Thesis (doctoral)--University of Tartu, 2006. / Thesis is based on 3 papers.

Made available in DSpace on 2014-06-11T19:35:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-12Bitstream added on 2014-06-13T20:26:53Z : No. of bitstreams: 1 blanch_gt_dr_arafo.pdf: 1786222 bytes, checksum: ad1e37adb10eabe9c9ee8dd690c60759 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O sistema nervoso central (SNC) tem um papel fundamental na regulação de mecanismos que controlam a osmolaridade dos líquidos corporais. O núcleo do trato solitário (NTS) é o sítio primário das aferências cardiovasculares e de osmorreceptores periféricos e se projeta à áreas prosencefálicas envolvidas com a regulação cardiovascular e do equilíbrio hidroeletrolítico. Demonstramos anteriormente que animais com lesão da porção comissural do NTS (commNTS) tem maior ingestão de água, natriurese e resposta pressora frente a sobrecarga intragástrica (ig) de NaCl 2 M. Os mecanismos responsáveis por estas alterações ainda não foram determinados. Uma vez que o estímulo com NaCl 2 M ig (2 ml) ativa osmorreceptores centrais e periféricos, não sabemos até o momento os efeitos da lesão do commNTS sobre as respostas observadas após a estimulação específica de osmorreceptores periféricos, que pode ser feita com NaCl 0,6 M ig. (2 ml). Desta forma os nossos objetivos foram: a) estudar os mecanismos que medeiam o aumento da pressão arterial e da natriurese após a sobrecarga de NaCl 2 M em animais com lesão do commNTS; b) verificar as alterações na expressão da proteína c-Fos após NaCl 2 M ig em ratos com lesão fictícia (sham) ou lesão do commNTS; c) verificar as alterações na expressão gênica no PVN após NaCl 2 M ig, d) estudar os efeitos na pressão arterial, na ingestão de água e na excreção renal subseqüentes a administração de NaCl 0,6 M ig, bem como os mecanismos responsáveis pelas alterações, em ratos com lesão fictícia (sham) ou lesão do commNTS; e) verificar as alterações na expressão da proteína c-Fos após NaCl 0,6 M ig em ratos com lesão fictícia (sham) ou lesão do commNTS. Ratos Holtzman (280-320 g) foram utilizados. A lesão eletrolítica... / The central nervous system has an important role controlling the mechanisms involved in the regulation of body fluid osmolality. The nucleus of the solitary tract (NTS) is the primary site of cardiovascular and peripheral osmoreceptors afferents and projects to prosencephalic areas involved in hydroelectrolytic balance and cardiovascular regulation. We have demonstrated that commissural NTS (commNTS) lesioned rats had an increase in arterial pressure and a greater increase in water intake and natriuresis after 2 M NaCl intragastric (ig) load. The mechanisms involved in these responses are not known. Since 2 M NaCl ig (2 ml) activates central and peripheral osmoreceptors, it is not known the effects of the commNTS lesion on the responses induced only by the activation of the peripheral osmoreceptors, which can be done by 0.6 M NaCl (2 ml) ig. Thus, the aims of this study were: a) to study the mechanisms involved in the increase of the arterial pressure and natriuresis in commNTS lesioned rats after 2 M NaCl ig; b) to verify the changes in c-Fos expression after 2 M NaCl ig in sham and commNTS lesioned rats; c) to verify the changes in gene expression in PVN after 2 M NaCl ig in naïve rats; d) to study the effects on arterial pressure, water intake and renal excretion after 0.6 M NaCl, as well as, the mechanisms involved in these responses, in sham and in commNTS lesioned rats; e) to verify the changes in c-Fos expression after 0.6 M NaCl ig in sham and commNTS lesioned rats. Male Holtzman rats (280-320 g) were used. Electrolytic lesion of the commNTS and all experiments were be performed in chronic period of lesion (14 to 21 days. For the lesion, a partial craniotomy of the occipital bone was performed, and the dorsal surface of the brainstem was exposed. The electrolytic lesion was performed using... (Complete abstract click electronic access below)

