Dans les leucémies aiguës myéloïdes (LAM), l'activation anormale de nombreuses voies de signalisation intracellulaires favorise la croissance et la survie des cellules tumorales. L'amélioration des connaissances biologiques de ces pathologies hétérogènes, dont le pronostic est réservé, devrait permettre le développement de thérapies ciblées. La kinase oncogénique mTOR est présente au sein de deux complexes, parmi lesquels mTORC1, activé constitutivement dans la majorité des blastes primaires de patients porteurs de LAM, qui contrôle la synthèse protéique, et mTORC2 activé constitutivement dans 50% des LAM. Les inhibiteurs allostériques de mTORC1 (la rapamycine et ses dérivés) n'inhibent pas la phosphorylation du répresseur traductionnel 4E-BP1, ne diminuent pas la traduction et induisent peu d'apoptose in vitro dans les LAM. Utilisés en monothérapie, leur effet est décevant. De plus ces inhibiteurs n'agissent pas sur le complexe mTORC2. J'ai étudié l'effet d'un inhibiteur catalytique de mTOR, l'AZD8055, actif sur les deux complexes. In vitro, l'AZD8055 inhibe efficacement la signalisation en aval de mTORC1 et de mTORC2, dont les sites de phosphorylation de 4E-BP1 résistants à la rapamycine, ainsi que la synthèse protéique. Il diminue la prolifération, bloque le cycle cellulaire en phase G0G1 et induit une apoptose caspase-dépendante dans les blastes primaires de LAM. Il diminue également la clonogénicité des progéniteurs leucémiques, sans affecter celle des cellules CD34+ normales. Dans un modèle murin de xéno-transplantation, l'AZD8055 inhibe la croissance tumorale et améliore la survie des souris traitées. Je me suis également intéressée à la régulation de l'activité de mTORC1 par les acides aminés. Dans les cellules de mammifères, l'activation de mTORC1 nécessite la présence de glutamine et de leucine qui agissent en coopération via deux transporteurs membranaires, SLC1A5 et SLC7A5/SLC3A2. J'ai montré que la privation en glutamine, obtenue par l'activité glutaminase de la drogue L-asparaginase ou par l'utilisation de milieux de culture spécifiques dépourvus sélectivement en acides aminés, inhibe l'activation de mTORC1 et induit de l'apoptose dans diverses lignées leucémiques et dans les blastes primaires de LAM. La L-asparaginase inhibe la synthèse protéique et ses effets fonctionnels sont liés à son activité glutaminase. J'ai pu également constater une augmentation de l'expression protéique de la glutamine synthase induite par la Lasparaginase, dont l'inhibition majore l'apoptose induite par la L-asparaginase dans certaines lignées leucémiques. J'ai également étudié l'effet de l'inhibition spécifique par un shARN inductible du transporteur SLC1A5, qui permet l'import de glutamine. L'inhibition de SLC1A5 bloque la réactivation de mTORC1 par l'association leucine/glutamine après privation et induit de l'apoptose dans la lignée leucémique MOLM14. Cette inhibition diminue la croissance tumorale dans un modèle de xénogreffe

Oxidative stress has been associated with many disease conditions in adults and neonates based on clinical and post mortem studies. Trauma is the commonest cause of oxidative stress. However a gap in knowledge of the natural history of oxidative stress in humans was identified as most studies have been post mortem or in animals. <p>The aim of this research is to understand treat and oxidative stress in trauma and surgical patients. The study involved three components including: the development and evaluation of the novel oxistress assay; study of clinical trauma and oxidative stress; and clinical trial of alanyl-glutamine supplementation following major surgery. The novel oxistress assay was used on urine samples in the normal population to determine reference values and subsequently on hospital patients to determine sensitivity and specificity. The study of clinical trauma and oxidative stress evaluated plasma antioxidants (FRAP assay), red cell glutathione (Asensis method), plasma and urine protein carbonyl (Levines method) and total oxidants in plasma and urine (oxistress assay) over 7 day period following trauma. The clinical trial was a double blind study of 69 major surgery patients evaluating biochemical and clinical parameters over 7 day period in comparison with pre-operative status. <p>The novel oxistress assay proves to be a sensitive and accurate bedside diagnostic tool for oxidative stress. It can also be used in the laboratory setting. Oxidative stress is associated with increased trauma severity resulting in antioxidant depletion, strong oxidant production and protein degradation. The presence of pre-morbid medical factors also increased oxidative stress in trauma patients. Oral alanyl-glutamine supplementation (0.3 g/kg) increased plasma glutamine and antioxidant levels while decreasing urine oxidant levels. It significantly reduced hospital stay in non-cancer and higher disease complexity patients. The intervention also reduced the resource intensity weighting (RIW) score. <p>Oxidative stress is a clinical problem in surgery and trauma patients that can now be easily diagnosed at the bedside using the novel oxistress assay. Treatment with alanyl-glutamine is effective in reducing oxidative stress and improving clinical outcome. It is highly recommended probably at a higher dose in order to achieve optimal results.

