Les oeufs d’escargot terrestre de l’espèce petit-gris Helix aspersa (syn. Cantareusaspersus) peuvent être utilisés pour évaluer l’écotoxicité de substances chimiques pures ou enmélange. La mesure des effets embryotoxiques classiquement réalisée est le succès d’éclosionaprès 15 à 20 jours d’exposition (Druart et al., 2012). Cependant, les mécanismes impliquésdans la mise en place des effets toxiques à différents niveaux d’organisation biologique chezl’embryon ne sont pas connus. Des oeufs d’escargots ont été exposés à des solutions decontaminants métallique (Cd) ou organiques (pesticides: le Round Up® flash, le Corail® et laBouillie Bordelaise) selon deux modalités différentes (en continu sur la totalité dudéveloppement embryonnaire ou sur une période de 24 heures) afin de 1/ déterminer denouveaux paramètres de mesure au cours du développement embryonnaire pouvant rendrecompte d’un effet toxique, 2/ détecter des effets génotoxiques de divers contaminants(solution métallique de Cd ou de formulations commerciales de pesticides) par la méthodeRandom Amplified Polymorphic DNA (RAPD) et 3/ d’étudier des systèmes de défense métalspécifiques(métallothionéines).Les paramètres morphologiques et physiologiques suivis au cours d’expositionscontinues au Cd ont montré des effets néfastes sur le rythme cardiaque, la durée del’incubation, la taille et le poids à l’éclosion chez les exposés à la plus forte concentrationtestée. Chez ces derniers des signes de fragmentation de l’ADN ont également été détectés enfin d’exposition. Le couplage de la méthode RAPD avec un système d’électrophorèse hauterésolution (SHR) a permis de détecter des effets génotoxiques suite à des expositionscontinues au Cd, au Round Up® et au Corail®. L’étude par PCR quantitative de l’expressiondes gènes des métallothionéines (MTs) a mis en évidence une expression constitutive des MTsainsi qu’un haut niveau d’expression du gène mixte CdCuMT chez les embryons non exposés.Chez les embryons exposés au Cd durant 24 heures, une surexpression du gène spécifiqueCdMT a été mise en évidence alors qu’aucune augmentation significative des taux detranscrits des 2 autres isogènes étudiés (CuMT et CdCuMT) n’a été démontrée.Les résultats de toxicité du Cd basés sur le taux d’éclosion et l’expression des gènes desMTs ont démontré que des facteurs comme le régime d’exposition (24 heures ou en continu)ou le stade de développement (âge des embryons lors de l’exposition) peuvent modulerl’embryotoxicité des substances chimiques.206Les données obtenues durant cette étude intégrative permettent de proposer un largepanel de paramètres de mesure des effets toxiques des substances chimiques chez l’embryond’escargot terrestre H. aspersa au niveau individuel (rythme cardiaque, taille, durée dedéveloppement et succès d’éclosion) et au niveau moléculaire (expression de gènes dessystèmes de défense, détection des signes de génotoxicité et de la fragmentation de l’ADN)pour l’évaluation de la toxicité des substances chimiques. L’approche RAPD-SHR, bien quenécessitant une certaine expertise pour l’analyse des profils d’amplifications obtenus, apparaîtadaptée pour une détection rapide et efficace du potentiel embryogénotoxiques de substancesvariées (métaux, pesticides. / The land snail species Helix aspersa (syn. Cantareus aspersus) eggs can be used to assess theecotoxicity of chemicals. Measurement of embryotoxic effect is classically based on hatching successafter 15-20 days of exposure (Druart et al., 2012). However, the mechanisms involved in toxic effectsin embryos at different levels of biological organization are not known. Eggs of snails were exposedto solutions metallic contaminants (Cd) or organic (pesticides: Round Up® Flash, Corail® andBordeaux Mixture) in two different regimes (continuous over the entire embryonic development orduring a period of 24 hours), in order to 1 / identify of new endpoints of toxic effect measurementsduring embryonic development, 2 / detect of genotoxic effects of metal solution (Cd) or threepesticides commercial formulations by Random Amplified Polymorphic DNA method (RAPD) and 3 /study metal-specific defense systems (metallothionein).Morphological and physiological parameters monitored during Cd continuous exposures showedadverse effects on heart rate, duration of incubation, size and weight of new hatchlings exposed to thehighest concentration tested. In the latter, signs of DNA fragmentation were detected at the end ofexposure. Coupling the RAPD with a high-resolution electrophoresis system (SHR) has enabled todetect genotoxic effects of Cd, Round Up® and Corail® after continuous exposures. Quantitative PCRstudy of metallothioneins (MTs) gene expression has showed constitutive expression of MTs genesand a high level of mRNA for the mixed gene CdCuMT in unexposed embryos. In embryos exposedto Cd for 24 hours, an overexpression of the specific gene CdMT has been demonstrated whereas thetwo other isogenes (CuMT and CdCuMT) didn’t show significant induction of expression rates.The toxicity results based on the hatching rate and MTs genes expression obtained with Cd haveshowed that factors such as the exposure regime (24 hours or continuous) or the stage of development(age of embryos upon exposure) can modulate embryotoxicity of chemicals. This thesis provides awide range of endpoints usable at the individual level (heart rate, height, hatching monitoring) and atthe molecular level (gene expression of defense systems, detection of genotoxicity signs and DNAladdering) for the assessment of the ecotoxicity of chemical substances. The RAPD-SHR, althoughrequiring some expertise to analyze profiles obtained, appears suitable for rapid and efficientdetection of potential embryogenotoxic effects of various substances (metals, pesticides).

O Transtorno de Déficit de Atenção e Hiperatividade (TDAH) é um dos transtornos neuropsiquiátricos mais prevalentes da infância caracterizado pelos sintomas de desatenção, hiperatividade e impulsividade. O TDAH é uma desordem neurocomportamental heterogênea e fenotipicamente complexa e sua etiologia ainda não foi completamente esclarecida, mas sabe-se que a interação de fatores ambientais e genéticos e o acúmulo de seus efeitos possivelmente aumenta a vulnerabilidade ao transtorno. Nesse estudo, foram investigados o imunoconteúdo de proteínas sinápticas e do desenvolvimento a partir de neurônios da região do córtex pré-frontal de animais SHR, um dos modelos animais mais validados para o estudo do TDAH. Também foi realizada uma análise morfomética do padrão de desenvolvimento dessas células ao longo de diferentes dias in vitro e o papel do BDNF, fator neurotrófico crucial para a sobrevivência e maturação das sinapses, no desenvolvimento dos neurônios SHR. A análise do imunoconteúdo da SNAP-25 mostrou aumento nos níveis dessa proteína no 2º DIV e diminuição no 5º DIV nos neurônios SHR em relação ao controle WKY, sem alterações entre as cepas nos outros dias analisados. Em relação aos níveis de sinaptofisina nos neurônios SHR, foi observado aumento somente no 5º DIV. A análise do proBDNF mostrou diminuição nos neurônios SHR no 5º DIV e aumento no 8º DIV. A imunodetecção do CREB mostrou que os neurônios SHR apresentam níveis diminuídos dessa proteína somente no 1º DIV. O receptor TrkB também apresentou alterações no seu imunoconteúdo, com aumento no 2º DIV e diminuição no 5º DIV nos neurônios SHR. O imunoconteúdo do BDNF e do TrkB fosforilado não apresentaram alterações entre as linhagens nos dias analisados. Além disso, foi realizada uma análise morfométrica de diferentes parâmetros de desenvolvimento dos neurônios ao longo de diferentes dias in vitro por meio da marcação da proteína da região somatodendrítica MAP-2. Foi observada diminuição no comprimento total dos neuritos dos neurônios SHR no 5º DIV em relação aos neurônios WKY. Também foi verificado redução no número de raízes no 2º DIV e redução no número de pontos de ramificação no 5º DIV nos neurônios SHR. As alterações observadas em proteínas que são relacionadas aos processos de sinapses e de desenvolvimento neuronal podem auxiliar na compreenssão das diferenças encontradas no padrão de desenvolvimento dos neurônios