Trypanosoma cruzi, utiliza os aminoácidos como importantes metabólitos para sua manutenção e viabilidade, dentre eles glutamato e glutamina que participam no suprimento energético, metaciclogênese e síntese de pirimidinas. Neste trabalho a glutamina sintetase de T. cruzi (TcGS) foi estudada sob a hipótese de contribuir com a detoxificação de NH4+. Extratos de T. cruzi e a enzima recombinante pura foram utilizados na caracterização cinética e determinação da conformação oligomérica. Na forma amastigotas, observamos que TcGS é mais expressa e ativa. Além disso, a metionina sulfoximina (MS) inibiu TcGS de maneira competitiva e com alta afinidade e reduziu a proliferação de epimastigotas em excesso extracelular de NH4+, relacionando TcGS com a detoxificação dessa molécula. Assim, o tratamento com MS ao longo do ciclo intracelular reduziu a eclosão de formas tripomastigotas de células CHO-K1, ressaltanto a importância da GS no metabolismo de nitrogênio e no ciclo de vida de T. cruzi. / Trypanosoma cruzi uses amino acids as important metabolites for its maintenance and viability, glutamate and glutamine are involved in energy supply, metacyclogenesis and synthesis of pyrimidines. It was described here the glutamine synthetase of T. cruzi (TcGS), and its relation with the ammonium detoxification. Extracts of T. cruzi and pure recombinant enzyme were used to determine the kinetic parameters and the oligomeric conformation of the enzyme. In amastigote forms, we observed that TcGS is more expressed and active. In addition, methionine sulfoximine (MS) inhibited competitively the TcGS, with high affinity and reduced the epimastigotes proliferation exposed to extracellular ammonium excess, connecting TcGS to the ammonium detoxification. Therefore, the treatment with MS along the intracellular cycle reduced the trypomastigote bursting from the CHO-K1 cells, demonstrating the importance of GS on the T. cruzi life cycle establishment and nitrogen metabolism.

O objetivo neste trabalho foi avaliar a concentração de aminoácidos essenciais (Lisina e Metionina) considerada na literatura como ideal, de acordo com a via de fornecimento (sucedâneo ou concentrado inicial), e sua associação com a suplementação de Glutamato e Glutamina, para bezerros em dois sistemas de aleitamento: convencional ou programado. No primeiro experimento foi realizado o levantamento da composição bromatológica e em aminoácidos dos principais sucedâneos comercializados no Brasil. Nos segundo e terceiro experimentos foram utilizados 45 bezerros da raça Holandês, em delineamento de blocos casualizados distribuídos nos tratamentos: 1) Controle: sem suplementação; 2) Suplementação de lisina e metionina para atingir consumo de 17 e 5,3 g/d, respectivamente, com correção feita com base na análise do sucedâneo, 3) Suplementação de lisina e metionina, para atingir consumo de 17 e 5,3 g/d, respectivamente, com correção feita com base na análise do concentrado inicial. A diferença entre os experimentos foi o sistema de aleitamento ao qual os bezerros foram submetidos: no segundo experimento os animais receberam 6L/d de sucedâneo; enquanto no terceiro estudo os animais foram submetidos ao sistema de aleitamento programado (4L/d até a 2ª semana; 8L/d da 3ª a 6ª semana; 4L/d da 7ª a 8ª semana). No quarto experimento o mesmo delineamento foi utilizado para avaliar, em sistema de aleitamento convencional, os tratamentos: 1) Controle: sem suplementação; 2) Sucedâneo lácteo suplementado com Lisina e Metionina, para atingir consumo de 17 e 5,3g/d, respectivamente + 1% na MS de produto contendo 10% de glutamato e de glutamina; 3) Sucedâneo lácteo suplementado com Lisina e Metionina para atingir consumo de 17 e 5,3g/d + 0,6% na MS de produto contendo 10% de glutamato e de glutamina. Os animais foram alojados em abrigos individuais, com livre acesso a água e concentrado inicial. O consumo de concentrado inicial e o escore fecal dos animais foram registrados diariamente. Semanalmente, foram realizadas pesagens e medidas de crescimento corporal. Nas semanas 2, 4, 6, 8 e 10 de vida foram realizadas colheitas de sangue para determinação de metabólitos como marcadores do status protéico dos animais (albumina, proteína total, N-ureico), status energético (glicose e ?