Partition of proteases from Lentinus citrinus DPUA 1535 by the Peg/Phosphate Aqueous Two-Phase System

A full two-level factorial design was employed to study the influence of PEG molar mass (MM PEG), PEG concentration (C PEG) and phosphate concentration (C PHOSPH) on proteases partition by Lentinus citrinus DPUA 1535 in a PEG/phosphate aqueous two-phase system (ATPS). For all ATPS studied, proteases partitioned for the top phase and the best proteases extraction condition was obtained with MM PEG = 6000 g mol-1, C PEG = 17.5% (w/w) and C PHOSPH = 25% (w/w) with (1.1) purification factor and (151%) activity yield. Findings reported here demonstrate a practical strategy that serves as a first step for proteases purification from crude extract by L. citrinus.

Inertization of small-scale chemical wastes using iron phosphate glass

The viability of small-scale heavy-metal waste immobilization into iron phosphate glasses was investigated. Several waste forms containing different amounts of heavy-ion wastes were evaluated (5%, 10%, 15%, 20%, 26%, 33%, 40% and 50% by mass) and their X-ray diffraction patterns revealed that no crystallization occurred in glasses with waste concentrations up to 26%. The dissolution rates for all of the reported glass compositions (ca. 10-8 g cm-2 min-1) are similar to those reported for the materials most commonly used for waste vitrification. Iron phosphate glasses thus proved to be very useful for the immobilization of heavy-metal wastes, exhibiting good contention and chemical durability comparable to that of borosilicate glasses.

Crystal structures of 5-chloro-2-methoxybenzoates of Mn(II), Co(II), Ni(II) and Zn(II)

The crystal and molecular structures of [bis(5-chloro-2-methoxybenzoate)tetraaquamanganese(II)], [pentaaqua(5-chloro-2-methoxybenzoato)cobalt(II)] (5-chloro-2-methoxybenzoate), [pentaaqua(5-chloro-2-methoxybenzoato)nickel(II)] (5-chloro-2-methoxybenzoate) and [aquabis(5-chloro-2-methoxybenzoate)zinc(II)] monohydrate were determined by a single-crystal X-ray analysis. Mn(H2O)4L2 (where L = C8H6ClO3) crystallizes in the monoclinic system, space group P21/c. [Co(H2O)5L]L and [Ni(H2O)5L]L both are isostructural, space group P212121. The crystals of [Zn(H2O)L2] H2O are monoclinic, space group Pc. Mn(II) ion is positioned at the crystallographic symmetry center. Mn(II) and Co(II) ions adopt the distorted octahedral coordination but Zn(II) tetrahedral one.The carboxylate groups in the complexes with M(II) cations function as monodentate, bidentate and/or free COO-groups. The ligands exist in the crystals as aquaanions. The complexes of 5-chloro-2-methoxybenzoates with Mn(II), Co(II) and Zn(II) form bilayer structure.

A Modified phosphate-carrier protein theory is proposed as a non-target site mechanism For glyphosate resistance in weeds

Glyphosate is an herbicide that inhibits the enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs) (EC 2.5.1.19). EPSPs is the sixth enzyme of the shikimate pathway, by which plants synthesize the aromatic amino acids phenylalanine, tyrosine, and tryptophan and many compounds used in secondary metabolism pathways. About fifteen years ago it was hypothesized that it was unlikely weeds would evolve resistance to this herbicide because of the limited degree of glyphosate metabolism observed in plants, the low resistance level attained to EPSPs gene overexpression, and because of the lower fitness in plants with an altered EPSPs enzyme. However, today 20 weed species have been described with glyphosate resistant biotypes that are found in all five continents of the world and exploit several different resistant mechanisms. The survival and adaptation of these glyphosate resistant weeds are related toresistance mechanisms that occur in plants selected through the intense selection pressure from repeated and exclusive use of glyphosate as the only control measure. In this paper the physiological, biochemical, and genetic basis of glyphosate resistance mechanisms in weed species are reviewed and a novel and innovative theory that integrates all the mechanisms of non-target site glyphosate resistance in plants is presented.

Inhibition of carbachol-induced inositol phosphate accumulation in the embryonic retina promoted by kainate and veratridine

In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 µM carbachol was used, the estimated IC50 value for kainate was 0.2 µM and the maximal inhibition of ~50% was obtained with 1 µM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 µM veratridine, but not 50 mM KCl, inhibited ~65% of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 ± 38.0 to 2044.5 ± 299.9 cpm/mg protein, retinal response decreased to 861.6 ± 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.

Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37ºC, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37ºC. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 µM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 µM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25ºC in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium

Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186%, L-leucine _ 155% and L-phenylalanine - 168%) and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20%, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166%) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Effects of nickel on the mineral composition of Fleischmann’s yeast (Saccharomyces cerevisiae)

Sugar cane juice containing 12% (w.w -1) of total reducing sugars and 0.0 to 5.0 mmol of nickel L-1, with pH ranging from 3.5 to 6.5, was inoculated with Fleischmann’s yeast (Saccharomyces cerevisiae) (10% w.w -1). Six hours after fermentation, the yeast’s cellular viability and trehalose content were evaluated. The resulting must was centrifuged and the raw yeast was analyzed by atomic absorption spectroscopy to evaluate the intracellular levels of calcium, copper, iron, magnesium, manganese, nickel, phosphorus, potassium, sulfur and zinc. The intracellular levels of iron, magnesium and calcium were affected and the yeast’s susceptibility to nickel was enhanced by the decrease in pH. The yeast’s growth was not affected by nickel at high pH, but the toxic effects of nickel were potentiated at low pH.

Novel cancer-related regulatory targets for the signalling sphingolipids sphingosine-1-phosphate and sphingosylphosphorylcholine

The signalling sphingolipid sphingosine-1-phosphate (S1P) is necessary for development of the immune system and vasculature and on a cellular level regulates migration, proliferation and survival. Due to these traits S1P has an important role in cancer biology. It is considered a primarily cancer-promoting factor and the enzyme which produces it, sphingosine kinase (SphK), is often over-expressed in tumours. S1P is naturally present in the blood, lymph, tissue fluids and cell cytoplasm and functions through its cell surface receptors (S1P1-5) and as an intracellular second messenger. Sphingosylphosphorylcholine (SPC) is closely related to S1P and has similar regulatory functions but has not been extensively studied. Both S1P and SPC are able to evoke either stimulatory or inhibitory effects on cancer cells depending on the context. The aim of this thesis work was to study novel regulatory targets of S1P and SPC, which mediate the effects of S1P/SPC signalling on cancer cell behaviour. The investigated targets are the transcription factor hypoxia-inducible factor 1 (HIF-1), the intermediate filament protein vimentin and components of the Hippo signalling pathway. HIF-1 has a central role in cancer biology, as it regulates a multitude of cancer-related genes and is potently activated by intratumoural hypoxia through stabilization of the regulatory subunit HIF-1α. Tumours typically harbour high HIF-1α levels and HIF-1, in turn, facilitates tumour angiogenesis and metastasis and regulates cancer cell metabolism. We found S1P to induce follicular thyroid cancer cell migration in normal oxygen conditions by increasing HIF-1α synthesis and stability and subsequently HIF-1 activity. Vimentin is a central regulator of cell motility and is also commonly over-expressed in cancers. Vimentin filaments form a cytoskeletal network in mesenchymal cells as well as epithelial cancer cells which have gone through epithelial-mesenchymal transition (EMT). Vimentin is heavily involved in cancer cell invasion and gives tumours metastatic potential. We saw both S1P and SPC induce phosphorylation of vimentin monomers and reorganization of the vimentin filament network in breast and anaplastic thyroid cancer cells. We also found vimentin to mediate the anti-migratory effect of S1P/SPC on these cells. The Hippo pathway is a novel signalling cascade which controls cancer-related processes such as cellular proliferation and survival in response to various extracellular signals. The core of the pathway consists of the transcriptional regulators YAP and TAZ, which activate predominantly cancer-promoting genes, and the tumour suppressive kinases Lats1 and Lats2 which inhibit YAP/TAZ. Increased YAP expression and activity has been reported for a wide variety of cancers. We found SPC to regulate Hippo signalling in breast cancer cells in a two-fold manner through effects on phosphorylation status, activity and/or expression of YAP and Lats2. In conclusion, this thesis reveals new details of the signalling function of S1P and SPC and regulation of the central oncogenic factors HIF-1 and vimentin as well as the novel cancer-related pathway Hippo.

Timing of di-ammonium phosphate addition to fermenting chardonnay must : effect on nitrogen utilization, ethyl-carbamate formation, and CAR1 expression by yeast /

