Neutrophils are significant players in both acute and chronic inflammatory conditions, and function in infectious and autoimmune ailments. MicroRNAs regulate homeostasis in health and disease by fine tuning the expression of a network of genes through post transcriptional regulation. Many microRNAs are expressed in restricted tissues, regulated by physiological conditions such as stress and disease, and are emerging as mediators for intercellular communication that shape the tissue environments. MicroRNA profiles have been recently utilized as biomarkers for diagnosis and prognostic purposes for their stability in plasma and significant correlation with the disease progress. In addition, several microRNAs are in clinical trials for infectious diseases, cardiovascular disorders and cancer. As for neutrophil biology, microRNAs that regulate hematopoiesis and neutrophil development are well known. However, only a few microRNAs are characterized in the context of neutrophil migration and activation by loss of function studies either in mice or in cell culture. In this work we characterize the role of total microRNA regulation on neutrophil function through whole body and neutrophil specific Dicer1 knockout, and identify a microRNA regulator of neutrophil motility. MiR-722 downregulates the transcript level of rac2 through binding to a seed match in the rac2 3'UTR. Furthermore, miR-722 over-expressing larvae display improved outcomes in both sterile and bacterial systemic models. Finally, the miR-722 mimics protect zebrafish from lethal LPS challenge, providing evidence and mechanism of an anti-inflammatory microRNA that restrains detrimental systemic inflammation. We further investigated the role of the inflammatory response in an ischemia-reperfusion model. Extensive future work is required, especially in animal models, to illustrate the pivotal and complex microRNA mediated regulatory network in neutrophils, which is expected to provide the foundation for highly selective microRNA based therapy to control neutrophil behavior in infection and inflammatory disorders.

Introdução: O pré-condicionamento isquêmico tem a capacidade de minimizar as lesões decorrentes do processo de isquemia e reperfusão. O pós-condicionamento isquêmico, descrito posteriormente, apresenta resultados semelhantes na proteção contra as lesões por isquemia e reperfusão em miocárdio, cérebro, rins, músculo esquelético e medula espinhal. Objetivo: Avaliar o efeito do pré e do pós-condicionamentos isquêmicos bem como sua associação sobre a lesão tecidual em músculo esquelético de ratos submetidos ao processo de isquemia e reperfusão. Método: Foram utilizados 50 ratos Wistar, distribuídos em cinco grupos de 10 animais: grupo Controle, em que se realizou isquemia parcial por clampeamento aórtico infra-renal (240 min) e reperfusão (60 min); Grupo Sham, fez-se o mesmo procedimento cirúrgico, porem sem o clampeamento da aorta abdominal; grupo Pós-condicionamento, onde precedendo o início da reperfusão, foi realizado o pós-condicionamento isquêmico (três ciclos de um minuto de reperfusão intercalados por três ciclos de um minuto de isquemia); grupo Pré-condicionamento, precedendo ao período isquêmico procedeu-se ao pré-condicionamento isquêmico(três ciclos de 5 min de isquemia intercalados por três ciclos de 5 min de reperfusão); grupo Pré e Pos-condicionamento, neste grupo associaram-se os dois métodos. Avaliaram-se os resultados pela dosagem de enzimas tissulares (aspartato aminotransferase-AST, creatinofosfoquinase-CK e desidrogenase lática-DHL), malondialdeído (MDA) e glicogênio tissular com tratamento estatístico. Resultado: Houve elevação significativa dos níveis de CK em todos os grupos em relação ao grupo sham. A AST elevou-se no grupo pré+pós-condicionamento em relação ao grupo controle. O comportamento da LDH foi semelhante entre os grupos. O marcador de lesão de membrana celular representado pelo MDA mostrou-se elevado em todos os grupos, exceto no grupo pós-condicionamento em relação ao grupo sham. Não ocorreu diferença entre os grupos isquêmicos e reperfundidos. Quanto à reserva energética ocorreu queda significativa do glicogênio no grupo controle em relação aos grupos sham, pré-condicionamento e pré+pós-condicionamento, exceto no grupo pós. Conclusão: Concluiu-se que o pré-condicionamento bem como a associação do pré com o pós-condicionamento isquêmico foram capazes de minimizar a lesão tecidual em musculatura estriada de ratos submetidos à isquemia e reperfusão, porém a associação dos métodos não trouxe vantagens adicionais sobre os mesmos aplicados isoladamente / Background: Ischemic preconditioning has the ability to reduce injuries resulting from ischemia and reperfusion. Ischemic post-conditioning, described later, shows similar protective results against injury by ischemia and reperfusion in myocardium, brain, kidney, skeletal muscle, and spinal cord. Objective: To evaluate the effect of ischemic pre- and post-conditioning and their combination on protection against skeletal muscle tissue injury in rats subjected to ischemia and reperfusion. Methods: We used 50 Wistar rats divided into five groups of 10 animals each: Control group, subjected to partial ischemia by infrarenal aortic clamping (240 min) and reperfusion (60 min); Sham group, subjected to the same surgical procedure but without clamping of the abdominal aorta; Post-conditioning group, in which ischemic post-conditioning was performed before the onset of reperfusion (three cycles of one min of reperfusion interspersed with three cycles of one min ischemia); pre-conditioning group, subjected to ischemic pre-conditioning prior to the ischemic period (three cycles of 5 min ischemia interspersed with three cycles of 5 min reperfusion); pre-and post-conditioning group, in which the two methods were used in combination. Results were evaluated by measuring levels of tissue enzymes (aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH), malondialdehyde (MDA) and tissue glycogen, with statistical analysis. Results: There was a significant elevation of CK levels in all groups compared to the sham group. AST increased in the pre- + post-conditioning group compared to the control group. The behavior of LDH was similar for all groups. The marker of cell membrane damage represented by MDA was high in all groups except the post-conditioning one compared to the sham group. No significant difference was observed between the ischemic and reperfused groups. As to the energy reserve (glycogen), a significant decrease occurred in the control group compared to the sham, pre-conditioning and pre- + post-conditioning groups, but not in the post-conditioning group. Conclusion: We conclude that pre-conditioning and combined pre - and post-conditioning were able to minimize tissue injury in the skeletal muscle of rats subjected to the ischemia and reperfusion process, but the combined methods did not bring about additional advantages compared to each method applied aloneT