Οι ασθενείς με αποφρακτικό ίκτερο έχουν αυξημένη συχνότητα επιπλοκών και θανάτου ιδιαιτέρως μετά από επεμβατικούς διαγνωστικούς ή θεραπευτικούς χειρισμούς. Ο νετερικός βλεννογόνος αποτελεί ανατομικό και λειτουργικό φραγμό ο οποίος περιορίζει εντός του αυλού τα βακτηρίδια και τις ενδοτοξίνες εμποδίζοντας τους την δίοδο στη συστηματική κυκλοφορία και την διασπορά τους. Υπάρχουν όμως παθολογικές καταστάσεις κατά τις οποίες ο εντερικός βλεννογόνος ανεπαρκεί επιτρέποντας την είσοδο στην κυκλοφορία και την διασπορά ενδοτοξινών και ενερικών μικροβίων – το φαινόμενο είναι γνωστό με τον όρο βακτηριακή μετακίνηση (bacterial translocation) – με αποτέλεσμα σηπτικές επιλοκές, ανεπάρκεια πολλαπλών οργάνων και υψηλή θνητότητα. Πειραματικές και κλινικές μελέτες έδειξαν ότι ο αποφρακτικός ίκτερος προκαλεί δυσλειτουργία του βλεννογόνιου εντερικού φραγμού με αποτέλεσμα την ενδοτοξιναιμία και βακτηριακή μετακίνηση που διαδραματίζουν κεντρικό ρόλο στην ανάπτυξη επιπλοκών στους ασθενείς με αποφρακτικό ίκτερο. Η έκθεση στην ενδοτοξίνη είναι σημαντική γιά την ανάπτυξη του ανοσοποιητικού συστήματος του ξενιστή, αλλά όταν εκτεθεί σε υψηλές συγκεντρώσεις τα αποτελέσματα είναι επιβλαβή. Υπό φυσιολογικές συνθήκες ο εντερικός φραγμός εμποδίζει την δίοδο των μικροβίων στο στείρο περιβάλλον της περιτοναικής κοιλότητας, της πυλαίας φλέβας και της συστηματικής κυκλοφορίας. Στο ήπαρ ο πληθυσμός των κυττάρων Kuppfer αποτελεί τον κύριο όγκο του δικτυοενδοθηλιακού συστήματος κι είναι στρατηγικά τοποθετημένος στην συμβολή του συστήματος της πυλαίας για την εξουδετέρωση κι απομάκρυνση ενδοτοξινών και βακτηριδίων της πυλαίας. Λειτουργικές διαταραχές είτε του εντερικού βλεννογόνου είτε των κυττάρων Kuppfer -λόγω κατασταλμένης εκκαθαριστικής ικανότητας εξαιτίας της χολόστασης - έχει ως αποτέλεσμα την διαφυγή ενδοτοξινών και μικροβίων αρχικά στους μεσεντέριους λεμφαδένες, στην πυλαία και στην συνέχεια μέσω της συστηματικής κυκλοφορίας σε απομακρυσμένα όργανα. Ο σκοπός της διατριβής αυτής είναι η επιβεβαίωση και η μελέτη της ενδοτοξαιμίας και βακτηριακής μετακίνησης στο πειραματικό μοντέλο του αποφρακτικού ικτέρου , καθως και η προσπάθεια αποτροπής του φαινομένου μέσω χορήγησης τροφικών κι ανοσοδιεγερτικών παραγόντων και συγκεκριμένα γλουταμίνης κι αργινίνης. Τα αποτελέσματα της παρούσας μελέτης επιβεβαίωσαν την ύπαρξη πυλαίας και συστηματικής ενδοτοξιναιμίας και βακτηριακής μετακίνησης στην εξωηπατική απόφραξη των χοληφόρων. Παρατηρήθηκε επίσης αύξηση της απόπτωσης στον εντερικό βλεννογόνο όπως τεκμηριώθηκε με βάση την μορφομετρική ανάλυση και τις μετρήσεις DNA και πρωτείνης, μηχανισμός που οδηγεί στην περαιτέρω ατροφία του βλεννογόνου πιθανώτατα μέσω διαταραχής στην αναλογία κυτταρικού πολλαπλασιασμού και κυτταρικού θανάτου. Η γλουταμίνη διαδραματίζει σημαντικό ρόλο στον εντερικό μεταβολισμό, δομή και λειτουργία όπως και σε αλλα ταχέως πολλαπλασιαζόμενα κύτταρα Η χορήγησή της φαίνεται να ελαττώνει την ΒΜ και να βελτιώνει την επιβίωση σε αρκετά μοντέλλα σήψης εντερικής προελεύσεως πιθανώς με τροφική δράση στον εντερικό βλεννογόνο και στα κύτταρα του ανοσοποιητικού Η αργινίνη, ειναι σημαντικό αμινοξύ για την λειτουργια των λεμφοκυττάρων και βελτιώνει την κυτταρική απάντηση του ανοσοποιητικού όταν χορηγηθεί τοσο σε ανθρώπους όσο και σε πειραματοζωα. Η αργινίνη όμως, επιπλέον συγκεντρώνει το ενδιαφέρον των μελετητών γιατι αποτελεί πρόδρομο μόριο του μονοξειδίου του αζώτου, μόριο με πολλαπλές λειτουργίες τόσο στην ανοσία και φλεγμονή όσο και στη εντερική διαπερατότητα