Certaines cyanobactéries photoautotrophes sont capables de fixer l’azote atmosphérique grâce à des cellules spécialisées, les hétérocytes. De plus, en aérobiose, comme ces cellules peuvent excréter de l’ammonium lorsque leurs activités glutamine synthétase sont partiellement inhibées. Elles sont considérées comme usines cellulaires potentielles pour une bioproduction d’engrais azoté. Nous utilisons une souche mutante de Anabaena variabilis PCC 7937-C9, cyanobactérie hétérocytée à taux de croissance élevé, pour étudier la capacité à produire de l’ammonium en photobioréacteurs. Les caractéristiques de croissance de cette souche ne différent pas significativement de celles de la souche sauvage, avec un taux de croissance spécifique maximal de 3.0 j–1 à 30°C. Nous montrons qu’une partie de l’azote excrété dans le milieu de culture est entrainé sous forme de NH3 par la phase gazeuse, expliquant ainsi des sous-estimations antérieures. Cette production dépend de la température, l’irradiance, le taux d’aération et la concentration en MSX. Des études cinétiques confirment que la production d’azote ammoniacal en phase liquide et en phase gazeuse est corrélée aux variations de pH. Une régulation pulsée de pH permet d’accroitre la production de NH3. Des cultures en chemostat confirment que les productions de NH3 gazeux sont maximales à pH 8.8. Une variation cyclique des teneurs en NH4 +/NH3 dissous semble réguler les teneurs en NH4 +/NH3 en dessous d’un seuil critique de 1.5 mmol L–1 via une consommation par les cellules végétatives. Ces caractéristiques physiologiques sont analysées pour une application potentielle à la fourniture d’azote à des cultures de microalgues oléagineuses. / Some photoautotrophic cyanobacteria species are able to fix dinitrogen thanks to specialized cells, the heterocyts. Moreover, these cells are known to secrete ammonia when the glutamine synthase activity is partially inhibited under aerobic conditions. They are considered as potential cell factories for fertilizer. The present study uses a mutant strain of Anabaena variabilis PCC 7937-C9, a fast-growing heterocytous cyanobacterium, to investigate the potential use of diazotrophic cyanobacteria in photobioreactors for ammonium production. The growth characteristics of this strain cultivated in chemostat cultures are not significantly different from those of the wild strain, with a maximal specific growth rate of 3.0 d–1 at 30°C. A part of the combined nitrogen excreted in the culture medium is shown to be stripped through the aeration of the cultures as NH3, indicating previous underestimation of NH4 +/NH3 excretion. This process is shown to be affected by parameters such as temperature, irradiance, gas flow rate and MSX concentrations. Kinetics study reveals that the dissolved NH4 +/NH3 as well as the gaseous NH3 productions are correlated to pH variations production; a pulse regulation of pH is used to increase the NH3 production. Chemostat cultures with pH regulation are used to confirm that maximal gaseous NH3 is produced at pH 8.8. A cyclic variation of dissolved NH4 +/NH3 seems to regulate the NH4 +/NH3 concentrations under a threshold level of 1.5 mmol L–1; uptake of NH4 + by vegetative cells seems to be involved. These physiological features are discussed in view of operative conditions for efficient nitrogen supply for production by oleaginous microalgae.

DL-Methionine (Met) has been conventionally used in swine diets with assumption of similar bioefficacy with L-Met. However, because L-Met is the form that is utilized by animals for protein synthesis, L-Met could, theoretically, be more available. Four experiments were conducted to evaluate L-Met bioavailability in nursery pigs with 21-day growth trials. A total of 105,105,112 and 84 crossbred pigs were used in Exp. 1, 2, 3 and 4, respectively. Each experiment had a low Met basal diet and 3 levels of the Met sources (DL-Met and L-Met). In addition to the basal diet, supplementation levels were 0.053%, 0.107% and 0.160% in Exp. 1, 0.040%, 0.080% and 0.120% in Exp. 2, 0.033%, 0.067% and 0.100% in Exp.3, 0.040%, 0.080% and 0.120% in Exp. 4. Body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), gain: feed (G:F) were measured and plasma urea nitrogen (PUN) was analyzed in blood samples weekly. In Exp. 3 and 4, preference studies were conducted with the basal diet and the second highest level of each Met source. When additional DL-Met or L-Met were supplemented to the basal diet, BW, ADG, ADFI, and G:F ratio increased (P < 0.05). In the comparison between the DL-Met and L-Met diets in Exp. 1, pigs in the L-Met group had greater ADG and G:F ratios in the d 0-7 (P < 0.05) period than those in the DL-Met group. However, there were no differences for the overall experimental period. In Exp. 2, pigs in the DL-Met group had greater BW (P < 0.05), ADG (P < 0.05) and ADFI (P < 0.05) than those in the L-Met group for the overall period whereas no differences were observed in G:F ratios and PUN concentrations. In Exp. 3 and 4, there were no differences in BW, ADG, ADFI, G:F ratios or PUN concentrations between L-Met and DL-Met groups for the overall period. There was no preference exhibited for either the DL-Met or L-Met diet. In the results of relative bioavailability of L-Met to DL-Met, the values was 111.1% for d 0-14 based on the estimation by ADG in Exp. 1; L-Met bioavailability was lower than DL-Met based on all response measures in Exp. 2. However, in Exp. 3, relative bioavailability of L-Met to DL-Met was 100.4, 147.3, and 104.1% for d 0-14 ADG, G:F ratio and PUN concentrations. In Exp 4, the relative bioavailability of L-Met was 92.9, 139.4 and 70.4% for d 0-14 ADG, G:F ratio and PUN concentrations. In conclusion, using L-Met in the nursery diet demonstrated no consistent beneficial effect on ADG, G:F ratio or relative bioavailability compared to conventional DL-Met.