-hidroxibutirato), crescimento ósseo (fosfatase alcalina) e crescimento muscular (creatinina). A composição em aminoácidos dos sucedâneos comercializados no Brasil foi menor que o esperado para dieta que substitui o leite integral. Nos experimentos 2 e 3 a suplementação com lisina e metionina no sucedâneo ou no concentrado inicial para bezerros não resultou em benefícios no desempenho ou no metabolismo. No estudo 4, a suplementação do sucedâneo com lisina, metionina em associação com glutamato e glutamina não alterou o desempenho, a saúde intestinal ou o metabolismo de bezerros leiteiros. / The aim in this work was to evaluate the concentration of essential amino acids (Lysine and Methionine) considered in the literature as ideal, according to feeding route (milk replacer or starter concentrate), and its association with the supplementation of glutamate and glutamine to calves in two feeding systems: conventional or step-down. In the first study, the chemical composition was analyzed and in amino acids of main milk replacer marketed in Brazil. In the second and third studies, 45 Holstein calves were used, in randomized blocks distributed in treatments: 1) Control: without supplementation; 2) Supplementation with lysine and methionine to reach consumption of 17 and 5.3 g/d, respectively, with correction based on the analysis basis of the milk replacer, 3) Supplementation of lysine and methionine to reach consumption of 17 and 5.3 g/d, respectively, with correction based on the analysis basis of starter concentrate. The difference between the experiments was the feeding system applied to the calves: in the second study, the animals received 6 L/d of milk replacer; while in the third study, the animals were submitted to the step-down system (4L/d until the 2nd week; 8L/d of the 3nd to 6th week; 4L/d of the 7th to 8th week). In the fourth study, the same experimental design was used to evaluate, in a conventional feeding system, treatments: 1) Control: without supplementation; 2) AminoGut 0.6%: milk replacer supplemented with Lysine and Methionine, to reach consumption 17 and 5.3g/d, respectively + 0.6% product containing 10% of glutamate and glutamine; 3) AminoGut 1%: milk replacer supplemented with Lysine and Methionine to reach consumption 17 and 5.3g/d + 0.6% product containing 10% of glutamate and glutamine. The animals were housed in individual hutches, with free access to water and starter concentrate. The consumption of starter concentrate and fecal scores of animals were monitored daily. Body growth was weighed and measured weekly. In weeks 2, 4, 6, 8 and 10, blood samples were collected to determine the metabolites as markers of protein status of animals (albumin, total protein, N-urea), energy status (glucose and BHBA), bone growth (alkaline phosphatase) and muscular growth (creatinine). The composition of amino acids of the milk replacer marketed in Brazil was lower than expected for diet that replaces the whole milk. In study 2 and 3, the supplementation of the milk replacer or starter concentrate with lysine and methionine resulted in no benefit on dairy calves performance or metabolism. In study 4 the supplementation of the milk replacer with lysine and methionine in association with glutamate and glutmine had no effect on performance, gut health nor metabolism of dairy calves.