και βακτηριακή μετακίνηση Η χορήγηση των δύο αυτών ανοσοθρεπτικών αμινοξέων μείωσε την ενδοτοξιναιμία και βακτηριακή μετακίνηση δρώντας πιθανώς τόσο στο ανοσοποιητικό σύστημα όσο και στην ακεραιότητα του εντερικού βλεννογόνου με μείωση της απόπτωσης και στην περίπτωση της γλουταμίνης παράλληλη αύξηση του κυτταρικού πολλαπλασιασμού, όπως αναδείχθηκε στην μέτρηση των μιτώσεων και στην αύξηση του εντερικού DNA και πρωτείνης. Σε περιπτώσεις εξωηπατικής απόφραξης των χοληφόρων έχει παρατηρηθεί σημαντική καταστολή του ανοσοποιητικού συστήματος με επίδραση τόσο στα Τ-κύτταρα όσο και στα κύτταρα Kuppfer. Προηγούμενες αναφορές στη δυσλειτουργία της κυτταρικής ανοσίας εισηγούνται ότι η καταστολή της δραστηριότητας των Τ-κυττάρων έχουν άμεσο αποτέλεσμα στη συστηματική ενδοτοξιναιμία, ενώ η καταστολή του μιτογονικού ερεθίσματος των Τ- κυττάρων συμβαίνει δευτερογενώς ως αποτέλεσμα βακτηριακής μετακίνησης. Στη δική μας μελέτη η χορήγηση των ανοσοθρεπτικών αυτών πεπτιδίων ελάττωσε την βακτηριακή μετακίνηση και βελτίωσε σημαντικά τις ιστολογικές αλλοιώσεις που προκαλεί η εξωηπατική χολόσταση στα πυλαία διαστήματα του ήπατος με πιθανώτερο αιτιοπαθογενετικό μηχανισμό την μείωση της ενδοτοξιναιμίας στην πυλαία φλέβα. Συμπερασματικά τα αποτελέσματα της παρούσας μελέτης επιβεβαιώνουν την ενδοτοξιναιμία και βακτηριακή μετακίνηση στον πειραματικό αποφρακτικό ίκτερο και δείχνουν πως η χορήγηση ανοσοθρεπτικών αμινοξέων όπως η γλουταμίνη και αργινίνη περιορίζουν το φαινόμενο με παράλληλη βελτίωση της ιστολογικής εικόνας τόσο τού ήπατος όσο και του λεπτού εντέρου. / Invasive diagnostic and therapeutic procedures in the presence of obstructive jaundice are associated with a high morbidity and mortality rate, primarily due to septic complications and renal impairement. Bacterial translocation is known as the passage of viable bacteria or endotoxins from the gut to the mesenteric lymph nodes and other organs which may commence or exacerbate the septic state. This study was undertaken to investigate the influence of experimental obstructive jaundice in wistar rats on portal and systematic (aortal) endotoxaemia, on bacterial translocation (ΒΤ) to mesenteric lymph nodes and distant organs, and on liver and ileal histology and apoptosis. In addition, in an attempt for therapeutic intervention we have explored the possible beneficial effect of immunonutrition in obstructive jaundice by administration of glutamine or arginine per os. The amino-acid glutamine although non essential and in abundance, is required as an enterocyte fuel while it seems to possess anabolic properties for the liver, skeletal muscle, intestinal mucosa and the immune system (e.g. lymphocytes, macrophages) and its supplementation has resulted in beneficial clinical outcomes. Glutamine supplements have been used to protect the intestinal mucosa and enhance the immune system. Various authors have postulated that glutamine may prevent or attenuate BT. Arginine plays an important role in many functions including protein synthesis and katabolism. Arginine is important in the function of lymphocytes and improves cellular immune response when administered. In addition it is the sole precursor amino acid in the generation of the immunity-enhancing agent nitric oxide (NO) by NO-synthase. In animals and humans in sepsis plasma L-arginine concentrations are markedly low, may become an indispensable amino acid, and low arginine concentrations are correlated with a worse prognosis. Arginine