The enzyme L-methionine γ-lyase (MGL) utilizes a pyridoxal-5’-phosphate-cofactor in order to convert L-methionine to α-ketobutyrate, ammonia and methyl mercaptan. MGL is proposed to be a potential drug target since it is expressed in the human pathogens, Trichomonas vaginalis and Entamoeba histolytica, but not in humans. There is currently a need to find alternative drug targets for these pathogens, because the misuse and overuse of the currently prescribed drugs of choice, metronidazole and tinidazole, have lead to drug resistance. The overall goal of this thesis was to examine the chemistry of MGL 1 from T. vaginalis (TvMGL1) by probing the active site by site-directed mutagenesis and with fluorinated methionine analogs. The mutation of the active site residue Cys113 to Ser led to a 5-fold decrease in turnover rate for L-methionine relative to the wild-type enzyme. The results suggest that the active site C113 residue plays an important role in catalysis and is consistent with literature reports for MGL homologs from Pseudomonas putida and E. histolytica. Probing the active site of TvMGL1 with the fluorinated methionine analogs, L-difluoromethionine (DFM) and L-trifluoromethionine (TFM), were found to increase the turnover rate of the enzyme with an increase in fluorine substitution. The results suggest that the bulky fluorine atoms do not interfere with the Michaelis-Menten kinetics of the enzyme, and the γ-elimination step is rate determining. The second goal of this thesis was to identify the reactive intermediates generated by the processing of TFM and the uninvestigated DFM by TvMGL1, and to investigate the theoretical and experimental chemistry and biochemistry of these fluorinated groups (CF3S- and CF2HS-). The reactivity of the intermediates, generated from the processing of DFM by TvMGL1 was correlated to the cytotoxicity observed in model organisms expressing TvMGL1, and consistent with the hypothesis that the intermediates will result in the thioformylation of primary amines. The results suggest that cytotoxicity requires thioacylation of a single primary amine, while sequential cross-linking of primary amines is not an absolute requirement. The relationship between the chemical structure of the reactive intermediates produced from the enzymatic processing of these analogs and their cellular toxicity is discussed. Attempts at the synthesis of 3,3-difluoro-O-methyl-L-homoserine were undertaken in order to examine the catalytic mechanism of TvMGL1, since the compound is expected to inhibit the enzyme. To ensure that the oxo moiety does not impede the chemistry of the enzyme, the analog, O-methyl-L-homoserine was examined as a potential substrate for TvMGL1. Several synthetic routes to 3,3-difluoro-O-methyl-L-homoserine were examined; however, attempts to fluorinate the β-carbon atom of the starting material were unsuccessful.

