As células T regulatórias (Tregs) são essenciais para a manutenção da tolerância periférica, prevenção de doenças autoimunes e limitantes nas doenças inflamatórias crônicas. Além disso, essas células exercem um papel fundamental no controle da rejeição de transplantes. Diferentes protocolos mostraram que é possível obter Tregs a partir de células T naive CD4+ in vitro. Para tal, é consenso que o TGF-? e a interleucina-2 (IL-2) são capazes de direcionar as células T naive CD4+ a se tornarem regulatórias após um estímulo antigênico (anti-CD3/CD28). Nosso grupo recentemente notou que, durante a imunomodulação de linfócitos T pelas células estromais mesenquimais (CTMs), estas eram capazes de produzir adenosina que, por sua vez, participa do processo de imunorregulação. Outros trabalhos indicam que as CTMs suprimem a proliferação dos linfócitos T pela geração de Tregs e que as CTMs induzem a geração de Tregs através da regulação negativa da via TCR e da via AKTmTOR. Evidências apontam que a adenosina pode atuar regulando negativamente a via mTOR. Portanto, acredita-se que a adenosina possa participar do processo de geração de Tregs através da modulação da via mTOR. Além disso, estudos recentes indicam que a ativação de receptores de adenosina, mais especificamente A2a, com agentes agonistas, leva ao aumento da produção de células Tregs, enquanto que a utilização de agentes antagonistas destes receptores leva à diminuição da diferenciação de Tregs. Porém, estes estudos mostram a geração de Tregs a partir de células T naive de camundongos. Visto a grande importância das Tregs no contexto imunológico, a produção eficiente de Tregs in vitro tem importância fundamental para o desenvolvimento de novos protocolos terapêuticos para o tratamento de doenças autoimunes e no combate à rejeição de transplantes. Assim, o objetivo central deste trabalho foi avaliar a participação de agonistas e antagonistas de receptores de adenosina na indução de células T regulatórias geradas in vitro (iTreg) pela ativação de células T CD4+ naive isoladas de sangue de cordão umbilical (SCU) humano. Para isso, células mononucleares foram isoladas de bolsas de SCU e as células T naive foram isoladas imunomagnéticamente. Essas células foram ativadas com beads ligadas a anticorpos anti-CD2/CD3/CD28 e cultivadas por cinco dias na presença de IL-2 e diferentes concentrações de drogas agonistas e antagonistas de receptores de adenosina. Em seguida, foram avaliados os principais marcadores de células T regulatorias por meio de citometria de fluxo e o meio de cultura foi coletado ao final da geração para quantificação de citocinas. Além disso, o RNA total foi extraído de todas as condições de cultivo para a análise da expressão de genes envolvidos na geração e desenvolvimento das Tregs, por PCR quantitativo. O potencial de supressão de células T efetoras também foi avaliado. / Regulatory T cells (Tregs) are essential for the maintenance of peripheral tolerance, prevention of autoimmune and limiting diseases in chronic inflammatory diseases. In addition, these cells play a key role in the control of transplant rejection. Different protocols have shown that it is possible to obtain Tregs from naive CD4+ T cells in vitro. To this end, there is consensus that TGF-? and interleukin-2 (IL-2) are capable of directing the naive CD4 + T cells to become regulatory following an antigenic stimulus (anti-CD3/CD28).. Our group recently noted that during the immunomodulation of T lymphocytes by mesenchymal stromal cells (MSCs), they were able to produce adenosine which in turn participates in the immunoregulation process. Other studies indicate that MSCs suppress the proliferation of T lymphocytes by generation of Tregs and that MSCs induce generation of Tregs by downregulation of the TCR pathway and the AKT-mTOR pathway. Evidence indicates that adenosine may act by downregulating the mTOR pathway. Therefore, it is believed that adenosine may participate in the generation of Tregs by modulating the mTOR pathway. In addition, recent studies indicate that activation of adenosine receptors, more specifically A2a, with agonist agents, leads to increased production of Treg cells, whereas the use of antagonistic agents of these receptors leads to a decrease in Treg differentiation.. However, these studies show the generation of Tregs from naive T cells of mice. In view of the great importance of Tregs in the immunological context, the efficient production of Tregs in vitro is of fundamental importance for the development of new therapeutic protocols for the treatment of autoimmune diseases and in the fight against transplant rejection. Thus, the central objective of this study was to evaluate the participation of adenosine receptor agonists and antagonists in induction of regulatory T cells generated in vitro (iTreg) by the activation of naive CD4+ T cells isolated from human umbilical cord blood (SCU). For this, mononuclear cells were isolated from SCU and naive T cells were immunomagnetic isolated. These cells were activated with beads bound to anti-CD2/CD3/CD28 antibodies and cultured for five days in the presence of IL-2 and different concentrations of agonist drugs and antagonists of adenosine receptors. Next, the major regulatory T-cell markers were assessed by flow cytometry and the culture medium was collected at the end of the generation for quantification of cytokines. In addition, total RNA was extracted from all culture conditions for the analysis of the expression of genes involved in the generation and development of Tregs by quantitative PCR. The potential for suppression of effector T cells was also evaluated.

