Essentiel au maintien d’un organisme sain, la division cellulaire est un processus biologique composée de deux phases : la mitose et la cytokinèse. Au cours de la mitose, un fuseau mitotique bipolaire est assemblé et les chromosomes s’alignent au niveau de la plaque métaphasique par l’attachement des kinétochores aux microtubules du fuseau. Une fois les chromosomes alignés, les chromatides soeurs sont séparées par les microtubules pendant l'anaphase et sont ségréguées entre les cellules filles. La cytokinèse est initiée peu après le début de l'anaphase, marquant ainsi la fin de la division cellulaire en séparant le cytoplasme en deux nouvelles cellules filles. Une exécution précise de la mitose et de la cytokinèse est essentielle pour le maintien de l'intégrité du génome. L'échec de l'un de ces processus affecte la fidélité génétique. Les erreurs de ségrégation des chromosomes durant la mitose peuvent entraîner un gain ou une perte de chromosomes entiers, appelé aneuploïdie. Tandis que l'échec de la cytokinèse conduit à la formation d'une cellule binucléée avec un génome entièrement dupliqué, appelé tétraploïdie. Dans les cellules somatiques, la tétraploïdie peut conduire à l'arrêt du cycle cellulaire, à la mort cellulaire, ou provoquer une instabilité chromosomique (CIN), favorisant ainsi la prolifération de cellules avec un potentiel tumorigène. Par conséquent, il est essentiel de bien comprendre la régulation et les causes potentielles de l’échec de la cytokinèse en particulier dans le contexte des systèmes multicellulaires comme l’embryon. En effet, dans ces systèmes, la réduction progressives de la taille des cellules coïncident avec les principaux évènements du développement. De plus, la binucléation est fréquemment observée dans les cliniques de fertilité chez les embryons humains. Cependant, l’impact de la binucléation sur les divisions préimplantatoires demeure inexpliqué à ce jour. Afin de déterminer les conséquences de la tétraploïdie, nous avons utilisé l'embryon de souris pour modèle et réalisé des expériences d'immunofluorescence à haute résolution et une imagerie sur cellules vivantes. Nous avons découvert que la tétraploïdie chez les embryons de souris provoque une CIN et l'aneuploïdie par un mécanisme différent de celui des cellules somatiques. Dans les cellules somatiques, la formation des fuseaux multipolaires causée par des centrosomes surnuméraires est le principal mécanisme conduisant à la tétraploïdie et ainsi, à une CIN. En revanche, chez les embryons de souris, qui ne possèdent pas de centrosomes, la tétraploïdie ne conduit pas à la formation des fuseaux multipolaires. Les embryons tétraploïdes de souris développent une CIN en raison d’une réduction du renouvellement des microtubules et d’une altération de l’activité de correction d’erreurs dans l’attachement des kinétochores aux microtubules. Ainsi, une mauvaise correction de l’attachement des kinétochores aux microtubules entraîne des niveaux élevés d'erreurs de ségrégation chromosomique. Dans le cadre d'une étude de suivi, nous avons ensuite utilisé des différentes expériences d'imageries sur des cellules vivantes et d'immunofluorescences. Celles-ci furent couplées à des micromanipulations de la taille des cellules, des techniques modifiant l'adhésion cellulaire et des approches de knock-down des protéines pour étudier les mécanismes de régulation de la cytokinèse. Les expériences d'imageries sur cellules vivantes et les micromanipulations du volume cytoplasmique ont démontré que la taille des cellules détermine la vitesse de constriction de l'anneau contractile, c'est-à-dire que la vitesse de constriction devient progressivement plus lente à mesure que la taille des cellules diminue. Cependant, ce phénomène n'a lieu que lorsque les embryons atteignent le stade de 16 cellules ce qui suggère qu'une limite supérieure de vitesse de constriction peut exister pour restreindre l’augmentation de cette vitesse quand les cellules sont trop grandes. La taille des cellules étant un déterminant de la progression de la cytokinèse, nos expériences de knock-down des protéines ont, de plus, démontré que la formation de la polarité cellulaire a un impact négatif sur l'assemblage et la constriction de l'anneau contractile dans les cellules externes au stade de morula. Plus précisément, nous avons constaté que la polarité limite le recrutement des composants de la cytokinèse spécifiquement d'un côté de l'anneau contractile, provoquant ainsi un déséquilibre de l’ingression du sillon de clivage et réduisant la vitesse de constriction dans les cellules externes. Nous spéculons que la polarité cellulaire agit comme un obstacle à la progression de la cytokinèse, rendant ainsi les cellules externes plus sensibles à un échec de la cytokinèse. Ces études ont démontré un nouveau mécanisme par lequel la tétraploïdie conduit à l’instabilité chromosomique et à l’aneuploïdie chez les embryons. Ainsi un défaut de la dynamique de correction de l’attachement des kinétochores aux microtubules entraîne une mauvaise ségrégation des chromosomes indépendamment à la formation des fuseaux multipolaires. Ce travail a mis en évidence un rôle inhibiteur de la polarité apicale inattendu sur la machinerie cytokinétique. Cette inhibition pourrait fournir une explication mécanistique de l’incidence élevée de la binucléation dans le trophectoderme. Dans l'ensemble, ces résultats contribuent à notre compréhension du contrôle spatio-temporel