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The tyrosine kinase receptor Tie-2 is expressed on the surface of endothelial cells and is necessary for angiogenesis and vascular stability. To date, the best characterized ligands for Tie-2 are Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2). Ang-1 has been identified as the main activating ligand for Tie-2 while the role of Ang-2 has been controversial since its discovery; some studies reported Ang-2 as a Tie-2 antagonist while others described Ang-2 as a Tie-2 agonist. The purpose of this thesis was to understand: (1) how the receptor Tie-2 is regulated by Ang-1 and Ang-2 in endothelial cells, (2) to compare the effects of Ang-1 and Ang-2, and (3) to determine the arrangement and distribution of Tie-2 in endothelial cells. The research presented in this thesis indicates that Tie-2 is arranged in variably sized clusters on the endothelial cell surface. Clusters of Tie-2 were expressed on all surfaces of cells: on the apical plasma membrane, on the tips of microvilli, and on the basolateral plasma membrane. When endothelial cells were stimulated with Ang-1, Tie-2 was rapidly internalized and degraded. Upon Ang-1 stimulation, Tie-2 localized to clathrin-coated pits on all surfaces of endothelial cells indicating that one pathway mediating Tie-2 internalization is through clathrin-coated pits. After activation of Tie-2, Ang-1 dissociates from the endothelial cell surface and accumulates in the surrounding medium. When experiments were repeated with Ang-2, it was discovered that Ang-2 induced all of the same effects on Tie-2 as Ang-1 but at a much reduced level and rate, indicating that Ang-2 likely functions as a partial agonist for Tie-2 in endothelial cells.

Oestrogen receptors (ER) have classically been described as ligand-inducible nuclear transcription factors. The pleiotrophic effects of ER function have a predominant role in the direct regulation of the growth, differentiation and development of tissues of the human reproductive system. There are two ER subtypes, ER and ER which differ in their specificity for ligand and the consequent actions they orchestrate. Moreover, the latter exists in multiple splice variants of which ER is the only fully functional homologue. Research into the underlying differences in subtype responses to ligand has involved examination of the intranuclear dynamics of individual receptor subtypes. Studies into the mobility of ER in response to ligand have exclusively focused on studies of full length ER and ER independently in transfected cell lines. The studies described in this thesis have investigated the kinetics of ER using Fluorescence Recovery After Photobleaching (FRAP) in infected cell lines which lends itself to more precise expression of the subtype of interest. The morphological impact of natural oestrogenic and synthetic ligands on ERs was examined and the influence on the intranuclear dynamics assessed. Further to this, the effect of co-expression of different ER subtype combinations was examined. Studies on the intranuclear mobility of ER have confirmed and extended the findings of others. Previous work on the development of ER agonists and antagonists has been to target specific overexpressing ER subtypes in a physiological setting. In this study, we demonstrated for the first time an overwhelming ER -selective effect in slowing the rate of mobility within the nucleus, suggesting the study of intranuclear dynamics is an important parameter for the examination of efficacy of a compound. Differential responses to ligand based on co-infected partnerships indicate that heterodimerisation has a profound effect in augmenting ligand-dependent regulation and activity.

Ca2+ influx through ionotropic glutamate receptors regulates a variety of developmental processes including neurite outgrowth and naturally occurring cell death. In the CNS, NMDA receptors were originally thought to be the sole source of Ca2+ influx through glutamate receptors; however, AMPA receptors also allow a significant influx of Ca2+ ions. The Ca2+ permeability of AMPA receptors is regulated by the insertion of one or more edited GluR2 subunits into the receptors. Although Ca2+-permeable AMPA receptors are a familiar feature in developing neurons, the developmental function of these receptors during the formation of the nervous system has yet to be established. This study was designed to investigate the expression and functional role of Ca2+-permeable AMPA receptors in developing chicken spinal motoneurons. Our results demonstrate that chicken lumbar motoneurons express functional AMPA receptors as early as embryonic day (E) 5. Electrophysiological recordings of kainate-evoked currents indicate a significant reduction in the Ca2+ permeability of AMPA receptors between E6 and E11. During this developmental period, the Ca2+ permeability of AMPA receptors decreases three-fold. Reduction in the Ca2+ permeability of AMPA receptors is accompanied by increased expression of GluR2 mRNA in the spinal motoneuron pool. Changes in GluR2 mRNA expression occur in parallel to changes in GluR2 protein expression in the chicken ventral spinal cord. Changes in the Ca2+-permeability of AMPA receptors are not mediated by age-dependent changes in the editing pattern of GluR2 subunits. At early stages of development, functional AMPA receptors were composed of a combination of GluR3 and GluR4 subunits. mRNA analysis indicates that GluR4 is the most abundant subunit in the chicken ventral spinal cord between E6 and E11. Immunohistochemistry analysis of spinal cord sections also demonstrated that both GluR3 and GluR4 proteins are expressed at E6 and E11. Expression of Ca2+-permeable AMPA receptors regulates the maturation of dendritic outgrowth in developing spinal motoneurons. Measurements of dendritic length and branching pattern demonstrate significant changes in the dendritic morphology of spinal motoneurons between E6 and E11. Blockade of AMPA receptor activation with CNQX between E5 and E8 causes a significant increase in dendritic outgrowth in lumbar motoneurons, when compared with vehicle-treated embryos. Treatment of chicken embryos with CNQX between E8 and E11, when AMPA receptors become Ca2+-impermeable, has no affect on dendritic morphology. However, blockade of NMDA receptor activation with MK-801 causes a significant reduction in dendritic outgrowth of lumbar motoneurons by E11. These findings indicate that AMPA receptor activation between E5 and E8 limits dendritic outgrowth in developing motoneurons, whereas NMDA receptor activation is involved in dendritic remodeling after the establishment of synaptic contacts with sensory afferents.

