Urotensin-II (U-II), a peptide with multiple vascular effects, is detected in cholinergic neurons of the rat brainstem and spinal cord. Here, the effects of U-II on [Ca2+]i, was examined in dissociated rat spinal cord neurons by fura 2 microfluorimetry. The neurons investigated were choline acetyltransferase-positive and had morphological features of motoneurons. U-II induced [Ca2+]i, increases in these neurons with a threshold of 10-9 M, and a maximal effect at 10-6 M with an estimated EC50 of 6.2 × 10-9 M. The [Ca2+]i increase induced by U-II was mainly caused by Ca2+ influx from extracellular space, as the response was markedly attenuated in a Ca2+-free medium. Omega-conotoxin GVIA (10-7 M), a N-type Ca2+ channel blocker, largely inhibited these increases, whereas the P/Q Ca2+ channel blocker, omega-conotoxin GVIIC (10-7 M) and the L-type Ca2+ channel blocker, verapamil (10-5 M) had minimal effects. Down-regulation of protein kinase C by 4-α-phorbol 12-myristate 13-acetate (10-6 M) or enzyme inhibition using the specific inhibitor bisindolylmaleimide I (10-6 M) did not inhibit the observed effects. Similarly, inhibition of protein kinase G with KT5823 (10-6 M) or Rp-8-pCPT-cGMPS (3 × 10-5 M) did not modify U-II-induced [Ca2+]i increases. In contrast, protein kinase A inhibitors KT5720 (10-6 M) and Rp-cAMPS (3 × 10-5 M) reduced the response to 25 ± 3% and 42 ± 8%, respectively. Present results demonstrate that U-II modulates [Ca2+]i, in rat spinal cord neurons via protein kinase A cascade.

Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngIIi) is unclear. Besides direct effects of AngIIi on cellular processes one could hypothesise a possible role of AngIIi in modulation of cellular responses induced after heterologous receptor stimulation. We therefore examined if AngIIi influences [Ca2+]i in A7r5 smooth muscle cells after serotonin (5HT) or UTP receptor stimulation. Application of AngIIi using liposomes, markedly inhibited 45Ca2+ influx after receptor stimulation with 5HT or UTP. This inhibition was reversible by intracellular administration of the AT1-antagonist losartan and not influenced by the AT2-antagonist PD123319. Similar results were obtained in single cell [Ca2+]i measurements, showing that AngIIi predominantly influences Ca2+ influx and not Ca2+ release via AT1-like receptors. It is concluded that AngIIi modulates signal transduction activated by heterologous receptor stimulation.

Philosophiae Doctor - PhD / Neurodegenerative disorders are characterised by progressive loss of the brain’s physiological functions as a result of gradual degeneration of neurons in the central nervous system. Even though they are classified as diseases of the elderly, occurrence earlier in life is possible, but that would suggest the influence of genetic and/or environmental factors. Due to the continuous rise in modernisation and industrialisation over the years, there has been an increase in incidence and prevalence of neurodegenerative disorders. With the advances in technology and life expectancy, the rates of the common forms (Alzheimer’s disease and Parkinson’s disease), are expected to increase exponentially by 2050. Unfortunately, there is still no clinically approved treatment or therapy to slow down or halt the degenerative process as most registered drugs only offer symptomatic relief. Confounding this issue is the lack of definite mechanism of neurodegeneration, which is still poorly defined and not completely understood. Nonetheless, the pathology of most neurodegenerative disorders is believed to be a combination of interrelated processes that eventually leads to neuronal cell death. Among the postulated processes, the impact of excitotoxicity mediated by NMDA receptor over-activation is prominent and it is implicated in virtually all neurodegenerative disorders. With this basic insight, it is believed that molecules capable of inhibiting NMDA receptors and associated calcium channels, without affecting the normal physiological functions of the brain, could potentially serve as good neuroprotective drugs. Competitive and uncompetitive blockers (MK-801 and ketamine) have been explored, but none were clinically accepted due to undesirable side effects such as hallucinations, sedation and depression. However, NGP1-01, a polycyclic cage molecule, has been shown to be neuroprotective through modulation of NMDA receptors and voltage gated calcium channels and attenuation of MPP+ -induced toxicity. A similar approach could be useful in the design and development of new neuroprotective drugs. The aim of this study was to synthesise a series of open and rearranged cage-like molecules and explore their neuroprotective potential in neuroblastoma SH-SY5Y cells. The proposed structures, with norbornane scaffolds that contained different moieties, were designed to structurally resemble NGP1-01 and MK-801. Once synthesised, the compounds were purified and characterised, and were evaluated for their biological activities. Compounds were first screened for cytotoxicity at different concentrations. Thereafter, they were evaluated for neuroprotective effects against MPP+ -induced excitotoxicity and for calcium flux modulatory effects on NMDA receptor and voltage gated calcium channels. The norbornane derivatives were synthesised and characterised, and all final products were afforded in sufficient yields. All compounds with the exception of two compounds displayed good cytotoxic profiles towards the SH-SY5Y neuroblastoma cells at 10 µM, 50 µM and 100 µM concentrations as they demonstrated percentage cell viabilities close to 100% (control treated cells). Only two compounds showed percentage cell viability of 51% and 59% at 100 µM. Utilising the same cell line, all compounds, tested at 10 µM, attenuated MPP+ -induced toxicity after 24 hours of exposure to a neurotoxin. This was evident in the 23% to 53% enhancement (significant with p < 0.05) in cell viability when compared to the MPP+ only treated cells. In comparison to known NMDA receptor and/or voltage gated calcium channel blockers (MK-801, NGP1-01 or nimodipine), the synthesised compounds demonstrated mono or dual inhibition of calcium channels as they effectively attenuated calcium influx by blocking NMDA receptors and/or voltage gated calcium channels expressed in neuroblastoma SHSY5Y cells. This group of compounds were found to be more potent NMDA receptor inhibitors, probably due to similarities with MK-801 and memantine, than voltage gated calcium channel inhibitors. All compounds demonstrated moderate to good calcium inhibitory effects at NMDA receptors in the range of 23% to 70% while a selected few displayed very little or no activity at the voltage gated calcium channels. In conclusion, 27 compounds with norbornane scaffolds were successfully synthesised and evaluated for cytotoxicity and neuroprotection. The abilities of the synthesised compounds to protect neurons from the neurotoxin MPP+ and reduce calcium flux into neuronal cells were successfully demonstrated. These characteristics are essential in neuroprotection as they may prove significant in halting or slowing down the disease progression. The compounds showing a good cytotoxicity profile, neuroprotective effects and ability to reduce calcium overload, could potentially act as neuroprotective agents with good safety profiles or contribute as lead structures to the development and design of structurally related molecules that could clinically benefit people with neurodegenerative disorders.

