A glândula pineal de mamíferos sintetiza o hormônio melatonina exclusivamente durante o período noturno. A síntese é regulada primordialmente pela via retino-hipotalâmico-pineal e modulada por vários fatores, incluindo o sistema peptidérgico. Assim, o papel da insulina na regulação da síntese de melatonina foi estudado a partir da realização de culturas de glândulas pineais estimuladas com noradrenalina, insulina e noradrenalina associada à insulina, em culturas temporizadas ou não pela noradrenalina, avaliando: a produção de melatonina por HPLC com detecção eletroquímica; as atividades das enzimas envolvidas na síntese da melatonina, por radiometria; assim como, a expressão gênica das enzimas quantificada por Real-Time PCR. Os resultados sugerem uma interação entre as vias de sinalização da noradrenalina e da insulina, com a respectiva potencialização da síntese da melatonina, induzida por noradrenalina, observada pela adição da insulina, efeito esse, que se dá, provavelmente através de mecanismos pós-transcricionais. / The mammalian pineal gland synthesizes the neurohormone melatonin exclusively during the dark phase. Its synthesis is primarily regulated via a retino-hypothalamic-pineal pathway and modulated by many factors, including the peptidergic system. Thus, the role of insulin on the regulation of melatonin synthesis was studied using cultured gland treated with norepinephrine, insulin and norepinephrine associated to insulin. The cultures were also synchronized or not by norepinephrine. Melatonin content was assayed by HPLC (High Performance Liquid Chromatography) with electrochemical detection, melatonin synthesis enzymes activities by radiometry and enzymes gene expressions by Real-Time PCR. The results suggest an interaction between norepinephrine and insulin signaling pathway, with insulinic potentialization on melatonin synthesis norepinephrine-mediated, and this effect, seems to accurs potentially through post-transcriptional events.

Plant promoters are the principle cis-acting regulatory sequences responsible for the temporal and spatial expression of genes. One method for isolating plant promoters is based on the ability of a common soil bacterium, <i> Agrobacterium tumefaciens </i>, to transfer a specific segment of DNA (T-DNA) into plant cells. This specific T-DNA has been shown to integrate stably into the recipient plant genome. If the T-DNA contains a promoterless marker gene, then T-DNA integration events occurring adjacent and downstream to a promoter region can be detected by the activation of the marker gene. These T-DNA-mediated gene fusions, consisting of an unknown plant promoter sequence and the coding sequence of a marker gene, can be isolated using the marker gene as a promoter tag. The key objective of this work was to develop a novel, bifunctional selectable marker gene and assess its use as: a selectable marker gene in bacterial and plant transformation systems, and as a promoter tag for T-DNA promoter-tagging studies in dicots. A bifunctional fusion gene was produced between phosphinothricin acetyltransferase and neomycin phosphotransferase (PAT::NPT II), by fusing an NPT II coding sequence to the 3' terminus of the PAT gene. The PAT gene product confers tolerance to a non-selective herbicide L-phosphinothricin (Ignite, Hoechst AG). The neomycin phosphotransferase ('npt II') gene allows for direct selection of transformed cells with the antibiotic, kanamycin. Using an <i>in vivo Escherichia coli </i> selection system, a translational fusion gene between these two reporter genes was achieved. The resulting protein had activities of both parent enzymes. This was demonstrated both in transformed <i>Escherichia coli</i> and in transformed <i>Nicotiana tabacum</i> and <i>Brassica napus</i> plants. Using this bifunctional selectable marker gene, a T-DNA promoter tagging vector, pBAU2, was constructed and its utility was demonstrated in <i>Nicotiana tabacum</i>. One of the <i>N. tabacum</i> promoter tagged events was selected for subsequent promoter isolation studies. The promoter from this regenerant was isolated by screening a Lambda subgenomic library and also by thermal asymmetric interlaced (TAIL-)PCR. The isolated upstream regulatory sequence was fused to a reporter gene, â-glucuronidase ('gus'), and subjected to a preliminary evaluation in <i> Nicotiana tabacum</i> and in <i>Brassica napus</i>.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