Information is transferred between neurons in the brain via electrochemical transmission at specialized cell-cell junctions called synapses. These structures are far from being static, but rather are influenced by plasticity mechanisms that alter features of synaptic transmission as means to build routes of information flow in the brain. Hebbian forms of synaptic plasticity – long-term potentiation and long-term depression – have been well studied and are considered to be the cellular basis of learning and memory, although their positive feedback nature is prone to instability. Neurons are also endowed with homeostatic mechanisms of synaptic plasticity that act to stabilize neural network functions by globally tuning synaptic drive. Precisely how neurons orchestrate this adaptive homeostatic response and how it influences Hebbian forms of synaptic plasticity, however, remains only partially understood. Using a combination of whole-cell electrophysiology, two-photon imaging and glutamate uncaging in organotypic hippocampal slices, I have expanded upon the known repertoire of homeostatic mechanisms that increase excitatory synaptic drive when CA1 hippocampal neurons experience a prolonged period of diminished activity. I found that the subunit composition of AMPA and NMDA receptors, the two major glutamate receptor subtypes at excitatory synapses, are altered which, in addition to increasing synaptic strength, are predicted to change the signaling and integrative properties of synaptic transmission. Moreover, I found that the amount of glutamate released from presynaptic terminals during evoked-transmission is enhanced and that this mechanism might, in part, underlie the uniform cell-wide homeostatic increase in synaptic strengths. Lastly, I found that homeostatic strengthening of synaptic transmission reduced the potential for CA1 synapses to exhibit long-term potentiation, and that this was caused by altered presynaptic release dynamics that impeded plasticity induction. Together, this work highlights several mechanistic strategies employed by neurons to increase excitatory synaptic drive during periods of activity deprivation which, in addition to balancing cellular excitability, alters the metaplastic state of synapses.

Thesis (M.D.)--Boston University

The long terra effects of glucose on the rate of (pro) insulin biosynthesis and the amount of preproinsulin mRNA in rat islets maintained in tissue culture were investigated. The rate of (pro)insulin synthesis was 35 times greater in islets cultured for 6 days in 8 mM glucose than it was in islets cultured in 4 mM glucose. The preproinsulin mRNA content at this time was 2 fold greater in islets incubated with 8 mM glucose compared to 4 mM glucose. The rate of (pro)insulin synthesis and the preproinsulin mRNA content of islets cultured at 8 mM glucose were maximal since no further significant increases were observed in islets cultured at 16 mM glucose for 6 days. These results indicate that the long term effects of glucose on the rate of (pro)insulin synthesis in rat islets of Langerhans is mediated both by transcriptional and translational events and that translational events exert the major controlling influence.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2007. / Title from second page of PDF document. Includes bibliographical references.

Yes / Wnt signalling and its downstream effectors are well known for their roles in embryogenesis and tumourigenesis, including the regulation of cell proliferation, survival and differentiation. In the nervous system, Wnt signalling has been described mainly during embryonic development, although accumulating evidence suggests that it also plays a major role in adult brain morphogenesis and function. Studies have predominantly concentrated on memory formation in the hippocampus, although recent data indicate that Wnt signalling is also critical for neuroendocrine control of the developed hypothalamus, a brain centre that is key in energy balance regulation and whose dysfunction is implicated in metabolic disorders such as type 2 diabetes and obesity. Based on scattered findings that report the presence of Wnt molecules in the tanycytes and ependymal cells lining the third ventricle and arcuate nucleus neurones of the hypothalamus, their potential importance in key regions of food intake and body weight regulation has been investigated in recent studies. The present review brings together current knowledge on Wnt signalling in the hypothalamus of adult animals and discusses the evidence suggesting a key role for members of the Wnt signalling family in glucose and energy balance regulation in the hypothalamus in diet-induced and genetically obese (leptin deficient) mice. Aspects of Wnt signalling in seasonal (photoperiod sensitive) rodents are also highlighted, given the recent evidence indicating that the Wnt pathway in the hypothalamus is not only regulated by diet and leptin, but also by photoperiod in seasonal animals, which is connected to natural adaptive changes in food intake and body weight. Thus, Wnt signalling appears to be critical as a modulator for normal functioning of the physiological state in the healthy adult brain, and is also crucial for normal glucose and energy homeostasis where its dysregulation can lead to a range of metabolic disorders.

