Σκοπός της παρούσης εργασίας είναι η στατιστική ανάλυση της σχέσης δομής-δράσης των χημικών ενώσεων που παράγονται από οξινδόλια και πουρίνες πάνω στα σύμπλοκα CDKs/Cyclins και η εξαγωγή συμπερασμάτων πάνω στις σχέσεις αυτές. Για το σκοπό αυτό ανατρέξαμε στην διεθνή βιβλιογραφία για να συλλέξουμε όσες πληροφορίες έχουν δημοσιευτεί έως τώρα για τις δύο αυτές ομάδες ενώσεων. Έπειτα μέσω ειδικών προγραμμάτων έγινε η επεξεργασία τους και η ανάλυση τους. Σκοπός είναι να συσχετίσουμε τη δομή των ενώσεων αυτών με τη δράση και τη βιολογική τους απόκριση πάνω στα σύμπλοκα των CDKs με τις κυκλίνες. / -

La contaminación con nitratos de las aguas subterráneas es una preocupación a nivel mundial, que ha ido tomando mayor importancia en el último tiempo. El ciclo del nitrógeno involucra la atmósfera, el suelo y el agua, además de una serie de procesos y organismos. De este ciclo y los factores que influyen en él depende el equilibrio del nitrógeno en el ambiente. El hombre a través de diferentes actividades ha provocado cambios en el equilibrio y en el ciclo del nitrógeno, aumentando las entradas de este nutriente al suelo y el agua. La aplicación de fertilizantes y/o purines en cultivos corresponde a una de las fuentes más importantes de entrada de N al suelo y las aguas subterráneas. Por otra parte la ingesta de nitratos en el agua de consumo puede causar importantes problemas de salud en la población. Diferentes estudios han probado que altas dosis de nitratos pueden provocar metahemoglobinemia. Además los compuestos son considerados cancerigenos finales. A través de distintos estudios y experiencias realizadas en terreno acerca de la lixiviación con nitratos se han definido los factores más relevantes que determinan la tasa de lixiviación de nitratos, estos factores son la tasa de aplicación de N, los suelos, las precipitaciones y la proporción de las distintas formas de N en el purín. Existen distintas metodologías para estudiar y cuantificar la contaminación de las aguas subterráneas con nitratos, en diferentes condiciones. Por un lado están los índices de vulnerabilidad, que mediante la evaluación de factores calculan un puntaje comparativo entre distintos sitios y por otro lado están los modelos de simulación o estadísticos, que calculan tasas de lixiviación. Se aplicó un índice de contaminación intrínseca, en el área norte de Santiago, donde la entrada de nitrógeno se debe al riego con aguas servidas de las zonas agrícolas. La comparación de los resultados obtenidos con las concentraciones de NO3 de las aguas subterráneas en el sector mostró una clara diferencia, permitiendo concluir que la contaminación de la zona no se relacionaba con la agricultura. Debido a la extensión del área de estudio no se pudo realizar una correlación adecuada de los puntajes obtenidos con el índice y las concentraciones de NO3. El índice de contaminación resulta una herramienta útil siempre y cuando se aplique a escala regional y/o nacional y se pueda comparar entre distintos lugares. Se realizó un análisis de sensibilidad a dos modelos de simulación seleccionados, MANNER y DNDC. El análisis arrojó que ambos programas pese a tener características muy distintas se comportan de manera similar frente a variaciones en las condiciones o factores requeridos para la simulación. Además se aplicaron ambos programas a tres escenarios representativos de las condiciones: más favorable, media y más desfavorable a la contaminación con nitratos por aplicación de purines en Chile. De la aplicación se concluyó que para el escenario medio y más favorable a la contaminación la lixiviación de nitratos resultaba muy alta, pronosticando un importante impacto en la calidad de las aguas subterráneas. Según estos resultados y la comparación de las tasas de aplicación registradas en Chile es claro que debemos preocuparnos y desarrollar medidas de manejo que prevenga la contaminación de los acuíferos. Finalmente, los índices de contaminación y por sobretodo los modelos de simulación son una importante herramienta que nos puede ayudar en el futura a desarrollar políticas de manejo que nos permitan un desarrollo sustentable de la agricultura.

No description available.

No description available.

No description available.

