In present time, to be mobile, to be able to physically move from point A to point B is something taken for granted by a small minority of the world’s population, while the vast majority are caught within or between borders. How did we reach to this point? This study examines the lived experiences of (im)mobility in Jordan. It captures the experiences of Syrians who live in Jordan and who have been denied mobility because of their flight from their homeland. By means of an ethnographical approach this study challenges conventional conceptions of what it means to be displaced. Situating the understanding of displacement in relation to the modern nation state, territorial boundedness, national identity and geographical categorizations it ultimately lays the foundation for conceptualizing the relative human (im)mobility and its links to a historical past. Through travelling and living in Jordan periodically between 2017-2018, totalizing approximately 6 months, conducting semi-structured interviews and engaging in everyday social and contextual encounters, this study offers a more multifaceted understanding of what it means to be (im)mobile in present time, as additional to conventional scholarship. It ultimately demonstrates that displacement as we understand it today is inadequate and simplified, and as we reassess its components we are able to reconceptualize the understandings of relative human (im)mobility.

Thesis advisor: Charles S. Hoffman / Glucose detection in yeast occurs via a cAMP signaling pathway that is similar to that of other signaling pathways in humans. The presence of glucose in the environment ultimately represses, as a result of cAMP signaling, the transcription of the gene fbp1. Adenylate cyclase is known to convert ATP to cAMP, and is thus a central protein in the propagation of the signal. Mutant forms of the adenylate cyclase gene (git2) have been found by the inability for the organism to repress fbp1 transcription in the presence of glucose. In this study, two questions were under investigation. The first was focused on the ability of the mutations to affect the dimerization of the catalytic domain. The second investigated multiple protein-protein interactions in the leucine rich-repeat (LRR) domain of adenylate cyclase. Both domains contain mutations that confer an activation defect, and they are thus are thought to have a relationship. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

O óxido nítrico (NO) é um mensageiro químico gerado pela atividade da enzima óxido nítrico sintase (NOS) a qual foi detectada em vários órgãos incluídos os do sistema reprodutor e parece estar envolvido na maturação oocitária. No entanto, há poucos estudos sobre o papel desse sistema em oócitos da espécie bovina. Sabe-se que o NO atua pela via da guanilato ciclase (GC) estimulando a produção do nucleotídeo GMPc, que por sua vez é capaz de influenciar nos níveis de outro nucleotídeo (AMPc) via fosfodiesterases (PDE). O AMPc é um importante elemento da via de sinalização do FSH nos complexos cumulus-oócitos e no controle da maturação oocitária. O objetivo do presente projeto foi investigar a influência do FSH na via do NOS/NO e seus componentes em oócitos bovinos maturados in vitro e o envolvimento das células do cumulus (CC) na via de sinalização. Para tanto, complexos cumulus-oócito (CCO) e oócitos desnudos (OD - maturados sem células do cumulus) foram maturados in vitro por 24h na presença ou ausência de FSH. As amostras foram avaliadas quanto a: 1) taxa da maturação nuclear; 2) níveis de produção de NO; 3) níveis de AMPc e GMPc; 4) abundância relativa de RNAm de NOS2, PDE5A, PDE6C, PKG1, PKG2, ADCY6, ADCY9, PDE3A e PKA1. O FSH, na concentração de 0,05UI/mL, estimulou positivamente a maturação nuclear em CCO e OD, com 80,6 e 89% de oócitos maturados, respectivamente. Quando comparados diretamente os grupos CCO e OD, o FSH não influenciou as taxas de maturação (71 e 71,3%, p>0,05), nem os níveis de produção de NO (12,8 e 7,4 µM/mL, p>0,05). Os níveis de GMPc em CCO aumentaram após 1 e 3 h de MIV na presença de FSH (266,3 e 187,2 pmol/pool com FSH e 240,5 e 168,5 pmol/pool sem FSH, respectivamente, p<0,05). Após 6 h os níveis de GMPc declinaram de forma mais acentuada no grupo sem FSH (46,3 e 106,9 pmol/pool, com e sem FSH, respectivamente, p<0,05). Os níveis de AMPc em CCO também foram mais elevados na presença de FSH à 1 e 3 h de MIV (7,60 e 7,81 pmol/pool, respectivamente) em comparação com CCO maturados sem FSH (0.30 e 0,76 pmol/pool, respectivamente, p<0,05). Após 6h, os níveis declinaram e foram similares para ambos os grupos (0,43 pmol/pool, p>0,05). Em relação à expressão dos genes selecionados, todos foram detectados nos oócitos (CCO e OD), porém, em células do cumulus, foram detectados apenas PDE5A, ADCY6, ADCY9 e PKA1. Quando observados os resultados do grupo CCO, apenas os genes PKG1, ADCY6 e PDE3A sofreram influência do FSH (p<0,05), apresentando um aumento destes transcritos. No grupo OD, apenas o gene PKG1 sofreu influência do FSH, também apresentando um aumento destes