The enzyme L-methionine γ-lyase (MGL) utilizes a pyridoxal-5’-phosphate-cofactor in order to convert L-methionine to α-ketobutyrate, ammonia and methyl mercaptan. MGL is proposed to be a potential drug target since it is expressed in the human pathogens, Trichomonas vaginalis and Entamoeba histolytica, but not in humans. There is currently a need to find alternative drug targets for these pathogens, because the misuse and overuse of the currently prescribed drugs of choice, metronidazole and tinidazole, have lead to drug resistance. The overall goal of this thesis was to examine the chemistry of MGL 1 from T. vaginalis (TvMGL1) by probing the active site by site-directed mutagenesis and with fluorinated methionine analogs. The mutation of the active site residue Cys113 to Ser led to a 5-fold decrease in turnover rate for L-methionine relative to the wild-type enzyme. The results suggest that the active site C113 residue plays an important role in catalysis and is consistent with literature reports for MGL homologs from Pseudomonas putida and E. histolytica. Probing the active site of TvMGL1 with the fluorinated methionine analogs, L-difluoromethionine (DFM) and L-trifluoromethionine (TFM), were found to increase the turnover rate of the enzyme with an increase in fluorine substitution. The results suggest that the bulky fluorine atoms do not interfere with the Michaelis-Menten kinetics of the enzyme, and the γ-elimination step is rate determining. The second goal of this thesis was to identify the reactive intermediates generated by the processing of TFM and the uninvestigated DFM by TvMGL1, and to investigate the theoretical and experimental chemistry and biochemistry of these fluorinated groups (CF3S- and CF2HS-). The reactivity of the intermediates, generated from the processing of DFM by TvMGL1 was correlated to the cytotoxicity observed in model organisms expressing TvMGL1, and consistent with the hypothesis that the intermediates will result in the thioformylation of primary amines. The results suggest that cytotoxicity requires thioacylation of a single primary amine, while sequential cross-linking of primary amines is not an absolute requirement. The relationship between the chemical structure of the reactive intermediates produced from the enzymatic processing of these analogs and their cellular toxicity is discussed. Attempts at the synthesis of 3,3-difluoro-O-methyl-L-homoserine were undertaken in order to examine the catalytic mechanism of TvMGL1, since the compound is expected to inhibit the enzyme. To ensure that the oxo moiety does not impede the chemistry of the enzyme, the analog, O-methyl-L-homoserine was examined as a potential substrate for TvMGL1. Several synthetic routes to 3,3-difluoro-O-methyl-L-homoserine were examined; however, attempts to fluorinate the β-carbon atom of the starting material were unsuccessful.

Schizophrenia postmortem brain is characterized by γ aminobutyric acid downregulation and by decreased dendritic spine density in frontal cortex. Protracted L-methionine treatment exacerbates schizophrenia symptoms, and our earlier work (Tremolizzo et al. and Dong et al.) has shown that L-methionine decreases reelin and GAD67 transcription in mice which is prevented by co-administration of valproate. In this study, we observed a decrease in spine density following L-methionine treatment, which was prevented by co-administration of valproate. Together with our earlier findings conducted under the same experimental conditions, we suggest that downregulation of spine density in L-methionine-treated mice may be because of the decreased expression of reelin and that valproate may prevent spine downregulation by inhibiting the methylation induced decrease in reelin.

The N-acetyl-L-methionine (NALM) molecule is a methionine (Met) derivative produced via acetylation of the L-Met α-amino group with an N-acetyl group. This molecule has been shown to be bioavailable and capable of fulfilling the dietary requirement for Met in animals and humans. The current experiment was conducted to test a hypothesis that lactating dairy cows fed with NALM would increase milk production by increasing N and energy utilization efficiencies in a dose dependent manner. Eight multiparous Holstein cows that were mid lactation (124 ± 13 days-in-milk) with similar milk production were used in a 4 x 4 Latin square design for 84 d. A developmental NALM product from CJ CheilJedang (Seoul, South Korea) was used as the supplemental source of rumen-protected Met in the present study. Four dietary treatments included 0 g (control), 15 g, 30 g, and 45 g/d/cow of NALM supplementation. Supplementing NALM significantly increased dry matter intake (linear effect; P < 0.01), while milk yield tended to increase quadratically (P = 0.07). A linear decrease in milk fat concentration was seen due to supplementation of NALM in relation to the control ration (P = 0.02). However, milk fat yield was similar across treatments. A trend toward an increase in milk protein yield was observed between the control ration and the ration supplemented with 45 g of NALM (1.18 vs. 1.21 kg/d; P = 0.10). There were no differences in energy-corrected or 3.5% fat-corrected milk yields in response to treatments. It is likely that the supplementation of NALM to mid to late lactating dairy cows may have shifted nutrient and energy utilization toward tissue gain and lactation, which resulted in a decrease in feed efficiency for lactation (P = 0.02). Overall results from the present study suggest that supplementing NALM to mid to late lactating cows can increase milk yield in a dose dependent manner with a shift of net energy partitioning toward milk production and body weight gain. In addition, supplementing NALM increased milk nitrogen (N) output without affecting urinary N excretion.

No description available.