In recent years it has been shown conclusively that at least two cannabinoid receptors, termed CB1 and CB2, exist in mammalian tissues. Previous studies using the mouse isolated vas deferens have yielded results which suggest that this tissue contains cannabinoid CB1 receptors which, when activated, can mediate inhibition of electrically-evoked contractions. However, there is evidence which indicates that several of the cannabinoid receptor agonists investigated in this study may exert their effects via non-CB, or even non- cannabinoid mechanisms. In the present study, this evidence was further investigated using the cannabinoid-mediated inhibition of electrically-evoked contractions in the mouse isolated vas deferens as a model of study. The results obtained from studies using the cannabinoid receptor antagonists O-1184 and the CB1-selective SR141716A highlighted the existence of a level of agonist-dependent antagonism in mouse isolated vas deferens. This was indicated by discrepancies obtained in the pKB values of these antagonists against the compounds under investigation. In this series of investigations it was observed that the endogenous cannabinoid receptor agonist, anandamide and the capsaicin-anandamide hybrid compound, arvanil were less potently antagonised by the CB1selective antagonist/inverse agonist, SR141716A than the highly CB1-selective agonist methanandamide. Such discrepancies in pKB values indicate that anandamide and arvanil may be acting on a receptor type distinct from the cannabinoid CB1 receptor. Additionally this series of studies indicated that anandamide and WIN55212-2 were more potently antagonised when non-cumulative responses to these compounds were constructed, indicating the possibility of tolerance developing to these compounds during the construction of cumulative concentration response curves. Several, more recent studies have indicated that anandamide and its metabolically more stable analogue methanandamide may exert their actions in part through vanilloid VR1 receptors. Upon further investigation using the vanilloid VR1 receptor antagonist capsazepine in addition to SR141716A, it was observed that the effects of anandamide, methanandamide, and the capsaicin-anandamide hybrid arvanil could be attenuated by both antagonists. These results indicate that these three agonists can act through both receptor types to mediate their effects in the mouse isolated vas deferens. In this study the putative water-soluble cannabinoid receptor agonist, O-1057 was shown to inhibit the of electrically-evoked contractions in the mouse isolated vas deferens when only water was used as a vehicle. This effect was inhibited by the cannabinoid receptor antagonists O-1184 and SR141716A, providing evidence that this novel water-soluble compound was acting through the CB1 receptor. In a further study the ability of the endogenous compound palmitoylethanolamide and a range of cannabinoids which can act on the CB2 in addition to the CB1 receptor, to downregulate mast cell degranulation was investigated. It was observed that PEA, CP55940 and WIN55212-2 but not the highly CB2 receptor-selective L759656 could exert this effect. It was not possible to investigate the effects of the CB2 receptor antagonist/inverse agonist SR144528 at this time.

ABSTRACT Background: The Peripheral Benzodiazepine Receptor (PBR) can be classified as a distinct receptor from the central benzodiazepine receptor. The PBR gene has been located to chromosome 22q13.31 in humans and has been found to consist of four exons, with the first and half of the fourth exon being untranslated to form the PBR protein. PBR is involved in numerous biological conditions including the regulation of cellular proliferation and apoptosis, steroidogenesis, heme biosynthesis, anion and porphyrin transport and mitochondrial functions such as oxidative phosphorylation and translocation of cholesterol from the outer to the inner mitochondrial membrane. Recent studies showed that the expression of PBR correlated with tumour malignancy and patient survival. Aim: The objectives of this research were to determine the expression pattern and level of PBR mRNA in various types of human normal and cancer tissues and to isolate the PBR protein.