transcritos (p<0,05). Em células do cumulus, os genes ADCY6 e ADCY9 sofreram influência do FSH, sendo que para a ADCY6 provocou um aumentos destes transcritos, e para a ADCY9 provocou uma queda dos mesmos (p<0,05). Em conclusão, o FSH pode exercer influência positiva na maturação nuclear de oócitos bovinos, agindo sobre os níveis de GMPc e AMPc, mas não sobre o NO. O FSH pode influenciar a expressão gênica em oócitos e em células do cumulus de bovinos. / Nitric oxide (NO) is a chemical messenger generated by the nitric oxide synthase (NOS) enzyme, which was detected in several organs including the reproductive system and appears too involved in oocyte maturation. However, there are few studies on the role of this system in bovine oocytes. NO is known to act via guanylate cyclase (GC) stimulating the production of the nucleotide cGMP, which in turn is capable of influencing the levels of another nucleotide, cAMP via phosphodiesterases (PDE). cAMP is an important factor in FSH signaling in cumulus-oocyte complexes (COC) for the control of maturation. The aim of the present work was to investigate the influence of FSH on the NOS/NO pathway and its components in bovine oocytes matured in vitro and the involvement of cumulus cells (CC) in the signaling pathway. COC and denuded oocytes (DO - matured without cumulus cells) were matured in vitro for 24 h with or without FSH. Samples were assessed for: 1) maturation rate; 2) levels of NO production; 3) levels of cGMP and cAMP; 4) relative abundance for mRNA of NOS2, PDE5A, PDE6C, PKG1, PKG2, ADCY6, ADCY9, PDE3A and PKA1. FSH positively stimulated oocyte maturation at 0.05UI/mL concentration for both COC and OD (80.6 and 89% maturation rates, respectively). When COC and OD were compared directly, FSH did not affect maturation rates (71 and 71.3%, p>0.05) nor NO production levels (12,8 and 7,4 µM/mL), p>0.05). cGMP levels increased after 1 and 3 h in vitro maturation (IVM) with FSH (266.3 and 187.2 pmol/pool with FSH and 240.5 and 168.5 pmol/pool without FSH, respectively, p<0.05). After 6 h IVM, cGMP levels in COC declined more in the group cultured with FSH (46.3 and 106.9 pmol/pool, with and without FSH, respectively, p<0.05). cAMP levels in COC were also increased in the presence of FSH at 1 and 3 h IVM (7.60 and 7.81 pmol/pool, respectively) in comparison to COC cultured without the hormone (0.30 and 0.76 pmol/pool, respectively, p<0.05). After 6 h, the levels declined and were similar for both groups (0.43 and 0.02 pmol/pool, p>0.05). Regarding mRNA expression for the selected genes, all of them were detected in oocytes, but only four of them were detected in cumulus cells: PDE5A, ADCY6, ADCY9 and PKA1. For COC only PKG1, ADCY6 and PDE3A were influenced by FSH (p<0.05), with an increase in transcript relative abundance, For DO, only PKG1 was influenced by FSH and also showed an increase in these transcripts (p<0.05). In cumulus cells, ADCY6 and ADCY9 were affected by FSH, with an increase for ADCY6 and a decrease in ADCY9 transcripts (p<0.05). In conclusion, FSH may positively influence nuclear maturation, acting on cGMP and cAMP levels, but not on NO. FSH may also influence gene expression in bovine oocytes and cumulus cells.

Considerando-se como fonte primária os manuais escolares brasileiros de Matemática, o saber a ser ensinado ainda situa uma apresentação dual, polarizado no viés pragmático ou teórico, ao que se segue um procedimento temático padrão que privilegia o desenvolvimento operatório envolvendo contextos exatos, finitos e determinísticos. Em particular, essas características se acentuam gravemente no momento de introdução dos números irracionais no ensino básico, o que ocasiona uma abordagem restritiva. Para superar este quadro, Bruner (1987) fundamenta que não devemos adiar o ensino de assuntos essenciais com base na crença de que são difíceis demais, pois as ideias fundamentais de qualquer assunto podem ser ensinadas na escolaridade básica, porém demanda um trabalho para além dos aspectos técnicos, o que equivale a retomada de características ligadas à compreensão. Neste trabalho, tivemos por hipótese que os pares discreto/contínuo; exato/aproximado; finito/infinito, presentes na análise da evolução epistemológica dos números reais e descritos em Machado (2009), se constituem em pilares conceituais essenciais para fundamentar um panorama favorável a uma abordagem significativa do tema dos números irracionais, de modo a compor um amálgama entre os aspectos técnicos e semânticos. Em face da necessária reflexão, em nível educacional, em torno de tal tema, delimitamos inicialmente um contexto investigativo pautado em um estudo qualitativo orientado pela questão Como são abordados os números irracionais no ensino básico, considerando-se como fonte o livro didático de Matemática?, a fim de mapear a apresentação deste assunto no Ensino Fundamental II e no Ensino Médio. O fundamento metodológico se inspirou nos núcleos de significação, descritos em Aguiar&Ozella (2006), que buscou apreender os sentidos que constituem o conteúdo do discurso