deficiency was demonstrated in bile duct ligated rats. Dietary L- arginine has been shown to enhance the immune system and wound healing. In obstructive jaundice the absence of intraluminal bile deprives the gut from its bacteriostatic, endotoxin neutralizing and mucosal trophic effect leading to increased intestinal bacterial and endotoxin load together with mucosal atrophy. These alterations promote bacterial and endotoxin translocation into the portal circulation and subsequently, through a decreased clearance capacity of Kupffer cells due to cholestasis, into the systemic circulation. Systemic endotoxaemia activates further a systemic inflammatory response, with dysfunctions of remote organs which in a vicious circle aggrevates the intestinal barrier dysfunction and liver injury. Sevenry-five male Wistar rats were randomly divided in four groups (15 each): I controls, II sham operated, III bile duct ligation (BDL), IV BDL + glutamine (3%in drinking water) and V BDL + arginine (2% in drinking water). Ileal samples for histology, DNA and protein content, liver biopsies, mesenteric lymph nodes (MLN) for cultures, portal and systemic blood samples for endotoxin measurements were obtained 10 days later. Compared to control, a significant increase in contaminated MLN and liver samples and in endotoxaemia was noted in group III (P < 0.01) which was significantly reduced in group IV and V (P < 0.05). Group IV presented also a significantly higher number of mitoses/crypt (M/c) , less apoptotic body counts (as did group V) and a higher DNA content compared to group III (P < 0.05). Liver biopsies from group III displayed typical changes of large duct obstruction that were significantly improved after glutamine or arginine treatment, with decreased ductular proliferation. Two main features highlight the forementioned dual role of the liver in sepsis: the first is the importance of the liver blood supply and particularly the portal flow, which arises from the splachnomesenteric vascular bed, a region especially subject to vasoconstriction and bacterial translocation. The second is the cellular heterogenicity of the liver, which is mainly hepatocytes, Kupffer cells, and endothelial sinusoidal cells. All of these cell types are involved in the immune, anti-infectious, and metabolic responses through multiple cell cross-talk and interactions. We had a beneficial effect on alterations of portal triads in the liver of glutamine or arginine supplemented rats. The mechanisms involved are not fully elucidated but possible explanations could be either the reduction of BT or an immunostimulatory up regulation by administered two immunonutrients. In conclusion, the present study verifies the increased incidence of bacterial and endotoxin translocation in experimental obstructive jaundice. Apoptosis in the small bowel of bile duct ligated rats is also increased. Oral administration of glutamine or arginine was shown to reduce both BT and endotoxaemia, improved histology in the terminal ileum and liver and also decreased apoptosis in the small bowel in extrahepatic experimental jaundice. These findings offer further insight in the pathophysiology of obstructive jaundice and suggest important biological effects of immunonutrition. Understanding the pathophysiology of barrier alterations in obstructive jaundice at the cellular and molecular level will allow novel treatment approaches and a better prognosis for patients in clinical settings.