<p>In plants, S-adenosyl-L-methionine-dependent phospho-base <em>N</em>-methyl transferases catalyze the three sequential methylations of phosphoethanolamine to phosphocholine, the precursor for choline and the major membrane phospholipid phosphatidylcholine. The enzyme phosphoethanolamine <em>N</em>-methyltransferase (PEAMT) catalyzes the first and committing step in choline synthesis, a step for which no known by-pass has been found. In <em>Arabidopsis thaliana</em> there are two loci annotated as encoding PEAMT and a putative PEAMT, At3g18000 (<em>NMT1</em>) and<em> </em>At1g73600 (<em>NMT3</em>), respectively. A related gene product that catalyzes the last two methylations is encoded by locus At1g48600 (<em>NMT2</em>). The objective of this study was to investigate the role of <em>NMT3 </em>in <em>Arabidopsis</em>. Three SALK lines carrying independent T-DNA insertions in At1g73600 were used: SALK_062703, SALK_016929c and SALK_120703c.</p> <p>Genomic DNA was used for PCR and sequence analysis of the products established the insertion of T-DNA in the protein coding region of At1g73600 for all three lines. Gene expression was analyzed by q-PCR. Primer design was particularly important for <em>NMT3 </em>transcript quantification by q-PCR. In SALK_062703 <em>nmt3 </em>mutants, the T-DNA is in exon 8 and in the SALK_120703c line it is in intron 6. In both cases, no <em>NMT3 </em>transcripts were detected using primers that annealed to sites 3’ to the position of the T-DNA in the gene. However, low levels of transcripts were detected using primers that annealed at positions 5’ to the site of T-DNA insertion. In the SALK_016929c line the position of the T-DNA insertion was in exon 2 and primers annealing near the site of the T-DNA insertion showed no <em>NMT3 </em>expression in this mutant but amplifying the mid portion of the gene showed WT levels of <em>NMT3 </em>transcripts. Thus all the mutants produce truncated <em>NMT3 </em>transcripts and by avoiding areas that overlap truncated transcript regions we could differentiate between <em>NMT3</em> knock-out or knock-down expression.</p> <p>Wild-type (<em>NMT3</em>) and <em>nmt3 </em>seedlings from the three lines grown on defined media plates showed no difference with respect to primary root length, number or density of lateral roots, and total root length. Exposing seedlings to salt (50 or 75 mM NaCl) led to reductions in root growth but again, roots of wild-type plants were indistinguishable from the mutant seedlings. One anomaly relates to the <em>nmt3</em> SALK_120703c<em> </em>line which showed two root phenotypes. On saline media most of the seedlings had longer roots that resembled the wild-type and other mutant lines and about a third had shortened roots. Whether the seedlings had long or short roots on salt, they all lacked <em>NMT3 </em>transcripts. This line is likely carrying another insertion that yields a salt-sensitive root phenotype. Mutant plants at four-weeks looked like wild-type plants and time of flowering was not reproducibly delayed or accelerated in mutant plants relative to wild-type.</p> <p>In wild-type seedlings the relative expression level of the three <em>NMT </em>genes is similar at day or night with transcript abundance ranked in the order <em>NMT3</em> > <em>NMT2 </em>> <em>NMT1. nmt3 </em>seedlings harvested midday showed no detectable <em>NMT3</em> expression but the abundance of <em>NMT1 </em>transcripts was 6.2-fold and 1.7-fold higher relative to wild-type in shoots and roots, respectively. At night, <em>NMT1 </em>expression in shoots of<em> nmt3 </em>seedlings decreased 4.8-fold relative to the level of <em>NMT1 </em>expression at midday while transcripts detected in roots increased slightly (1.3-fold). Using SALK_036291 <em>nmt1 </em>seedlings we found that <em>NMT3 </em>expression in shoots and roots was modestly up-regulated in the absence of <em>NMT1 </em>expression and the expression of <em>NMT3 </em>is lower at night than during the day. Also, regardless of the genotype or time of day, <em>NMT2 </em>expression remained constant even when <em>NMT1 </em>and <em>NMT3 </em>transcripts underwent several-fold changes in abundance. Interestingly, four-week old <em>nmt3 </em>plants of the SALK_062703 line showed that <em>NMT3 </em>expression is knocked-out in leaves but only knocked-down in roots.</p> <p><em> NMT3 </em>was the most highly expressed of the three <em>NMT </em>genes monitored by q-PCR. Nonetheless, three independent T-DNA insertion lines defective for <em>NMT3</em> expression were wild-type by appearance and as such, offer compelling evidence that NMT3 is not required by <em>Arabidopsis. </em>The increased expression of <em>NMT1 </em>in <em>nmt3 </em>plants and <em>NMT3</em> in <em>nmt1 </em>plants strongly suggests that plants compensate for the loss of one gene by up-regulating, to varying extents, the expression of the remaining <em>NMT </em>gene. If this is the case, a reasonable prediction made for a cross between <em>nmt1 </em>and <em>nmt3 </em>plants is that it would be lethal unless plants have yet another way to circumvent the loss of an essential enzyme for this committing metabolic bottleneck in choline synthesis.</p> / Master of Science (MSc)