ADAR (Adenosine Deaminases acting on RNA) family proteins are double-strand RNA binding proteins that deaminate specific adenosines into inosines. This A-to-I conversion is called A-to-I RNA editing and is well conserved in the animal kingdom from nematodes to humans. RNA editing is a pre-splicing event on nascent RNA that may affect alternative splicing when the editing occurs in the exon-intron junction or in the intron. Also, editing may change biological function of small RNAs by editing the premicroRNAs or other noncoding RNAs. Editing also alters protein amino acid sequences because inosine in the mRNA base pairs with cytosine and is therefore read as guanosine. In mammals, there are three ADAR family proteins, ADAR1, ADAR2, and ADAR3, encoded by three different genes. So far, no enzymatic activity of ADAR3 is detected. The most frequently edited targets of ADAR1 and ADAR2 are regions covering copies of Alu transposable elements in primates. In addition, loss of some specific editing events leads to profound phenotypes when the editing does not occur correctly. For example, some human neural disorders – such as epilepsy, forebrain ischemia, and Amyotrophic Lateral Sclerosis – are known to be associated with abnormally edited ion channel transcripts. Drosophila has a single ADAR protein (encoded by the Adar gene) that is highly conserved with human ADAR2 (encoded by the ADARB1 gene). To date, 972 editing sites have been identified in 597 transcripts in Drosophila, and approximately 20% of AGO2-associated esiRNAs are edited. Similar to mammals, many ion channel-encoding mRNA transcripts undergo ADAR-mediated A-to-I editing in Drosophila. While Adar1 null mice die at the embryonic stage and Adar2 null mice die shortly after birth due to seizures, Adar null flies are morphologically normal and have normal life span under ideal conditions. However, Adar null flies exhibit severe neurodegeneration and locomotion defects from eclosion, whilst Adar overexpression (OE) is lethal. To better understand the physiological role of RNA editing and ADAR, and to shed light on ADAR-related human disease, I used Drosophila Adar mutant flies as a model organism to investigate phenotypes, and to find chromosomal deletions and specific mutations that rescue the neural-behavioural phenotype of the Adar null mutant flies. Using the publicly available chromosomal deletions collectively covering more than 80% of the euchromatic genome of Chromsome III, I performed a genetic screen to find rescuers of the lethality caused by Adar overexpression. I confirmed that mutation in Rdl (Resistant to dieldrin, the gene encoding GABAA receptor main subunit) rescues. This rescue was not likely caused by effects on Adar expression level or activity. Driven by the hypothesis that the rescue may be due to reduction in GABAergic input to neurons, I recorded spontaneous firing activity of Drosophila larval aCC motor neurons using in vivo extracellular current recording technique. As expected, the neurons overexpressing Adar had much less activities compared with wild type neurons. Also, I found that Adar null fly neurons fired much more and showed epilepsy-like increased excitability. Although feeding PTX (Picrotoxin), a GABAA receptor antagonist, failed to rescue the lethality, reducing the expression of GAD1 to reduce synthesis of GABA was able to rescue the ADAR overexpression lethality. These results suggest that ADAR may finetune neuron activity synergistically with the GABAergic inhibitory signal pathway. I used MARCM (mosaic analysis using a repressible cell marker) to detect cellautonomous phenotypes in Adar null cells in otherwise wild type flies. Although neurodegeneration, observed as enlarged vacuoles formation in neurophils, was detected both in histological staining and EM images, the Adar null neurons marked with GFP from early developmental stages were not lost with age. Nevertheless, swelling in the axons or fragmentation of the axon branches of Adar null neurons was sometimes observed in the midbrain. By comparing the Poly-A RNA sequencing data from Adar null and wild type fly heads, we detected significant upregulation of innate immune genes. I confirmed this by qRT PCR and found that inactive ADAR reduces the innate immune gene transcript levels almost as much as active ADAR does. Further, using the locomotion assay, I confirmed that reintroducing inactive ADAR into Adar null flies can improve the flies’ climbing ability. Based on the Adar null flies having comparatively low viability, I performed a second deficiency screen to find rescuers of Adar null low viability using the same set of deficiencies as in the lethality rescue screen described above. I found seven deletions removing 1 to 37 genes that significantly increased the relative viability of the Adar null flies. However, not all the rescuing deficiencies also improved the Adar null locomotion. One rescuing gene, CG11357 was mapped from one of the rescuing deficiencies, and some mutant alleles of cry, JIL-1 and Gem3 also showed significant effects on the Adar null fly viability. The single gene viability rescuers were also not necessarily locomotion or neurodegeneration rescuers. Although the initial aim was to find neural-behavioural rescuing genes from the viability screen, the viability rescuers found in the screen are more likely to play a role in different aspects of stress response for survival.