Cyclooctyne and cycloocten-5-yne undergo, at room temperature, a 1,3-dipolar cycloaddition with dialkyl acetylenedicarboxylates 1a,b to generate furan-derived short-lived intermediates 2, which can be trapped by two additional equivalents of 1a,b or alternatively by methanol, phenol, water or aldehydes to yield polycyclic products 3b–d, orthoesters 4a–c, ketones 5 or epoxides 6a,b, respectively. Treatment of bis(trimethylsilyl) acetylenedicarboxylate (1c) with cyclooctyne leads to the ketone 7 via retro-Brook rearrangement of the dipolar intermediate 2c. In all cases, the products are formed with perfect atom economy.

Das dreistufige Flüssigsilicierverfahren (LSI) stellt eine Methode dar, siliciumcarbidbasierte faserverstärkte Keramiken herzustellen. Ausgangspunkt ist ein faserverstärkter Kunststoff, der über Pyrolyse (Konvertierung des Matrixpolymers in Kohlenstoff) und Silicierung (Siliciuminfiltration und Reaktion zu Siliciumcarbid) keramisiert wird. In der vorliegenden Arbeit werden die Matrix mittels der verwendeten Matrixpolymere (Matrixdesign) und das Faser/Matrix-Interface durch das Aufbringen von Faserbeschichtungen (Interfacedesign) definiert gestaltet. Die in der Arbeit eingesetzten Matrixpolymere beeinflussen durch eine unterschiedliche Poren- und Rissbildung in der Kohlenstoffmatrix die Siliciuminfiltration und die damit verbundene Siliciumcarbidbildung. Die Matrixpolymere erzeugen einerseits eine C-SiC-Dualphasenmatrix, wie diese bei den C/C-SiC-Verbunden angestrebt wird. Andererseits kann eine weitestgehend einphasige SiC-Matrix eingestellt werden, welche für die Herstellung von SiC/SiC-Verbunden interessant ist. Bei diesen Verbundwerkstoffen ist eine zusätzliche Faserbeschichtung entscheidend, um die Faser/Matrix-Bindung zu reduzieren und die Fasern vor dem Siliciumangriff während der Herstellung zu schützen. Als Faserbeschichtung werden eine BNx-Schicht und eine SiNx-Schicht entwickelt, die in einer BNx/SiNx-Doppelschicht kombiniert werden. Die Schichtherstellung erfolgt mittels chemischer Gasphasenabscheidung (CVD) auf einem kommerziellen SiC-Fasergarn (Tyranno SA3). Die amorphe BNx-Schicht ist innerhalb des Fasergarnes sehr homogen. Dahingegen besitzt die amorphe SiNx-Schicht einen Gradient in der Schichtdicke sowie in der chemischen Zusammensetzung. Bei der thermischen Auslagerung bleibt die BNx-Schicht stabil. Die SiNx-Schicht kristallisiert und es bilden sich Poren und Siliciumausscheidungen innerhalb der Schicht. Zudem entstehen teilweise Risse und Schichtabplatzungen. Weitere alternative Schichtkonzepte werden vorgeschlagen. / The liquid silicon infiltration (LSI) process is used to produce silicon carbide (SiC) based fiber reinforced ceramics and consists of three stages. Starting point is a fiber reinforced plastic, which is ceramized by means of pyrolysis (conversion of the matrix polymer to carbon) and siliconization (silicon infiltration and reaction to form silicon carbide). In the present work, the matrix and the fiber/matrix interface are designed by utilizing special matrix polymers and fiber coatings, respectively. The used matrix polymers lead to different pore and crack formation in the carbon matrix affecting the liquid silicon infiltration and the silicon carbide formation. The polymers not only create a dual phase C-SiC matrix, which is aspired for the production of C/C-SiC composites, but also form a single phase SiC matrix favorable for the SiC/SiC composite production. An additional coating of the fibers for these composite materials is crucial to reduce the fiber/matrix bonding and to protect the fibers from corrosive silicon attack. Separate BNx and SiNx single coatings are developed, which are combined to a double coating. The coating process is realized by chemical vapor deposition (CVD) on a commercial SiC fiber yarn (Tyranno SA3). The amorphous BNx coating is very uniform within the yarn, whereas the amorphous SiNx coating is characterized by a gradient regarding the layer thickness as well as the chemical composition. During the high temperature heat treatment the BNx coating remains stable. The SiNx coating crystallizes and pores as well as silicon precipitations are formed. Moreover, the coating partially ruptures. In this work, some additional alternative coating concepts are also proposed.