Glutamine (Gln) is a critical intermediate in nitrogen metabolism in all organisms. Here, a whole cell biosensor (GlnLux) for Gln was constructed by transforming a bacterial Gln auxotroph with a constitutive lux reporter. The biosensor was optimized for sensitivity, linearity, efficiency, specificity and robustness to permit detection of Gln in vitro and in vivo. The optimized GlnLux biosensor achieved nanomolar sensitivity with Gln standards. Extracts from only 1 mg of maize (Zea mays L.) leaf tissue were sufficient for Gln detection by GlnLux. Measurements of Gln in leaf extracts by GlnLux correlated with quantification by high performance liquid chromatography (Spearman r = 0.95). GlnLux permitted indirect in planta imaging of Gln using a CCD camera, enabling identification of plants that had been fertilized with nitrogen. Imaging using GlnLux also resolved predicted spatial differences in leaf Gln concentration. In a second application, it was demonstrated that GlnLux embedded into agar permits non-destructive screening of co-inoculated bacterial colonies for biological nitrogen fixation (BNF). GlnLux agar was able to distinguish a Bradyrhizobium japonicum wild type strain (nif+) from a mutant strain defective in nitrogenase (nif-) following ≥8 h of co-incubation. The technology was used to screen a bacterial endophyte diversity library cultured from Zea mays (L.) seeds for biological nitrogen fixation. / OMAFRA

The pathway of seawater ammonium assimilation and influence of light on amino acid synthesis remain unresolved in cnidarian symbioses. Labeled ammonium (10 μM 15NH4Cl) in seawater was used to trace the pathway of the incorporation into amino acids in host tissue, Zoanthus sp., and zooxanthellae, Symbiodinium microadriaticum. Freshly isolated zooxanthellae were exposed to 20 μM 15NH4Cl with coral homogenate to evaluate the role of host factors on amino acid synthesis. High performance liquid chromatography and mass spectrometry was used to measure percent labeling and concentrations of amino acids. In zooxanthellae, ammonium was assimilated into glutamine likely via glutamine synthetase and into glutamate via glutamine 2-oxoglutarate amidotransferase. Interrupting photosynthesis with DCMU did not inhibit glutamine and tryptophan synthesis however reduced the 15N-enrichment of glutamate, aspartate, and ornithine in zooxanthellae, as well as arginine, ornithine, and lysine in host tissue. Coral homogenate had little effect on the 15N-enrichment of glutamine, aspartate, and alanine in freshly isolated zooxanthellae. Evidence is presented to support the uptake of ammonium ions and data shows that glutamine and not glutamate is translocated to the coral host.

Le but de cette étude consiste à démontrer l’impact positif d’une supplémentation en glutamine chez des nageurs élites, afin d’améliorer le statut immunitaire et d’évaluer si les changements plasmatiques de la glutamine peuvent expliquer l’incidence d’infections des voies respiratoires (IVRS). En parallèle, ce projet évalue si les apports alimentaires influencent la glutamine plasmatique et l’incidence d’IVRS. L’étude s’est effectuée auprès de 14 athlètes élites (8 hommes, 6 femmes). Chaque athlète a participé aux deux conditions expérimentales : un supplément de glutamine et une solution placebo isocalorique. Les périodes de supplémentation se déroulaient sur sept jours, incluant trois journées consécutives de compétition. Le profil hématologique, après les compétitions, montre qu’un supplément de glutamine n’améliore pas significativement la concentration plasmatique en glutamine ni les niveaux de cytokines comparativement à une solution placebo. Bien que les résultats soient semblables sous les deux conditions, les niveaux post-compétition ont tendance à être supérieurs aux valeurs pré-supplémentation, lorsqu’un apport exogène en glutamine est fourni à l’organisme alors que les concentrations plasmatiques de glutamine tendent à diminuer lorsqu’une solution placebo est administrée (p=0.067). L'incidence d’IVRS ne peut être expliquée par une faible concentration plasmatique de glutamine ni par un apport exogène de glutamine. On observe cependant une augmentation d’IVRS suite aux compétitions, soient de 8 athlètes pour le groupe placebo contre 3 au groupe glutamine. Les athlètes atteints d'IVRS semblent consommer moins d'énergie totale (kcal) et de protéines que les athlètes sains (p=0.060). Les résultats obtenus ne démontrent pas qu’une supplémentation en glutamine améliore le profil immunitaire et ne prévienne l’incidence d’IVRS, mais ils soulèvent l’hypothèse qu’un apport exogène en glutamine stabilise les niveaux plasmatiques de glutamine, permettant aux athlètes de poursuivre leurs entraînements et de récupérer efficacement. / The purposes of this study were to determine the positive impacts of glutamine supplementation upon immune system status and to determine whether changes in plasma glutamine relate to the appearance of upper respiratory tract infections (URTI) in elite swimmers. Furthermore, this study evaluated dietary intakes and its influence on immune parameters and URTI incidence. Fourteen athletes (8 men, 6 women) took part of the study. Each athlete participated in both experimental conditions: glutamine supplement and an isocaloric solution placebo. The supplementation period lasted seven day, including three consecutive competing days. Post competing hematologic profils of swimmers show that glutamine supplement does not significantly improve plasma glutamine neither cytokines levels, compared to a placebo solution. Even if plasma glutamine concentrations are similar with both conditions, the post competiting levels tend to be higher than pre competing values, when glutamine is supplemented. Futhermore, plasma glutamine levels show a decreasing trend under control conditions (p=0.060). In this study, URTI can not be explained by low plasma glutamine or supplemented glutamine. However, URTI incidence is higher after competitions, where 8 athletes showed symptoms (control group) and 3 only in the experimental group. Athletes with URTI seem to consume less energy and proteins than healthy athletes (p=0.060). These data does not suggest that glutamine supplementation improves immune function or prevents URTI in highly trained swimmers during competition. However, results support the hypothesis that exogenous glutamine stabilizes plasma glutamine levels, allowing athletes to tolerate training workload and recover properly.