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O acidente vascular cerebral, doenÃa incapacitante e terceira causa de morte em paÃses desenvolvidos à caracterizada pela interrupÃÃo ou reduÃÃo do fluxo sangÃÃneo para o cÃrebro capaz de causar alteraÃÃo na funÃÃo cerebral. Sabe-se que o receptor A1 da adenosina possui um papel chave na neuroproteÃÃo devido à diminuiÃÃo da liberaÃÃo de glutamato e hiperpolarizaÃÃo neuronal. O objetivo desse trabalho foi determinar os efeitos do prÃ-condicionamento por estresse de contensÃo em ratos submetidos à isquemia cerebral transitÃria (ICT) por oclusÃo bilateral das carÃtidas e a participaÃÃo dos receptores A1 da adenosina nesse processo. Inicialmente, ratos Wistar machos, entre 200-240g, foram submetidos ao estresse de contensÃo (ST) em cilindros por 2h e imediatamente depois submetidos à ICT pela oclusÃo de ambas as artÃrias carÃtidas durante 30min. Um dos grupos dos animais foi prÃ-tratado com o antagonista do receptor A1 da adenosina, DPCPX, antes do estresse de contensÃo nas doses de 0,1mg/kg ou 1mg/kg. A temperatura retal foi monitorada e mantida a 37ÂC atravÃs de uma luz incandescente. Vinte e quatro horas depois do tÃrmino da ICT os animais foram sacrificados, tiveram seus cÃrebros dissecados, seccionados e imersos em soluÃÃo de Cloreto de 2,3,5-Trifeniltetrazol (TTC) a 1% por 30 min. para analise da viabilidade do tecido cerebral. Os testes comportamentais foram efetuados 72h apÃs a ICT e consistiram em Teste do Campo Aberto para a atividade locomotora, Labirinto em Y para a memÃria operacional ou de procedimento e Esquiva Passiva para aferiÃÃo da memÃria aversiva de curta e longa duraÃÃo. Os animais submetidos à ICT tiveram dano no tecido cerebral (FO= 10,36  0,75%; ISQ= 18,52  2,62%) alÃm de diminuiÃÃo no comportamento exploratÃrio de rearing (no de eventos: FO= 5,00 1,23; ISQ= 1,50  0,72) e dÃficit da memÃria aversiva de longa duraÃÃo (FO= 271,2  17,61s; ISQ= 108,4 67,64s). Nenhuma diferenÃa significativa foi encontrada no nÃmero de cruzamentos em Campo Aberto (FO= 15,71 2,02; ISQ= 11,00 2,13), na memÃria de procedimento (FO= 70,16  5,77; ISQ= 71,37  7,94), ou na memÃria aversiva de curta duraÃÃo (FO= 145,9  42,75; ISQ= 113,1  64,97).Os animais prÃ-condicionados por estresse tiveram uma reduÃÃo na taxa de infarto cerebral (FO= 10,36  0,75%; ISQ= 18,52  2,62%; ISQ+ST= 12,59  0,87%) e um retorno aos nÃveis normais do comportamento de rearing observado no teste do campo aberto (FO= 5,00 1,23; ISQ= 1,50 0,72; ISQ+ST= 6,091 1,443). No teste de esquiva passiva, observamos uma tendÃncia à melhora da memÃria aversiva de longa duraÃÃo (FO= 271,2  17,61s; ISQ= 108,4 67,64s; ISQ+ST= 156,1Â45,81s). Quando tratados com o DPCPX na dose de 1mg/kg, os animais tiveram um bloqueio da neuroproteÃÃo obtida com o prÃ-condicionamento (ISQ= 18,52  2,62%; ISQ+ST= 12,59  0,87%; ISQ+ST+DPCPX 1= 19,95  3,38%), aumento no nÃmero de rearings que havia sido normalizada pela contensÃo (ISQ= 1,50 0,72; ISQ+ST= 6,091 1,443; ISQ+ST+DPCPX 1= 3,20 0,90) e uma tendÃncia à reversÃo dos efeitos do prÃ-condicionamento na memÃria aversiva de longa duraÃÃo (ISQ= 108,4 67,64s; ISQ+ST= 156,1Â45,81s; ISQ+ST+DPCPX 1= 88,61 38,83s). O estresse de contensÃo conferiu neuroproteÃÃo aos animais submetidos à ICT e tal neuroproteÃÃo foi perdida pelo tratamento prÃvio com DPCPX. Esses achados apontam para a participaÃÃo do receptor A1 da adenosina na proteÃÃo conferida por estresse de contensÃo por mecanismos que ainda precisam ser esclarecidos. / Stroke,as disabling disease and as third cause death in developed countries, is characterized for the interruption of cerebral blood flow capable to cause alteration on brain functions. It is well established that the activation of A1 adenosine receptor confers neuroprotection against acute noxious brains stimuli. The aim of this study was to investigate the effects of preconditionnement by restraint stress on rats subjected to transient cerebral ischemia (TCI) and the participation of A1 receptor in this process. Firstly, Wistar male rats weighing 200-240g were exposed to immobilisation stress for 2 hours followed to TCI by occlusion of both carotid arteries for 30 minutes. Group of animals were pretreated with A1 receptor antagonist DPCPX (0,1mg/kg or 1 mg/kg. i.p.) before immobilisation stress. Retal temperature was monitored and 37ÂC were maintened during cirurgical procedure using a heating light. Infarct size was determined by TTC staining 24h after TCI and the behavioral tests were performed after 72 hours. Open field test were used to assess locomotor activity, Y-maze test for working memory and passive avoidance test to aversive short and long term memory evaluation. Our results showed that TCI caused damage on brain tissue (sham operated= 10.36  0.75%; ISC= 18.52  2.62%), decreased the vertical exploratory behavior (number of events: sham= 5.00  1.23; ISC= 1.50  0.72) and deficit on long term aversive memory (sham= 