Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Durante a anidrobiose (um estado ametabólico muito estável), o nematóide Panagrolaimus superbus tolera vários tipos de estresses físicos. A fim de melhor compreender essa extremotolerância, P. superbus foi submetido a regimes de hiperaceleração (RHA) de até 400.000 x g. Surpreendentemente, foi observado que esse verme tolera a exposição à RHA tanto dessecado (i.e., em anidrobiose) como hidratado. Para verificar se esse fenômeno era específico para essa espécie ou algo observável em outros organismos, os mesmos procedimentos experimentais foram realizados no organismo modelo Caenorhabditis elegans. Intrigantemente, C. elegans também mostrou o mesmo perfil de sobrevivência. Ademais, o desenvolvimento, comportamento, morfologia e crescimento populacional desse nematóide também foram analisados após a exposição ao RHA, não sendo observada quaisquer mudanças nesses parâmetros em função da exposição à hipergravidade. Em seguida, foram realizadas buscas (tBLASTn) no genoma de C. elegans por homólogos de genes relacionados ao gravitropismo que são naturalmente encontrados em plantas. Essa busca resultou nos genes dnj- 16 (homólogo ao ARG1), ipla-1 (homólogo ao SGR2) e uma sequência não caracterizada (homóloga a TWD1). Especial atenção foi despendida ao gene dnj-16, uma vez que é o mais conservado entre eles. As análises de RT-qPCR revelaram que o dnj-16 é ligeiramente regulado para baixo durante o RHA, o que não era esperado caso ele possuísse função semelhante ao seu homólogo em plantas. A análise do estado metabólico desse nematoide durante o RHA lançou luz sobre os dados de RT-qPCR, mostrando que a queda na expressão de dnj-16 é provavelmente devida à centrifugação. Posteriormente, diversas análises in silico foram realizadas a fim de caracterizar o gene dnj-16 e a sua respectiva proteína. Inicialmente, a análise comparativa dos domínios DnaJ, transmembrana e coiled coil das proteínas dnj-16, ARG1, ARL1 e ARL2 apontou para uma grande semelhança não apenas na sequência como na estrutura dessas proteínas. Essa grande similaridade motivou análises para desvendar o papel e a origem do gene dnj-16. Três hipóteses ((i) homologia, (ii) convergência e (iii) transferência gênica horizontal (TGH)) foram consideradas na investigação desse intrigante gene. Os resultados obtidos nas análises in silico apontaram para uma TGH mediada por RNA, potencialmente ocorrida a 1325 m.a., como a hipótese mais plausível para explicar a origem de dnj-16 e algumas espécies parasitas do gênero Phytophthora como prováveis mediadores dessa transferência. Dessa forma, os dados apresentados nessa tese mostram pela primeira vez que C. elegans é tolerante a RHA ordens de magnitude mais altas do que se pensava serem compatíveis com a vida multicelular. Além disso, os dados sugerem que dnj-16 foi transferido horizontalmente de plantas para nematoides e que a ultracentrifugação leva a uma redução no metabolismo de C. elegans, o que ajudaria a explicar a sua sobrevivência sob tal condição extrema. Por fim, o conjunto de dados desse trabalho representa contribuições originais para a compreenção da biologia, da genética e da evolução de C. elegans. / During anhydrobiosis (a very stable ametabolic state), the nematode Panagrolaimus superbus tolerates many types of physical stresses. In order to better understand this extremotolerance, P. superbus underwent hyperacceleration regimes (RHA) of up to 400,000 x g. Surprisingly, it was observed that this worm tolerated RHA exposure both dried (i.e., during anhydrobiosis) and hydrated. In order to verify if this phenomenon was specific for this species or something observable in other organisms, the same experimental procedures were performed in the model organism Caenorhabditis elegans. Intriguingly, C. elegans also showed the same survival profile. In addition, the development, behavior, morphology and population growth of this nematode were also analyzed after the exposure to RHA and no changes were observed in these parameters due to the hypergravity exposure. Thereafter, searches (tBLASTn) were performed on C. elegans genome by homologs of gravitropism related genes that are naturally found in plants. These