expresso nos textos dos livros didáticos. O percurso dos núcleos de significação confirmou que, nos livros didáticos analisados, a apresentação dos números irracionais ocorre de modo polarizado: alguns optam por um viés empírico e outros pela definição formal. Verificou-se que, após uma abordagem inicial, não ocorre intercâmbio destas opções, o que acarreta um rápido esgotamento das ferramentas para se desenvolver as temáticas, limitando a compreensão da complexidade dos números irracionais no ensino básico. A partir das hipóteses e da pesquisa empírica, nos propusemos a delinear as contribuições presentes no movimento dialético entre os pares discreto/contínuo, finito/infinito e exato/aproximado, cujas mútuas conexões permeiam um espaço de significações, um campo que possibilita organizar, tecer e ampliar a rede de significados, conforme Machado (1995), favorecendo um quadro de maior compreensão à apresentação dos números irracionais. O enfoque epistemológico realizado revelou uma multiplicidade de relações envolvendo os números irracionais e diversos assuntos do currículo de Matemática, não devidamente caracterizadas e exploradas no ensino básico, o que serviu de mote para a apresentação de algumas situações de ensino para ilustrar os aportes orientadores sugeridos. Acreditamos que o caminho epistemológico trilhado viabilizou uma abertura para ampliar o quadro de significados em relação a outros tópicos presentes na Matemática Elementar, considerando-se como suporte a potencialidade presente nos eixos discreto/contínuo; exato/aproximado; finito/infinito, assim como no par determinístico/aleatório. / Considering Brazilian mathematics school textbooks as a primary research source, the knowledge to be taught still has a dual presentation, polarized in a pragmatic or theoretical way, what follows a thematic procedure pattern that favors an operational development involving exact, finite and deterministic contexts. In particular, these characteristics are seriously accentuated by the time of irrational numbers introduction at basic education, which leads to a restrictive approach. To overcome this situation, Bruner (1987) states that we should not postpone teaching key issues based on the belief that they are too hard, because the fundamental ideas of any subject can be taught at basic education, but it demands a work that overcome technical aspects, considerations that are equivalent to the resumption with aspects related to understanding. In this work, we had by hypothesis that the tension inherent on discrete/continuous, exact/approximate, finite/infinite pairs, extracted from analyses on real numbers epistemological evolution and described at Machado (2009), constitutes an essential conceptual pillar to establish a helpful framework to enable a significant irrational numbers approach, in order to compose an amalgam between technical and semantic aspects. Considering the necessary educational discussion involving this theme, we initially delimited an investigative context based on a qualitative study guided by the question How irrational numbers are approached in basic education, considering mathematics textbook as a source?\' in order to map this subject presentation at Middle and High School. The methodological foundation was inspired in meaning core, described in Aguiar and Ozella (2006), which aims to capture the sense that constitutes the speech content expressed inside mathematics scholar textbooks. The analysis from meaning core route reveals that, in the textbooks examined, the most known irrational numbers introduction occurs in a polarized way: some opt for a pragmatic bias and others by formal definition. However, it was found that after an initial approach, there is no further relationship between these options, which causes a rapid depletion of the tools to develop these themes, which limits the complexity understanding of irrational numbers in basic education. From the hypotheses and the empirical research, we intended to delineate contributions presented on the dialectical movement between discrete/continuous, finite/infinite and exact/approximate pairs, whose mutual connections permeate a \'space of meanings\', a field that allows to organize, to weave and to expand a network of meanings, as Machado (1995), favoring a framework for better understanding the irrational numbers development in basic school. The epistemological approach performed revealed a multiplicity of relationships involving irrational numbers and various subjects of mathematics curriculum, not properly characterized and exploited in basic education, references which served as contexts for the presentation of some teaching situations to illustrate the contributions guidance suggested. We believe that the epistemological path trodden enables an opening to increase possibilities of meanings in relation to other topics of Elementary Mathematics, considering as support the capability constituents presented in discrete/continuous, exact/approximate, finite/infinity axis, as well as in deterministic/random pair.

