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No description available.

No description available.

The regulation of the immune system is complex, with many factors involved in controlling immune cell development, activation and homeostasis. These factors include neuropeptides as well as classic immunoregulatory molecules such as cytokines, chemokines and hormones. Neuropeptides and tachykinins in particular are known to be involved in immune response modulation through a cascade of events including vasodilation, plasma extravasation, the activation of immune cells, the secretion of pro-inflammatory cytokines and the recruitment of more immune cells. Furthermore, there is growing evidence that tachykinins play a role in hematopoiesis with Substance P as the proposed effector molecule. In 2000, our lab discovered a new tachykinin with remarkable structural similarity to SP and SP-like neurokinin receptor binding affinity. This molecule was designated Hemokinin-1 due to its expression in hematopoietic cells and its function in B cell development. Further gene expression analysis of HK-1 reveals a wide expression pattern although HK-1 transcripts are found predominantly in peripheral tissues while SP is mainly expressed in neuronal tissue. Based on this differential expression pattern, it has been suggested that HK-1 may act as the peripheral tachykinin and may have functions distinct from SP. In addition, given the crossreactivity of the SP antibodies to HK-1, it is important to determine whether HK-1 is the actual mediator of some functions previously attributed to SP. In this thesis, we examine the differential expression pattern of HK-1 to determine molecular mechanisms of regulation of HK-1 transcription and ultimately provide clues to its function in the immune system. In our analysis of the HK-1 promoter, we found a major difference in the basic transcriptional control of HK-1 and SP at the level of transcription initiation and identified several transcription factors including CREB and NFκB involved in regulating TAC4 gene expression in immune cells. Data presented in this thesis also reveal that the HK-1 gene is a direct target of Early B-cell Factor, a transcription factor known to activate B cell-specific genes as well as genes involved in adipogenesis and neuronal development. Our results show EBF regulates HK-1 gene expression in differentiating B cells as well as a monocytic cell line. Our data indicate EBF may also be responsible for the high levels of HK-1 transcript in the olfactory epithelium, suggesting a bridge between the nervous system and the immune system.

The regulation of the immune system is complex, with many factors involved in controlling immune cell development, activation and homeostasis. These factors include neuropeptides as well as classic immunoregulatory molecules such as cytokines, chemokines and hormones. Neuropeptides and tachykinins in particular are known to be involved in immune response modulation through a cascade of events including vasodilation, plasma extravasation, the activation of immune cells, the secretion of pro-inflammatory cytokines and the recruitment of more immune cells. Furthermore, there is growing evidence that tachykinins play a role in hematopoiesis with Substance P as the proposed effector molecule. In 2000, our lab discovered a new tachykinin with remarkable structural similarity to SP and SP-like neurokinin receptor binding affinity. This molecule was designated Hemokinin-1 due to its expression in hematopoietic cells and its function in B cell development. Further gene expression analysis of HK-1 reveals a wide expression pattern although HK-1 transcripts are found predominantly in peripheral tissues while SP is mainly expressed in neuronal tissue. Based on this differential expression pattern, it has been suggested that HK-1 may act as the peripheral tachykinin and may have functions distinct from SP. In addition, given the crossreactivity of the SP antibodies to HK-1, it is important to determine whether HK-1 is the actual mediator of some functions previously attributed to SP. In this thesis, we examine the differential expression pattern of HK-1 to determine molecular mechanisms of regulation of HK-1 transcription and ultimately provide clues to its function in the immune system. In our analysis of the HK-1 promoter, we found a major difference in the basic transcriptional control of HK-1 and SP at the level of transcription initiation and identified several transcription factors including CREB and NFκB involved in regulating TAC4 gene expression in immune cells. Data presented in this thesis also reveal that the HK-1 gene is a direct target of Early B-cell Factor, a transcription factor known to activate B cell-specific genes as well as genes involved in adipogenesis and neuronal development. Our results show EBF regulates HK-1 gene expression in differentiating B cells as well as a monocytic cell line. Our data indicate EBF may also be responsible for the high levels of HK-1 transcript in the olfactory epithelium, suggesting a bridge between the nervous system and the immune system.