No description available.

Made available in DSpace on 2014-06-11T19:30:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-13Bitstream added on 2014-06-13T19:00:53Z : No. of bitstreams: 1 delbin_ma_dr_rcla.pdf: 750379 bytes, checksum: 217de471de3341cdcc9171a5f69905f5 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste trabalho foi o de avaliar o efeito do treinamento físico na reatividade vascular de artérias mesentérica e pulmonar de ratos submetidos à isquemia/reperfusão (IR) pulmonar. Ratos wistar foram utilizados nesse estudo, e divididos em cinco grupos: controle sedentário (C/SD); controle treinado (C/TR); falso operado sedentário (SHAM/SD); isquemia/reperfusão sedentário (IR/SD) e isquemia/reperfusão treinado (IR/TR). O treinamento físico consistiu em corrida em esteira, sessões de 60 min/dia, 5 vezes por semana durante 8 semanas (velocidade 1,2 km/h e 0% de inclinação). O processo de IR pulmonar foi realizado através de oclusão total da artéria pulmonar, veia pulmonar e brônquio esquerdo por 90 minutos e reperfusão de 120 minutos. Em seguida, as artérias mesentérica e pulmonar foram isoladas e curvas concentração-resposta à acetilcolina (ACh), histamina (HIST), nitroprussiato de sódio (SNP) foram obtidas na presença e na ausência de endotélio. Curvas concentração-resposta à fenilefrina (PHE) e ao análogo do tromboxano A2 (U46619) também foram obtidas na presença de endotélio. Análise do extravasamento de proteínas plasmáticas, atividade da mieloperoxidase pulmonar (MPO), nitrato e nitrito (NOx-), atividade da enzima superóxido dismutase (SOD), interleucina-6 (IL-6) plasmáticas e expressão de proteína arterial da eNOS, nNOS, Cu/Zn SOD-1, p47phox e TNF- α foram avaliados. Verificou-se aumento nos valores de NOx-, SOD, IL-6 plasmático, atividade da MPO e extravasamento de proteínas nos grupos IR/SD e IR/TR quando comparados aos demais grupos. O treinamento físico não alterou nenhum destes parâmetros. Em artéria mesentérica, houve diminuição na potência à ACh e a PHE no grupo IR/SD quando comparado aos grupos C/TR e IR/TR, sem alterações nos valores de resposta máxima. Não foram verificadas alterações na resposta ao U46619... / The aim of this work was to evaluate the effect of physical training in the responsiveness of rat mesenteric and pulmonary rings submitted to lung ischemia/reperfusion (IR). Rats were divided into five groups named: control sedentary (C/SD); control trained (C/TR); sham operated sedentary (SHAM/SD); ischemia/reperfusion sedentary (IR/SD) and ischemia/reperfusion trained (IR/TR). Run training was performed for 5 days/week, each session of 60 minutes, during 8 weeks (speed of 1.2 km/h and 0% grade). Left pulmonary IR was performed by occluding the pulmonary artery, bronchus and pulmonary vein for 90 minutes and reperfusion for 120 minutes. Concentration-response curves to acetylcholine (ACh), histamine (HIST), sodium nitroprusside (SNP) with intact and denuded endothelium were obtained. Contractile response curves were performed for phenylephrine (PHE) and tromboxane A2 analogue (U46619). The pulmonary plasma protein extravasation, lung myeloperoxidase activity (MPO), plasma levels of nitrite/nitrate (NOx-), superoxide dismutase activity (SOD), interleukin-6 (IL-6) and the protein expression for eNOS, nNOS, Cu/Zn SOD-1, p47phox and TNF-α were evaluated. The levels of NOx-, SOD, IL-6, MPO activity and pulmonary plasma protein extravasation were markedly increased in IR/SD and IR/TR compared to others groups which were not modified by exercise training. In mesenteric rings a decreased of potency to ACh and PHE in IR/SD compared to C/TR and IR/TR without changes in the maximal response were observed. No changes to U46619 were seen in all groups. The nNOS, Cu/Zn SOD-1 and p47phox protein expression were not modified. In pulmonary rings the potency to PHE was decreased in IR/SD and IR/TR without changes in the maximal response. No changes to U46619 were seen in all groups. Neither pulmonary IR nor exercise training changed the protein expression for eNOS, nNOS, Cu/Zn SOD-1 and p47phox... (Complete abstract click electronic access below)