de la cytokinèse au cours du développement embryonnaire et fournissent de nouvelles informations mécanistiques sur les origines et les conséquences biologiques de la tétraploïdie chez les embryons préimplantatoires. Les résultats présentés dans cette thèse ont des implications cliniques importantes, puisqu’ils fournissent des preuves définitives que la tétraploïdie générée par un échec de la cytokinèse est délétère pour le développement embryonnaire. Ces travaux mettent ainsi en lumière que la binucléation est un critère de sélection embryonnaire important à considérer lors des traitements de fertilité. / Cell division is comprised of mitosis and cytokinesis and is an essential biological process for the maintenance of healthy organisms. During mitosis, a bipolar spindle is assembled, and the chromosomes are aligned at the metaphase plate via the attachment of kinetochores to spindle microtubules. Once chromosome alignment is achieved, the sister chromatids are pulled apart by the microtubules during anaphase and segregated into the nascent daughter cells. Cytokinesis is initiated after anaphase onset and marks the completion of cell division by partitioning the cytoplasm of the dividing cell into two new daughter cells. Successful and timely completion of both mitosis and cytokinesis is key for the maintenance of genome integrity, and failure in either one of these processes affects genetic fidelity. Whereas chromosome segregation errors in mitosis can lead to whole chromosome gains or losses, termed aneuploidy, cytokinesis failure leads to the formation of a binucleated cell with an entirely duplicated genome, termed tetraploidy. In somatic cells, tetraploidy can either lead to cell cycle arrest and death or cause chromosomal instability (CIN), thereby promoting the proliferation of cells with high tumorigenic potential. Therefore, understanding cytokinesis regulation and the potential causes of cytokinesis failure is key, especially in the context of multicellular embryonic systems, wherein progressive cell size reductions coincide with developmental transitions. Moreover, binucleation is frequently observed in human embryos in fertility clinics, and whether binucleation impacts early divisions remains elusive. To elucidate the consequences of tetraploidy, we used the mouse embryo as a model and employed high-resolution immunofluorescence and live-cell imaging experiments. We found that tetraploidy in mouse embryos causes CIN and aneuploidy by a mechanism distinct from that of somatic cells. Whereas in somatic cells multipolar spindle formation caused by supernumerary centrosomes is the major mechanism by which tetraploidy leads to CIN, in mouse embryos - which are acentriolar – tetraploidy does not lead to multipolar spindle formation. Instead, mouse tetraploid embryos develop CIN due to reduced microtubule turnover and impaired error correction activity, which prevents the timely resolution of kinetochore-microtubule mis-attachments, thereby leading to high levels of chromosome segregation errors. As a follow-up study, we next employed live imaging and immunofluorescence experiments, coupled with micromanipulations of cell size, cell adhesion and protein knockdown approaches to investigate the regulatory mechanisms of cytokinesis. Live imaging experiments and micromanipulations of cytoplasmic volume demonstrated that cell size determines the speed of contractile ring constriction i.e., constriction speed becomes progressively slower as the cells decrease in size. However, this phenomenon takes place only when embryos reach the 16-cell stage, suggesting that an upper limit of constriction speed may exist to restrict the scalability of ring constriction to cell size. In addition to cell size being a powerful determinant of cytokinesis progression, our loss-of-function experiments revealed that the emergence of cell polarity negatively impacts contractile ring assembly and constriction in outer cells at the morula stage. More specifically, we found that polarity limits the recruitment of cytokinesis components specifically to one side of the contractile ring, thereby causing unbalanced furrow ingression and reducing constriction speed in outer cells. We speculate that cell polarity may act as an obstacle for cytokinesis progression and render outer cells to be more susceptible to cytokinesis failure. These studies have revealed a novel mechanism by which tetraploidy leads to chromosomal instability and aneuploidy in embryos, wherein defective kinetochore-microtubule dynamics cause chromosome mis-segregation in a manner independent of multipolar spindle formation. In addition, this work unravelled an unexpected inhibitory role of apical polarity on the cytokinetic machinery that might provide a mechanistic explanation for the high incidences of binucleation in the outer layer of blastocysts. Altogether, these findings contribute to our understanding of the spatiotemporal control of cytokinesis during embryonic development and provide new mechanistic insights into the origins and biological consequences of tetraploidy in preimplantation embryos. The results presented in this thesis have substantial clinical implications, as they provide definitive evidence that tetraploidy generated by cytokinesis failure is deleterious to embryonic development, therefore underlining binucleation as an important embryo selection criterion to be considered during fertility treatments.