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This paper basically tries to seek if there are any tendencies or possibilities that the power of colonizer still remains in the colonized country in terms of migration policy regarding migrant worker rights. In this paper, the author focuses on the policy of both Malaysia and the United Kingdom as a host country regarding the rights of migrant as the topic of the paper. In this paper the author uses comparative analysis by comparing the migration policy of Malaysia and the United Kingdom and focuses on the migrant worker rights to see if there are any possibilities or tendencies that Malaysia adopted the migration policy of the United Kingdom. In conclusion, the United Kingdom policy regarding the system and recruitment of migrant workers in order to limit the influx of migrant workers influenced Malaysia to adopt similar policy. But Malaysia has different implementation of policy regarding migrant worker rights comparing to the United Kingdom.

Les canaux TRP (Transient Receptor Potential) sont des acteurs clés de l'homéostasie calcique. Plusieurs de ces canaux interviennent dans l'influx calcique des cellules épithéliales bronchiques, notamment TRPC6, qui est impliqué dans un couplage fonctionnel avec le canal Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Les mutations du CFTR (F508del et G551D) sont à l'origine de la mucoviscidose (Cystic Fibrosis (CF)), qui aboutit à l'augmentation de l'influx calcique dans les cellules CF. L'objectif de ce travail a été d'étudier l'implication des canaux TRP dans la dérégulation de l'influx calcique des cellules épithéliales bronchiques CF. Nous avons mis en évidence que CFTR régulait négativement l'activité de TRPC6, tandis que l'influx calcique via TRPC6 permettait de potentialiser l'activité du canal muté CFTR-G551D, activé au préalable par le VX-770. Nous proposons donc une nouvelle stratégie thérapeutique, combinant un potentiateur de CFTR et un activateur spécifique de TRPC6. Nous nous sommes ensuite intéressés au rôle des canaux TRPV, en particulier TRPV5 et TRPV6, dans l'influx calcique des cellules épithéliales bronchiques. Nous avons observé que l'influx Ca2+ constitutif, attribuable à ces deux canaux, était doublé dans les cellules CF, dû à une augmentation de l'activité de TRPV6. En effet, l'expression de la PLC-δ1, une enzyme régulant négativement TRPV6, est dramatiquement réduite dans les cellules CF. La correction de l'adressage du F508del-CFTR a permis de normaliser l'activité de TRPV6 sans restaurer l'expression de la PLC-δ1 dans les cellules CF, suggérant un contrôle plus complexe de TRPV6 dans les cellules épithéliales bronchiques. / TRP (Transient Receptor Potential) channels are keys actors of Ca2+ homeostasis. Several of these channels are involved in the Ca2+ influx of bronchial epithelial cells, including TRPC6 which is implicated in a functional coupling with the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) channel. CFTR mutation leads Cystic Fibrosis (CF) disease and causes abnormal Ca2+ homeostasis trought an increased of Ca2+ influx in CF bronchial epithelial cells. Our objective is to investigate the implication of TRP channels in abnormal Ca2+ influx of CF bronchial epithelial cells.We showed that CFTR down regulates TRPC6 activity whereas Ca2+ influx through TRPC6 potentiates G551D-CFTR, activated by VX-770. We propose a new therapeutic strategy that combines a CFTR potentiator and a specific activator of TRPC6. Then, we focused on the role of TRPV channels, particularly TRPV5 and TRPV6, in Ca2+ influx of bronchial epithelial cells. We observed that constitutive Ca2+ influx, related to TRPV5/TRPV6 activity, was twice higher in CF cells due to the increase of TRPV6 activity. The expression of PLC-δ1, an enzyme that negatively regulates TRPV6 activity, is dramatically decreased in CF cells. The correction of F508del-CFTR trafficking allows TRPV6 activity normalization but do not restore PLC-δ1 expression level in CF cells, suggesting a more complex control of TRPV6 in bronchial epithelial cells.