L’infarctus du myocarde constitue la première cause de mortalité cardiovasculaire. Ainsi, toute stratégie permettant de moduler la vulnérabilité du coeur à l’ischémie-reperfusion (IR) peut représenter un intérêt majeur de santé publique. L’exercice en endurance est reconnu comme une stratégie de cardioprotection efficace dont les mécanismes cellulaires restent néanmoins peu connus. Les objectifs de ce travail de thèse sont donc i) d’évaluer le rôle préventif de l’exercice sur le développement d’un phénotype sensible à l’IR myocardique, et ii) de tenter de mieux comprendre le rôle de la eNOS dans la radioprotection par l’exercice. Dans la première partie de ce travail, nous avons mis en évidence que l’exercice permet de prévenir le développement d’un phénotype pathologique cardiomyocytaire,par une amélioration du statut antioxydant et un maintien de l’homéostasie calcique cellulaire, et ainsi permet de normaliser la sensibilité du coeur à l’IR chez une population à risque. Dans un second temps, les travaux réalisés ont permis de mettre en avant le rôle majeur de la eNOS dans la cardioprotection par l’exercice. Cette cardioprotection est associée à une diminution du niveau de phosphorylation (Ser1177) et surtout de l’état de découplage de cette enzyme au cours des premières minutes de reperfusion. Ces modifications, associées à l’amélioration du statut antioxydant cardiaque par l’exercice, sont à l’origine d’une diminution du stress nitro-oxydant au cours de la reperfusion,expliquée par une moindre synthèse de NO et une meilleure capacité à éliminer l’O2.-, permettant ainsi de limiter la synthèse de ONOO-. L’ensemble de ce travail de thèse a ainsi permis de mettre en évidence la complexité de la cardioprotection par l’exercice, nécessitant l’interaction entre différents mécanismes cellulaires tels que l’amélioration du statut enzymatique antioxydant, le découplage de la eNOS au cours de la reperfusion précoce et la régulation de l’homéostasie calcique intracellulaire. Ce travail à d’autre part permis de mieux appréhender le rôle complexe de la voie de synthèse du NO parla eNOS dans la modulation de la vulnérabilité du coeur à un stress tel que l’IR / Exercise training is recognized as an efficient way to protect the myocardium against ischemiareperfusion(IR). However, mechanisms responsible for such cardioprotection remain still unclear. Theaims of this work were then i) to evaluate the preventive effect of exercise on a model highly sensitiveto myocardial IR, and ii) to investigate the role of eNOS in exercise-induced cardioprotection. In a firstpart we showed that regular boots of exercise, by its beneficial effects on calcium handling andenzymatic antioxidant status, prevents the highly sensitive phenotypical remodeling of the heart andthen normalized heart vulnerability to IR. Then, in a second part of this work, we showed that exerciseinducedcardioprotection was associated with a decrease of eNOS phosphorylation at Ser1177 andespecially its uncoupling during early reperfusion. Such phenomenon, associated with increased heartantioxidant capacity was responsible for reduced nitro-oxidative stress. Indeed, reduced NOSdependentNO synthesis associated with the improved capacity to scavenge O2.- contribute to preventthe formation of ONOO-. Altogether, these results showed that exercise-induced cardioprotection is acomplex mechanism requiring interactions between antioxidant capacity improvement, eNOSuncoupling during reperfusion and intracellular calcium homeostasis. Finally, this work opens newperspectives regarding the role of NO synthesis modulation to impact heart sensitivity to IR