Esta pesquisa se insere no campo da Psicologia Cultural do Desenvolvimento Humano e tem como objetivo investigar o compartilhamento da educação e do cuidado de crianças pequenas (1 a 5 anos) em um assentamento e um acampamento rural. Consideramos como compartilhamento da educação a participação de um grupo de pessoas, adultos ou crianças, nos momentos cotidianos da criança. A hipótese inicial da investigação estava relacionada a uma aparente coletivização da educação no acampamento e uma individualização no assentamento, determinados, sobretudo, pelas práticas culturais de cada contexto. Participaram da investigação 10 crianças (5 moradoras de um acampamento rural e 5 de um assentamento rural). A metodologia consistiu em observação participante com a imersão da pesquisadora por uma média de 6 dias no cotidiano familiar e comunitário de cada criança, com anotação em diário de campo das atividades, parceiros e cenários de compartilhamento do cuidado e da educação da criança. Os resultados apontam para: um intenso compartilhamento no grupo de vizinhança no acampamento e no grupo de família extensa no assentamento; a importância do cuidado e educação exercido entre as crianças, especialmente em situações de brincadeira; o histórico de migração como condição relacionada à maior busca pelo compartilhamento; a mulher como principal cuidadora, aspecto intimamente relacionado ao trabalho doméstico e na agricultura.O trabalho propicia reflexões acerca da diversidade dos modos de vida das crianças nestes contextos, sendo que há potencialidades educacionais ali construídas, fruto de uma riqueza cultural latente. Aponta ainda para considerações acerca das políticas públicas e garantia de direitos às crianças. / This research is within the field of Cultural Psychology of Human Development and aims to investigate the sharing of education and care of young children (1-5 years) in a settlement and in a rural campsite. We consider education as a share of the participation of a group of people, adults or children, in the child\'s everyday moments. The initial hypothesis of the research was related to an apparent collectivization of education in the camp and an individualization in the settlement, determined primarily by cultural practices of each context. Ten children participated in the research (5 residents of a rural camp and 5 of a rural settlement). The methodology consisted in an active observation with the immersion of the researcher by an average of 6 days in family and community daily life of every child, taking field notes in a diary of activities, partners and children\'s care and education sharing scenarios. The results show: an intense sharing in the neighborhood group in the camp and extended family group in the settlement; the importance of care and education carried out among children, particularly in play situations; the migration history as a related condition to the search for greater sharing; the woman as main caregiver, closely related to domestic work aspect and agriculture. The work provides reflections on the diversity of ways of life of children in these contexts, therefore there\'s educational possibilities being built, result from a latent cultural wealth. It also points to considerations about public policies and guarantee of rights to children.

Estudos anteriores de nosso laboratório demonstraram que a estimulação aguda dos receptores 2-adrenérgicos (2-AR) atenua a perda de massa muscular induzida pela desnervação motora (DEN) por meio de uma via dependente de AMPc/PKA. No entanto os mecanismos moleculares envolvidos na ativação crônica destes receptores ainda são pouco conhecidos. Por outro lado, a ativação desta via de sinalização também está envolvida no controle da estabilidade dos receptores nicotínicos (AChR) na junção neuromuscular (JNM), sugerindo que a densidade dos AChR possa estar sob controle neuro-humoral. Desta forma, aventou-se a possibilidade de que além dos efeitos protetores na massa muscular, a ativação dos receptores 2-AR pudesse mediar a estabilização dos AChR na placa motora. Para testar essa hipótese, camundongos foram submetidos à DEN através da secção do nervo ciático, um protocolo clássico de indução de atrofia muscular e desestabilização dos AChR, e tratados com salina ou clembuterol (CB), um 2-agonista seletivo, por até 14 dias. Após 3 dias de DEN, observou-se redução da massa muscular e aumento do conteúdo proteico e expressão do RNAm de genes relacionados à ativação do sistema Ubiquitina-Proteassoma (atrogina-1 e MuRF1) e do sistema autofágico/lisossomal (catepsina L e LC3). A DEN também promoveu aumento no turnover dos AChR, no número de vesículas endocíticas e na expressão do RNAm para a subunidade 1 dos AChR. Após 7 dias, a DEN reduziu a expressão dos genes relacionados à atrofia e aumentou a atividade da via do AMPc/PKA independentemente do tratamento com CB. Na tentativa de elucidar os sinais extracelulares que produziam esta resposta adaptativa, foi demonstrado que neurônios catecolaminérgicos trafegam ao longo do nervo ciático e sua ablação pela DEN reduziu o conteúdo de noradrenalina muscular. Baseados nestes resultados, foi postulado a existência de uma hipersenbilidade às catecolaminas em músculos desnervados cronicamente. O tratamento com CB por 3 dias aboliu o aumento da expressão dos atrogenes induzido pela DEN e este efeito foi associado ao maior conteúdo de AMPc e de substratos fosforilados pela PKA. Além disso, o CB diminuiu a hiperexpressão do RNAm para catepsina L e LC3 induzida pela DEN de 7 dias. Embora