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

A biossíntese de MEL pela pineal envolve a conversão da serotonina a NAS pela enzima AA-NAT, seguida demetilação da NAS pela enzima HIOMT gerando a MEL. A ativação β-adrenérgica é essencial e a co-estimulação de receptores P2Y1 potencia a produção de NAS induzida por noradrenalina. Neste trabalho avaliamos o efeito da estimulação de receptores P2Y1 sobre a produção de MEL induzida pela estimulação β-adrenérgica. A co-estimulação purinérgica com ADP, levou a potenciação da produção de NAS induzida por ISO e a inibição do conteúdo de MEL de maneira dependente de concentração. Em extratos nucleares de pineais estimuladas com ADP, a translocação nuclear de AP-1 foi observada, não havendo alteração significativa na translocação nuclear dos dímeros NF-κB p50/p50 e p50/RelA. PDTC, inibidor de NF-κB, não alterou o conteúdo de NAS e MEL em pineais em cultura estimuladas com ISO e ADP. A expressão gênica e a atividade enzimática de AA-NAT e HIOMT não foram alteradas pela co-estimulação com ISO e ADP. O bloqueio seletivo dos receptores P2Y1 por A3\'P5\'P inibiu de maneira dependente de concentração o efeito potenciador do ADP sobre a produção de NAS induzida por ISO, mas não reverteu a inibição observada nos níveis de melatonina. Estes resultados apontam para um mecanismo diferencial de modulação da produção de NAS e MEL abrindo novas perspectivas ao estudo dos mecanismos regulatórios da produção de MEL e seus metabólitos / The biosynthesis of MEL by the pineal gland involves the conversion of serotonin to NAS by the enzyme AA-NAT, followed by methylation of NAS by the enzyme HIOMT generating MEL. The activation of β-adrenergic receptors is essential and the co-stimulation of P2Y1 receptors potentiates noradrenaline-induced NAS production. In this study we evaluated the effect of P2Y1 receptors stimulation on the production of MEL synthesis induced by β-adrenergic stimulation. Purinergic co-stimulation with ADP potentiated ISO-induced NAS production and inhibited melatonin content in a concentration dependent manner. In nuclear extracts from stimulated pineal glands with ADP, the nuclear translocation of AP-1 was observed, with no significant change in the nuclear translocation of the NF-κB dimers p50/p50 and p50/RelA. PDTC, an inhibitor of NF-κB, did not alter the content of NAS and MEL in cultured pineal glands co-stimulated with ISO and ADP. ISO and ADP co-stimulation did not alter the transcript and enzyme activity of AA-NAT and HIOMT. The selective blockade of P2Y1 receptors by A3\'P5\'P inhibited, in a concentration-dependent manner, the potentiating effect of ADP on ISO-induced NAS production but did not change the inhibition observed on MEL levels. These results suggest a differential mechanism on the modulation of NAS and MEL production opening new perspectives to the study of the regulatory mechanisms involved in the biosynthesis of MEL and its metabolites

The sphincter of Oddi (SO) is a neuromuscular structure located at the junction of the bile and pancreatic ducts with the duodenum. The primary functions of the SO are to regulate the delivery of bile and pancreatic juice into the duodenum, and to prevent reflux of duodenal contents into the biliary and pancreatic systems. Neural, hormonal or functional disturbances of biliary motility can lead to painful and sometimes life threatening clinical conditions, such as SO dysfunction and acute pancreatitis. Clearly understanding the regulation of biliary and duodenal motility patterns is necessary and may provide useful pharmacological sites for drug development to aid in the treatment of these diseases. Spontaneous activity of the SO is regulated by complex interactions between the enteric nervous system, hormones, possibly interstitial cells of Cajal and other bioactive agents, together with modulation via neural reflexes between the duodenum, common bile duct/gallbladder, and stomach. Purines are one group of neurotransmitters/regulatory agents that have been shown to effect gastrointestinal motility, however their functions in the regulation of SO motility have not been elucidated. The studies described in this thesis used in vitro organ bath techniques and in vivo preparations to determine the effects of exogenous purines on possum SO and duodenal motility. The possum SO has been extensively characterized and is an excellent model for motility studies. In vitro, exogenous adenosine was found to decrease spontaneous activity in both the SO and duodenum. In contrast exogenous ATP induced both excitatory and inhibitory responses in the SO and duodenum. Interestingly, the adenosine and ATP-induced effects were predominantly exhibited by the proximal portion of the SO (proximal-SO), with no or little effect observed in the distal portion of the SO (distal-SO). These data support the hypothesis that the SO is comprised of different functional components that can act differently in response to certain stimuli, and highlights the importance of studying each of the SO components. Agonists and antagonists, together with immunohistochemical studies, were used in an attempt to identify the P1 and P2 receptor sub-types responsible for mediating the adenosine- and ATP-induced responses. In the duodenum the adenosine-induced decrease in spontaneous activity was likely to be mediated by A2A and A3 receptors, but the receptors mediating the proximal-SO response could not be identified. In the duodenum ATP induced a complex non-neural response consisting of a P2X1, and P2Y2 and/or P2Y4 mediated immediate inhibition. This was followed by a return to baseline activity or small excitation. The response concluded with a late inhibitory response, likely to be mediated by P2Y1 receptors, but the effects of other P2Y receptors could not be excluded. In contrast, ATP application to the proximal-SO evoked a partially neurally mediated early excitation, likely via P2X receptors, followed by an inhibition of activity, likely via activation of non-neural P2Y2 and/or P2Y4 receptors. In vivo studies with exogenous application of adenosine and ATP to the SO activated neural pathways to produce increased motor activity. Characterisation of these neural pathways found ATP and/or adenosine to activate excitatory cholinergic motor neurons. ATP also activated an inhibitory nicotinic/nitrergic pathway. This is the first comprehensive investigation of the possible involvement of purines in the regulation of SO motility. These studies demonstrate that exogenous purines influence SO and duodenal motility, inducing complex neural and non-neural responses, acting via multiple P1 and P2 receptors. It now remains to be determined if endogenously released purines induce similar responses, together with elucidation and location of the receptor sub-types involved.