Doctor of Philosophy (PhD) / Little is known about the role that tachykinins, such as substance P and its receptor, the neurokinin-1 receptor, play in the generation of sympathetic nerve activity and the integration within the ventrolateral medulla (VLM) of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The studies described in this thesis investigate these autonomic functions and the role of tachykinins through physiological (response to hypercapnoea, chapter 3), anatomical (neurokinin-1 receptor immunohistochemistry, chapter 4) and microinjection (neurokinin-1 receptor activation and blockade, chapters 5 and 6) experiments. In the first series of experiments (chapter 3) the effects of chemoreceptor activation with hyperoxic hypercapnoea (5%, 10% or 15% CO2 in O2) on splanchnic sympathetic nerve activity and sympathetic reflexes such as the baroreflex and somato-sympathetic reflex were examined in anaesthetized rats. Hypercapnoea resulted in sympatho-excitation in all groups and a small increase in arterial blood pressure in the 10 % CO2 group. Phrenic nerve amplitude and phrenic frequency were also increased, with the frequency adapting back to baseline during the CO2 exposure. Hypercapnoea selectively attenuated (5% CO2) or abolished (10% and 15% CO2) the somato-sympathetic reflex while leaving the baroreflex unaffected. This selective inhibition of the somato-sympathetic reflex while leaving the baroreflex unaffected was also seen following neurokinin-1 receptor activation in the rostral ventrolateral medulla (RVLM) (see below). Microinjection of substance P analogues into the RVLM results in a pressor response, however the anatomical basis for this response is unknown. In the second series of experiments (chapter 4), the distribution of the neurokinin-1 receptor in the RVLM was investigated in relation to catecholaminergic (putative sympatho-excitatory “C1”) and bulbospinal neurons. The neurokinin-1 receptor was demonstrated on a small percentage (5.3%) of C1 neurons, and a small percentage (4.7%) of RVLM C1 neurons also receive close appositions from neurokinin-1 receptor immunoreactive terminals. This provides a mechanism for the pressor response seen with RVLM microinjection of substance P analogues. Neurokinin-1 receptor immunoreactivity was also seen a region overlapping the preBötzinger complex (the putative respiratory rhythm generation region), however at this level a large percentage of these neurons are bulbospinal, contradicting previous work suggesting that the neurokinin-1 receptor is an exclusive anatomical marker for the propriobulbar rhythm generating neurons of the preBötzinger complex. The third series of experiments (chapter 5) investigated the effects of neurokinin-1 receptor activation and blockade in the RVLM on splanchnic sympathetic nerve activity, arterial blood pressure, and autonomic reflexes such as the baroreflex, somato-sympathetic reflex, and sympathetic chemoreflex. Activation of RVLM neurokinin-1 receptors resulted in sympatho-excitation, a pressor response, and abolition of phrenic nerve activity, all of which were blocked by RVLM pre-treatment with a neurokinin-1 receptor antagonist. As seen with hypercapnoea, RVLM neurokinin-1 receptor activation significantly attenuated the somato-sympathetic reflex but did not affect the sympathetic baroreflex. Further, blockade of RVLM neurokinin-1 receptors significantly attenuated the sympathetic chemoreflex, suggesting a role for RVLM substance P release in this pathway. The fourth series of experiments (chapter 6) investigated the role of neurokinin-1 receptors in the RVLM, caudal ventrolateral medulla (CVLM), and nucleus tractus solitarius (NTS) on regional cerebral blood flow (rCBF) and tail blood flow (TBF). Activation of RVLM neurokinin-1 receptors increased rCBF associated with a decrease in cerebral vascular resistance (CVR). Activation of CVLM neurokinin-1 receptors decreased rCBF, however no change in CVR was seen. In the NTS, activation of neurokinin-1 receptors resulted in a biphasic response in both arterial blood pressure and rCBF, but no significant change in CVR. These findings suggest that in the RVLM substance P and the neurokinin-1 receptor play a role in the regulation of cerebral blood flow, and that changes in rCBF