displayed increased sensitivity to the aspirin analogues compared to the ADC cell lines (flo-1 and oe33). The anticancer properties of these novel aspirin compounds appear to not involve the COX-enzymes at the tested concentrations. These initial findings support further studies into the potential of these aspirin analogues as chemotherapeutic agents against oesophageal cancer.

Mucins are heavily glycosylated epithelial proteins. Under or overexpression of mucins may lead to several different types of dysfunctions. Mucins are under investigation as possible diagnostic markers for malignancies and other disease. This study examined the over/under expression of Mucin-3 in the presence of IP6. Three groups of six mice were fed varying levels of phytate (inositol 6-phosphate, IP6) for six days before being subjected to carbon dioxide asphyxiation. Sections of the duodenum, jejunum, ileum, stomach, and colon were collected, lysed, and the tissue proteins were prepared and analyzed by Western Blot to determine Mucin-3 expression. The hypothesis was that the presence of phytate in the digestive tract would modulate the expression of Mucin 3. Results showed that on average, Mucin 3 expression in untreated tissue was higher in areas of the duodenum and jejunum and lower in the ileum and stomach. Presence of IP6 decreased expression of Mucin 3 in the stomach by one percent in the 1g/kg phytate group while increasing in the 2g/kg phytate group by one percent. The expression of Mucin 3 in the duodenum increased fifty-five and thirty-nine percent in the 1g/kg and 2g/kg phytate groups, respectively. Jejunum Mucin 3 expression was increased by ninety percent in both 1g/kg and 2g/kg phytate groups. The ileum 0g/kg and 2g/kg phytate groups had the same expression of Mucin 3, but the 1g/kg group had a thirty-six percent decrease.