<p>The kidney is the major organ responsible for the regulation of the composition and volume of the body fluids, which is essential for homeostasis. The glycosaminoglycan hyaluronan (HA), with extreme water-binding capacity, is present in the interstitium of the kidney with a heterogenous distribution. The importance of HA in renal water-handling is unknown and was the focus of the present investigation.</p><p>Acute water-loading in rats caused the amount of papillary HA to increase and during water deprivation, the amount was reduced. Gerbils, with extreme urine concentrating capacity, have less HA in the renal papilla in normal conditions and responded diametrically different to water-loading (reduction in HA). Renomedullary interstitial cells (RMICs), which are probably the main producers of HA in the renal medulla, were cultured at different media osmolalities to mimic the milieu of the medulla during variations in the water balance. The amount of HA found in the media was decreased at high osmolalities and increased at low osmolalities, thereby strengthening the <i>in vivo</i> results. CD44, an HA-receptor involved in the uptake and degradation of HA, was expressed on RMICs in an osmolality dependent manner. During high media osmolality, the CD44 expression increased and at lower osmolalities, the opposite occurred, probably due to the need for uptake and degradation of HA.</p><p>Renal ischemia-reperfusion injury causes a cortical accumulation of HA, up-regulation of CD44, and a depression of functional parameters. The time periods of ischemia correlated with the accumulation of HA which, in turn, was inversely correlated to GFR. Hyaluronidase injections in this setting failed to reduce HA levels and significantly improve renal function.</p><p>In conclusion, the results from the present study suggest an important role for HA and RMICs in renal water-handling and that the intrarenal distribution of HA is altered after ischemia-reperfusion injury, which correlates with renal dysfunction.</p>

<p>Diabetes mellitus (DM) is one of the leading causes of end stage renal failure. An increased susceptibility to renal ischemia/reperfusion (I/R)-injury was found in DM rats. Unilateral renal ischemia for as short as 20 minutes led to an irreversible progressive injury in DM kidneys, whereas the injury in non-DM kidneys was almost reversible. The renal I/R injury was characterized by anuria, infiltration of inflammatory cells, tubular atrophy, dilation of the remaining tubuli and tubulointerstitial fibrosis. Necrotic areas were found in the inner parts of the outer medulla and in the papilla. The renal medulla was the most vulnerable part of the kidney. This was seen both by the extent of fibrosis four and eight weeks after I/R and by the presence of TUNEL-positive (apoptotic) cells 6h after ischemia. Increased accumulation of HA and enhanced CD44 expression was seen after I/R in DM kidneys.</p><p>Treatment with long acting insulin 7-14 days before I/R, decreased the number of apoptotic cells in the renal medulla and protected renal function and morphology after the insult, while insulin treatment after the injury did not have any protective effect. Short acting insulin given 2-6 hours before I/R partially protected renal function but did not improve the morphological picture.</p><p>Treatment with the angiotensin II receptor type 1 blocker candesartan, the PAF-antagonist UR-12670, the immunosuppressive agents tacrolimus and cyclosporin A, or prednisolone did not improve the outcome of the renal I/R injury in DM. Injection of cobalt protoporphyrin (CoPP) intraperitoneally in order to induce an over-expression of heme oxygenase-1 (HO-1) resulted in a trend towards a better function in DM kidneys after I/R. However, the induction of HO-1 by intraperitoneal CoPP injection was not achieved in all rats, when examined by western blot.</p><p>In conclusion, unilateral renal I/R leads to a severe progressive injury in DM kidneys. Insulin treatment before ischemia, but not after, reduces the renal injury in DM rats. Studies using a more reliable administration of CoPP are required to decide if induction of HO-1 protects against renal I/R injury in DM.</p>