A non-reactive model system comprising a highly concentrated and unstable oil-in-water emulsion was used to investigate the retention of oil by the membrane in producing biodiesel with a membrane reactor. Critical flux was identified using the relationship between the permeate flux and transmembrane pressure along with the separation efficiency of the membrane. It was shown that separation efficiencies above 99.5% could be obtained at all operating conditions up to the critical flux. It was observed that the concentration of oil in all collected permeate samples using the oil-water system was below 0.2 wt% when operating at a flux below the critical flux. Studies to date have been limited to the characterization of low concentrated emulsions below 15 vol.%. The average oil droplet size in highly concentrated emulsions was measured as 3200 nm employing direct light scattering (DLS) measurement methods. It was observed that the estimated cake layer thickness of 20 to 80 mm was larger than external diameter of the membrane tube i.e. 6 mm based on a large particle size. Settling of the concentrated emulsion permitted the detection of a smaller particle size distribution (30-100 nm) within the larger particles averaging 3200 nm. It was identified that DLS methods could not efficiently give the droplet size distribution of the oil in the emulsion since large particles interfered with the detection of smaller particles. The content of the smaller particles represented 1% of the total weight of oil at 30°C and 5% at 70°C. This was too low to be detected using DLS measurements but was sufficient to affect ultrafiltration. In order to study the critical flux in the presence of transesterification reaction and the effect of cross flow velocity on separation, various oils were transesterified in another membrane reactor providing higher cross flow velocity. higher cross flow velocity provides better separation by reducing materials deposition on the surface of the membrane due to higher shearing. The oils tested were canola, corn, sunflower and unrefined soy oils (Free Fatty Acids (FFA< 1%)), and waste cooking oil (FFA= 9%). The quality of all biodiesel samples was studied in terms of glycerine, mono-glyceride, di-glyceride and tri-glyceride concentrations. The composition of all biodiesel samples were in the range required by ASTM D6751 and EN 14214 standards. A critical flux based on operating pressure in the reactor was reached for waste cooking and pre-treated corn oils. It was identified that the reaction residence time in the reactor was an extremely important design parameter affecting the operating pressure in the reactor. / Natural Sciences and Engineering Research Council of Canada (NSERC)

Les cyclopropanes sont des motifs d’une grande importance puisqu’ils sont présents dans plusieurs molécules biologiquement actives en plus d’être de puissants intermédiaires dans la synthèse de molécules complexes. Au cours de cet ouvrage, nous avons développé une nouvelle méthode générale pour la synthèse d’ylures d’iodonium de malonates, soit d’importants précurseurs d’esters cyclopropane-1,1-dicarboxyliques. Ainsi, à l’aide de ces ylures, une méthode très efficace pour la synthèse d’esters cyclopropane-1,1-dicarboxyliques racémiques a été développée. Des travaux ont aussi été entrepris pour la synthèse énantiosélective de ces composés. Par ailleurs, les esters cyclopropane-1,1-dicarboxyliques ont été utilisés dans le développement de deux nouvelles méthodologies, soit dans une réaction de cycloaddition (3+3) avec des imines d’azométhines et dans la formation d’allènes par l’addition-1,7 de cuprates. Nous avons aussi poursuivi l’étude synthétique du cylindrocyclophane F impliquant l’utilisation de cyclopropanes pour le contrôle des centres chiraux. Ainsi l’addition-1,5 d’un cuprate sur un ester cyclopropane-1,1-dicarboxylique a été utilisée comme l’une des étapes clés de notre synthèse. L’autre centre chiral a pu être contrôlé par l’hydrogénolyse sélective d’un cyclopropylméthanol. Ces études ont, par ailleurs, mené au développement d’une nouvelle réaction d’arylcyclopropanation énantiosélective utilisant des carbénoïdes de zinc générés in situ à partir de réactifs diazoïques. Cette méthode permet d’accéder très efficacement aux cyclopropanes 1,2,3-substitués. De plus, nous avons développé la première réaction de Simmons-Smith catalytique en zinc menant à un produit énantioenrichi. / Cyclopropanes are important scaffolds as they are present in many biologically actives compounds and they are