Thèse (Ph.D.)--Université Laval, 2006. / Bibliogr. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Made available in DSpace on 2015-07-22T20:49:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-09-24 / Vários estudos têm demonstrado a relação entre sistema imunológico e hormonal com o exercício físico e poucos estudos têm analisado esta relação em diferentes intensidades de esforço na natação. O objetivo desta pesquisa foi verificar as alterações metabólicas, hormonais e imunológicas antes e depois de um exercício de natação intermitente agudo com sessões diferentes durante o programa de treinamento. Dezessete nadadores do sexo masculino foram analisados em 3 sessões diferentes de treinamento, utilizando intensidades de 90% (potência anaeróbica – Pan), 70% (potência aeróbica – Pae) e 98% (capacidade anaeróbica – CAn) da velocidade máxima do melhor tempo de prova, resultado proveniente da melhor performance de competição. Amostras sanguíneas foram coletadas no pré e imediatamente após o exercício. Foi encontrado aumento significativo no lactato pós-exercício das três sessões de treinamento, na glicose pósexercício nas sessões de Pan e PAe, respectivamente. A glutamina aumentou significativamente no PAe e CAn. Foi observado um aumento nas concentrações de cortisol em PAe e em Pan. Os leucócitos aumentaram significativamente depois das três diferentes sessões. Não foram observadas diferenças significativas na concentração das imunoglobulinas e na contagem diferencial dos linfócitos, neutrófilos e basófilos. Os eosinófilos apresentaram diminuição significativa no pós-exercício de PAe e CAn em relação ao PAn e os monócitos não apresentaram alterações significativas no pósexercício nas três sessões, entretanto entre os tipos de treinamentos ocorreu uma diminuição significativa no PAe. Vários trabalhos revelam evidências das alterações nas concentrações e funções do sistema imune em decorrência do exercício físico. Esta apresentou protocolo de treinamento aplicado nos nadadores são adequados para alterar alguns componentes metabólicos, hormonais e leucocitários. / The effect of physical exercise in immune function has been extensively studied. The intensity and duration of physical exercise have considerable influence in immunologic parameters. However, few studies have compared different exercise intensities in different stages of a physical training program. To this point, we aimed to verify the metabolic, hormonal and immunologic changes before and after acute intermittent swimming exercise following different stages of training program. Seventeen male swimmers were analyzed in three stages of training, using the intensity of the three sessions was 90% (anaerobic potency – ANP), 70% (aerobic potency - AEP) and 98% (anaerobic ability – ANA) of the maximal speed from the best time of the distance, resulted from peak performance in competition. Blood samples were collected pre and immediately after exercise. Lactato increased significantly after the three different stages of training program, glucose post exercise in the ANP and AEP sessions, respectively. Glutamine increased significantly in AEP and ANA. Increased cortisol levels were also observed in AEP and in ANP. Leukocytes increased significantly after the three different sessions. There were no significant differences in lymphocytes, neutrophils e basophils. The eosinophils decreased in AEP and ANA. Monocytes no significant differences after the three different stages of training program, however decreased significantly on AEP. The immunoglobulins did not change of the three exercise patterns of pre and post-exercise. In summary, the protocoll intermittent swimming exercise following different stages of training the alterations capacity metabolic, hormones parameters and leukocytes any populations, favoring however health condition, so illnesses to not present. / TEDE / BV UNIFESP: Teses e dissertações

Introdução: A lesão de isquemia e reperfusão (I/R) intestinal pode causar danos celular e tecidual local e em órgãos a distância. Alguns fatores podem estar envolvidos nesses processos, tais como: a geração de espécies reativas de oxigênio, mediadores inflamatórios, óxido nítrico (NO) e estresse do retículo endoplasmático (RE). Devido ao envolvimento do estresse oxidativo nas lesões de I/R intestinal, algumas opções terapêuticas com antioxidantes estão sendo estudadas e testadas nas lesões de I/R intestinal. Objetivo: Avaliar o efeito local e sistêmico da glutamina no intestino e fígado de animais submetidos à I/R intestinal. Métodos: Foram utilizados 20 ratos wistar machos divididos em quatro grupos: Sham operated (SO), Glutamina+Sham operated (G+SO), Isquemia e reperfusão intestinal (I/R); Glutamina+Isquemia e reperfusão intestinal (G+I/R). Os animais foram anestesiados e, após, realizada a laparotomia mediana e identificação da artéria mesentérica superior. A artéria foi clampeada por 30 e após esse tempo, os animais foram mantidos por mais 15 minutos em reperfusão