The kidney is the major organ responsible for the regulation of the composition and volume of the body fluids, which is essential for homeostasis. The glycosaminoglycan hyaluronan (HA), with extreme water-binding capacity, is present in the interstitium of the kidney with a heterogenous distribution. The importance of HA in renal water-handling is unknown and was the focus of the present investigation. Acute water-loading in rats caused the amount of papillary HA to increase and during water deprivation, the amount was reduced. Gerbils, with extreme urine concentrating capacity, have less HA in the renal papilla in normal conditions and responded diametrically different to water-loading (reduction in HA). Renomedullary interstitial cells (RMICs), which are probably the main producers of HA in the renal medulla, were cultured at different media osmolalities to mimic the milieu of the medulla during variations in the water balance. The amount of HA found in the media was decreased at high osmolalities and increased at low osmolalities, thereby strengthening the in vivo results. CD44, an HA-receptor involved in the uptake and degradation of HA, was expressed on RMICs in an osmolality dependent manner. During high media osmolality, the CD44 expression increased and at lower osmolalities, the opposite occurred, probably due to the need for uptake and degradation of HA. Renal ischemia-reperfusion injury causes a cortical accumulation of HA, up-regulation of CD44, and a depression of functional parameters. The time periods of ischemia correlated with the accumulation of HA which, in turn, was inversely correlated to GFR. Hyaluronidase injections in this setting failed to reduce HA levels and significantly improve renal function. In conclusion, the results from the present study suggest an important role for HA and RMICs in renal water-handling and that the intrarenal distribution of HA is altered after ischemia-reperfusion injury, which correlates with renal dysfunction.

Diabetes mellitus (DM) is one of the leading causes of end stage renal failure. An increased susceptibility to renal ischemia/reperfusion (I/R)-injury was found in DM rats. Unilateral renal ischemia for as short as 20 minutes led to an irreversible progressive injury in DM kidneys, whereas the injury in non-DM kidneys was almost reversible. The renal I/R injury was characterized by anuria, infiltration of inflammatory cells, tubular atrophy, dilation of the remaining tubuli and tubulointerstitial fibrosis. Necrotic areas were found in the inner parts of the outer medulla and in the papilla. The renal medulla was the most vulnerable part of the kidney. This was seen both by the extent of fibrosis four and eight weeks after I/R and by the presence of TUNEL-positive (apoptotic) cells 6h after ischemia. Increased accumulation of HA and enhanced CD44 expression was seen after I/R in DM kidneys. Treatment with long acting insulin 7-14 days before I/R, decreased the number of apoptotic cells in the renal medulla and protected renal function and morphology after the insult, while insulin treatment after the injury did not have any protective effect. Short acting insulin given 2-6 hours before I/R partially protected renal function but did not improve the morphological picture. Treatment with the angiotensin II receptor type 1 blocker candesartan, the PAF-antagonist UR-12670, the immunosuppressive agents tacrolimus and cyclosporin A, or prednisolone did not improve the outcome of the renal I/R injury in DM. Injection of cobalt protoporphyrin (CoPP) intraperitoneally in order to induce an over-expression of heme oxygenase-1 (HO-1) resulted in a trend towards a better function in DM kidneys after I/R. However, the induction of HO-1 by intraperitoneal CoPP injection was not achieved in all rats, when examined by western blot. In conclusion, unilateral renal I/R leads to a severe progressive injury in DM kidneys. Insulin treatment before ischemia, but not after, reduces the renal injury in DM rats. Studies using a more reliable administration of CoPP are required to decide if induction of HO-1 protects against renal I/R injury in DM.

No description available.