o CB não tenha alterado a meia-vida dos AChR em músculos inervados e desnervados, houve um total bloqueio do aumento do número de vesículas endocíticas contendo o AChR em músculos desnervados e tratados com CB. Corroborando estes dados, o CB aumentou a incorporação de AChR novos nas JNM e este efeito foi também associado à maior expressão do RNAm para a subunidade 1-AChR em músculos desnervados. Esta ação do CB no turnover dos AChR parece ser direta uma vez que neuroniôs catecolaminérgicos presentes no nervo ciático ativam receptores 2-ARe a produção de AMPc especificamente na JNM. Em estudos in vitro, foi demonstrado que a estimulação colinérgica produzida pelo carbacol (10-4M) diminuiu a velocidade de síntese de proteínas, aumentou a proteólise total e a atividade do sistema proteolítico Ca2+-dependente em músculos soleus de ratos por meio da ativação dos receptores nicotínicos. Este efeito catabólico do carbacol foi completamente bloqueado pela adição de CB (10-4M) ao meio de incubação. Os dados obtidos no presente estudo permitem sugerir que a estimulação crônica dos 2-AR no músculo esquelético induz um efeito anti-catabólico pela supressão dos sistemas proteolíticos proteassomal e lisossomal, provavelmente através da via de sinalização do AMPc/PKA. A inibição destes sistemas pode estar relacionada ao aumento do turnover dos AChR, uma vez que a velocidade de incorporação destes receptores na JNM foi aumentada pelo CB. Além disso, os achados que mostram a associação entre neurônios noradrenérgicos e colinérgicos no nervo ciático, que conjuntamente inervam as JNM, e a co-localização de receptores 2-AR e AChR na sinapse permitem sugerir a existência de uma interação funcional entre o sistema colinérgico e adrenérgico na manutenção da massa muscular e da placa motora. / Previous studies from our laboratory have shown that the acute stimulation of 2-adrenergic receptor (2-AR) attenuates the muscle loss induced by motor denervation (DEN) through a cAMP/PKA dependent pathway. However, the molecular mechanisms involved in the chronic activation of these receptors are poorly understood. Furthermore, the activation of this signaling pathway is also involved in controlling the stability of nicotinic receptors (AChR) at the neuromuscular junction (NMJ), suggesting that the density of AChR may be under neurohumoral control. Thus, we postulated that besides the protective effects on muscle mass the activation of 2-AR receptors could mediate the stabilization of AChR in the motor plate. To test this hypothesis, mice were submitted to DEN through of the sciatic nerve section, a classical protocol of induction muscle atrophy and destabilization of AChR, and were treated with saline or clenbuterol (CB), a selective 2-agonist for 14 days. DEN decreased the muscle mass and increased the protein content and mRNA expression of genes related to the activation of the ubiquitin-proteasome system (atrogin-1 and MuRF1) and autophagic/lysosomal system (cathepsin L and LC3). DEN also promoted an increase in the turnover of AChR, number of endocytic vesicles and the expression of mRNA for the 1 subunit of AChR. Interestingly, chronic DEN induced down-regulation of atrophy related-genes, and increased the activity of cAMP/PKA pathway independently of CB treatment. In an attempt to elucidate the extracellular signals that produced this adaptive response, it was demonstrated that catecholaminergic neurons travels along the sciatic nerve and its ablation by DEN reduces muscle norepinephrine content. Based on these results, it was postulated the existence of a muscle adrenergic hypersensitivity to circulating catecholamines induced by chronic DEN. CB treatment for 3 days completely abolished the higher expression of atrogenes and this effect was associated with increased Camp content and PKA phosphorylated substrates. Furthermore, CB decreased the DEN-induced hyperexpression of cathepsin L and LC3 mRNA at 7 days. Although CB has not altered the half-life of AChR in innervated and denervated muscles, it produced a total blockage of the increased number of endocytic vesicles containing the AChR in denervated muscles. Consistently, CB increased the incorporation of new AChR and this effect was associated with an increased expression of the 1-subunit AChR mRNA in denervated muscles. This action of CB on AChR turnover appears to be direct, since catecholaminergic neurons are present in the sciatic nerve stimulating 2-AR and cAMP production specifically in the NMJ. Furthermore, in vitro studies demonstrated that cholinergic stimulation produced by carbachol (10-4M) decreased the rate of protein synthesis and increased the proteolytic activity of Ca2+-dependent system in rat soleus muscle through activation of nicotinic receptors. This catabolic effect of carbachol was completely blocked by the addition of CB (10-4M) to the incubation medium. These data suggest that chronic stimulation of 2-AR in skeletal muscle induces an anti-catabolic effect by suppressing proteasomal and lysosomal proteolytic systems, probably through the cAMP/PKA signaling. The inhibition of these systems seems to be related to the increased AChR incorporation into NMJ induced by CB treatment. Moreover, the association between noradrenergic and cholinergic neurons in the sciatic nerve, both of which innervate the motor endplates, and the co-localization of AChR and 2-ARat the synapse suggest the existence of a functional interaction between cholinergic and adrenergic systems in the maintenance of muscle mass and motor endplate.