<p> Some plants such as spinach, sugar beet, and wheat accumulate the quaternary ammonium compound glycine betaine when exposed to stresses in their environment. Environmental stress can be in the form of an excess or deficiency of water, high salt content, and/or exposure to excessively low or high temperatures and many if not all of these stresses are associated with cell dehydration. </p> <p> Glycine betaine is an organic solute that is believed to help restore the osmotic potential of a cell undergoing dehydration by reducing water loss and preventing damage to the structure and function of macromolecules. However, many plants such as Arabidopsis, tobacco, and rice do not accumulate glycine betaine. Given the perceived benefits of glycine betaine production by plants under stress, studies have been carried out to identify factors regulating its production. </p> <p> Glycine betaine is synthesized by the two-step oxidation of choline. The capacity to synthesize phosphocholine for choline production has been found to limit the production of glycine betaine in non-accumulating plants such as tobacco. As such, genetic engineering has been used to enhance the production of choline to up-regulate the synthesis of glycine betaine. This strategy has required knowledge of the enzyme(s) catalyzing the three N-methylation steps of the phosphocholine biosynthetic pathway. </p> <p> This study focused on a gene product identified as putative-phosphoethanolamine N-methyltransferase (putative PEAMT) based upon its similarity to a spinach Nmethyltransferase known to convert phosphoethanolamine to phosphocholine. This gene is located at the locus Atlg73600 on chromosome I of Arabidopsis and its predicted amino acid sequence has high similarity to two other genes encoding N-methylating enzymes located at At3 g 18000 (a biochemically confirmed PEAMT) and At 1 g48600 (annotated as a putative PEAMT). </p> <p> In this study, publicly available microarray data was examined to identify an expression profile of transcripts associated with the Atlg73600 gene in organs and tissues of Arabidopsis at various developmental stages. A summary of the micro array data shows the highest abundance of transcripts for Atlg73600 to be in the rosette leaves of Arabidopsis at 18.0- 20.9 days of growth. </p> <p> Arabidopsis plants grown from seeds from four SALK lines reported to have a TDNA insert in the Atlg73600 gene were screened for the presence of a T-DNA tag using a three primer PCR design strategy. Individual plants from two of the lines were found to have a T-DNA insert present. RT-PCR was then used to analyze the expression of transcripts associated with the Atlg73600 gene in these mutant lines. Transcripts were not detected among the amplified products from eDNA produced from the SALK line designated 062703 but they were found at reduced levels in eDNA of SALK line 016929c. </p> <p> In future studies the two T -DNA mutant lines identified in this study can be used to assign a biological role for the product of the Atlg73600 gene by examining the phenotype of these mutant plants relative to that of wild-type plants under normal and/or stressed conditions. The line found with no expression associated with the Atlg73600 gene will be useful in crosses with T -DNA knock-out mutants of genes at loci At3g18000 and Atlg48600. Systematic knock-outs for each of the genes in isolation and in combination will help discern whether there is functional redundancy in their biological roles or if their individual expression contributes uniquely towards the development of a plant or its stress response. Given the associated role for PEAMT in phosphatidylcholine metabolism, lipidomics could be used to determine if the composition of the plant membranes is altered relative to wild-type when the Atlg73600 gene is knocked-out. </p> / Thesis / Master of Science (MSc)

L to D isomerization of amino acids in a commercially prepared alkali-treated food-grade soy isolate was investigated. A soy flour product was also investigated as a control which had not been alkali treated. Methionine and valine collected from two soy flour and two soy isolate samples were coupled with L-leucine-N-carboxyanhydride. If both D- and L-forms were present this would produce diastereomeric dipeptide derivatives. The derivatives were separated by ion-exchange chromatography using a TSM Amino Acid Analyzer. Comparison of the sample methionine and valine dipeptide peaks to standard dipeptide peaks revealed the presence of L-valine, L-methionine and D-methionine in both soy flour and soy isolate samples. Methionine was substantially isomerized in both products. D-valine was not observed in either soy flour or soy isolate samples. Thus, isomerization of methionine appeared to occur at a faster rate than valine. Acid hydrolysis, toasting of soy meal, and alkaline treatment are all conditions which may be responsible for the isomerization of methionine. / Master of Science