evoked in the CVLM and NTS are most likely secondary to changes in arterial blood pressure. Substance P and neurokinin-1 receptors in the RVLM, CVLM and NTS do not appear to play a role in the brainstem regulation of tail blood flow. In the final chapter (chapter 7), a model is proposed for the role of tachykinins in the brainstem integration of the sympathetic baroreflex, sympathetic chemoreflex, cerebral vascular tone, and the sympatho-excitation seen following hypercapnoea. A further model for the somato-sympathetic reflex is proposed, providing a mechanism for the selective inhibition of this reflex seen with hypercapnoea (chapter 3) and RVLM neurokinin-1 receptor activation (chapter 5). In summary, the ventral medulla is essential for the generation of basal sympathetic tone and the integration of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The tachykinin substance P, and its receptor, the neurokinin-1 receptor, have a role to play in many of these vital autonomic functions. This role is predominantly neuromodulatory.

Doctor of Philosophy (PhD) / Little is known about the role that tachykinins, such as substance P and its receptor, the neurokinin-1 receptor, play in the generation of sympathetic nerve activity and the integration within the ventrolateral medulla (VLM) of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The studies described in this thesis investigate these autonomic functions and the role of tachykinins through physiological (response to hypercapnoea, chapter 3), anatomical (neurokinin-1 receptor immunohistochemistry, chapter 4) and microinjection (neurokinin-1 receptor activation and blockade, chapters 5 and 6) experiments. In the first series of experiments (chapter 3) the effects of chemoreceptor activation with hyperoxic hypercapnoea (5%, 10% or 15% CO2 in O2) on splanchnic sympathetic nerve activity and sympathetic reflexes such as the baroreflex and somato-sympathetic reflex were examined in anaesthetized rats. Hypercapnoea resulted in sympatho-excitation in all groups and a small increase in arterial blood pressure in the 10 % CO2 group. Phrenic nerve amplitude and phrenic frequency were also increased, with the frequency adapting back to baseline during the CO2 exposure. Hypercapnoea selectively attenuated (5% CO2) or abolished (10% and 15% CO2) the somato-sympathetic reflex while leaving the baroreflex unaffected. This selective inhibition of the somato-sympathetic reflex while leaving the baroreflex unaffected was also seen following neurokinin-1 receptor activation in the rostral ventrolateral medulla (RVLM) (see below). Microinjection of substance P analogues into the RVLM results in a pressor response, however the anatomical basis for this response is unknown. In the second series of experiments (chapter 4), the distribution of the neurokinin-1 receptor in the RVLM was investigated in relation to catecholaminergic (putative sympatho-excitatory “C1”) and bulbospinal neurons. The neurokinin-1 receptor was demonstrated on a small percentage (5.3%) of C1 neurons, and a small percentage (4.7%) of RVLM C1 neurons also receive close appositions from neurokinin-1 receptor immunoreactive terminals. This provides a mechanism for the pressor response seen with RVLM microinjection of substance P analogues. Neurokinin-1 receptor immunoreactivity was also seen a region overlapping the preBötzinger complex (the putative respiratory rhythm generation region), however at this level a large percentage of these neurons are bulbospinal, contradicting previous work suggesting that the neurokinin-1 receptor is an exclusive anatomical marker for the propriobulbar rhythm generating neurons of the preBötzinger complex. The third series of experiments (chapter 5) investigated the effects of neurokinin-1 receptor activation and blockade in the RVLM on splanchnic sympathetic nerve activity, arterial blood pressure, and autonomic reflexes such as the baroreflex, somato-sympathetic reflex, and sympathetic chemoreflex. Activation of RVLM neurokinin-1 receptors resulted in sympatho-excitation, a pressor response, and abolition of phrenic nerve activity, all of which were blocked by RVLM pre-treatment with a neurokinin-1 receptor antagonist. As seen with hypercapnoea, RVLM neurokinin-1 receptor activation