L’objectif de cette thèse était de caractériser le rôle des différentes familles de phosphodiestérases (PDEs), les enzymes de dégradation du 3'-5'- adénosine monophosphate cyclique (AMPc), dans la régulation de la signalisation de l’AMPc dans deux types de cellules musculaires lisses (CMLs), l’aorte de rat (CMLAR) et la vessie de rat néonatal (CMLVRN). Dans les CMLARs en culture, nous avons déterminé le profil d’expression et d’activité des PDE-AMPc. Nous avons alors montré, à l’aide de la technique de FRET basée sur une sonde sensible à l’AMPc pour mesurer l’AMPc en temps réel dans une cellule isolée, que l’inhibition de la PDE4 démasque un effet d’hydrolyse de l’AMPc cytosolique par la PDE1 et la PDE3, alors que les PDE3 et PDE4 agissent de façon synergistique dans le compartiment sous-membranaire. Les mécanismes de cette compartimentation subcellulaire des signaux restent à caractériser.Dans les CMLVRNs, les PDE3 et PDE4 régulent les contractions phasiques, par des mécanismes différents. L’inhibition de la PDE4 limite les contractions stimulées par le carbachol par un mécanisme dépendant de la protéine kinase A, impliquant une augmentation de la fréquence des sparks calciques, qui entrainent l’activation des canaux potassiques BK, assurant en final une diminution des transitoires calciques. Au contraire, l’effet de l’inhibition de la PDE3 implique la protéine kinase G mais par un mécanisme qui reste à définir.En conclusion, ce travail montre que dans les CMLs, les différents familles de PDE-AMPc sont douées de spécificité de fonction et/ou de mécanisme d’action, et participent ainsi à une compartimentation subcellulaire des voies de signalisation. / The aim of the present thesis was to characterize the role of the different families of phosphodiesterases (PDEs), the enzymes degrading 3'-5'-cyclic adenosine monophosphate (cAMP), in controlling the cAMP signalling in two distinct smooth muscle cells (SMCs), the rat aorta SMC (RASMCs) and the rat bladder SMC (RBSMCs).In cultured RASMCs, we firstly characterized the pattern of cAMP-PDE expression and activity. We then showed, by using a FRET-based cAMP sensor to explore cAMP signals in living cells, that PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, whereas PDE3 and PDE4 act synergistically at the submembrane compartment. The mechanisms of this subcellular compartmentation need to be characterized. In neonatal RBSMCs, we showed that both PDE3 and PDE4 are involved in regulating the phasic contractions albeit through distinct mechanisms. PDE4 inhibition inhibits the carbachol-enhanced contractions through a protein kinase A-dependent pathway involving an increase in Ca2+ sparks frequency which activates BK channels to ultimately decrease Ca2+ transients, whereas PDE3 inhibition acts through a protein kinase G-dependent pathway through a still unknown mechanism.In conclusion, our work shows that in the SMC, the different cAMP-PDE families exhibit a specificity in their function and/or mechanism of action, thus participating to a subcellular signaling compartmentation.

L'AMP cyclique (AMPc) et le GMP cyclique (GMPc) sont des seconds messagers essentiels pour la régulation de la fonction cardiaque. Leurs niveaux sont régulés par l’adénylate cyclase et la guanylate cyclase, respectivement, et par les phosphodiestérases (PDEs). Cependant, une telle régulation est altérée dans l'insuffisance cardiaque (IC). En effet, la diminution de la signalisation de l’AMPc et l’augmentation de celle du GMPc est caractéristique des cœurs défaillants.Parmi la superfamille des PDEs, la PDE2 a la particularité d'être stimulée par le GMPc, conduisant ainsi à une augmentation remarquable de l'hydrolyse de l'AMPc. Ceci semble induire une interaction entre les voies de signalisation de l’AMPc et du GMPc. Cependant, le rôle de la PDE2 dans le cœur défaillant est très peu connu.Dans ce contexte, nous avons examiné si la PDE2 cardiaque est modifiée dans l’IC chez l’Homme et chez les modèles animaux d’IC, et déterminé le rôle de la PDE2 dans la signalisation β-adrénergique dans les cardiomyocytes. Grâce à l’utilisation de Western blot, de technique radioenzymatique, d’imagerie basée sur le FRET, de la planimétrie, de la microscopie à épifluorescence et des mesures du courant calcique de type L, réalisés sur les tissus myocardiques humains et/ou dans des cardiomyocytes isolés de cœurs des modèles animaux d’IC, respectivement, nous avons montré que l’expression et l’activité de la PDE2 sont augmentées dans les cœurs défaillants. Cette augmentation réduit l’effet d’une stimulation β-adrénergique aiguë, contribuant à la désensibilisation β-adrénergique observée dans l’IC. En accord avec ces résultats, la surexpression de la PDE2 dans des cardiomyocytes sains, réduit l’augmentation des taux d'AMPc et l’amplitude du courant ICa,L et abolit l'effet inotrope positif suite à une stimulation β-adrénergique aiguë, sans affecter la contractilité basale. Plus important, les cardiomyocytes surexprimant la PDE2, montrent une protection contre les réponses hypertrophiques induites par la noradrénaline et contre les arythmies induites par l'isoprotérénol.En conclusion, ce travail met en évidence l'altération de la PDE2 dans l’IC et nous laisse suggérer que l’augmentation de la PDE2 dans l’IC peut constituer un mécanisme de défense important dans des conditions de stress cardiaque, notamment en antagonisant la suractivation de la voie β-adrénergique. Ainsi, l'activation