searches resulted in the genes dnj-16 (homologous to ARG1), ipla-1 (homologous to SGR2) and an uncharacterized sequence (homologous to TWD1). Special attention was given to dnj-16 gene, since it is the most conserved among them. RT-qPCR analyzes revealed that dnj-16 is slightly down regulated during RHA, which was not expected if it had similar function to its homologue in plants. Analysis of the metabolic status of this nematode during RHA shed light on the RT-qPCR data, showing that the decrease in dnj-16 expression was probably due to centrifugation. Subsequently, several in silico analyzes were performed in order to characterize the dnj-16 gene and its respective protein. Initially, the comparative analyzis of the DnaJ, transmembrane and coiled coil domains of dnj-16, ARG1, ARL1 and ARL2 proteins pointed to a great similarity not only in the sequence as well as in the structure of these proteins. This great similarity motivated analyzes in order to uncover the real nature of the dnj-16 gene. Three hypotheses ((i) homology, (ii) convergence and (iii) horizontal gene transfer (HGT)) were considered in the investigation of this intriguing gene. The results obtained in the in silico analyzes indicated an RNA mediated TGH, potentially occurred 1325 my (millions of years), as the most plausible hypothesis to explain the origin of dnj-16 and some parasitic species of the Phytophthora genus as probable mediators of this transference. Therefore, data presented in this thesis show for the first time that C. elegans tolerates RHA of magnitude orders higher than it was thought to be compatible with multicellular life. In addition, the data suggest that dnj-16 was transferred horizontally from plants to nematodes and that ultracentrifugation leads to a reduction in C. elegans metabolism, which would help explain its survival under such extreme condition. Finally, the data set of this work represent original contributions to the understanding of the C. elegans\' biology, genetics and evolution.

Le système nerveux autonome (SNA) contribue de façon importante aux régulations des fonctions cardiovasculaires. Pendant des décennies, les chercheurs ont essayé de comprendre comment la variabilité de la fréquence cardiaque (VFC) et le gain du baroréflexe cardiaque pouvaient être utilisés comme marqueurs significatifs du contrôle neurovégétatif cardiaque, et parfois de son altération. L'objectif général de cette thèse est de mieux comprendre le rôle du SNA dans la modulation et les adaptations des fonctions cardiaques et vasculaires. Le projet comprenait quatre études.Dans la première étude, nous avons analysé les effets de l'accélération gravitationnelle graduées sur la régulation neurovégétative de la fréquence cardiaque et de la vasomotricité artériolaire. Dans ces expositions expérimentales des variables cardiovasculaires et respiratoires ont été modifiées de façon spécifique. Nos résultats ne sont pas compatibles avec la mise en jeu d’une régulation sympathique au niveau cardiaque en situation d’hypergravité brève. Nous avons supposé que seule la branche sympathique du SNA était active durant une exposition à une accélération de gravité élevée. La réponse adaptative de la vasomotricité artérielle vasculaire est observée en condition de grande décharge des barorécepteurs. Notre deuxième travail eu pour objet l'effet de la dénervation pulmonaire sur la VFC, et a donc été conduit chez des patients ayant subi une greffe pulmonaire complète. Le greffon n'étant plus relié au SNA, il s'agit d'un excellent modèle expérimental pour l'étude de la régulation cardiovasculaire en l’absence de modulation de l'activité cardiaque par des afférences nerveuses pulmonaires parasympathiques et/ou sympathiques. Puisque la VFC dans les hautes fréquences est reconnue comme largement déterminée par le profil ventilatoire, on s’attend à ce que la composante à haute fréquence de la VFC soit absente chez les sujets transplantés bi-pulmonaires. Les résultats montrent que cette dénervation pulmonaire implique une forte réduction de la VFC totale et dans les deux bandes de fréquence étudiées, hautes et basses. Cela indique donc qu’une large contribution de la modulation nerveuse de la VFC répond aux afférences