Tumourigenic cells utilize aberrant metabolic process that supports the biosynthetic requirements for hyperproliferation, survival and prolonged maintenance characterised by glucose metabolism to lactate dehydrogenase independent of oxygen availability (Warburg effect). In addition, tumourigenic cells exert increased glycolytic- and glutaminolytic activity in order to provide increased quantities of adenosine triphosphate. The aim of this research project was to investigate the influence of glutamine deprivation on proliferation, morphology, oxidative stress, mitochondrial membrane potential, cell cycle progression, antioxidant defences, deoxyribonucleic acid (DNA) damage, energy status, cell survival signaling and cell death induction in tumourigenic- and non-tumourigenic breast cell lines. In this study it was found that glutamine deprivation results in differential antiproliferative activity where the MCF-7 cell line was the most affected with decreased cell growth to 61% after 96 h of glutamine deprivation. Aberrant redox activity was most prominently observed in the MCF-7 cell line accompanied with biphasic mitochondrial membrane potential- and reactive oxygen species production. The MCF-7 cell line showed significant mitochondrial membrane depolarisation after 24 h and 96 h deprivation from glutamine (1.5- and 1.37 fold). Cell cycle progression analysis illustrated an increase in the amount of cells present in the S-phase in the MCF-7 cell line after 72 h of glutamine deprivation. The MDA-MB-231 cell line resulted in a significant increase in cells occupying the G2/M phase after 24 h of glutamine deprivation. Glutamine deprivation in the BT-20 cell line resulted in a significant increase in cells occupying G1 phase after 72 h of glutamine deprivation. The MCF-7 cell line demonstrated the least amount of viable cells when analysing apoptosis induction, when compared to the MDA-MB-231-, MCF-10A- and BT-20 cell lines after glutamine deprivation suggesting that the MCF-7 cell line is the most affected cell line. Analysis of antioxidant mechanism via superoxide dismutase (SOD) inhibition illustrated increased SOD activity in the MCF-7 cell line (9.1%) after 72 h of glutamine deprivation. Evaluation of catalase protein concentration indicated that the MCF-7 catalase expression increased to 1.28 fold after 24 h of glutamine deprivation when compared to cell propagated in complete growth medium. DNA damage was demonstrated by visualising the presence of fluorescent 8-hydroxydeoxyguanosine and showed that the MCF-7 cell line presented with significant 8-hydroxydeoxyguanosine staining. Survival signaling was also evaluated through visualising extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) signaling which demonstrated increased ERK activation in the non-tumourigenic MCF-10A cell line and decreased PI3K activation. This study provides evidence that there are differential- and time-dependent responses in breast tumourigenic cells versus non-tumourigenic cells, to glutamine deprivation thus unraveling the crosstalk between glutamine deprivation, oxidative stress and cell death and different cell types will enable us to better understand the basics of tumour cell metabolism and thus develop therapeutics that provide promising pre-sensitization potential for chemotherapeutic agents. / Dissertation (MSc)--University of Pretoria, 2020. / Physiology / MSc / Unrestricted

In recent years, U.S. swine producers have received pressure from consumers to reduce antibiotic usage. With the increased consumer pressure, pork producers have sought out other technologies, including feed additives, to reduce antibiotic usage in commercial pork production. Therefore, the objective of Chapter 2 was to determine whether supplementing L-glutamine at cost-effective levels can replace dietary antibiotics to improve pig welfare and productivity following weaning and transport. Based on previous research, we hypothesized that withholding dietary antibiotics would negatively affect pigs while diet supplementation with 0.20% L-glutamine (GLN) would have similar effects on pig performance and health as antibiotics. Mixed sex pigs (N = 480; 5.62 ± 0.06 kg BW) were weaned (18.4 ± 0.2d of age) and transported for 12 h in central Indiana, for two replicates, during the summer of 2016 and the spring of 2017. Pigs were blocked by BW and allotted to 1 of 3 dietary treatments [n = 10 pens/dietary treatment/replicate (8 pigs/pen)]; antibiotics [A; chlortetracycline (441 ppm) + tiamulin (38.6 ppm)], no antibiotics (NA), or GLN fed for14 d. On d 15 to 34, pigs were provided common antibiotic free diets in two phases. Data were analyzed using PROC MIXED in SAS 9.4. Day 14 BW and d 0 to 14 ADG weregreater (P = 0.01) for A (5.6% and 18.5%, respectively) and GLN pigs (3.8% and 