de PDE2 myocardique peut représenter une nouvelle stratégie thérapeutique anti-adrénergique intracellulaire dans l’IC. / Cyclic AMP (cAMP) and cyclic GMP (cGMP) are critical second messengers for the regulation of cardiac function. Their levels are regulated by adenylyl and guanylyl cyclases, respectively, and by cyclic nucleotides phosphodiesterases (PDEs). However, such regulation is altered in heart failure (HF). Indeed diminished cAMP- and augmented cGMP-signaling is characteristic of failing hearts.Among the PDE superfamily, PDE2 has the unique property to be stimulated by cGMP, thus leading to a remarkable increase in cAMP hydrolysis. This appears to mediate a negative cross-talk between cAMP- and cGMP signaling pathways. However, the role of PDE2 in the failing heart is only poorly understood.In this context, we investigated whether myocardial PDE2 is altered in human and experimental HF and determined PDE2 mediated effects on β-adrenoceptor (β-AR) signaling in cardiomyocytes. Using immunoblotting, radioenzymatic- and FRET-based assays, video-edge-detection, epifluorescent microscopy and L-type Ca2+ current measurements, performed in myocardial tissues and/or isolated cardiomyocytes from human and/or experimental HF, respectively, we showed that PDE2 is markedly upregulated in failing hearts. This reduces the effect of an acute β-adrenergic stimulation, and contributes to the β-adrenergic desensitization which is a characteristic feature in HF. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and ICa,L amplitude and abolished the inotropic effect following acute β-AR stimulation, without affecting basal contractility. Importantly, PDE2-overexpressing cardiomyocytes showed marked protection from norepinephrine-induced hypertrophic responses and from isoproterenol-induced arrhythmias.In conclusion, this work highlights the alteration of PDE2 in HF and lets us assume that PDE2 upregulation in HF may constitute an important defence mechanism during cardiac stress, e.g. by antagonizing excessive β-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular anti-adrenergic therapeutic strategy in HF.

Esta pesquisa se insere no campo da Psicologia Cultural do Desenvolvimento Humano e tem como objetivo investigar o compartilhamento da educação e do cuidado de crianças pequenas (1 a 5 anos) em um assentamento e um acampamento rural. Consideramos como compartilhamento da educação a participação de um grupo de pessoas, adultos ou crianças, nos momentos cotidianos da criança. A hipótese inicial da investigação estava relacionada a uma aparente coletivização da educação no acampamento e uma individualização no assentamento, determinados, sobretudo, pelas práticas culturais de cada contexto. Participaram da investigação 10 crianças (5 moradoras de um acampamento rural e 5 de um assentamento rural). A metodologia consistiu em observação participante com a imersão da pesquisadora por uma média de 6 dias no cotidiano familiar e comunitário de cada criança, com anotação em diário de campo das atividades, parceiros e cenários de compartilhamento do cuidado e da educação da criança. Os resultados apontam para: um intenso compartilhamento no grupo de vizinhança no acampamento e no grupo de família extensa no assentamento; a importância do cuidado e educação exercido entre as crianças, especialmente em situações de brincadeira; o histórico de migração como condição relacionada à maior busca pelo compartilhamento; a mulher como principal cuidadora, aspecto intimamente relacionado ao trabalho doméstico e na agricultura.O trabalho propicia reflexões acerca da diversidade dos modos de vida das crianças nestes contextos, sendo que há potencialidades educacionais ali construídas, fruto de uma riqueza cultural latente. Aponta ainda para considerações acerca das políticas públicas e garantia de direitos às crianças. / This research is within the field of Cultural Psychology of Human Development and aims to investigate the sharing of education and care of young children (1-5 years) in a settlement and in a rural campsite. We consider education as a share of the participation of a group of people, adults or children, in the child\'s everyday moments. The initial hypothesis of the research was related to an apparent collectivization of education in the camp and an individualization in the settlement, determined primarily by cultural practices of each context. Ten children participated in the research (5 residents of a rural camp and 5 of a rural settlement). The methodology consisted in an active observation with the immersion of the researcher by an average of 6 days in family and community daily life of every child, taking field notes in a diary of activities, partners and children\'s care and education sharing scenarios. The results show: an intense sharing in the neighborhood group in the camp and extended family group in the settlement; the importance of care and education carried out among children, particularly in play situations; the migration history as a related condition to the search for greater sharing; the woman as main caregiver, closely related to domestic work aspect and agriculture. The work provides reflections on the diversity of ways of life of children in these contexts, therefore there\'s educational possibilities being built, result from a latent cultural wealth. It also points to considerations about public policies and guarantee of rights to children.