The maximum amount of ethyl carbamate (EC), a known animal carcinogen produced by the reaction of urea and ethanol, allowed in alcoholic beverages is regulated by legislation in many countries. Wine yeast produce urea by the metabolism of arginine, the predominant assimilable amino acid in must. This action is due to arginase (encoded by CARl). Regulation of CARl, and other genes in this pathway, is often attributed to a well-documented phenomenon known as nitrogen catabolite repression. The effect of the timing of di-ammonium phosphate (DAP) additions on the nitrogen utilization, regulation of CARl, and EC production was investigated. A correlation was found between the timing of DAP addition and the utilization of nitrogen. When DAP was added earlier in the fermentations, less amino nitrogen and more ammonia nitrogen was sequestered from the media by the cells. It was also seen that early DAP addition led to more total nitrogen being used, with a maximal difference of ~25% between fermentations where no DAP was added versus addition at the start of the fermentation. The effect of the timing ofDAP addition on the expression of CARJ during fermentation was analyzed via northern transfer and the relative levels of CARl expression were determined. The trends in expression can be correlated to the nitrogen data and be used to partially explain differences in EC formation between the treatments. EC was quantified at the end of fermentation by GC/MS. In Montrachet yeast, a significant positive correlation was found between the timing of DAP addition, from early to late, and the final EC concentration m the wine (r = 0.9226). In one of the fermentations, EC levels of 30.5 ppb was foimd when DAP was added at the onset of fermentation. A twofold increase (69.5 ppb) was observed when DAP was added after 75% of the sugars were metabolized. When no DAP was added, the ethyl carbamate levels are comparable at a value of 38 ppb. In contrast, the timing of DAP additions do not affect the level EC produced by the yeast ECU 18 in this manner. The study of additional yeast strains shows that the effect of DAP addition to fermentations is strain dependent. Our results reveal the potential importance of the timing of DAP addition to grape must with respect to EC production, and the regulatory effect of DAP additions on the expression of genes in the pathway for arginine metabolism in certain wine yeast strains.

Dynamic magnetic resonance spectroscopy of phosphate energetics during muscle exercise and recovery

Entailing of phosphorus exchanges in most bio-chemicals as a key factor in disease, increases researcher’s interest to develop the technologies capable of detecting this metabolite. Phosphorus magnetic resonance spectroscopy is able to detect key metabolites in a non-invasive manner. Particularly, it offers the ability to measure the dynamic rate of phosphocreatine(PCr) degeneration through the exercise and recovery. This metric as a valid indication of mitochondrial oxidative metabolism in muscle, differentiate between normal and pathological state. To do magnetic resonance imaging and spectroscopy, clinical research tools provide a wide variety of anatomical and functional contrasts, however they are typically restricted to the tissues containing water or hydrogen atoms and they are still blind to the biochemicals of other atoms of interests. Through this project we intended to obtain the phosphorus spectrum in human body – specificadenerativelly in muscle – using 31P spectroscopy. To do so a double loop RF surface coil, tuned to phosphorus frequency, is designed and fabricated using bench work facilities and then validated through in vitro spectroscopy using 3 Tesla Siemens scanner. We acquired in vitro as well as in vivo phosphorus spectrum in a 100 mM potassium phosphate phantom and human calf muscle in rest-exercise-recovery phase in a 3T MR scanner. The spectrum demonstrates the main constituent in high-energy phosphate metabolism. We also observed the dynamic variation of PCr for five young healthy subjects who performed planter flexions using resistance band during exercise and recovery. The took steps in this project pave the way for future application of spectroscopic quantification of phosphate metabolism in patients affected by carotid artery disease as well as in age-matched control subjects.

Fonction de la protéine Ceroid lipofuscinosis neuronal 5 (CLN5) dans le tri et le recyclage à l’endosome