useful intermediates in the synthesis of complex molecules. In this thesis, we developed a novel general method for the synthesis of iodonium ylides of malonates, which are important precursors in the synthesis of cyclopropane-1,1-dicarboxylic esters. From these ylides, a method to generate racemic cyclopropane-1,1-dicarboxylic esters very efficiently was developed. Further works was also achieved on an asymmetric version of this reaction. Cyclopropane-1,1-dicarboxylic esters were used to develop two new methods: a (3+3) cycloaddition reaction with azomethine imines and the formation of allenes by the 1,7-addition of cuprates. We also continued our studies towards the total synthesis of cylindrocyclophane F, which use the cyclopropanes to control all chiral centers. The 1,5-addition of a cuprate on a cyclopropane-1,1-dicarboxylic ester was utilized as one of the key steps of our synthesis. The other chiral centre was controlled by the hydrogenolysis of a cyclopropylmethanol. Moreover, these studies led to the development of a novel highly enantioselective arylcyclopropanation reaction using zinc carbenoids generated in situ from diazo compounds. This method allows the efficient access to 1,2,3-substituted cyclopropanes. Moreover, we developed the first Simmons-Smith reaction using a catalytic amount of zinc to produce an enantioenriched product.

Les cyclopropanes sont des motifs d’une grande importance puisqu’ils sont présents dans plusieurs molécules biologiquement actives en plus d’être de puissants intermédiaires dans la synthèse de molécules complexes. Au cours de cet ouvrage, nous avons développé une nouvelle méthode générale pour la synthèse d’ylures d’iodonium de malonates, soit d’importants précurseurs d’esters cyclopropane-1,1-dicarboxyliques. Ainsi, à l’aide de ces ylures, une méthode très efficace pour la synthèse d’esters cyclopropane-1,1-dicarboxyliques racémiques a été développée. Des travaux ont aussi été entrepris pour la synthèse énantiosélective de ces composés. Par ailleurs, les esters cyclopropane-1,1-dicarboxyliques ont été utilisés dans le développement de deux nouvelles méthodologies, soit dans une réaction de cycloaddition (3+3) avec des imines d’azométhines et dans la formation d’allènes par l’addition-1,7 de cuprates. Nous avons aussi poursuivi l’étude synthétique du cylindrocyclophane F impliquant l’utilisation de cyclopropanes pour le contrôle des centres chiraux. Ainsi l’addition-1,5 d’un cuprate sur un ester cyclopropane-1,1-dicarboxylique a été utilisée comme l’une des étapes clés de notre synthèse. L’autre centre chiral a pu être contrôlé par l’hydrogénolyse sélective d’un cyclopropylméthanol. Ces études ont, par ailleurs, mené au développement d’une nouvelle réaction d’arylcyclopropanation énantiosélective utilisant des carbénoïdes de zinc générés in situ à partir de réactifs diazoïques. Cette méthode permet d’accéder très efficacement aux cyclopropanes 1,2,3-substitués. De plus, nous avons développé la première réaction de Simmons-Smith catalytique en zinc menant à un produit énantioenrichi. / Cyclopropanes are important scaffolds as they are present in many biologically actives compounds and they are useful intermediates in the synthesis of complex molecules. In this thesis, we developed a novel general method for the synthesis of iodonium ylides of malonates, which are important precursors in the synthesis of cyclopropane-1,1-dicarboxylic esters. From these ylides, a method to generate racemic cyclopropane-1,1-dicarboxylic esters very efficiently was developed. Further works was also achieved on an asymmetric version of this reaction. Cyclopropane-1,1-dicarboxylic esters were used to develop two new methods: a (3+3) cycloaddition reaction with azomethine imines and the formation of allenes by the 1,7-addition of cuprates. We also continued our studies towards the total synthesis of cylindrocyclophane F, which use the cyclopropanes to control all chiral centers. The 1,5-addition of a cuprate on a cyclopropane-1,1-dicarboxylic ester was utilized as one of the key steps of our synthesis. The other chiral centre was controlled by the hydrogenolysis of a cyclopropylmethanol. Moreover, these studies led to the development of a novel highly enantioselective arylcyclopropanation reaction using zinc carbenoids generated in situ from diazo compounds. This method allows the efficient access to 1,2,3-substituted cyclopropanes. Moreover, we developed the first Simmons-Smith reaction using a catalytic amount of zinc to produce an enantioenriched product.