In this thesis the enriched stable isotope marking technique was examined to determine its validity and effectiveness in marking of Lake Sturgeon fin rays. The enriched stable isotope marking technique uses stable isotopes of elements that replace calcium in boney tissue, which are fixed in nature, however in laboratory settings can be easily manipulated. Manipulated ratios of naturally occurring isotopes can be determined from a small sample of the fin ray, thus allowing for the discrimination of fish stocks. Further, the thesis examined the underlying assumptions for strontium and barium into the Lake Sturgeon. For the marking technique to work, strontium and barium must be taken up from the environment and accumulated long-term in boney tissue. Net flux and tissue retention of strontium is reported and the research has demonstrated that enriched stable isotopes can be successfully used to mark the fin ray of Lake Sturgeon for long-term stock discrimination. / October 2015

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Made available in DSpace on 2015-07-22T20:50:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-24 / O ultrassom (US) vem sendo amplamente utilizado para melhorar a eficiência de transfecção de vetores não-virais. No entanto, o mecanismo pelo qual o US promove a entrega de DNA nas células ainda é pouco entendido. Este fenômeno é normalmente atribuído a sonoporação. Porém, com base em experimentos anteriores realizados em nosso laboratório, suspeitamos que outro mecanismo esteja envolvido no processo de captação de DNA. Para estudar o mecanismo de entrega, um vetor plasmideal expressando EGFP (pEGFP-N3, 4,7 kb) foi utilizado para transfectar células NIH3T3 com um aparelho de US terapêutico sem a adição de microbolhas. Em condições de insonação de 2 W/cm2, duty cycle de 20% por 30s o US promoveu cerca de 40% de eficiência de transfecção, mas com 1 W/cm2 resultou em níveis muito baixos de transfecção. Fixados esses parâmetros, também foi avaliada a produção de espécies reativas de oxigênio (ROS), o aumento da concentração intracelular de cálcio ([Ca2+]i) e as alterações no potencial de membrana através de microscopia confocal. A produção de ROS foi aumentada durante a insonação, sendo interrompida logo que o US foi desligado. A [Ca2+]i também foi aumentada durante a exposição ao US, mas seus níveis não retornaram ao basal durante os 3 minutos de observação. Porém, 1 W/cm2 não foi suficiente para mobilizar o cálcio durante a insonação, e o influxo de cálcio teve início apenas 12 segundos após o término do US. Quando expostas ao US, as células também apresentaram mudanças no potencial de membrana atingindo um estado de hiperpolarização, retornando ao estado normal logo que o US foi desligado. A alteração desses três parâmetros pelo US sugere que a entrega de DNA plasmideal deva ocorrer por endocitose. Por fim, utilizando DNA plasmideal fluorescente, mostramos que esta molécula entra na célula via endocitose mediada por clatrina. / Ultrasound (US) has been widely used to improve the efficiency of non-viral vector transfection. However, the mechanism that enables the uptake of plasmid DNA in cells by US insonation is poorly understood, but it is typically attributed to sonoporation. Based on our previous results, we hypothesized that other mechanisms, such as endocytosis, are involved in this process. To explore the mechanism of plasmid DNA uptake, a plasmid vector expressing EGFP (pEGFP-N3: 4.7 kb) was used to transfect NIH3T3 cells using a therapeutic US without microbubbles and was monitored in real-time using a confocal microscope. We achieved about 40% transfection efficiency when we applied 2 W/cm2 with 20% of duty-cycle for 30 s, but 1 W/cm2 resulted in a very low level of transfection. In these experiments, the production of reactive oxygen species was augmented during the insonation but was stopped soon after turning off the US. Calcium influx was also augmented during the insonation, but its level did not return to basal levels following the 3-min observation period. However, 1 W/cm2 was not sufficient to mobilize calcium influx during the insonation, and calcium influx began 12 s after turning off the US. US insonation also changed the cell membrane potential to promote a hyperpolarization state, which returned to the normal state soon after turning off the US. The alteration of these parameters by US indicates the uptake of plasmid DNA by endocytosis. Finally, using a fluorescently labeled plasmid, we showed that this molecule enters into cells via clathrin-mediated endocytosis, not via caveolin-1. / TEDE / BV UNIFESP: Teses e dissertações