Neural activation is thought to be essential for the expression of maximal muscle performance, but the exact contribution of neural mechanisms such as the level of agonist, antagonist and stabiliser muscle activation to muscle strength is not fully understood. Explosive neuromuscular performance, including the ability to initiate (the electromechanical delay, EMD) and develop force rapidly (termed, rate of force development, RFD) are considered essential for the performance of explosive sporting tasks and joint stabilisation and thus injury avoidance. The thesis aimed to improve our understanding of the contribution of neural factors to muscle performance, with a specific focus on explosive neuromuscular performance. The work in this thesis utilised a range of approaches to achieve this aim. Initially, the association between muscle activation and rate of force development and EMD was established. Comparison of unilateral and bilateral actions was then undertaken. Finally interventions with the aim to both negatively affect and improve muscle strength, which included fatigue and resistance training (RT), respectively was undertaken and the neural contributions to changes in performance established. Agonist activation during the early phase of voluntary force production was shown to be an important determinant of voluntary EMD, explaining 41% of its inter-individual variability. Agonist activation was an important determinant of early, but not late phase RFD. Use of bilateral actions resulted in a reduction in explosive strength, which was thought to be due to differences in postural stability between unilateral and bilateral strength tasks. The level of stabiliser activation was strongly related to the level of agonist activation during the early phase of explosive force development and had a high association with explosive force production. Task-specific adaptations following isoinertial RT, specifically, the greater increase in isoinertial lifting strength than maximal isometric strength were due to training-specific changes in the level of agonist activation. High-intensity fatigue achieved a more substantial decline in explosive than maximal isometric strength, and this was postulated to be due to neural mechanisms, specifically decreased agonist activation. This work provides an in depth analysis of the neural contributions to maximal muscle performance.

Résumé : La compréhension des contraintes qui régissent l'assemblage des machineries supramoléculaires – qu'elles soient solubles ou bien ancrées dans les membranes biologiques – est un enjeu scientifique majeur.Le système de de sécrétion type VI (T6SS) est un organelle bactérien récemment mis en évidence qui a pour particularité de posséder une origine évolutive commune avec le bactériophage T4. En raison de cette origine évolutive commune, certaines sous unités du T6SS et du bactériophage T4 présentent des structures comparables. Cependant, un grand nombre des sous unités du T6SS reste à caractériser. Parmi celles-ci, les protéines SciB et SciC sont retrouvées dans tous les systèmes de sécrétion de type VI suggérant que ces deux protéines participent à la formation du "core-complexe": le complexe minimal requis pour le fonctionnement du T6SS. / The recently identified type VI secretion system has been demonstrated to be involved in most of these processes. The T6SS is a highly complex macromolecular machine that allows Gram-negative bacteria to deliver effector proteins to both prokaryotic and eukaryotic cells in a contact-dependent manner. The T6SS promotes therefore antibacterial competition, virulence towards eukaryotes or even both. The T6SS is composed of a minimal set of 13 subunits, which are currently believed to form the core apparatus. They assemble two distinct sub-complexes: one is a cytosolic contractile structure related to the tail of contractile bacteriophages, whereas the other spans the whole cell envelope. Therefore, the T6SS is generally depicted as an inverted phage tail anchored to the cell envelope through its membrane-associated complex. Contractile tails are currently thought to assemble from four structural elements: the baseplate, the internal tube, the contractile sheath and the tail terminator. The aim of my Ph.D. work was to further characterize the assembly and function of the T6SS phage tail-like complex in enteroaggregative E. coli. In this thesis document, I provide evidence that the internal tube assembles from Hcp hexamers stacked in a head-to-tail manner and that this internal cylinder is used as a template during sheath assembly. I also characterized a sub-complex of three proteins (TssEFG) that forms the baseplate of the T6SS and controls the polymerization of the tube and sheath. Finally, I recently showed that the T6SS functions like a nano-crossbow to kill target cells as the contraction of the T6SS results in prey cell death during interbacterial competition.