La cartographie peptidique est une méthode qui permet entre autre d’identifier les modifications post-traductionnelles des protéines. Elle comprend trois étapes : 1) la protéolyse enzymatique, 2) la séparation par électrophorèse capillaire (CE) ou chromatographie en phase liquide à haute performance (HPLC) des fragments peptidiques et 3) l’identification de ces derniers. Cette dernière étape peut se faire par des méthodes photométriques ou par spectrométrie de masse (MS). Au cours de la dernière décennie, les enzymes protéolytiques immobilisées ont acquis une grande popularité parce qu’elles peuvent être réutilisées et permettent une digestion rapide des protéines due à un rapport élevé d’enzyme/substrat. Pour étudier les nouvelles techniques d’immobilisation qui ont été développées dans le laboratoire du Professeur Waldron, la cartographie peptidique par CE est souvent utilisée pour déterminer le nombre total de peptides détectés et leurs abondances. La CE nous permet d’avoir des séparations très efficaces et lorsque couplée à la fluorescence induite par laser (LIF), elle donne des limites de détection qui sont 1000 fois plus basses que celles obtenues avec l’absorbance UV-Vis. Dans la méthode typique, les peptides venant de l’étape 1) sont marqués avec un fluorophore avant l’analyse par CE-LIF. Bien que la sensibilité de détection LIF puisse approcher 10-12 M pour un fluorophore, la réaction de marquage nécessite un analyte dont la concentration est d’au moins 10-7 M, ce qui représente son principal désavantage. Donc, il n’est pas facile d’étudier les enzymes des peptides dérivés après la protéolyse en utilisant la technique CE-LIF si la concentration du substrat protéique initial est inférieure à 10-7 M. Ceci est attribué à la dilution supplémentaire lors de la protéolyse. Alors, afin d’utiliser le CE-LIF pour évaluer l’efficacité de la digestion par enzyme immobilisée à faible concentration de substrat,nous proposons d’utiliser des substrats protéiques marqués de fluorophores pouvant être purifiés et dilués. Trois méthodes de marquage fluorescent de protéine sont décrites dans ce mémoire pour étudier les enzymes solubles et immobilisées. Les fluorophores étudiés pour le marquage de protéine standard incluent le naphtalène-2,3-dicarboxaldéhyde (NDA), la fluorescéine-5-isothiocyanate (FITC) et l’ester de 6-carboxyfluorescéine N-succinimidyl (FAMSE). Le FAMSE est un excellent réactif puisqu’il se conjugue rapidement avec les amines primaires des peptides. Aussi, le substrat marqué est stable dans le temps. Les protéines étudiées étaient l’-lactalbumine (LACT), l’anhydrase carbonique (CA) et l’insuline chaîne B (INB). Les protéines sont digérées à l’aide de la trypsine (T), la chymotrypsine (CT) ou la pepsine (PEP) dans leurs formes solubles ou insolubles. La forme soluble est plus active que celle immobilisée. Cela nous a permis de vérifier que les protéines marquées sont encore reconnues par chaque enzyme. Nous avons comparé les digestions des protéines par différentes enzymes telles la chymotrypsine libre (i.e., soluble), la chymotrypsine immobilisée (i.e., insoluble) par réticulation avec le glutaraldéhyde (GACT) et la chymotrypsine immobilisée sur billes d’agarose en gel (GELCT). Cette dernière était disponible sur le marché. Selon la chymotrypsine utilisée, nos études ont démontré que les cartes peptidiques avaient des différences significatives selon le nombre de pics et leurs intensités correspondantes. De plus, ces études nous ont permis de constater que les digestions effectuées avec l’enzyme immobilisée avaient une bonne reproductibilité. Plusieurs paramètres quantitatifs ont été étudiés afin d’évaluer l’efficacité des méthodes développées. La limite de détection par CE-LIF obtenue était de 3,010-10 M (S/N = 2,7) pour la CA-FAM digérée par GACT et de 2,010-10 M (S/N = 4,3) pour la CA-FAM digérée par la chymotrypsine libre. Nos études ont aussi démontrées