The research was designed to investigate the effects of AIT-082, a derivative of the purine hypoxanthine containing a para-amino benzoic acid moiety, on neural cells. AIT-082 has been shown to possess a number of neurotrophic and neuroprotective properties and to enhance memory. Furthermore, AIT-082 is undergoing clinical trials as a potential treatment for Alzheimers disease.<p>The first part of the study investigated the ability of AIT-082 to influence cellular ATP levels in cortical neurons. Decreased energy metabolism is a key point in Yings (Ying, 1996a) theory of the development of Alzheimers disease. Previous work with AIT-082 had shown that it could protect hippocampal neurons from cellular damage caused by sublethal doses of glutamate. Specifically, AIT-082 prevented neurite degeneration. Also, AIT-082 was shown to increase mitochondrial membrane potential, especially at the distal tips of the neurites, in hippocampal neurons. I hypothesized that AIT-082 was protecting the neurons by increasing the ability of the mitochondria to generate ATP and thereby increasing the amount of ATP available to the cell. ATP was collected and measured from cortical neuron cultures that were exposed to glutamate, AIT-082, glutamate and AIT-082. The ATP levels were compared to the ATP levels from cortical neuron cultures that were exposed to vehicle for glutamate and AIT-082. The results did not significantly increase ATP levels in cortical neurons following glutamate exposure. <p>The next set of experiments involved investigations into the ability of AIT-082 to influence phosphorylation events in neural cells. AIT-082 shares some neurotrophic and neuroprotective properties with a group of drugs called the immunophilin ligands. The neuroprotective properties of the immunophilin ligands are mainly due to their ability to influence protein phosphorylation by inhibiting the activity of calcineurin a protein phosphatase. The first set of experiments used western blot techniques to measure serine peptide and threonine peptide phosphorylation levels in proteins from whole brain homogenates that were incubated with vehicle, AIT-082, and GMP. Both AIT-082 and GMP caused an increase in the level of serine peptide phosphorylation compared to vehicle but only the increase caused by GMP treatment proved to be significant. Further, threonine phosphorylation levels were significantly increased by GMP but not AIT-082. Phosphorylation levels of short peptide sequences containing either a phosphorylated serine or threonine residue were also measured in neuronal and astrocytic cultures. The neuronal cultures were exposed to 4 h of hypoxia to mimic the conditions of reduced energy availability observed in Alzheimers disease brains. Astrocyte cultures were exposed to 4 h of hypoxia/ischemia for the same reason. Both cell types were allowed to recover for 0, 1, 4, 12 and 24 hours with or without AIT-082 following the insult. AIT-082 treatment did not significantly affect phosphorylation levels of proteins harvested from either neuron or astrocyte cultures at any time period. I conclude therefore, that AIT-082 is not able to influence phosphorylation of the short amino acid sequences containing phosphorylated serine or threonine residues that could be detected by the primary antibodies used in my experiments.