Os diferentes taxa de Arrojadoa exibem uma variedade de estruturas subterrâneas, tanto de origem caulinar como radicular. O desenvolvimento de estruturas subterrâneas de origem caulinar em Arrojadoa é uma característica única entre as Cactaceae do leste do Brasil. No entanto, não há estudos sobre a diversidade morfológica das estruturas subterrâneas encontradas neste gênero, nem na sua utilidade para ajudar a resolver a taxonomia do grupo. Por isso, foi realizada uma investigação morfológica e anatômica das estruturas subterrâneas em doze taxa de Arrojadoa sensu lato. Para a análise anatômica, amostras das regiões apical, mediana e basal das diferentes estruturas subterrâneas foram fixadas em FAA 50 (formaldeído, ácido acético glacial e etanol 50º GL) e armazenadas em etanol 70º GL. Seções transversais e longitudinais foram realizadas com uso de micrótomo rotativo e à mão. O gênero revelou uma grande diversidade de estruturas subterrâneas: tubérculos simples e ramificados, caules subterrâneos curtos, caules subterrâneos longos (sóboles), raízes contráteis (estas espessadas) e raízes comuns (fibrosas e não fibrosas); sendo que uma mesma espécie pode apresentar mais de um tipo de estrutura em seu sistema subterrâneo. O estudo morfológico propõe que sejam reconhecidas oito espécies e cinco subespécies de Arrojadoa, além de uma espécie de colocação genérica duvidosa. Quanto às análises anatômicas este estudo confirmou a natureza caulinar dos tubérculos, sóboles e caules subterrâneos curtos, sendo que apenas no tubérculo de A. hofackeriana foi verificada a participação da região de transição na sua estrutura. Também foi observado, nas raízes espessadas (raízes contráteis) de Arrojadoa um mecanismo de contração semelhante ao relatado para outra espécie da família, sendo o xilema secundário com lenho \"WBT\" (\"wide-band tracheids\") o principal tecido responsável pelo espessamento do órgão. Já nos caules (tubérculos, sóboles e caules curtos), embora haja uma contribuição do parênquima no xilema secundário com o lenho do tipo \"WBT\" e/ou associado a elementos de vaso, a região cortical é responsável pela maior parte do espessamento do órgão relacionado à suculência e armazenamento. / The different Arrojadoa taxa display variety of underground structures, formed both from the stem and the root. The development of these structures from stem in Arrojadoa is unique among the Cactaceae of the East of Brazil. However, there are no studies about the morphologic diversity of underground structures found in this genus, or about their use to help solve the group taxonomy. Thus, a morphologic and anatomic investigation of the underground structures was made in twelve taxa of Arrojadoa sensu lato. For the anatomical analysis, samples of the apical, median and basal portions of the different underground structures were fixed in FAA 50 (formaldehyde, glacial acetic acid and ethanol 50º GL), and stored in ethanol 70º GL. Longitudinal and transversal sections were made with rotative microtome and by free-hand. The genera revealed a great diversity of underground structures: simple and branched tubers, short underground stem, long underground stem (soboles), contractile roots (thickened) and common roots (fibrous and non-fibrous); and the same species can display more than one type of structure in its underground system. The morphologic study proposes that should be recognized eight species and five sub-species of Arrojadoa, besides one that is dubious in the genera aspect. About the anatomical analysis, this study confirmed the stem origin of the tubers, soboles and short underground stem, and only in the A. hofackeriana tuber it was observed the participation of the root-stem transition region. It was also observed in the thickened roots (contractile roots) of Arrojadoa a contraction mechanism similar to what is reported to other species of the family, with the secondary xylem with WBT (wide-band tracheids) wood the major responsible for thickening of the organ. In the stems (tubers, soboles and short stems), although the parenchyma contributes with the secondary xylem with the WBT and/or associated with vessel elements, the cortical region is the responsible for mostly all thickening of the organ related to succulence and storage.

Die vorliegende Untersuchung sollte die kontraktile Funktion von humanen Vorhoftrabekeln und linksventrikulären Papillarmuskeln der Ratte während Hypoxie / Reoxygenierung als Hauptkomponenten von Ischämie / Reperfusion charakterisieren. Weitere Merkmale der Ischämie wurden durch erhöhte extrazelluläre K+-Konzentration und Azidose simuliert. Einblicke in die zelluläre Ca2+-Regulation ergaben sich aus Aktionspotential-(AP)-messungen, der SR- Ca2+-ATPase-Aktivität und Kraft-Intervall- Beziehungen. Die Rolle des Energiestoffwechsels und der endogenen antioxidativen Kapazität für die kontraktile Funktion von infarktbedingt hypertrophiertem Rattenmyokard während Hypoxie / Reoxygenierung ist durch Messung der Kreatinkinase-(CK)-Aktivität, ihrer Isoenzymverteilung und der Aktivitäten von Superoxiddismutase (SOD) und Glutathionperoxidase (GSH-Px) charakterisiert worden. Der Einsatz der Radikalfänger Histidin und Butylhydroxytoluen während Hypoxie und schneller Reoxygenierung an Rattenpapillarmuskeln sollte zur Protektion gegen den toxischen Effekt unterschiedlicher reaktiver Sauerstoffspezies dienen. In den durchgeführten Experimenten zeigte sich eine geringere Empfindlichkeit des humanen Vorhofmyokards gegenüber reduzierter O2-Versorgung und Reoxygenierung als im Rattenmyokard. Die während simulierter Ischämie im humanen Myokard auftretende Azidose hat einen günstigen Effekt auf die Wiederherstellung der isometrischen Kontraktionskraft nach Reoxygenierung, was jedoch mit einer gestörten Regulation der kontraktilen Funktion verbunden ist. Hypertrophiertes Myokard in der chronischen Phase nach Infarkt zeigt eine verminderte Empfindlichkeit gegenüber Hypoxie / Reoxygenierung, was auf adaptive Veränderungen im Energiestoffwechsel (erhöhte CK-MB und