271.2  17.61s; ISC= 108.4  67.64s). No differences were found on the crossing behavior (sham= 15.71  2.02; ISC= 11.00  2.13), working memory (sham= 70.16  5.77; ISC= 71.37  7.94) neither short term memory (sham= 145.9  42.75; ISC= 113.1  64.97). The infarct volume rates on restraint stress (RS) group were significantly less than ischemic (ISC) group (sham= 10.36  0.75%; ISC= 18.52  2.62%; RS= 12.59  0.87%) while the number of rearing were significantly higher (sham= 5.00 1.23; ISC= 1.50 0.72; RS= 6.091 1.443). On the passive avoidance test, restraint stress tend to impair the ischemic damage on the long term memory (sham= 271.2  17.61s; ISC= 108.4  67.64s; RS= 156.1  45.81s). When treated with DPCPX (1mg/kg) the infarct size show an increase (ISC= 18.52  2.62%; RS = 12.59  0.87%; DPCPX= 19.95  3.38%) suggesting a blockade of neuroprotection action achieved by restraint stress. DPCPX also decreased the number of rearing on the open field test (ISC= 1.50  0.72; RS= 6.091 1.443; DPCPX = 3.20  0.90) and tend to reverse the improvement of long term aversive memory accessed by restraint stress (ISC= 108.4  67.64s; RS= 156.1  45.81s; DPCPX 1= 88.61 38.83s). This work showed a neuroprotection of pre conditioning restraint stress against cerebral ischemia and the blockade of this action by a previously administration of DPCPX, A1 adenosine antagonist. These findings point to the involvement of the A1 adenosine receptor in the protection conferred by restraint stress by mechanisms that still need to be clarified.

A hipertensão arterial é um problema de saúde pública no Brasil, pois aproximadamente 20 % da população adulta desenvolve hipertensão essencial, cujas causas ainda não são conhecidas. No entanto, sua gênese pode estar relacionada com disfunção nas áreas do sistema nervoso central (SNC) que regulam o sistema cardiovascular. O núcleo do trato solitário (NTS) e o bulbo ventrolateral são áreas importantes no controle neural da pressão arterial. Os receptores de adenosina A2a (rA2a) são encontrados em todo o SNC e estão relacionados com estudos terapêuticos de diversas doenças. No NTS a estimulação dos rA2a provoca ajustes pontuais em outros sistemas de neurotransmissão, além de diminuir a pressão arterial. A nicotina é uma molécula com uma vasta faixa de efeitos modulatórios em nosso organismo. Entre esses efeitos se destacam a capacidade de interagir com diversos sistemas de neurotransmissão nas áreas do bulbo relacionadas com a regulação da pressão arterial e de antecipar e/ou intensificar o desenvolvimento da hipertensão em sujeitos com pré-disposição genética. Desta forma, o objetivo do presente trabalho é avaliar o efeito modulatório da nicotina sobre o rA2a em cultura mista de neurônios e células gliais da porção dorso-medial do bulbo de ratos geneticamente hipertenso (SHR) e normotensos (WKY). Para isso, utilizaram-se técnicas como a de PCR em tempo real, Western Blotting e análise de ligação do receptor. Nossos resultados demonstraram que: 1) em condição basal células de ratos normotensos apresentam maior ligação do rA2a do que células de ratos hipertensos; 2) tratamento com nicotina resultou na diminuição da ligação do receptor em ambas as cepas, com um efeito de maior magnitude em células de ratos WKY; 3) nas duas linhagens o tratamento com nicotina alterou os níveis protéicos do rA2a, assim como o RNAm do receptor; 4) a linhagem e o tratamento separadamente, como a interação entre ambos influenciaram na expressão do RNAm , níveis protéicos e ligação do rA2a nas células dos ratos WKY e SHR. Por fim, os resultados apresentados aqui indicam que o rA2a em células de ratos hipertensos tem sua função deprimida em comparação com as células de ratos normotensos; e que a nicotina foi capaz de modular o funcionamento do rA2a, o qual pode influenciar no controle da pressão arterial. Esses dados são bastante interessantes, pois abrem novas perspectivas de análise dos mecanismos intracelulares envolvidos na modulação dos rA2a pela nicotina, assim como a importância desse sistema no desenvolvimento da hipertensão / Hypertension is one of the most common worldwide diseases afflicting humans. Because of the associated with morbidity and mortality and the cost to the society, it became an important public health challenge in Brazil. The mechanisms involved in development of hypertension still remain unclear However, hypertension can result from neuronal network imbalance in areas of the central nervous system that control blood pressure. The nucleus tractus solitarius (NTS) plays an important role in cardiovascular control. Within the NTS there are several neurotransmitters and neuromodulatory substances, such as adenosine, which acts on purinoreceptors A2a (A2ar). The A2ar modulates neurotransmission in the NTS and its activation may induce decrease in blood pressure by different mechanisms. Nicotine is a molecule that cross the blood-brain barrier and acts in several areas of central nervous system including the NTS. In this nucleus, nicotine is able to interact with some neurotransmitter systems and contributes for the development of hypertension in subjects with genetic predisposition to this disease. The goal of