La mouche Drosophila melanogaster est un modèle de choix dans l’étude des grandes fonctions neurophysiologiques notamment en raison de la disponibilité d’une importante variété d’outils disponibles (approches génétiques, pharmacologiques et comportementales). Le cerveau de la mouche, malgré sa simplicité apparente, est capable de traiter des fonctions complexes d’intégration des différents paramètres environnementaux nécessaires à sa survie. Dans le cerveau drosophile, les corps pédonculés (CP) sont des structures impliquées dans de nombreuses fonctions neurophysiologiques de premier plan telles que l'apprentissage et la mémoire olfactive, la régulation de l’activité locomotrice, l'orientation spatiale, la régulation du sommeil ou encore la prise de décision. Il a été montré par des approches associant essentiellement observations comportementales et outils génétiques que la voie de signalisation de l'AMPc joue un rôle crucial dans la réalisation des fonctions diverses des CP. Les cellules de Kenyon (CK) qui sont les cellules intrinsèques des CP, reçoivent principalement des afférences du système olfactif par l’intermédiaire des neurones de projections (PN) en provenance des lobes antennaires et des afférences neuromodulatrices (dopaminergiques et octopaminergiques). Les synapses entres PN et CK se font sur un mode cholinergique grâce à des récepteurs canaux à l’acétylcholine de type nicotinique (nAchR). Nous avons utilisé une technique récente d’imagerie calcique par bioluminescence utilisant une protéine recombinante, la GFP-Aequorine. Cette technique nous a permis de suivre l’activité cellulaire calcique consécutive à l’application de nicotine, un agoniste des nAchR. Grâce à l’observation de ces réponses suite à une combinaison d’approches génétiques corroborée par des approches pharmacologiques, nous avons pu mettre en évidence une modulation complexe et régionalisée de la réponse calcique dans les CP par l’AMPc et d’autres différents partenaires tels que des canaux K+ et Ca2+. Dans un premier temps, nous avons démonté l’existence d’une modulation directe de l’intensité de la réponse par l’AMPc. Nous avons également montré, pour la première fois, que des réponses Ca2+ « spontanées » peuvent être directement inductibles par augmentation de l’AMPc. Nous avons mis en évidence l’existence d’un nouveau partenaire de la modulation de la réponse des CP indépendant de la PKA : les CNG (Cyclic Nucleotides Gated Channels) dont le rôle n’avait jusqu’ici jamais été démontré dans les corps pédonculés. Enfin nous avons pu observer une régionalisation de la régulation de l’activité Ca2+ des CP par l’AMPc. Dans un deuxième temps nous nous somme intéressé aux principales conductances calciques et potassiques. Nous avons mis en évidence que différents canaux calciques voltages dépendants (VGCC) sont impliqués de façon régionalisée et séquentielle dans la formation de la réponse calcique. Il a pu également être démontré que le signal est modulé de façon différentielle dans les calices et les lobes par l’AMPc à travers différents canaux potassiques. Enfin des protocoles originaux ont été développés, tels que la micro application de drogue ou l’électrostimulation permettant d’étudier la neuromodulation dans les CP, à réutiliser pour des travaux ultérieurs du laboratoire. Ce travail est une première étape dans la compréhension des voies de signalisations et des mécanismes intracellulaires impliqués dans l’apprentissage et la mémoire olfactive. / In Drosophila, the Mushroom-Bodies (MBs) are implicated in multiple functions, as olfactory learning and memory, locomotor activity, spatial orientation, sleep, decision making, and up to now but indirectly, in various addiction. Notably, the MBs, which express the nAchR, receive their main inputs from the cholinergic olfactory pathways, through the Projections Neurons (PNs). In this thesis we characterized, at the cellular and molecular levels, the nicotine effect on the Kenyon cells (KCs: the intrinsic neurons) of the Mushroom-Bodies. We used the in-Vivo brain imaging approach, based on the Ca2+-Sensitive bioluminescent probe (GFP-Aequorin), to characterize the nicotinic induced Ca2+-Response on the KCs of the MBs. More specifically we searched the role of different partners involved in the cAMP pathway, in order to understand their roles in the different components of the response and in its modulation. First using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca2+-Response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca2+-Response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has a PKA-Independent effect through the cyclic nucleotide-Gated Ca2+-Channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects between the calyx/cell-Bodies and the lobes, suggesting a differential and regionalized regulation of ACSecond we exploited both genetic approaches to interfere with different types of Ca2+- and K+-Channels, first we show that the disruption of the VGCC, as cacophony, Dmcα1d and Dmcα1g lead to a striking decrease of the Ca2+-Response both in the CCB and the lobes. Moreover, for two of them, cacophony and Dmcα1d, the duration of the response is importantly increased. Second, the disruption of the fast inactivating K+-Currents, as shaker (sh), shaker-Like (shal) and slowpoke (slo) reveal that the knocked-Down of shal and slo lead to a striking decrease of the Ca2+-Response, while the knocked-Down of sh has only a mild effect. Interestingly, the stimulation of the adenylate cyclase (AC) by the forskolin with the various K+-Channels disruption show an antagonist effect of the cAMP in the CCB between sh (inhibitory) and slow (excitatory) while AC simulation mediate excitatory effects in the ML though both shal and sh. Finally, the knock-Down of the two slow inactivating K+-Currents as shaker w (shaw) and shaker b (shab) also yields to a strong decrease of the Ca2+-Response In conclusion, our results provide new insights into the complexity of the Ca2+-Response in the MBs and are a first step toward deciphering the roles of the VGCC and K+-Channels in the multiples roles of the MBs. Finaly we developed several original protocols to explore the role of the neuromodulation on the KC.This work constitutes an important step toward a better understanding of the pathway required in learning and memory.