CK-BB Isoenzyme mit kleinerem Km-Wert für Kreatinphosphat), in der endogenen antioxidativen Kapazität (Erhöhung der Aktivitäten von SOD und GSH-Px um 40% bzw. 50%) und in der Regulation der kontraktilen Funktion (verminderte SR Ca2+-ATPase-Aktivität und Isomyosinverschiebung von V1 nach V3) zurückgeführt werden kann. Eine bessere Erholung der kontraktilen Funktion nach Reoxygenierung kann durch schnellen pO2- Wiederanstieg erreicht werden. Der Einsatz von Pharmaka mit verschiedenen Angriffspunkten im Radikalstoffwechsel und besonders deren Kombination während Hypoxie / Reoxygenierung ermöglicht zusätzlich eine verbesserte Kardioprotektion. / This study characterizes the contractile function of human atrial trabeculae and rat left ventricular papillary muscles during hypoxia / reoxygenation as the major components of ischemia / reperfusion. Further characteristics of ischemia were simulated by increased extracellular K+ concentration and acidosis during hypoxia. Insights into the cellular Ca2+ regulation were obtained from action potential recordings, from measurements of sarcoplasmic reticulum (SR) Ca2+ transport, and from force-interval relations. We examined changes in SR calcium transport, creatine kinase (CK) system, the antioxidant enzymes glutathionperoxidase (GSH-Px) and superoxiddismutase (SOD) 6 wks. after infarction (MI) due to coronary ligation in rats. Phenotypic modifications vs. sham operation (SHAM) were related to the contractile response of hypertrophied papillary muscle to hypoxia / reoxygenation. The oxygen radical scavengers histidine and butylhydroxytoluene were applied during hypoxia and rapid reoxygenation to protect the myocardium against oxygen radical damage. Generally, human atrial trabeculae were less sensitive to reduced oxygen supply and reoxygenation when compared to rat papillary muscles. In human atrial trabeculae, isometric peak force development recovered better after simulated ischemia than after hypoxia but the regulation of contractile function was clearly disturbed. In rat papillary muscles, rapid reoxygenation caused a better recovery of contractile function after hypoxia. Application of the oxygen radical scavengers histidine, butylhydroxytoluene, and especially their combination during hypoxia / reoxygenation had additional cardioprotective effects. In MI vs. SHAM we observed under aerobic control conditions: decreses in isometric contraction and relaxation rate, a reduced Vmax-equivalent of sarcomeric shortening, a faster twitch-to- twitch decay of post-rest potentiation (PRC) which correlated closely to the decrease in SR Ca2+ uptake (-25%), a decrease in CK activity (-20%), reduced CK-MI and CK-MM, increased CK-MB and CK-BB, and enhanced activities of SOD (+40%) and GSH-Px (+50%). During hypoxia, an initial increase in peak force (PF) was followed by a slower PF decline in MI vs. SHAM. During reoxygenation, rates of contraction and relaxation recovered better in MI. In SHAM but not MI, twitch-to-twitch decay of PRC was accelerated after reoxygenation vs. aerobic control. The results suggest that adaptive changes in SR Ca2+ handling, CK isoenzymes, and antioxidant enzymes may contribute to higher resistance against reduced O2 supply and reoxygenation in hypertrophy due to MI.

Perivascular adipose tissue (PVAT) and vascular endothelial cells both have important structural and functional roles in blood vessels and are the focus of this doctoral thesis. Firstly, PVAT has been rediscovered as an endocrine organ, releasing vasorelaxing substances. Secondly, the endothelial monolayer functions as an important barrier, the role of which is to restrict the transfer of molecules or even blood-borne cells between the lumen of the blood vessel and the surrounding tissue. In my main study, the presence of PVAT caused 'anti-contractile' effects, which were reversed by nitric oxide synthase (NOS) inhibition in rat mesenteric arteries and were lost in adiponectin-knockout mice. The β3 adrenoceptor agonist CL-316,243 increased PVAT-dependent anti-contractile effects and caused myocyte hyperpolarisation. Hyperpolarisation to CL-316,243 could be mimicked by the adipokine, adiponectin, and by the 5'AMP kinase (AMPK) activator, A-769662. In addition, the AMPK inhibitor, dorsomorphin, and the selective BKCa channel blocker, iberiotoxin, each blocked hyperpolarisations to CL-316,243, adiponectin and A-769662. The anti-contractile effects of CL-316,243 could also be mimicked by A-769662 but were not blocked by dorsomorphin. Moreover CL-316,243 still had anti-contractile effects in adiponectin-knockout mice. However, inhibiting the production of both NO and hydrogen peroxide reduced anti-contractile effects of CL-316,243. In obese Sprague Dawley rats both the hyperpolarising and the anti-contractile effects to CL-316,243 were impaired, while hyperpolarisation to A-769662 were unchanged. Western blots revealed that NOS, a possible downstream target of AMPK, was phosphorylated in PVAT control samples, a form which was decreased in PVAT from obese rats. These results collectively indicate that the anti-contractile and hyperpolarising effects observed following stimulation with CL-316,243 are due to activation of different PVAT-dependent pathways, both of which probably contribute to vasodilatation in blood vessels. Understanding these pathways is crucial for the development of improved treatments for obesity and hypertension. During my work at Novartis, I found that activation of sphingosine-1-receptors type 1 (S1P1) with the activator FTY720 (Fingolimod, Novartis; used in multiple sclerosis treatment) caused closure of the endothelial barrier in human umbilical vein cells. This effect could be mimicked with a recombinant peptide of nectin, an adherens junction protein. The novel S1P1 antagonists 'A1' and 'A2' (Novartis) inhibited the effect of FTY720, but not those of nectin. The discovery of nectin as a potential barrier closure modulator might contribute to the development of additional treatments for use in multiple sclerosis.