this study was to analyze the modulatory effects of nicotine on A2ar in cultured neurons and glial cells from medulla oblongata of normotensive (WKY) and spontaneously hypertensive rats (SHR). By means of real time PCR, Western Blotting and binding receptor assay. We have demonstrated that in basal condition cells of WKY presents increased binding of A2ar than the cells of SHR. Nicotine treatment induced a decrease in the binding of A2ar in both strains, however, this response was more pronounced in cells of WKY than SHR. Changes in mRNA and protein levels of A2ar was also observed in response to nicotine treatment. The strains and treatment separately, as well as the interaction between them influenced mRNA expression, protein level and binding of A2ar in NTS cells of WKY and SHR rats. Finally, these results show for the first time changes in A2ar mRNA expression, protein level and binding in cells from the medulla oblongata of WKY and SHR rats, as well as, the nicotine modulation upon this system, which might influence cardiovascular control. These data open up new approaches for the study of intracellular mechanisms involved in the modulation of adenosine A2a receptor by nicotine, as well as the importance of this interaction in the development of hypertension.

Dans le cerveau adulte, la signalisation liée à l’adénosine facilite ou inhibe la libération vésiculaire de neurotransmetteurs suite à l’activation des récepteurs de l’adénosine de type 2A ou 1 (A2AR ou A1R), respectivement. Cependant, son rôle dans le développement est mal connu. Au cours de ma thèse, j’ai étudié le rôle de la signalisation adénosine dans la synaptogenèse GABAergiques de l’hippocampe. Nous avons mis en évidence (i) une sécrétion activité-dépendante accrue d’adénosine et d’ATP pendant la période de synaptogenèse, (ii) un pic d’expression de l’enzyme limitant la formation de l’adénosine à partir de l’ATP extracellulaire, l’ecto-5’-nucleotidase, aux synapses pendant cette période critique, et (iii) un pic d’expression péri/post- synaptique du A2AR concomitant de la période de synaptogenèse. Cette expression développementale des molécules clés de la signalisation adénosine dépendante du A2AR corrélait avec un rôle de ce récepteur dans la stabilisation des synapses GABAergiques naissantes, une régulation restreinte à la période de synaptogenèse. De plus, la suppression de A2AR par une approche shRNA dans des neurones isolés conduisait à une perte de synapses GABAergiques équivalente à celle observée après un blocage pharmacologique de l’activité du A2AR, signifiant que la stabilisation synaptique médiée par le A2AR est un processus « cellule autonome » indépendant de l’activité du réseau neuronal et qu’elle requiert l’activation du A2AR dans la cellule post-synaptique.L’ATP et l’adénosine sont secrétés par la glie et les neurones ; cependant, nous avons montré in vitro que la libération neuronale activité-dépendante suffit à stabiliser les synapses GABAergiques naissantes. En utilisant la vidéomicroscopie sur cellules vivantes, nous avons montré que la signalisation adénosine stabilise les synapses actives. Puis, nous avons caractérisé le mécanisme moléculaire sous-jacent. Nous rapportons la contribution de la cascade adénylate cyclase/adénosine monophosphate cyclique/protéine kinase A et nous avons identifié une ciblé clé, la géphrine, la molécule d’ancrage postsynaptique des récepteurs GABAA. Enfin, nous avons mis en évidence que la stabilisation de l’élément présynaptique requiert probablement le complexe trans-synaptique Slitrk3-PTPd.Puisque le GABA exerce une fonction similaire au cours du développement et que le GABA et l’adénosine sont co-libérés à certaines synapses, j’ai étudié l’interaction entre ces deux voies de signalisation. Mes résultats favorisent l’hypothèse que la signalisation GABA, en activant la calcium-calmoduline, converge vers la signalisation adénosine en potentiant les adenylates cyclases sensibles au calcium. Mon travail m’a permis de proposer que, au cours d’une période clé du développement, les A2ARs postsynaptiques agissent comme des senseurs de l’activité des terminaisons présynaptiques GABAergiques pour stabiliser les synapses actives. En absence d’activité et donc de libération d’adénosine/ATP, les synapses seraient éliminées. / In the adult brain, adenosine signaling facilitates or inhibits neurotransmitter vesicular release mainly through activation of type 2A or 1 adenosine receptors (A2AR or A1R), respectively. However, its role in development remains to be elucidated. During my PhD, I addressed the role of A2AR-mediated signalling in GABAergic synaptogenesis in the hippocampus. We found (i) a larger activity-dependent release of ATP and adenosine during the period of synaptogenesis in the hippocampus, (ii) a peak of expression of the ecto-5’-nucleotidase, the rate-limiting enzyme for the formation of adenosine from extracellular ATP in synapses during this critical period, and (iii) a peak of peri/post-synaptic expression of A2AR concomitant with the period of synaptogenesis. This developmental expression of the key molecules of the adenosine A2AR signalling pathway correlated with a role of A2AR