significantly attenuated the somato-sympathetic reflex but did not affect the sympathetic baroreflex. Further, blockade of RVLM neurokinin-1 receptors significantly attenuated the sympathetic chemoreflex, suggesting a role for RVLM substance P release in this pathway. The fourth series of experiments (chapter 6) investigated the role of neurokinin-1 receptors in the RVLM, caudal ventrolateral medulla (CVLM), and nucleus tractus solitarius (NTS) on regional cerebral blood flow (rCBF) and tail blood flow (TBF). Activation of RVLM neurokinin-1 receptors increased rCBF associated with a decrease in cerebral vascular resistance (CVR). Activation of CVLM neurokinin-1 receptors decreased rCBF, however no change in CVR was seen. In the NTS, activation of neurokinin-1 receptors resulted in a biphasic response in both arterial blood pressure and rCBF, but no significant change in CVR. These findings suggest that in the RVLM substance P and the neurokinin-1 receptor play a role in the regulation of cerebral blood flow, and that changes in rCBF evoked in the CVLM and NTS are most likely secondary to changes in arterial blood pressure. Substance P and neurokinin-1 receptors in the RVLM, CVLM and NTS do not appear to play a role in the brainstem regulation of tail blood flow. In the final chapter (chapter 7), a model is proposed for the role of tachykinins in the brainstem integration of the sympathetic baroreflex, sympathetic chemoreflex, cerebral vascular tone, and the sympatho-excitation seen following hypercapnoea. A further model for the somato-sympathetic reflex is proposed, providing a mechanism for the selective inhibition of this reflex seen with hypercapnoea (chapter 3) and RVLM neurokinin-1 receptor activation (chapter 5). In summary, the ventral medulla is essential for the generation of basal sympathetic tone and the integration of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The tachykinin substance P, and its receptor, the neurokinin-1 receptor, have a role to play in many of these vital autonomic functions. This role is predominantly neuromodulatory.

L’objectif de cette thèse est d’étudier le rôle de neuropeptides cérébraux, telle que la Neurokinin B codée par le gène tac3, dans le contrôle de la reproduction d’une espèce en danger, l’anguille Européenne, Anguilla anguilla. La maturation sexuelle de l’anguille est bloquée à un stade prépubertaire avant la migration océanique. Etant donnée sa position phylogénétique basale parmi les téléostéens, l’anguille est un modèle pertinent pour étudier l’évolution moléculaire et fonctionnelle de neuropeptides d’intérêt. Deux gènes paralogues tachykinine 3 (tac3) ont été identifiés dans le génome de l’anguille, chacun codant pour deux peptides. Ces gènes paralogues résultent de la duplication complète du génome spécifique aux téléostéens, comme le montrent les analyses phylogénétiques et synténiques. Les analyses de qPCR montrent que les deux gènes sont exprimés dans le cerveau. Les quatre peptides d’anguille ont été synthétisés et testés sur des cultures primaires de cellules hypophysaires d’anguille. Les quatre peptides inhibent l’expression de l’hormone lutéinisante et d’un récepteur à la gonadolibérine, révélant un double rôle inhibiteur dans le contrôle de la reproduction. / The aim of this PhD is to investigate the role of brain neuropeptides, such as neurokinin B, encoded by tac3 gene, in the control of reproduction of an endangered species, the European eel, Anguilla anguilla. The sexual maturation of the eel is blocked at a prepubertal stage before the oceanic migration. Due to its basal phylogenetic position among teleosts, the eel is also a relevant model for studying molecular and functional evolution of key neuropeptides. Two tachykinin 3 (tac3) paralogous genes were identified in the eel genome, each encoding two peptides. These paralogs result from the teleost-specific whole genome duplication, as shown by phylogeny and synteny analyses. Both genes are expressed in the brain as shown by qPCR. The four eel peptides were synthesized and tested on primary cultures of eel pituitary cells. The four peptides inhibited the expression of luteinizing hormone and gonadotropin-releasing hormone receptor, revealing a dual inhibitory role in the control of reproduction.