11β-Hydroxysteroid Dehydrogenase Type 2 (11βHSD2) catalyses the inactivation of cortisol. In aldosterone target tissues co-expression of 11βHSD2 and mineralocorticoid receptors (MR) protects the receptor from activation by glucocorticoids. In the syndrome of Apparent Mineralocorticoid Excess, mutations in the HSD11B2 gene cause hypertension, which is thought to be driven by volume expansion secondary to sodium retention. 11βHSD2 mice are indeed hypertensive but paradoxically volume contracted, suggestive of a urine-concentrating defect. The current studies were designed to evaluate sodium and water homeostasis in 11βHSD2-/- mice. 11βHSD2-/- mice developed a severe and progressive polyuric-polydipsic phenotype. Despite basal polyuria, at <100 days 11βHSD2-/- mice had a functional concentration response when challenged with 24 hours water deprivation. At >180 days the exacerbated polyuria was associated with severe medullary injury in the null mice. Basal aquaporin 2 (AQP2) abundance was reduced in the 11βHSD2-/- mice at both <100 and >180 days. Moreover, vasopressin 2 receptor (V2R) stimulation failed to normalize the impaired response to water deprivation in >180 day null mice. Consequently, a renal origin to the polyuria was postulated. Indeed, mice in which 11βHSD2 had been selectively targeted in the brain had a normal water turnover. A key finding from these studies is that functional deletion of 11βHSD2 in the brain, specifically the nucleus of the solitary tract (NTS), resulted in an increased salt appetite. Moreover, the mice displayed a preference for 1.5% NaCl over water. Blockade of mineralocorticoid receptors (MR) significantly reduced NaCl intake. This is the first demonstration of an increased salt appetite in a model with normal renal function and in the absence of sodium depletion. These data implicate activation of MR on 11βHSD2 positive neurons in the NTS in the behavioural drive to consume sodium.

Osmoregulation is critical to life and is tightly regulated by integrated physiological and behavioral responses to maintain the osmolality of body fluid. In particular, this involves recovery from dehydration both at the intracellular and extracellular levels. To achieve appropriate body fluid balance, three major hormones namely secretin (SCT), angiotensin II (ANGII) and vasopressin (VP) are responsible. Of note, SCT and ANGII share overlapping physiological roles including similar expression pattern within the brain, dipsogenic actions and activation of VP expression and/or release in mice. However, it remains unclear how their receptor pathways may cross-interact to aid osmoregulation. In recent years, G protein-coupled receptor (GPCR) oligomerization has been implicated to play roles in regulating processes such as expression, pharmacological diversity, signal transduction and internalization. Though not as extensively studied, class B GPCRs are also gaining merit in their oligomerization abilities, within which the wealth of available information is focused on SCT receptor (SCTR) homomers and heteromers. Moreover, there is also evidence indicating the ability for ANGII receptors to oligomerize. On the basis of this information, this project predominantly aims to explore the molecular association between SCTR and ANGII receptors via in vitro experiments and provide insights into its physiological relevance. In this study, bioluminescence resonance energy transfer (BRET) assays revealed SCTR and ANGII type 1a receptor (AT1aR) to form hetero-complexes. This oligomerization event was found by BRET competition to be contributed predominantly by transmembrane (TM) domain regions 2 and 4 in SCTR, and TM1 and TM4 in AT1aR. Within which, combinational use of mutant TM peptides and SCTR chimeras revealed the importance of lipid-exposed residues, particularly Leu204 and Ser205 in SCTR TM2 as key contact points for formation of the SCTR/AT1aR complex. Morphologically, the heteromers were visualized by confocal FRET imaging at the cell surface and found have a role in modulating AT1aR trafficking. It was also found that the SCTR/AT1aR complex affected Gαs signaling specifically, reducing maximal response values by 24.3 ± 2.8 % compared to CHO-K1 cells transfected with only SCTR. While, this negative effect could be abolished by co-application of SCT and ANGII peptides, use of constitutively active AT1aR mutants or disruption of the hetero-complex using SCTR mutants. Taken together, the SCTR/AT1aR complex was proposed to impose conformational restraints on the SCTR that could be overcome upon activation of the AT1aR. Physiologically, hyperosmolality isovolemic induced drinking could be attenuated by central administration of TM peptides and the protein kinase A pathway blocker H-89, indicating receptor oligomerization to have a role in neural osmoregulation via a Gαs dependent pathway. This study presents novel findings regarding the receptor oligomerization of SCTR and AT1aR, which may be the molecular basis to the overlapping roles of SCT and ANGII in water homeostasis. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

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