intestinal. A glutamina foi administrada por via intraperitoneal, na dose de 25 mg/Kg diluída em 1 mL de solução fisiológica. O tratamento foi realizado uma vez ao dia, durante 48 horas antes da indução da isquemia. Foram realizadas análises séricas para a função de integridade hepática através das enzimas aspartato aminotransferase (AST), alanina aminotransferase (ALT) e fosfatase alcalina (FA) e danos ao DNA pelo ensaio cometa. Realizamos a análise histológica dos tecidos através da coloração de Hematoxilina-Eosina e imunohistoquímica para avaliar a quantidade de células marcadas com os anticorpos monoclonais IL-1β, IL-6, TNF-α e NF-B no intestino e fígado. O homogeneizado do intestino e fígado foram utilizados para a avaliação dos níveis de lipoperoxidação (LPO) através das substâncias que reagem ao ácido tiobarbitúrico (TBARS), avaliação da atividade das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD) e glutationa peroxidase (GPx), determinação dos níveis de glutationa (GSH), avaliação dos metabólitos do óxido nítrico (nitritos/nitratos) e para as análises moleculares das proteínas iNOS, NF-B, Nrf2, Keap1, SOD, NQO1, HSP70, GRP78 e ATF-6 por Western Blot. Resultados: O pré-tratamento com glutamina reduziu os níveis de LPO, óxido nítrico, danos ao DNA, bem como as enzimas de integridade hepática. Observamos que a glutamina foi eficaz na preservação da arquitetura tecidual do intestino e fígado dos animais submetidos a I/R intestinal, reduzindo parâmetros como infiltrado inflamatório, perda das vilosidades intestinais e necrose. Constatou-se que a glutamina ativou a via do Nrf2 e as enzimas antioxidantes, reduziu o dano celular, e inibiu o estresse do RE, além de reduzir os mediadores do processo inflamatório. Conclusão: Neste estudo, sugerimos que o pré-tratamento com a glutamina desempenhou um papel protetor tanto no intestino como no fígado dos animais submetidos a I/R intestinal, demonstrado pelas análises estudadas, possivelmente pela sua ação antioxidante e anti-inflamatória. / Background: Injury by intestinal ischemia and reperfusion (I/R) can cause local and cellular damage to tissues and organs at distance. Some factors may be involved in those processes, such as the generation of reactive oxygen species, inflammatory mediators, nitric oxide (NO) and endoplasmic reticulum stress. Due to the involvement of oxidative stress in intestinal I/R lesions, some therapeutic options with antioxidants are being studied and tested in order to reduce these damages. Objective: To evaluate the local and systemic effect of glutamine in the intestine and liver of animals submitted to intestinal I/R. Methods: Twenty male Wistar rats were divided into four groups: Sham operated (SO), Glutamine+Sham operated (G+SO), Intestinal Ischemia and reperfusion (I/R); Glutamine+intestinal ischemia and reperfusion (G+I/R). The animals were anesthetized and after we performed the median laparotomy and identification of the superior mesenteric artery. The artery was clamped for 30 minutes and after that the animals were maintained for another 15 minutes in intestinal reperfusion. Glutamine was administered intraperitoneally at a dose of 25 mg/kg diluted in 1 ml of saline solution. Treatment was performed once daily for 48 hours prior to induction of ischemia. Serum samples for hepatic integrity were collected, and the enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (FA) were accessed. DNA damage was evaluated by the comet assay. We performed the histological analysis of the tissues through the staining of Hematoxylin-Eosin and immunohistochemistry, in order to evaluate the amount of cells labeled with the monoclonal antibodies IL-1β, IL-6, TNF-α and NF-B in the intestine and liver. The intestinal and liver homogenates were used to evaluate the levels of lipoperoxidation (LPO) through thiobarbituric acid reactive substances (TBARS), evaluation of the activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx), determination of glutathione levels (GSH) and nitric oxide (nitrites/nitrates), and for the molecular analyzes of the iNOS, NF-B, Nrf2, Keap1, SOD, NQO1, HSP70, GRP78 and ATF-6 proteins we performed Western blot analysis. Results: Pretreatment with glutamine reduced levels of LPO, nitric oxide, DNA damage, as well as liver integrity enzymes. We observed that glutamine was effective in preserving the intestinal and liver tissue architecture of animals submitted to intestinal I/R, reducing parameters such as inflammatory infiltrate, loss of intestinal villi and necrosis. It was found that glutamine activated the Nrf2 pathway and antioxidant enzymes, reduced cell damage, and inhibited endoplasmic reticulum stress in addition to reducing mediators of the inflammatory process. Conclusion: In this study, we suggest that pretreatment with glutamine played a protective role in both intestine and liver of animals submitted to intestinal I/R, demonstrated by the present analyzes, possibly for its antioxidant and anti-inflammatory action.