11.4%, respectively) compared to NA pigs, with no differences between A and GLN pigs. Day 0 to 14 ADFI increased for A (P < 0.04; 9.3%) compared to NA pigs; however, no differences were detectedcomparing GLN to A and NA pigs. Once dietary treatments ceased, no differences (P > 0.05) in growth performance amongdietary treatments were detected. On d 13, plasma tumor necrosis factor alpha (TNF-α) was reduced (P = 0.02) in A (36.7 ± 6.9 pg/ml) and GLN pigs (40.9 ± 6.9 pg/ml) versus NA pigs (63.2 ± 6.9 pg/ml). Aggressive behavior tended to be reduced overall (P = 0.09; 26.4%) in GLN compared to A pigs, but no differences were observed between A andGLN versus NA pigs. Huddling, active, and eating/drinking behaviors were increased overall (P < 0.02; 179, 37, and 29%, respectively) in the spring replicate compared to the summer replicate. A subset of pigs from Chapter 2 were utilized, in Chapter 3, toevaluate the dietary treatment effects on intestinal morphology and gene expression. On d 33, mast cells/mm2were increased (P= 0.05) in GLN and NA pigs vs. A pigs (22.2% and 19.7%, respectively). On d 33, villus height:crypt depth tended to be increased(P= 0.07; 7.0%) in GLN and A pigs vs. NA pigs.On d 33, glucagon-like peptide 2 (GLP-2)mRNA abundance was decreased (P= 0.01; 50.3%) in GLN and NA pigs vs. A pigs.Crypt depth was increased (P= 0.01; 16.2%) and villus height:crypt depthratiowas reduced (P= 0.01; 9.6%)during the spring replicate compared to the summer replicate on d 33. On d 13, TNF-α and occludin mRNA abundance wereincreased (P≤0.04; 45.9%and 106.5%, respectively)andzonula occludens-1(ZO-1)mRNA abundance tended to be increased (P= 0.10; 19.2%) in the spring replicate compared to the summer replicate. Previous research and the results of Chapter 2 indicates that supplementing nursery diets with 0.20% GLN provides similar growth and health benefits as dietary antibiotics, but it is unknown whether greater inclusion levels will provide additional benefits. Therefore, the objectiveof Chapter 4 was to evaluate the impact of replacing dietary antibiotics with increasing levels ofGLNon growth performance, health status, and production costs in pigs following weaning and transport. We hypothesized that diet supplementation with 0.20% to 1.00% GLNwould incrementally improvepig health and productivity compared to dietary antibiotics. Mixed sex pigs (N = 308; 5.64 ± 0.06 kg BW) were weaned (19.1 ± 0.2 d of age) and transported in central Indiana during the autumn of 2017. Pigs were blocked by BW and allotted to 1 of 7 dietary treatments (n = 8 pens/dietary treatment); A[chlortetracycline (441 ppm)+ tiamulin (38.6 ppm)], NA, 0.20% GLN, 0.40% GLN, 0.60% GLN, 0.80% GLN, or 1.00% GLNfed for 14 d. On d 15 to 35, pigs were provided NA diets in two phases. Data were analyzed using PROC MIXED in SAS 9.4. Overall, ADG (P= 0.04; 6.4%) and ADFI (P= 0.04; 6.9%) were reduced in NA pigs vs. 0.40% GLN or A pigs. Increasing GLN in the diet tended to increase (linear;P= 0.10) ADG. Overall, increasing GLN in the diet tended to increase (linear; P= 0.08) d 35 BW. Day 35 BW was greater (P= 0.01) in 0.80%GLN and A pigs compared to NA, 0.20% GLN, and 0.60% GLN pigs, but no BW differences were detected between 0.80% GLN and A and 0.40% GLN and 1.00% GLN pigs. In addition, d 35 BW was greater (P= 0.01) for 0.40% GLN and 1.00% GLN compared to 0.20% GLN. Overall income over feed and therapeutic injectable antibiotics cost for enteric and unthrifty challenges was greater (P= 0.02) in 0.80% GLN pigs compared to NA, 0.20% GLN, and 0.60% GLN pigs, but no income over feed and therapeutic injectable antibiotics cost for enteric and unthrifty challenges differences were detected between 0.80% GLN pigs and 0.40% GLN, 1.00% GLN, and A pigs. Health challenges in swine herds negatively impact swine growth rate and performance. Therefore,utilizing the pigs from Chapter 2, the study objective for Chapter 5 was to quantifythe impact of differences in rearing conditions through post hoc analysis on growth performance, tissue accretion rates, and production economics in pigs during different replicates(summer or spring). We hypothesized that pigs reared under health challenged conditions would have decreased growth performance and tissue accretion rates resulting in increased production costs compared to pigs reared with less health challenges. Data were analyzed using PROC MIXED and PROC NLMIXED in SAS 9.4. Therapeutic injectable antibiotics cost was reduced(P= 0.01; 246.7%) in the spring replicate compared to the summer replicate. Income over feed and therapeutic injectable antibiotics cost was greater (P= 0.01; 23.1%; $16.62/pig) in the spring replicate compared to the summer replicate.Predicted ADG was greater (P≤ 0.05) in spring replicate barrows compared to thesummer replicate barrows during the ranges