Neuromuscular explosive strength (defined as rate of force development; RFD) is considered important during explosive functional human movements; however this association has been poorly documented. It is also unclear how different variants of strength training may influence RFD and its neuromuscular determinants. Furthermore, RFD has typically been measured in isometric situations, but how it is influenced by the types of contraction (isometric, concentric, eccentric) is unknown. This thesis compared neuromuscular function in explosive power athletes (athletes) and untrained controls, and assessed the relationship between RFD in isometric squats with sprint and jump performance. The athletes achieved a greater RFD normalised to maximum strength (+74%) during the initial phase of explosive contractions, due to greater agonist activation (+71%) in this time. Furthermore, there were strong correlations (r2 = 0.39) between normalised RFD in the initial phase of explosive squats and sprint performance, and between later phase absolute explosive force and jump height (r2 = 0.37), confirming an association between explosive athletic performance and RFD. This thesis also assessed the differential effects of short-term (4 weeks) training for maximum vs. explosive strength, and whilst the former increased maximum strength (+20%) it had no effect on RFD. In contrast explosive strength training improved explosive force production over short (first 50 ms; +70%) and long (>50 ms; +15%) time periods, due to improved agonist activation (+65%) and maximum strength (+11%), respectively. Explosive strength training therefore appears to have greater functional benefits than maximum strength training. Finally, the influence of contraction type on RFD was assessed, and the results provided unique evidence that explosive concentric contractions are 60% more effective at utilising the available force capacity of the muscle, that was explained by superior agonist activation. This work provides a comprehensive analysis of the association between athletic performance and RFD, the differential effects of maximum vs. explosive strength training, and the influence of contraction type on the capacity for RFD.

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.

Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-06T13:43:25Z No. of bitstreams: 1 arquivototal.pdf: 2157033 bytes, checksum: c35af421ba5130163d96afa847c031de (MD5) / Made available in DSpace on 2017-09-06T13:43:25Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2157033 bytes, checksum: c35af421ba5130163d96afa847c031de (MD5) Previous issue date: 2014-08-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Abnormalities in intestinal contractile reactivity represent one of the pathophysiological processes that characterize intestinal colic, diarrhea and constipation. Exercise is an activity that affects all organs and tissues, resulting in many health benefits. E interferes with intestinal contractility in mice exercised on a treadmill, this decreasing contractility. The aim of this study was to evaluate the influence of chronic aerobic exercise swimming contractile reactivity, lipid peroxidation and morphology of the intestinal smooth muscle of rats. For this, we used Wistar rats (Rattus norvegicus) were divided into two groups, the sedentary group (SED), which was in contact with water for 2 minutes during the training of exercised rats. Exercised group (EX), who underwent a swim for an hour, being attached to the trunk of the animal a metal ring, corresponding to 3% of their body weight. The animals were divided into groups exercised that this procedure performed 5 days per week for 2 (EX2), 4 (EX4), 6 (EX6) or 8 (EX8) weeks. After the fifth day of training, the animals rested for 48 h. Then the animals were euthanized, ileum was removed and suspended in isolated organ baths and sinks to the isotonic contractions were recorded. All experimental protocols were previously approved by the Ethics Committee on Animal Use the CBiotec / UFPB (Protocol: 0907/13). Cumulative concentration-response curves to KCl were attenuated due to the exercise, as the values of Emax, which was reduced from 100% (SED) to 63.1 ± 3.9, 48.8 ± 3.8, 19.4 ± 1.8, 59.4 ± 2.8% in the groups exercised by 2, 4, 6 and 8 weeks, respectively. However, no significant difference in the power parameter. Similarly, the concentration-response curves to cumulative carbachol (CCh), were attenuated due to the exercise, as Emax values of 100% (SED) to 74.1 ± 5.4, 75.9 ± 5.2 62.9 ± 4.6% in the groups exercised by 2, 4 and 6 weeks, respectively. And EX8 group (E max = 89.7 ± 3.4%) was not significantly different from the SED group. However, the power of CCh contraction was not changed in relation to the SED group, but changed between groups EX2 (EC50 = 1.5 ± 0.5 x 10-6 M) EX8 (EC50 = 2.1 ± 0,4 x 10-7 M) EX6 (EC50 = 1.5 ± 0.3 x 10-6 M) EX8. The concentration of malondialdehyde (MDA), which indicates that lipid peroxidation was increased from 20.6 ± 3.6 mM/mL (SED) to 44.3 ± 4.4 mM/mL (EX4), but this was not peroxidation changed in EX6 (20.0 ± 3.6 mM/mL) and EX8 (17.2 ± 3.6 mM/mL) group. The total muscle layer (TML) was reduced from 75.5 ± 0.9 μm (SED) to 63.0 ± 1.3 μm (EX6), and no significant difference in the other exercise