A radioterapia é amplamente empregada no tratamento de tumores abdominais. Entretanto, o seu emprego provoca danos indesejáveis ao tecido sadio, especialmente o intestinal, refletidos por meio de manifestações clínicas e histológicas. Buscando minimizar esses efeitos colaterais, inúmeras alternativas vêm sendo estudadas, dentre elas a suplementação dietética com aminoácidos. O objetivo deste estudo foi determinar o efeito da suplementação dietética com os aminoácidos L-glutamina, L-arginina e glicina nas camadas submucosa e muscular do íleo de ratos submetidos a irradiação abdominal. Cinqüenta ratos Wistar machos adultos foram distribuídos aleatoriamente em cinco grupos: I Controle não irradiado e sem suplementação de aminoácidos; II Controle irradiado e sem suplementação de aminoácidos; III irradiado e suplementado com L-glutamina; IV irradiado e suplementado com glicina; V irradiado e suplementado com L-arginina. O período de suplementação dietética foi de 14 dias, com a irradiação ocorrendo no 8. dia do experimento. A irradiação provocou diminuição da espessura da camada submucosa dos animais do grupo II, como também do conteúdo de colágeno, em comparação ao grupo controle não irradiado. A suplementação com L-arginina e glicina provocou aumento expressivo da espessura da camada submucosa, enquanto a suplementação com L-glutamina manteve a espessura dessa camada similar à observada nos animais não irradiados. Os animais com dieta suplementada com L-arginina e glicina também apresentaram aumento da espessura da camada muscular circular interna, em comparação aos ratos dos grupos I, não se observando diferenças no que se refere à camada muscular longitudinal externa. A suplementação com aminoácidos levou a aumento na intensidade de imunomarcação de colágeno tipo I nos animais do grupo IV, menos marcante no grupo III, e o grupo V foi o que mais se assemelhou ao observado nos animais não-irradiados. No tocante ao colágeno tipo III, a imunomarcação foi mais intensa no grupo IV, menos marcante no grupo V, e no grupo de ratos suplementado com L-glutamina, foi similar à encontrada nos animais não-irradiados. Os resultados sugerem que a suplementação dietética com glicina e L-arginina leva a aumento da quantidade de colágeno na parede ileal de ratos submetidos a radioterapia abdominal, eventualmente predispondo ao surgimento de fibrose intestinal, enquanto que a suplementação com L-glutamina propicia a manutenção da espessura da camada submucosa e pode contribuir para a atenuação dos efeitos colaterais decorrentes da irradiação. / Radiotherapy is widely employed in the treatment of abdominal tumors. Nevertheless, its use damages healthy tissue, especially intestinal tissue, with consequent clinical and histological manifestations. In order to minimize these side effects, several alternatives are being studied, including dietary supplementation with amino acids. The aim of this study was to determine the effect of dietary supplementation with L-glutamine, L-arginine, and glycine on the submucosal and muscular layers of the ileum of rats submitted to abdominal radiation. Fifty male Wistar rats weighing 255 to 325 g were randomly divided into five groups: I Control not irradiated and not supplemented with amino acids; II Control irradiated but not supplemented with amino acids; III irradiated and supplemented with L-glutamine; IV irradiated and supplemented with glycine; V irradiated and supplemented with L-arginine. The period of dietary supplementation lasted 14 days, with radiation taking place on the 8th day of the experiment. Radiation determined a decrease in the thickness and in the collagen content of the submucosal layer in group II animals compared to the non-irradiated group. Supplementation with L-arginine and glycine induced a marked increase in the thickness of the submucosal layer, while supplementation with L-glutamine caused no changes compared to non-irradiated animals. The animals receiving a diet supplemented with L-arginine and glycine also showed increased thickness of the internal circular muscle layer compared to group I rats, with no difference in the external longitudinal muscle layer. Supplementation with amino acids led to increased immunostaining of type 1 collagen in group IV animals, with less intense staining in group III and no difference in group V compared to non-irradiated animals. Regarding type III collagen, immunostaining was more intense in group IV and less intense in group V, while in rats supplemented with L-glutamine there was no difference compared to non-irradiated animals. These results suggest that dietary supplementation with glycine and L-arginine induces an increase in the collagen content of the ileal wall of rats submitted to abdominal radiotherapy, eventually predisposing it to intestinal fibrosis, while supplementation with L-glutamine favors the maintenance of the submucosal layer thickness and can contribute to attenuating the side effects of radiation.