in the stabilization of nascent GABA synapses, a regulation restricted to the period of synaptogenesis. Furthermore, suppressing A2AR with a shRNA approach in isolated neurons led to a loss of synapses equivalent to that seen upon A2AR activity blockade, reporting that the A2AR-mediated synapse stabilization is a cell autonomous process that requires A2AR activation in the postsynaptic cell. ATP/adenosine can be secreted by both glia and neurons; however, we found that activity-dependent release of neuronal adenosine is sufficient to stabilize newly formed GABA synapses in vitro. Using live cell imaging, we showed adenosine signalling stabilizes active synapses. We then characterized the molecular mechanism downstream postsynaptic A2AR. We report the contribution of adenylyl cyclase/cyclic adenosine monophosphate/protein kinase A signalling cascade and we identified a key target, the postsynaptic scaffolding molecule gephyrin. We further showed the A2AR-mediated stabilization of the presynaptic compartment most probably requires the trans-synaptic Slitrk3-PTPd complex. Since GABA exerts a similar function during development and GABA and adenosine are co-released at some synapses, I further investigated the interplay between these two pathways. My results support the hypothesis that GABA signalling converge onto the adenosine signalling pathway by potentiating calcium-sensitive adenylyl cyclases through the activation of calmodulin.Altogether these results let us propose that, during a key developmental period, postsynaptic A2ARs act as sensors of the activity of GABAergic presynaptic terminals to stabilize active nascent GABAergic synapses. In absence of activity and therefore secretion of adenosine/ATP, synapses will be eliminated.

Abstract not available

Abstract not available

Purines as a class of compounds have been implicated in many biological systems, including as adenosine receptor antagonists. A method of synthesising 2,6,9-trisubstituted purines would be useful to produce small libraries of compounds for probing adenosine receptor selectivity. A library of trisubstituted purines has been achieved using a solid-phase methodology. The electronic properties of the substrate were found to result in difficulties with the loading of substrate onto the resin. Theoretical calculations provided the basis for mono-substitution in order to activate the substrate. This modified substrate has loaded onto the resin in reproducible and high yields. Amine and thiol, on-resin, C-2 substitution was shown to proceed at room temperature. This represents significantly milder conditions than are generally seen in the literature. This is due to the activating effect of the carbamate linker chosen on the pyrimidine ring. This also results in a faster reaction rate than is seen in the corresponding solution-phase reaction. This study showed that the electronic profile of the loaded substrate was responsible for the low alkylation on the carbamate nitrogen of loaded dichloro- or C-6 substituted chloropyrimidines. This reaction was modified by activating the pyrimidine ring via C-2 substitution and has been shown to go to completion with three different alkyl groups to give a clean product direct from resin cleavage. On-resin nitro reduction had been planned. The resin bound product would then be carried on to the next step of resin cleavage and cyclisation of the imidazole ring to give the final purine products. On resin reduction could not be achieved, however, cleavage of the compound from the resin and reduction in solution was found to be efficient as the cyclisation reagents could be included in this step without interfering with yield or purity of products and so this represents a clear improvement upon the planned synthesis. Efforts to fully characterise the library brought up issues of purine NMR. Extremely broad signals were observed in the proton spectra of many of the compounds making assignments difficult. Broad 13C NMR signals have also been observed. Restricted rotation about the substituent N-C bond is responsible for these problems. Crystal structure data has confirmed the double bond character of this bond with one of the substituted pyrimidines. High temperature NMR experiments have demonstrated how this can be overcome and the fine structure of the spectra observed. HMBC and COSY correlations have been used alongside the 1H and 13C spectra to allow full characterisation of the compounds wherever possible. Receptor homology models were created and updated for all four adenosine receptor subtypes. Known adenosine agonists and antagonists were created and minimised for use in docking experiments. Receptor docking experimental data is reported. Binding assays are being carried out by a third party and will be submitted for publication at a later date. A small library of 2,6,9-trisubstituted purines has been synthesised, exemplifying an efficient and robust method to achieve pure compounds for biological evaluation. A good level of diversity has been achieved at each combinatorial position (two substitutions and an N-alkylation). Final compounds have been isolated in good yields with a high level of purity.