groups. The circular muscle layer (CML) was reduced from 50.9 ± 0.3 μm (SED) to 44.0 ± 1.8, 43.5 ± 1.3, 35.5 ± 1.4 and 41.6 ± 0.6 μm in groups trained for 2, 4, 6 and 8 weeks, respectively. Already the longitudinal muscle layer (LML) was increased from 21.6 ± 0.3 μm (SED) to 31.8 ± 1.0, 36.2 ± 2.5, 29.6 ± 1.8 and 30.8 ± 1.3 μm in groups trained by 2, 4, 6 and 8 weeks, respectively. According to the results, aerobic exercise alters the response of the ileum to KCl and CCh, reducing the amplitude of the intestinal contraction. Reduces oxidative damage, decreasing lipid peroxidation. And promotes changes in tissue level to establish an adaptation of the body to exercise. The data indicate a direct relationship between swimming exercise and reactivity of the intestinal smooth muscle of rat, which should be better studied and thus clarify the mechanisms involved and possible therapeutic implications. / Anormalidades na reatividade contrátil intestinal representam um dos processos fisiopatológicos que caracterizam a cólica intestinal, diarreia e constipação. Analisando essa reatividade contrátil no íleo de camundongos exercitados em esteira, verificou-se que houve redução dessa reatividade. Diante disso, o objetivo desse trabalho foi avaliar a influência do exercício aeróbio crônico de natação na reatividade contrátil, peroxidação lipídica e morfologia do íleo de ratos. Todos os protocolos experimentais foram previamente aprovados pelo Comitê de Ética no Uso de Animais da UFPB (Protocolo: 0907/13). Para isso, utilizou-se ratos Wistar (Rattus norvegicus), inicialmente foi realizada uma padronização das intensidades de exercício para confirmar em quais intensidades o exercício era considerado aeróbico. Os animais foram divididos em 6 grupos, grupo controle (GC) e grupos exercitados, estes foram submetidos a uma sessão de exercício de natação, tendo fixado ao seu tronco um anel de metal com as seguintes intensidade: 3, 4, 5, 6 ou 8% do peso corporal do animal, durante 1 hora. Após esse protocolo foi coletado o sangue do animal e realizada dosagem de lactato, cujo limiar de lactato era de 5,5 mmol/L. Verificou-se que intensidade inferiores a 5% do peso corporal do animal submetido à natação, o exercício é do tipo aeróbico. A partir disso, foi escolhida a menor intensidade analisada para seguir com o exercício crônico. Os animais foram divididos nos seguintes grupos, sedentário (SED) e exercitado (EX), os animais do grupo exercitado foram submetidos a um exercício de natação por uma hora, sendo fixado ao tronco do animal um anel de metal, correspondente a 3% do seu peso corporal. Os animais exercitados realizaram um treinamento de 5 dias por semana, sendo os grupos exercitados por 2 (EX2), 4 (EX4), 6 (EX6) ou 8 (EX8) semanas. A cada cinco dias de treinamento, os animais descansaram por 48 h. Após a realização do protocolo, os animais foram eutanasiados, o íleo era retirado, suspenso em cubas de banhos para órgãos isolados e eram registadas as contrações isotônicas. Curvas concentrações-resposta cumulativas ao KCl foram atenuadas em função do exercício, conforme os valores de Emax, que foi reduzido de 100% (SED) para 63,1 ± 3,9, 48,8 ± 3,8, 19,4 ± 1,8, 59,4 ± 2,8% nos grupos exercitados por 2, 4, 6 e 8 semanas, respectivamente. Porém, não apresentou diferença significante no parâmetro de potência contrátil. De forma similar, as curvas concentrações-resposta cumulativas ao carbacol (CCh), foram atenuadas em função do exercício, conforme os valores de Emax de 100% (SED) para 74,1 ± 5,4, 75,9 ± 5,2, 62,9 ± 4,6% nos grupos exercitados por 2, 4 e 6 semanas, respectivamente. E o grupo EX8 (Emax = 89,7 ± 3,4%) não apresentou diferença significante do grupo SED. E a potência contrátil do CCh também não foi alterada em relação ao grupo SED, mas foi alterada entre grupos EX2 (CE50 = 1,5 ± 0,5 x 10-6 M) e EX8 (CE50 = 2,1 ± 0,4 x 10-7 M), EX6 (CE50 = 1,5 ± 0,3 x 10-6 M) e EX8. A concentração de malondialdeído (MDA) foi realizada através de dosagens bioquímicas, e foi aumentada de 20,6 ± 3,6 μM/mL (SED) para 44,3 ± 4,4 μM/mL (EX4), porém essa peroxidação não foi alterada no grupo EX6 (20,0 ± 3,6 μM/mL) e EX8 (17,2 ± 3,6 μM/mL. As medidas das camadas musculares foram registradas através de cortes histológicos, sendo a camada muscular circular (CMC) reduzida de 50,9 ± 0,3 μm (SED) para 44,0 ± 1,8, 43,5 ± 1,3, 35,5 ± 1,4 e 41,6 ± 0,6 μm, nos grupos exercitados por 2, 4, 6 e 8 semanas, respectivamente. Já a camada muscular longitudinal (CML) foi aumentada de 21,6 ± 0,3 μm (SED) para 31,8 ± 1,0, 36,2 ± 2,5, 29,6 ±1,8 e 30,8 ± 1,3 μm, nos grupos exercitados por 2, 4, 6 e 8 semanas, respectivamente. De acordo com os resultados, o exercício aeróbico: altera a reatividade contrátil do íleo ao KCl e CCh, diminuindo a amplitude da contração intestinal. Promove um aumento da peroxidação lipídica no grupo EX4, que pode ter servido como estímulo para célula produzir antioxidantes e inibir o estresse oxidativo. E promove alterações morfológicas para estabelecer uma adaptação do órgão ao exercício. A diminuição da reatividade contrátil e as alterações histológicas parecem não estar correlacionadas com a peroxidação lipídica. Dessa forma, demonstra-se pela primeira vez uma relação direta entre o treinamento de natação e a reatividade intestinal do músculo liso de rato.