pulmonaires. La sensibilité du baroréflexe est réduite. Le rapport plus élevé entre les basses et les hautes fréquences traduit une réduction de puissance totale principalement due à la diminution de la composante haute fréquence. Ces résultats montrent que les afférences pulmonaires contribuent largement à la à la modulation neurovégétative de la composante à hautes fréquences de la VFC. La variabilité de la pression artérielle est beaucoup moins modifiée que celle de la VFC par la transplantation bipulmonaire, ce qui met en évidence que les afférences pulmonaires contribuent spécifiquement à la modulation de la VFC. Cette observation est un argument fort pour reconnaître des voies de régulation différentes pour les variabilités de fréquence cardiaque d’une part et de pression artérielle d’autre part. Le troisième article traite des effets sur la modulation cardiovasculaire de blocages pharmacologiques du SNA sur les régulations de fréquence cardiaque et de vasomotricité périphérique, au repos et pendant l'exercice. / Autonomic nervous system (ANS) and cardiovascular regulation are closely linked. For decades, researches have tried to understand how heart rate variability (HRV) and baroreflexes can be used as significant markers of the autonomic nervous control, and sometimes of its impairments. The general aim of this thesis is to gain further insights into the role of ANS in modulating cardiac and vascular functions. The project consisted of four studies.In the first study, we analysed the effects of gravitational acceleration on cardiovascular autonomic control. This special condition showed interesting results for cardiorespiratory variables. Our results did not agree with the notion of sympathetic up-regulation in hypergravity. We speculated that only the sympathetic branch of the ANS might have been active during elevated gravitational acceleration exposure. Furthermore, the vascular response occurred in a condition of massive baroreceptor unloading.Our second work targeted the effect of lung denervation on HRV in bilateral lung transplant recipients. As the graft is no longer connected to the ANS, this is an excellent experimental model for the study of cardiovascular regulation without modulation of heart activity by parasympathetic and/or sympathetic lung afferents. The hypothesis was that the modulation of the high frequency component of HRV by the breathing frequency is mediated by the ANS. This hypothesis would be supported by the results if the high frequency component of HRV is suppressed in bilateral lung transplant recipients. Lung denervation implied strong HRV reduction, all indices being decreased, indicating that neural modulation from lung afferents contributes largely to HRV. Baroreflex sensitivity was reduced. The higher low-versus-high frequency ratio implied that the total power drop was mostly due to the high frequency component, indicating that neural modulation from lung afferents largely contributes to the high frequency component of HRV. The changes in blood pressure variability were smaller than those in HRV, suggesting that the effects of lung denervation were specific to HRV modulation. This finding confirms that blood pressure variability and HRV are under different control mechanisms.The third article concerns the effects of autonomic blockades on cardiovascular modulation, at rest and during exercise. We hypothesized that HRV should decrease with vagal or sympathetic blockades, and disappear during simultaneous blockade of both ANS branches. The results suggest that the parasympathetic outflow to the heart is the main determinant of HRV, while the role of the sympathetic branch is less important. Indeed, sympathetic blockades failed in changing HRV indices at rest, indicating that a selective blockade of cardiac ß-adrenergic receptors has no effects on spontaneous heart rate oscillations. These effects are specific to HRV, as the effects observed on blood pressure variability are indirectly related to the action of the administered drugs. The changes in baroreflex sensitivity were consistent with the changes in arterial blood pressure variability, suggesting that baroreflexes may modulate the LF power of arterial blood pressure.