Effects of substance P (SP) and selective tachykinin agonists on neurotransmission at guinea-pig intracardiac ganglia were studied in vitro. Voltage responses of neurons to superfused tachykinins and nerve stimulation were measured using intracellular microelectrodes. Predominant effects of SP (1 μM) were to cause slow depolarization and enable synaptic transmission at low intensities of nerve stimulation. Augmented response to nerve stimulation occurred with 29 of 40 intracardiac neurons (approx. 73%). SP inhibited synaptic transmission at 23% of intracardiac neurons but also caused slow depolarization. Activation of NK3 receptors with 100 nM [MePhe 7]neurokinin B caused slow depolarization, enhanced the response of many intracardiac neurons to low intensity nerve stimulation or local application of acetylcholine, and triggered action potentials independent of other stimuli in 6 of 42 neurons. The NK1 agonist [Sar 9,Met(O2)11]SP had similar actions but was less effective and did not trigger action potentials independently. Neither selective agonist inhibited cholinergic neurotransmission. We conclude that SP can function as a positive or negative neuromodulator at intracardiac ganglion cells, which could be either efferent neurons or interneurons. Potentiation occurs primarily through NK3 receptors and may enable neuronal responses with less preganglionic nerve activity. Inhibition of neurotransmission by SP is most likely explained by the known blocking action of this peptide at ganglionic nicotine receptors.

Em Triatoma infestans ainda não há pesquisas de isolamento de Peptídeos Antimicrobianas e como têm contato muitos microrganismos acreditamos que eles produzem esses componentes antimicrobianos. Neste estudo identificamos quatro peptídeos antimicrobianos principais. Os Tin-TK-I e Tin-TK-II que são similares com Taquicininas de insetos, não são hemolíticos e ativos contra três bactérias e três fungos, e são capazes de lisar membranas. Tin-TK-I é degradado por aminopeptidases e tem estrutura randômica. Tin-TK-II é degradado por carboxipeptidases e tem estrutura de hélice 310. A Triastina que é similar a uma proteína cuticular de T. infestans, é ativa contra duas bactérias e três fungos, não hemolítica, forma uma hélice 310, lisa membranas, e sofre ação de carboxi e aminopeptidases. E o Triatogênio que é similar ao fibrinopeptideo-A humano, e possui um análogo. É ativo contra três bactérias e seis fungos, e seu análogo contra três bactérias e três fungos. Não são hemolíticos, formam poros em membranas, formam alfa-hélices e são degradados por aminopeptidases. / Regarding new researches with Antimicrobial Peptides in triatomines, none have been developed with T. infestans, and due to its survival in a highly infectious habitat, we believe that it products antimicrobial molecules. On this study, four main AMPs have been identified. Tin-TK-I and II that were similar to Tachykinin-like proteins from insects, they were not hemolytic, and were active against three bacteria and three fungi on the antimicrobial assay. Tin-TK-I presents a random structure and was degraded by aminopeptidases, as Tin-TK-II presents a helix 310 structure and was affected by carboxipeptidases. Both of them can disrupt negatively charged membranes. Triastin that was similar to a cuticular protein from T. infestans, was active against two bacteria and three fungi, had no hemolytic activity, presents a helix 310 structure and can disrupt negatively charged membranes. Triatogen was similar to the human fibrinopeptide A, and it has an analogue. Triatogen was active against three bacteria and six fungi, and its analogue was active against three bacteria and three fungi. They were not active against human erythrocytes, both were degraded by aminopeptidases. They can generate pores on membranes and both have alfa-helix structure.