Os diferentes taxa de Arrojadoa exibem uma variedade de estruturas subterrâneas, tanto de origem caulinar como radicular. O desenvolvimento de estruturas subterrâneas de origem caulinar em Arrojadoa é uma característica única entre as Cactaceae do leste do Brasil. No entanto, não há estudos sobre a diversidade morfológica das estruturas subterrâneas encontradas neste gênero, nem na sua utilidade para ajudar a resolver a taxonomia do grupo. Por isso, foi realizada uma investigação morfológica e anatômica das estruturas subterrâneas em doze taxa de Arrojadoa sensu lato. Para a análise anatômica, amostras das regiões apical, mediana e basal das diferentes estruturas subterrâneas foram fixadas em FAA 50 (formaldeído, ácido acético glacial e etanol 50º GL) e armazenadas em etanol 70º GL. Seções transversais e longitudinais foram realizadas com uso de micrótomo rotativo e à mão. O gênero revelou uma grande diversidade de estruturas subterrâneas: tubérculos simples e ramificados, caules subterrâneos curtos, caules subterrâneos longos (sóboles), raízes contráteis (estas espessadas) e raízes comuns (fibrosas e não fibrosas); sendo que uma mesma espécie pode apresentar mais de um tipo de estrutura em seu sistema subterrâneo. O estudo morfológico propõe que sejam reconhecidas oito espécies e cinco subespécies de Arrojadoa, além de uma espécie de colocação genérica duvidosa. Quanto às análises anatômicas este estudo confirmou a natureza caulinar dos tubérculos, sóboles e caules subterrâneos curtos, sendo que apenas no tubérculo de A. hofackeriana foi verificada a participação da região de transição na sua estrutura. Também foi observado, nas raízes espessadas (raízes contráteis) de Arrojadoa um mecanismo de contração semelhante ao relatado para outra espécie da família, sendo o xilema secundário com lenho \"WBT\" (\"wide-band tracheids\") o principal tecido responsável pelo espessamento do órgão. Já nos caules (tubérculos, sóboles e caules curtos), embora haja uma contribuição do parênquima no xilema secundário com o lenho do tipo \"WBT\" e/ou associado a elementos de vaso, a região cortical é responsável pela maior parte do espessamento do órgão relacionado à suculência e armazenamento. / The different Arrojadoa taxa display variety of underground structures, formed both from the stem and the root. The development of these structures from stem in Arrojadoa is unique among the Cactaceae of the East of Brazil. However, there are no studies about the morphologic diversity of underground structures found in this genus, or about their use to help solve the group taxonomy. Thus, a morphologic and anatomic investigation of the underground structures was made in twelve taxa of Arrojadoa sensu lato. For the anatomical analysis, samples of the apical, median and basal portions of the different underground structures were fixed in FAA 50 (formaldehyde, glacial acetic acid and ethanol 50º GL), and stored in ethanol 70º GL. Longitudinal and transversal sections were made with rotative microtome and by free-hand. The genera revealed a great diversity of underground structures: simple and branched tubers, short underground stem, long underground stem (soboles), contractile roots (thickened) and common roots (fibrous and non-fibrous); and the same species can display more than one type of structure in its underground system. The morphologic study proposes that should be recognized eight species and five sub-species of Arrojadoa, besides one that is dubious in the genera aspect. About the anatomical analysis, this study confirmed the stem origin of the tubers, soboles and short underground stem, and only in the A. hofackeriana tuber it was observed the participation of the root-stem transition region. It was also observed in the thickened roots (contractile roots) of Arrojadoa a contraction mechanism similar to what is reported to other species of the family, with the secondary xylem with WBT (wide-band tracheids) wood the major responsible for thickening of the organ. In the stems (tubers, soboles and short stems), although the parenchyma contributes with the secondary xylem with the WBT and/or associated with vessel elements, the cortical region is the responsible for mostly all thickening of the organ related to succulence and storage.