A glândula pineal de mamíferos sintetiza o hormônio melatonina exclusivamente durante o período noturno. A síntese é regulada primordialmente pela via retino-hipotalâmico-pineal e modulada por vários fatores, incluindo o sistema peptidérgico. Assim, o papel da insulina na regulação da síntese de melatonina foi estudado a partir da realização de culturas de glândulas pineais estimuladas com noradrenalina, insulina e noradrenalina associada à insulina, em culturas temporizadas ou não pela noradrenalina, avaliando: a produção de melatonina por HPLC com detecção eletroquímica; as atividades das enzimas envolvidas na síntese da melatonina, por radiometria; assim como, a expressão gênica das enzimas quantificada por Real-Time PCR. Os resultados sugerem uma interação entre as vias de sinalização da noradrenalina e da insulina, com a respectiva potencialização da síntese da melatonina, induzida por noradrenalina, observada pela adição da insulina, efeito esse, que se dá, provavelmente através de mecanismos pós-transcricionais. / The mammalian pineal gland synthesizes the neurohormone melatonin exclusively during the dark phase. Its synthesis is primarily regulated via a retino-hypothalamic-pineal pathway and modulated by many factors, including the peptidergic system. Thus, the role of insulin on the regulation of melatonin synthesis was studied using cultured gland treated with norepinephrine, insulin and norepinephrine associated to insulin. The cultures were also synchronized or not by norepinephrine. Melatonin content was assayed by HPLC (High Performance Liquid Chromatography) with electrochemical detection, melatonin synthesis enzymes activities by radiometry and enzymes gene expressions by Real-Time PCR. The results suggest an interaction between norepinephrine and insulin signaling pathway, with insulinic potentialization on melatonin synthesis norepinephrine-mediated, and this effect, seems to accurs potentially through post-transcriptional events.

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor α2 (GFRα2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRα2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.

The distribution of urotensin-II-immunoreactivity (irU-II) was studied in the rat brainstem and spinal cord with the use of an antiserum against the human urotensin II (U-II) peptide. A population of ventral horn neurons in the spinal cord, hypoglossal nucleus, dorsal motor nucleus of the vagus, facial motor nucleus, nucleus ambiguus, abducens nucleus and trigeminal motor nucleus exhibited irU-II of varying intensities. The number of irU-II motor neurons was higher in the lumbar segments as compared to that of cervical, thoracic and sacral segments. Double-labeling the sections with U-II- and choline acetyltransferase (ChAT)-antisera revealed that nearly all irU-II ventral horn and brainstem neurons were ChAT-positive. The result provides the first immunohistochemical evidence of the presence of irU-II in cholinergic motoneurons of the rat spinal cord and brainstem.

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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Interleukin-1alpha (IL-1alpha) is a well-known proinflammatory mediator acting as a secreted molecule. However, in addition to its ability to activate its membrane-bound receptor, there is growing evidence on its noncanonical nuclear function, which classifies IL-1alpha as a "dual function cytokine". This nuclear action depends on the evolutionary conserved N-terminal domain of IL-1alpha. After proteolytic processing, the N-terminal domain of IL-1alpha translocates into nucleus. Histone acetyltransferase (HAT) complexes were previously identified as nuclear targets of IL-1alpha precursor. However, the specific protein which is responsible for the interaction between IL-1alpha and HAT complexes has not been identified yet. To dissect this interaction, the N-terminal domain of IL-1alpha was produced in yeast. Suitability of this experimental setup for testing the interaction between IL-1alpha and eukaryotic HAT complexes was evaluated in this study. IL-1alpha has been analyzed in this study using bioinformatics approaches as well. Putative amphipatic acidic helixes of IL-1alpha have been characterized. One of the potential binding partners of these domains is protein Ada2. Protein Ada2, mature IL-1alpha and IL-1alpha precursor in fusion with epitopes suitable for affinity purification were produced in...