of 22 to 38 and 119 to 177 days of age, respectively. Spring replicate gilts had greater ADG (P≤ 0.05) compared to summer replicate gilts during the ranges of 22 to 47 and 112 to 177 days of age, respectively. The maximum predicted empty body protein accretion rate for the summer replicate gilts and the spring replicate gilts is 145 and 156 g/d, respectively. In conclusion, GLN supplementation improved pig performance and health after weaning and transport similarly to A across studies and GLN shows promise as an antibiotic alternativewith 0.40% GLN appearing to be the optimal level. Health challenges in pigs can have profound negative impacts on tissue accretion ratesand key economic drivers for pork producers such as poorer feed efficiency and reduced hot carcass weight. The adverse health effects resulting in reduced growth performance, increased production costs($16.62/pig), and negatively impact producer profitability.<p> </p>

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20299号 / 薬科博第68号 / 新制||薬科||8(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 松﨑 勝巳, 教授 加藤 博章, 教授 石濱 泰 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

Field and greenhouse experiments were conducted to determine barnyardgrass control as affected by glufosinate and graminicide tank-mixtures, application timing of tank-mixtures of graminicides plus glufosinate and application time of day of tank-mixtures of glufosinate and clethodim. When increased rates of graminicide were tank-mixed with glufosinate, barnyardgrass control was unaffected by quizalofop-P plus glufosinate; however, clethodim plus glufosinate control in the field indicated the potential for reduced barnyardgrass control. When evaluating increasing glufosinate rates tank-mixed with graminicides, barnyardgrass control was not negatively affected by the combination of glufosinate and graminicides. The difference in soybean yield among the graminicides may indicate that the cyclohexanedione herbicides had a slight yield advantage over the aryloxyphenoxypropionate herbicides due to potential increased levels of barnyardgrass control. Applications of glufosinate alone provide variable control throughout the growing season in both field and greenhouse experiments. Although barnyardgrass control in the field was not affected by glufosinate application timing, data from the greenhouse indicates potential exists for reduced control if glufosinate is applied 1 or 3 d before graminicides. Clethodim was unaffected by application time of day; however, glufosinate applications at midnight reduced barnyardgrass control compared to applications made at noon and 6 P.M. Applications at 6 A.M. also reduced barnyardgrass efficacy compared to applications at 6 P.M. Environmental factors such as temperature and light at the time of application are likely responsible for the time of day effects observed in these studies. For maximum benefit from incorporating graminicides into a glufosinate weed control system, fluazifop-P, quizalofop-P, clethodim and sethoxydim should be applied with glufosinate at 594 or 890 g ai ha-1. Sequential treatments of glufosinate should be applied 7 d prior to a graminicide application or 1, 3 or 7 d after a graminicide application. To optimize barnyardgrass efficacy with tank mixtures of glufosinate and clethodim, applications should be made at noon or early evening to avoid potential time of day effects.

Metabolic reprogramming has been recognised as a hallmark in solid tumours. Malignant modification of the tumour’s bioenergetics provides energy for tumour growth and progression. Otto Warburg first reported these metabolic and biochemical changes in 1927. In prostate cancer (PCa) epithelial cells, the tumour metabolism also changes during development and progress. These alterations are partly driven by the androgen receptor, the key regulator in PCa development, progress, and survival. In contrast to other epithelial cells of different entities, glycolytic metabolism in prostate cells sustains physiological citrate secretion in the normal prostatic epithelium. In the early stages of PCa, citrate is utilised to power oxidative phosphorylation and fuel lipogenesis, enabling tumour growth and progression. In advanced and incurable castration-resistant PCa, a metabolic shift towards choline, amino acid, and glycolytic metabolism fueling tumour growth and progression has been described. Therefore, even if the metabolic changes are not fully understood, the altered metabolism during tumour progression may provide opportunities for novel therapeutic strategies, especially in advanced PCa stages. This review focuses on the main differences in PCa’s metabolism during tumourigenesis and progression highlighting glutamine’s role in PCa.

Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.