Adenosina e óxido nítrico, importantes neuromoduladores endógenos, atuam modulando finamente o controle neural cardiovascular no núcleo de trato solitário (NTS). Embora se tenha conhecimento sobre a relação entre adenosina e NO periférica e centralmente, em particular, no bulbo, os mecanismos pelos quais a adenosina interfere na dinâmica da neurotransmissão nitrérgica, ainda não são totalmente conhecidos. Logo, alterações na interação entre esses sistemas podem ser especialmente relevantes para indivíduos predispostos à hipertensão. Dessa forma, os objetivos do presente estudo foram estudar a interação entre o sistema adenosinérgico e nitrérgico em culturas de células da porção dorsomedial do bulbo de ratos normotenso Wistar Kyoto (WKY) e espontaneamente hipertensos (SHR). Para tal, utilizou-se técnicas para quantificação dos níveis de nitrito, PCR em tempo real e RNA de interferência. Foi observada uma redução e um aumento concentração-dependente nos níveis de nitrito e do mRNA da nNOS induzido pelos agonistas dos receptores A1(A1R) e A2A(A>sub>2AR), CPA e CGS21680, respectivamente. Os efeitos nos níveis de nitrito foram atenuados pela administração dos antagonistas seletivos dos A1R e A2AR, CPT e ZM241385. Knockdown dos A1R e A2AR mostraram que a redução da expressão desses receptores aumentaram e diminuíram os níveis de expressão da nNOS, respectivamente. Pré-tratamento com o inibidor não seletivo da nNOS, L-NAME, aboliu os níveis aumentados de nitrito desencadeados pelo CGS21680 em células de WKY e SHR. Por fim, é mostrado que a via cAMP-PKA está envolvida na sinalização que deflaga tantos os níveis reduzidos de nitrito, via A1R, quantos os níveis aumentados de nitrito, via A2AR, em culturas de WKY e SHR. Em síntese, nossos resultados destacam a influência da adenosina sobre a síntese de NO em culturas de células da porção dorsomedial do bulbo de ratos WKY e SHR. Pelo menos em parte, o perfil modulatório é diferenciado em ratos SHR / Adenosine and nitric oxide, important endogenous neuromodulators, act on the fine tuning regulation of neural cardiovascular control in the nucleus tractus solitarius (NTS). Although the relationship between adenosine and NO peripheral and centrally, is well established, in particular, in the oblongata medulla, the mechanisms by which adenosine interferes in the dynamics of nitrergic neurotransmission, is not completely understood. Thus, changes in the interaction between these systems may be especially relevant for individuals predisposed to hypertension. The aim of this study was to evaluate the interaction between the adenosinergic and nitrergic systems in cell culture from the dorsomedial medulla oblongata of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). This purpose was performed the quantification of nitrite level, RT-PCR analysis and RNA interference. We observed a concentration-dependent decrease and increase of nitrite and nNOS mRNA levels in cultured cells of WKY and SHR rats induced by agonists of adenosine A1 (A1R) and A2A receptor (A2AR), CPA and CGS21680, respectively. These effects in nitrite level were attenuated by the administration of the A1R and A2AR selective antagonist, CPT and ZM241385. Furthermore, knockdown of A1R and A2AR showed an increase and decrease of nNOS mRNA levels, respectively. The pretreatment with nonselective inhibitor of NOS, L-NAME, abolished nitrite-increased levels triggered by CGS 21680 in WKY and SHR cells. Finally, it is shown that the cAMP-PKA pathway is involved in A1R and A2AR -mediated decrease and increase in nitrite levels in SHR and WKY cells. In summary, our results highlight the influence of adenosine on nitric oxide levels in cultured cells from dorsal medulla oblongata of WKY and SHR rats. In part, the modulatory profile is different in the SHR strain