Les réseaux vasculaires cérébraux adaptent leur activité à la demande métabolique des neurones environnants, mais leur contribution fonctionnelle à la consolidation de la mémoire, processus par lequel les traces mnésiques se stabilisent dans le temps, reste inconnue. A l’aide d’un test de mémoire olfactif associatif couplé à des approches biochimiques et d’imagerie cérébrale chez le rat, nous avons étudié la dynamique des changements vasculaires au cours de la consolidation mnésique qui nécessite une interaction transitoire entre l'hippocampe et les régions corticales constituant les sites dépositaires des souvenirs. Nous montrons que la formation d’une mémoire durable est associée, dès l’encodage, à un signal hypoxique qui déclenche une angiogenèse transitoire dans des régions corticales spécifiques impliquées plus tard dans le stockage des souvenirs. Manipuler cette angiogenèse corticale précoce (ACP) par blocage ou stimulation spécifique de la voie de signalisation de l'angiopoïétine-2 perturbe, ou améliore, le rappel des informations anciennement acquises. Stimuler l’ACP chez un modèle de rats hypertendus présentant des déficits d’activation de la voie de l’angiopoïetine-2 et de formation de la mémoire pallie le déficit mnésique observé, confirmant l'importance fonctionnelle de l’ACP comme un prérequis à la formation des souvenirs. L'hypergravité, connue pour altérer les fonctions vasculaires, n’a pas modifié l'organisation de la mémoire. Nos résultats identifient l’ACP comme un processus neurobiologique crucial sous-tendant la formation et la stabilisation des souvenirs. Ils révèlent l'importance de la plasticité vasculaire dans la modulation des fonctions cognitives et suggèrent que les changements structurels précoces du réseau vasculaire cérébral constituent un mécanisme permissif pour la régulation de la plasticité neuronale au sein des réseaux corticaux impliqués dans la formation progressive et le stockage des souvenirs. / While the cerebral microvasculature is known to adapt its activity according to the metabolic demand of surrounding neurons, the functional contribution of vascular networks to memory consolidation, the process by which memory traces acquire stability over time, remains elusive. By using an associative olfactory memory task in rats coupled to biochemical and imaging techniques, we investigated the dynamics of vascular changes during memory consolidation which requires a transitory interaction between the hippocampus and distributed cortical regions that ultimately support storage of enduring memories. We found that remote memory formation was associated, upon encoding, with a hypoxic signal that triggered transitory angiogenesis in specific cortical regions which support memory storage and retrieval only weeks later. Manipulating early cortical angiogenesis (ECA) by selectively blocking or stimulating the angiopoietin-2 signaling pathway impaired or improved remote memory retrieval, respectively. Enhancing ECA in spontaneously hypertensive rats, which exhibit reduced angiopoietin- 2 expression when cognitively challenged and are unable to properly stabilize and/or retrieve remotely acquired information, was efficient in rescuing the observed memory deficit, thus confirming the functional importance of ECA as a prerequisite for the formation of remote memories. Hypergravity, known to impair vascular functions, failed to alter the organization of recent and remote memory. Altogether, our findings identify ECA as a crucial neurobiological process underlying the formation and stabilization of remote memory. They highlight the importance of vascular plasticity in modulating cognitive functions and suggest that the early structural changes within vascular networks constitute a permissive mechanism for the regulation of neuronal plasticity within cortical networks which support the formation and storage of enduring memories.

Le présent travail de Doctorat porte sur l’adaptation et le déconditionnement cardiovasculaire chez l’astronaute en microgravité réelle prolongée, en microgravité simulée de courte durée (avec et sans contremesures par hypergravité), et en confinement de longue durée. Afin de répondre à cette thématique, 3 études expérimentales sur l’humain ont été réalisées, et de fait, ce présent travail de Doctorat se divise en 3 parties distinctes. La première est la mission « Mars 500 » qui comprend un confinement de 520 jours de 6 sujets-volontaires. La seconde est le projet « Vessel Imaging » qui comprend un vol spatial respectif de 6 mois à bord de la « Station Spatiale Internationale (ISS) » de 10 sujets-astronautes. La troisième est l’étude «Short Time Bed-Rest (STBR)» (12 sujets) qui comprend une courte période de microgravité simulée par alitement prolongé à -6° (5 jours) avec et sans l’utilisation de deux contremesures cardiovasculaires par hypergravité (continue ou intermittente). / This PhD work focuses on astronaut cardiovascular adaptation and deconditioning in real prolonged microgravity, short simulated microgravity (with and without countermeasures) and long-term confinement. To answer to this topic 3 humans experimental studies have been performed, and thus the present PhD work is divided into 3 distinct parts . The first one is the mission « Mars 500 » which consists in 520-days confinement with 6 subjects-volunteers mission. The second is the project « Vessel Imaging » whitch consit in a 6-months spaceflight aboard the « International Space Station » with 10 subjects-astronauts. The third is the « Short Time Bed -Rest (STBR) » study (12 subjects) which consist in a short period of bedrest (-6°, 5 days) with and without the use of two cardiovascular countermeasures by hypergravity (continuous or intermittent).