La croissance et la capacité d'absorption d'azote de la microalgue d'eau douce Chlorella vulgaris co-immobilisée dans des billes d'alginate, en présence de la bactérie promotrice de croissance des microalgues Azospirillum brasilense Cd, ont été étudiés. La co-immobilisation des microalgues avec A. brasilense conduit à deux phénomènes indépendants directement affectés par des facteurs de culture, tels que la forme azotée, le pH et la présence d'une source de carbone. Premièrement, la croissance de la population de microalgues augmentait sans une augmentation de la capacité d'absorption d'azote par les cellules seules, ou deuxièmement, la capacité des cellules à absorber l'azote augmentait sans une augmentation de la population entière de microalgues. Ces phénomènes étaient dépendants de la densité de la population des microalgues, qui était à son tour affectée par les facteurs de culture. Cela renforce la conclusion selon laquelle la taille de la population de microalgues contrôle l'absorption d'azote chez les cellules de C. vulgaris -plus la population est élevée (peu importe les paramètres expérimentaux), moins les cellules absorbent d'azote. Les résultats ont montré que YAzospirillum affectait la croissance de la Chlorella vulgaris. L'un des processus proposés par lequel XAzospirillum promeut la croissance des plantes supérieures, est la production de phytohormones, principalement 1' acide 3-indole acétique (auxine ou AI A). Par conséquent, l'effet de cette phytohormone, produite par plusieurs souches de VAzospirillum et favorisant la croissance de microalgue, a été étudié. Cinq souches de type sauvage â!Azospirillum {A. brasilense souches Cd, Sp6, 8-1 et Sp245 et A. lipoferum JA4) et leurs mutants déficients en production d'AIA : A. brasilense SpM 7918 (modifié de A. brasilense Sp6), A. brasilense FAJ0009 (modifié de A. brasilense Sp245), A. brasilense S-l-gfp (modifié de A. brasilense 8-1) et A. lipoferum 3A4::ngfp\5 (modifié de A. lipoferum JA4) ont été co-immobilisés avec C. vulgaris en billes d'alginate. Aucune production d'AIA n'est possible sans l'apport de tryptophane, nos résultats corroborant des observations antérieures selon lesquelles la production d'AIA chez Azospirillum est induite par la présence de tryptophane. Les taux élevés de tryptophane étaient délétères sur la croissance des pousses de C. vulgaris utilisées. Cette recherche montre que que l'AIA produit par 1' Azospirillum spp. est impliqué dans la croissance du C vulgaris immobilisé dans des billes d'alginate, et que la co-immobilisation avec différentes souches d'Azospirillum spp. atténue les effets délétères du tryptophane sur le C. vulgaris. Co-immobilisation de Chlorella vulgaris avec Azospirillum brasilense affecte l'absorption d'ammonium par les microalgues par le biais de métabolismes indéfinis. Les résultats présentés dans ce travail montrent clairement qu'il y a un effet & Azospirillum sur les activités spécifiques de la Glutamine synthétase (GS) et de la Glutamate déshydrogénase (GDH) de Chlorella. Lorsque Chlorella coimmobilisée absorbe plus d'ammonium que Chlorella seule, les deux enzymes fonctionnent en même temps, et les activités spécifiques par cellule de la GS et de la GDH sont plus élevées. Toutefois, dans les cas où seule l'activité GS augmente, mais pas celle de la GDH, l'activité GS plus élevée n'est pas suffisante pour atteindre une absorption d'ammonium plus élevée au niveau de la culture. En résumé, il y a un effet positif d'A. brasilense sur l'activité de la glutamine synthétase et de la glutamate déshydrogénase chez Chlorella. La régulation et la relation de l'activité enzymatique avec la concentration initiale en ammonium demeurent floues .Les données présentées dans ce chapitre dans leur ensemble montrent que les activités plus élevées de GS et de GDH augmentent l'absorption d'ammonium par Chlorella. / Growth and capacity to absorb nitrogen in the freshwater microalgae Chlorella vulgaris when co-immobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense Cd and the mechanisms involved were studied in this work. Co-immobilization of the microalgae with A. brasilense leads to two independent phenomena directly affected by culturing factors, such as nitrogen species, pH, and presence of a carbon source. First, growth of the microalgae population increased without an increase in capacity of the single cells to absorb nitrogen; second, the capacity of cells to absorb nitrogen increased without increasing the microalgae population. These phenomena were dependent on population density of the microalgae, which was, in turn, affected by culturing factors. This supports the conclusion that the size of the microalgae population controls absorption of nitrogen in C. vulgaris cells—the higher the population (whatever the experimental parameters), the less nitrogen each cell takes up. The results show that Azospirillum