The sphincter of Oddi (SO) is a neuromuscular structure located at the junction of the bile and pancreatic ducts with the duodenum. The primary functions of the SO are to regulate the delivery of bile and pancreatic juice into the duodenum, and to prevent reflux of duodenal contents into the biliary and pancreatic systems. Neural, hormonal or functional disturbances of biliary motility can lead to painful and sometimes life threatening clinical conditions, such as SO dysfunction and acute pancreatitis. Clearly understanding the regulation of biliary and duodenal motility patterns is necessary and may provide useful pharmacological sites for drug development to aid in the treatment of these diseases. Spontaneous activity of the SO is regulated by complex interactions between the enteric nervous system, hormones, possibly interstitial cells of Cajal and other bioactive agents, together with modulation via neural reflexes between the duodenum, common bile duct/gallbladder, and stomach. Purines are one group of neurotransmitters/regulatory agents that have been shown to effect gastrointestinal motility, however their functions in the regulation of SO motility have not been elucidated. The studies described in this thesis used in vitro organ bath techniques and in vivo preparations to determine the effects of exogenous purines on possum SO and duodenal motility. The possum SO has been extensively characterized and is an excellent model for motility studies. In vitro, exogenous adenosine was found to decrease spontaneous activity in both the SO and duodenum. In contrast exogenous ATP induced both excitatory and inhibitory responses in the SO and duodenum. Interestingly, the adenosine and ATP-induced effects were predominantly exhibited by the proximal portion of the SO (proximal-SO), with no or little effect observed in the distal portion of the SO (distal-SO). These data support the hypothesis that the SO is comprised of different functional components that can act differently in response to certain stimuli, and highlights the importance of studying each of the SO components. Agonists and antagonists, together with immunohistochemical studies, were used in an attempt to identify the P1 and P2 receptor sub-types responsible for mediating the adenosine- and ATP-induced responses. In the duodenum the adenosine-induced decrease in spontaneous activity was likely to be mediated by A2A and A3 receptors, but the receptors mediating the proximal-SO response could not be identified. In the duodenum ATP induced a complex non-neural response consisting of a P2X1, and P2Y2 and/or P2Y4 mediated immediate inhibition. This was followed by a return to baseline activity or small excitation. The response concluded with a late inhibitory response, likely to be mediated by P2Y1 receptors, but the effects of other P2Y receptors could not be excluded. In contrast, ATP application to the proximal-SO evoked a partially neurally mediated early excitation, likely via P2X receptors, followed by an inhibition of activity, likely via activation of non-neural P2Y2 and/or P2Y4 receptors. In vivo studies with exogenous application of adenosine and ATP to the SO activated neural pathways to produce increased motor activity. Characterisation of these neural pathways found ATP and/or adenosine to activate excitatory cholinergic motor neurons. ATP also activated an inhibitory nicotinic/nitrergic pathway. This is the first comprehensive investigation of the possible involvement of purines in the regulation of SO motility. These studies demonstrate that exogenous purines influence SO and duodenal motility, inducing complex neural and non-neural responses, acting via multiple P1 and P2 receptors. It now remains to be determined if endogenously released purines induce similar responses, together with elucidation and location of the receptor sub-types involved.

Extracellular adenosine 5′-triphosphate (ATP) is an agonist for the P2Z receptor of human leukaemic lymphocytes and opens a Ca ²⁺-selective ion channel, which also conducts Ba²⁺, Sr²⁺ and the small fluorescent dye, ethidium⁺. A wide range of receptor agonists, many of which raise cytosolic [Ca²⁺] activate phospholipase D (PLD). In the present study, it was shown that both ATP and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) stimulated PLD activity in a concentration-dependent manner, and the inhibitory effects of suramin, oxidised ATP, extracellular Na⁺ and Mg²⁺ suggested that the effect of these agonists is mediated by P2Z receptors. The role of divalent cations in ATP-stimulated PLD activity was investigated. Several agonists (eg ATP, thapsigargin, ionomycin) stimulated a rise in cytosolic [Ca²⁺] in human lymphocytes, but only ATP and ionomycin stimulated PLD activity. When Ca²⁺ influx was prevented by EGTA, the majority of ATP-stimulated and all of ionomycin-stimulated PLD activity was inhibited. Preloading cells with the Ca²⁺ chelator, BAPTA, reduced cytosolic [Ca²⁺] and, paradoxically, ATP-stimulated PLD activity was potentiated. ATP-stimulated PLD activity was supported by both Ba²⁺ and Sr²⁺ when they were substituted for extracellular