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the growing population of elderly people. Although the etiology of the disease is yet to be fully elucidated, pathological hallmarks have been consistently described, including the accumulation of amyloid plaques, dysfunctional ionic homeostasis, synaptic disruption and neurodegeneration. The amyloid hypothesis postulates that aberrant production of amyloid-β (Aβ) proteins, which have a high propensity to aggregate, lies at the center of the pathological mechanism of AD. In particular, soluble oligomeric Aβ structures have been identified as primary toxic species. The interaction of these structures with several cellular targets, including ion channels such as nicotinic acetylcholine receptors (nAChR) and voltage operated Ca²⁺ channels (VOCC), has also been implicated in Aβ toxicity and AD. The aim of this thesis is to investigate how the acute and chronic actions of Aβ in vitro are affected by nicotinic ligands. Acute application of Aβ₁₋₄₂ to fluo-3-loaded PC12 cells potentiated Ca²₊ increases evoked by stimulation of nAChR and VOCC, while chronic application reduced redox potential, disrupted membrane integrity and initiated apoptosis in PC12 cells. In addition to mimicking the toxic responses of PC12 cells, Aβ₁₋₄₂ also reduced neurite outgrowth and synaptogenesis in rat primary cortical neurons. All actions of Aβ were prevented by inhibitors of Aβ₁₋₄₂ oligomerisation, including the hexapeptide KLVFFA. Neuroprotection afforded by (+)-nicotine also occurred via inhibition of Aβ₁₋₄₂ oligomerisation, rather than by a receptor-mediated mechanism. No other pharmacological approaches, including application of two novel ligands selective for α7 nAChR: the partial agonist SSR180711 and antagonist α-conotoxinArIB[V11L,V16D], characterized herein, protected against Aβ₁₋₄₂ toxicity. While inhibiting oligomerisation prevented the actions of Aβ₁₋₄₂, enhanced oligomerisation evoked amplified toxic responses. However, the potentiation of Ca²⁺ signalling diminished following enhanced oligomerisation. This, coupled with a lack of VOCC-involvement in Aβ toxicity and the differential actions of truncated Aβ peptides on toxicity and Ca²⁺ signaling, indicates that the acute disruption of Ca²⁺ signaling by Aβ does not underpin the chronic toxic effects of Aβ.

WW-domain binding protein-2 (WBP-2) was cloned as an E6-associated protein (E6-AP) interacting protein and its role in steroid hormone receptor (SHR) function was investigated. We show that WBP-2 differs from other SHR coactivators, as it specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER alpha), whereas it had no significant effect on the androgen receptor, glucocorticoid receptor or the activation functions of p53 or VP-16. We also demonstrated that, like other well characterized coactivators, WBP-2 contains an intrinsic activation domain. Depletion of endogenous WBP-2 with small interfering RNAs indicated that normal physiological protein level of WBP-2 was required for the proper functioning of ER alpha and PR. Moreover, chromatin immunoprecipitation (ChIP) assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. As we initially identified WBP-2 as an E6-AP interacting protein, we investigated whether WBP-2 and E6-AP function in concert. Our data shows that WBP-2 and E6-AP each enhance PR function and when co-expressed they additively enhance the transactivation functions of PR. However, WBP-2 was also able to enhance the transactivation functions of ER alpha and PR in mouse embryonic fibroblast cells generated from E6-AP knockout mice lines, suggesting that the coactivation functions of WBP-2 was not dependent on E6-AP. The further elucidate the molecular mechanism of action of WBP-2; we dissected the functional importance of the polyproline (PY) motifs contained within the WBP-2 protein. Mutational analysis suggests that one of three PY motifs, PY3 of WBP-2 was essential for its coactivation and intrinsic activation functions. In this study, we also demonstrate that the WBP-2 binding protein, Yes-kinase associated protein 1 (YAP1) acts as a secondary coactivator of ER alpha and PR. However, the coactivation function of YAP1 is revealed only in the presence of wild-type WBP-2 and not with the PY motif 3 mutant WBP-2. This is consistent with our observations that, unlike the wild-type WBP-2, the PY motif 3 mutant WBP-2 does not interact with YAP1. Our quantitative reChIP assays demonstrates an estrogen-dependent recruitment and association of ER alpha with both WBP-2 and YAP1. The hormone-dependent recruitment of YAP1 to ER alpha responsive promoter is dependent on the physiological expression levels of WBP-2. This is consistent with, our observation that the coactivation functions of YAP1 is dependent on WBP-2, and is also in agreement with other known secondary coactivators that get recruited to SHR responsive promoter via their interaction with primary coactivators. Surprisingly, the association of WBP-2 with ER alpha and its recruitment to the ER alpha target promoter was abrogated by YAP1 knock-down, suggesting that WBP-2 and YAP1 may stabilize each other at the promoter, and consequently, are functionally interdependent. Taken together our data establish the role of WBP-2 and YAP1 as selective coactivators for ER alpha and PR transactivation pathways.