affects the growth of Chlorella vulgaris. One of the mechanisms proposed by which Azospirillum promotes growth of higher plants is the production of phytohormones, mainly the auxin indole-3-acetic acid (IAA); therefore the effect of this phytohormone produced by several strains of Azospirillum in promoting growth of the microalgae was studied. Five wild type strains of Azospirillum {A. brasilense strains Cd, Sp6, 8-1, and Sp245 and A. lipoferum JA4) and their indole-3-acetic-acid déficient mutants, A. brasilense SpM 7918 (modifïed from A. brasilense Sp6), A. brasilense FAJ0009 (modified from A. brasilense Sp245), A. brasilense %-\-gfp (modified from A, brasilense 8-1), and A. lipoferum lA4::ngfp\5 (modified from A. lipoferum JA4) were coimmobilized with C. vulgaris in alginate beads. No IAA production occurred without tryptophan. Our results corroborate previous observations that IAA production in Azospirillum is induced by the presence of tryptophan. It was found that high levels of tryptophan are deleterious to the growth of the C. vulgaris strain we used. This study demonstrated that IAA produced by Azospirillum spp. is involved in growth promotion of C. vulgaris immobilized in alginate beads, and that co-immobilization with several strains of Azospirillum spp. alleviated the deleterious effects of tryptophan in C. vulgaris. Co-immobilization of Chlorella vulgaris with Azospirillum brasilense affects ammonium absorption by the microalgae via undetermined metabolism. The results presented in this work show clearly that there is an effect of Azospirillum on the specific activity of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella. When coimmobilized Chlorella absorbs more ammonium than Chlorella alone, both enzymes are operating at the same time, and the specific activity per cell of GS and GDH are higher. However, in cases when only the activity of GS increases, but not of GDH, this GS higher activity was not sufficient to yield a higher absorption of ammonium at the culture level. There is a positive effect of A. brasilense on the activity of glutamine synthetase and glutamate dehydrogenase in Chlorella. However, the regulation and the relation of the enzymatic activity with initial ammonium concentration are still unclear.

Changes in physiological function occurring during a body water deficit may result in significant decrements in performance, cognitive function and fine motor control during exercise. This may be due to the magnitude of the body water deficit. Rehydration strategies are important to prevent these deleterious effects in performance. The purpose of this study was to examine the changes before and after prolonged exercise of an alanine-glutamine dipeptide (AG) on cognitive function and reaction time. Twelve male endurance-trained runners (age: 23.5 [plus or minus] 3.7 y; height: 175.5 [plus or minus] 5.4 cm; weight: 70.7 [plus or minus] 7.6 kg) participated in this study. Participants were asked to run on a treadmill at 70% of their predetermined VO2max for 1 h and then run at 90% of VO2max until volitional exhaustion on four separate days (T1-T4). T1 was a dehydration trial and T2-T4 were all different hydration modalities (electrolyte drink, electrolyte drink with a low dose of AG, electrolyte drink with a high dose of AG, respectively) where the participants drank 250 mL every 15 min. Before and after each hour run, cognitive function and reaction tests were administered. Hopkins Magnitude Based Inferences were used to analyze cognitive function and reaction time data. Results showed that physical reaction time was likely faster for the low dose trial than the high dose trial. Dehydration had a possible negative effect on the number of hits in 60-sec compared to both the low and high dose trials. Comparisons between only the electrolyte drink and the high dose ingestion appeared to be possibly negative. Analysis of lower body quickness indicates that performance in both the low and high dose trials were likely improved (decreased) in comparison to the dehydration trial. Multiple object tracking analysis indicated a possible greater performance for dehydration and low dose compared to only the electrolyte drink, while there was a likely greater performance in multiple object tracking for the high dose trial compared to consumption of the electrolyte drink only. The serial subtraction test was possibly greater in the electrolyte drink trial compared to dehydration. Rehydration with the alanine-glutamine dipeptide during an hour run at a submaximal intensity appears to maintain or enhance subsequent visual reaction time in both upper and lower body activities compared to a no hydration trial. The combination of the alanine-glutamine dipeptide may have enhanced fluid and electrolyte absorption from the gut and possibly into skeletal tissue to maintain neuromuscular performance.