Le tri et le transport efficace des hydrolases acides vers le lysosome jouent un rôle critique pour la fonction des cellules. Plus de 50 maladies humaines sont dues à des mutations des enzymes lysosomales, des protéines régulant des processus-clés du transport vers le lysosome ou des enzymes effectuant des modifications posttraductionnelles importantes pour la fonction du lysosome. L’objectif de cette thèse est d’identifier des protéines et des mécanismes permettant à la cellule de réguler le transport des enzymes vers le lysosome. Nous avons formulé l’hypothèse que des protéines mutées dans des maladies lysosomales et dont les fonctions étaient inconnues pouvaient jouer un rôle dans le transport vers le lysosome. Les céroïdes-lipofuscinoses neuronales forment une famille de maladies lysosomales rares mais sont aussi les maladies neurodégénératives infantiles les plus fréquentes. Plusieurs gènes impliqués dans les NCL encodent des protéines aux fonctions inconnues. Les travaux présentés dans cette thèse ont identifié la protéine « ceroid lipofuscinosis neuronal-5 » (CLN5) qui est localisée à l’endosome et au lysosome comme élément nécessaire au recrutement et à l’activation de rab7. Rab7 est une protéine Rab-clé qui contrôle le trafic à l’endosome tardif. Cette petite GTPase est impliquée dans le recrutement de retromer, un complexe protéique qui régule le trafic de l’endosome vers l’appareil de Golgi des récepteurs de tri lysosomal comme sortilin et le récepteur du mannose-6-phosphate. Dans les cellules où CLN5 est déplété, les récepteurs de tri lysosomal sont moins recyclés plus rapidement dégradés. En utilisant des expériences de photomarquage nous avons aussi pu démontrer que Rab7 est moins activées en l’absence de CLN5. Pour exécuter leur fonction les protéines rabs doivent être recrutée à la membrane et activées par l’échange d’une molécule de GDP pour une molécule de GTP. Le recrutement des Rabs à la membrane nécessite une modification posttraductionnelle lipidique pour être facilités. En utilisant un modèle de levures nous avons démontré que l’homologue de Rab7, Ypt7 est palmitoylée. Nous avons aussi démontré que la palmitoyltransférase Swif1 est nécessaire au recrutement de Ypt7 à la membrane. Nous avons aussi remarqué que les sous- unités de retromer chez la levure sont moins recrutées lorsque les palmitoyltransférases sont déplétées. Dans les cellules de mammifères nous avons démontré que Rab7 est également palmitoylé et que cette palmitoylation est possiblement effectuée par les palmitoyltransférases DHHC1 et DHHC8. La palmitoylation de Rab7 a lieu sur les cystéines en C-terminal qui sont nécessaires au recrutement membranaire et qui auparavant étaient uniquement décrites comme prénylées. En utilisant la méthode de « click chemistry » nous avons découvert que lorsque la prénylation de Rab7 est bloquée le niveau de palmitoylation augmente. Pour caractériser l’interaction entre CLN5 et Rab7 nous avons performé des expériences afin d’établir définitivement la topologie de cette protéine. Nous avons ainsi démontré que CLN5 est une protéine hautement glycosylée qui est initialement traduite en protéine transmembranaire et subséquemment clivée par un membre de la famille des peptidase de peptide signal (SPP). Cette protéine soluble peut alors possiblement interagir avec CLN3 qui est aussi palmitoylée pour recruter et activer Rab7. Nos études suggèrent pour la première fois que CLN5 pourrait être un recruteur et un activateur de Rab7 qui agirait avec la protéine CLN3 pour séquestrer Rab7 avec les autres récepteurs palmitoylés et permettre leur recyclage vers l’appareil de Golgi.

Effets des recuits ultra-rapides (10^5 K/s) sur la formation des siliciures métalliques en phase solide

La synthèse de siliciures métalliques sous la forme de films ultra-minces demeure un enjeu majeur en technologie CMOS. Le contrôle du budget thermique, afin de limiter la diffusion des dopants, est essentiel. Des techniques de recuit ultra-rapide sont alors couramment utilisées. Dans ce contexte, la technique de nanocalorimétrie est employée afin d'étudier, in situ, la formation en phase solide des siliciures de Ni à des taux de chauffage aussi élevés que 10^5 K/s. Des films de Ni, compris entre 9.3 et 0.3 nm sont déposés sur des calorimètres avec un substrat de a-Si ou de Si(100). Des mesures de diffraction de rayons X, balayées en température à 3 K/s, permettent de comparer les séquences de phase obtenues à bas taux de chauffage sur des échantillons de contrôle et à ultra-haut taux de chauffage sur les calorimètres. En premier lieu, il est apparu que l'emploi de calorimètres de type c-NC, munis d'une couche de 340 nm de Si(100), présente un défi majeur : un signal endothermique anormal vient fausser la mesure à haute température. Des micro-défauts au sein de la membrane de SiNx créent des courts-circuits entre la bande chauffante de Pt du calorimètre et l'échantillon métallique. Ce phénomène diminue avec l'épaisseur de l'échantillon et n'a pas d'effet en dessous de 400 °C tant que les porteurs de charge intrinsèques au Si ne sont pas activés. Il est possible de corriger la mesure de taux de chaleur en fonction de la température avec une incertitude de 12 °C. En ce qui a trait à la formation des siliciures de Ni à ultra-haut taux de chauffage, l'étude montre que la séquence de phase est modifiée. Les phases riches en m étal, Ni2Si et théta, ne sont pas détectées sur Si(100) et la cinétique de formation favorise une amorphisation en phase solide en début de réaction. Les enthalpies de formation pour les couches de Ni inférieures à 10 nm sont globalement plus élevées que dans le cas volumique, jusqu' à 66 %. De plus, les mesures calorimétriques montrent clairement un signal endothermique à haute température, témoignant de la compétition que se livrent la réaction de phase et l'agglomération de la couche. Pour les échantillons recuits a 3 K/s sur Si(100), une épaisseur critique telle que décrite par Zhang et Luo, et proche de 4 nm de Ni, est supposée. Un modèle est proposé, basé sur la difficulté de diffusion des composants entre des grains de plus en plus petits, afin d'expliquer la stabilité accrue des couches de plus en plus fines. Cette stabilité est également observée par nanocalorimétrie à travers le signal endothermique. Ce dernier se décale vers les hautes températures quand l'épaisseur du film diminue. En outre, une 2e épaisseur critique, d'environ 1 nm de Ni, est remarquée. En dessous, une seule phase semble se former au-dessus de 400 °C, supposément du NiSi2.