Ca²⁺. Furthermore, both ATP-stimulated PLD activity and ATP-stimulated ¹³³Ba²⁺ influx showed a linear dependence on extracellular [Ba²⁺]. Thus it was concluded that ATP stimulated PLD activity in direct proportion to the influx of divalent cations through the P2Z ion channel and this PLD activity was insensitive to changes in bulk cytosolic [Ca²⁺]. The calmodulin (Ca²⁺/CaM) inhibitor, trifluoperazine (TFP) inhibited ionomycin- and ATP-stimulated PLD activity and ATP-stimulated apoptosis, but had no effect on PLD activity already activated by ATP. However, TFP inhibited ATP-stimulated Ca²⁺, Ba²⁺ and ethidium⁺ fluxes, at concentrations below those which inhibit Ca²⁺/CaM, suggesting that TFP inhibits the P2Z receptor. Similarly, the isoquinolinesulphonamide, KN-62, a selective inhibitor of Ca²⁺/CaM-dependent protein kinase II (CaMKII), also prevented ATP-stimulated apoptosis, but had no effect on pre-activated PLD. In addition, KN-62, and an analogue, KN-04, which has no effect on CaMKII, potently inhibited ATP-stimulated Ba²⁺ influx (IC₅₀ 12.7 ± 1.5 and 17.3 ± 2.7 nM, respectively), ATP-stimulated ethidium⁺ uptake (IC₅₀ 13.1 ± 2.6 and 37.2 ± 8.9 nM, respectively), ATP-stimulated phospholipase D activity (50% inhibition 5.9 ± 1.2 and 9.7 ± 2.8 nM, respectively) and ATP-induced shedding of the surface adhesion molecule, L-selectin (IC₅₀ 31.5 ± 4.5 and 78.7 ± 10.8 nM, respectively). They did not inhibit phorbol ester- or ionomycin-stimulated PLD activity or phorbol ester-induced L-selectin shedding. Neither KN-62 nor KN-04 (both 500 nM) have any effect on UTP-stimulated Ca²⁺ transients in fura-2-loaded human neutrophils, a response which is mediated by the P2Y₂ receptor, neither did they inhibit ATP-stimulated contractile responses mediated by the P2X₁ receptor of guinea pig urinary bladder. Thus, KN-62 and KN-04 are almost equipotent as P2Z inhibitors with IC₅₀s in the nanomolar, indicating that their actions cannot be due to CaMKII inhibition, but rather that they are potent and direct inhibitors of the P2Z receptor. Extracellular ATP-induced shedding of L-selectin from lymphocytes into the medium is a Ca²⁺-independent response. L-selectin is either cleaved by a metalloproteinase or a PLD with specificity for glycosylphosphatidylinositol (GPI). The novel hydroxamic acid-based zinc chelator, Ro-31-9790 blocks ATP-induced L-selectin shedding, but was without effect on ATP-induced Ba²⁺ influx or ATP-stimulated PLD activity. Furthermore, another zinc chelator, 1,10-phenanthroline, an inhibitor of a GPI-PLD, potentiated rather than inhibited ATP-stimulated PLD activity, suggesting that ATP-induced L-selectin shedding and ATP-stimulated PLD activity are independent of each other. Although extracellular ATP is the natural ligand for the lymphocyte P2Z receptor, it is less potent than BzATP in stimulating Ba²⁺ influx. Concentration-response curves for BzATP- and ATP-stimulated ethidium⁺ influx gave EC₅₀s 15.4 ± 1.4 µM and 85.6 ± 8.8 µM, respectively. The maximal response to ATP was only 69.8 ± 1.9% of that for BzATP. Hill coefficients were 3.17 ± 0.24 and 2.09 ± 0.45 for BzATP and ATP respectively, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z-operated ion channel. A rank order of agonist potency of BzATP > ATP = 2MeSATP > ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. When ATP (300 -1000 µM) was added simultaneously with 30 µM BzATP (EC₉₀), it reduced both ethidium⁺ and Ba²⁺ fluxes by 30 - 40% relative to values observed with BzATP alone. KN-62, previously shown to be a specific inhibitor of the lymphocyte P2Z receptor, was a less potent antagonist of BzATP-induced fluxes than ATP, when maximal concentrations of both agonists (50 and 500 µM respectively) were used. However, when BzATP (18 µM) was used at a concentration equiactive with a maximally effective ATP concentration, KN-62 showed the same inhibitory potency for both agonists. The ecto-ATPase antagonist, ARL-67156, inhibited both ATP- and BzATP-stimulated Ba²⁺ influx, suggesting that the lower efficacy of ATP compared with BzATP was not due to preferential hydrolysis of ATP. Thus, the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a full agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes. Both ATP- and BzATP-stimulated PLD activity were significantly inhibited (P < 0.05) when cells were suspended in iso-osmotic choline Cl medium. Choline⁺ was found to be a permeant for the P2Z ion channel, since ATP induced a large uptake of [¹⁴C]choline⁺ (60 to 150 µmol/ml intracellular water) during a 5 min incubation, which remained in the cells for several hours, and ATP was used to load cells with these levels of choline⁺. Intracellular choline⁺ inhibited ATP-, BzATP-, PMA- and ionomycin-stimulated PLD activity. Brief exposure of lymphocytes to ATP increased the subsequent basal rate of ethidium⁺ uptake, and this was prevented by intracellular choline⁺. It is proposed that P2Z-mediated Ca²⁺ influx in lymphocytes activates PLD leading to significantly changes of the phospholipid composition of the plasma membrane, which subsequently produces a permeability lesion, which in turn contributes to cell death.