que la courbe d’étalonnage était linéaire dans la région de travail (1,0×10-9-1,0×10-6 M) avec un coefficient de corrélation (R2) de 0,9991. / Peptide mapping is a routine method for identifying post-translational modifications of proteins. It involves three steps: 1) enzymatic proteolysis, 2) separation of the peptide fragments by capillary electrophoresis (CE) or high performance liquid chromatography (HPLC), 3) identification of the peptide fragments by photometric methods or mass spectrometry (MS). During the past decade, immobilized enzymes for proteolysis have been gaining in popularity because they can be reused and they provide fast protein digestion due to the high ratio of enzyme-to-substrate. In order to study new immobilization techniques developed in the Waldron laboratory, peptide mapping by CE is frequently used, where the total number of peptides detected and their abundance are related to enzymatic activity. CE allows very high resolution separations and, when coupled to laser-induced fluorescence (LIF), provides excellent detection limits that are 1000 times lower than with UV-Vis absorbance. In the typical method, the peptides produced in step 1) above are derivatized with a fluorophore before separation by CE-LIF. Although the detection sensitivity of LIF can approach 10 12 M for a highly efficient fluorophore, a major disadvantage is that the derivatization reaction requires analyte concentrations to be approx. 10 7 M or higher. Therefore, it is not feasible to study enzymes using CE-LIF of the peptides derivatized after proteolysis if the initial protein substrate concentration is <10-7 M because additional dilution occurs during proteolysis. Instead, to take advantage of CE-LIF to evaluate the efficiency of immobilized enzyme digestion of low concentrations of substrate, we propose using fluorescently derivatized protein substrates that can be purified then diluted. Three methods for conjugating fluorophore to protein were investigated in this work as a means to study both soluble and immobilized enzymes. The fluorophores studied for derivatization of protein standards included naphthalene-2,3-dicarboxaldehyde (NDA), fluoresceine-5-isothiocyanate (FITC) and 6-carboxyfluorescein N-succinimide ester (FAMSE). The FAMSE was found to be an excellent reagent that conjugates quickly with primary amines and the derivatized substrate was stable over time. The studied substrates were -lactalbumin (LACT), carbonic anhydrase (CA) and insulin chain-B (INB). The CE-LIF peptide maps were generated from digestion of the fluorescently derivatized substrates by trypsin (T), chymotrypsin (CT) or pepsin (PEP), either in soluble or insoluble forms. The soluble form of an enzyme is more active than the immobilized form and this allowed us to verify that the conjugated proteins were still recognized as substrates by each enzyme. The digestion of the derivatized substrates with different types of chymotrypsin (CT) was compared: free (i.e., soluble) chymotrypsin, chymotrypsin cross-linked with glutaraldehyde (GACT) and chymotrypsin immobilized on agarose gel particles (GELCT), which was available commercially. The study showed that, according to the chymotrypsin used, the peptide map would vary in the number of peaks and their intensities. It also showed that the digestion by immobilized enzymes was quite reproducible. Several quantitative parameters were studied to evaluate the efficacy of the methods. The detection limit of the overall method (CE-LIF peptide mapping of FAM-derivatized protein digested by chymotrypsin) was 3.010-10 M (S/N = 2.7) carbonic anhydrase using insoluble GACT and 2.010-10 M (S/N = 4.3) CA using free chymotrypsin. Our studies also showed that the standard curve was linear in the working region (1.0×10-9-1.0×10-6 M) with a correlation coefficient (R2) of 0.9991.

No description available.

Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.