SHR. Essas modificações a nível proteico podem estar alterando o crescimento e o padrão de arborização dendrítica e implicar em modificações na funcionalidade dessas células importantes para a melhor compreensão das bases neurobiológicas do TDAH. / Attention deficit hyperactivity disorder (ADHD) is one of the most common neuropsychiatric disorders of childhood characterized by symptoms of inattention, hyperactivity and impulsivity. ADHD is a heterogeneous and phenotypically complex neurobehavioral disorder with unknown etiology, but the interaction between environmental and genetic factors have been described to increase the vulnerability to the disorder. In this study, we investigated the immunocontent of synaptic and development proteins of prefrontal cortex neurons from one of the most validated animal models for the study of ADHD (SHR). We also performed a morphometric analysis along development of these cells at different days in vitro and the role of a neurotrofic factor (BDNF) in neuronal outgrowth. SNAP-25 immunocontent was increased at 2 DIV and decreased at 5 DIV in SHR neurons. Synaptophysin levels show increases only at 5 DIV in SHR neurons. The levels of proBDNF were decreased at 5 DIV and increased at 8 DIV in SHR neurons. CREB immunodetection showed that SHR neurons present decreased levels only at 1 DIV. The TrkB receptor also presented changes in immunocontent, with increase at 2 DIV and decrease at 5 DIV in the SHR neurons. Morphometric analysis during neuronal development by immunostaining with MAP-2 somatodendritic protein show decrease in total length at 5 DIV in SHR neurons in relation to WKY neurons. Besides that, SHR neurons exhibit reduction in number of roots at 2 DIV and number of branch points at 5 DIV. Changes in proteins related to synaptic processes and neuronal during development can help to understand differences found in the pattern of development of the neurons SHR. These changes at protein level may be altering neuronal outgrowth and dendritic arborization and possible involve modifications in functionality of these cells important for better understanding the neurobiological bases of ADHD.

Attention-deficit hyperactivity disorder (ADHD) is a common neuropsychiatric disorder with onset at preschool age Approximately 5-10% of school-aged children worldwide have ADHD. Psychostimulants are the most common treatments for ADHD, although the precise etiology and pathological mechanisms underlying ADHD are poorly understood. Animal models could help to elucidate and further the understanding of this disorder. Among the major rodent models of ADHD of the genetic and neurotoxin-exposed animal models, the spontaneously hypertensive rats (SHR) are more extensively studied. Nevertheless, the mechanism of ADHD is complex and the evidence of SHR model for ADHD has been conflicting. Objective. In this work, we combined behavioral, neurochemical, neuroimaging, pharmacological and molecular studies to examine SHR as an animal model of ADHD. At the same time, the results of our studies could help us to explore the potential mechanism of ADHD. Material and methods. We compared the locomotor activity, attention, inhibition, learning and memory of juvenile male SHR with those of age- and gender-matched genetic control Wistar-Kyoto rats (WKY) by using the open field test, Morris water maze and prepulse inhibition test. We employed magnetic resonance imaging (MRI) to measure potential morphological differences between different brain areas of SHR and WKY, and the functional MRI (fMRI) for functional differences in these brain areas. We also measured dopamine concentration and dopamine related genes expression in the different dopamine pathways by using enzyme-linked immunosorbent assay (ELISA) to measure the dopamine concentration and by using real time PCR to assay genes expression. We examined SHR responses to D-amphetamine (D-AMP), which is psychostimulant. These included locomotor activity and inhibition ability during D-AMP treatment, expression of dopamine related genes after D-AMP treatment measured by real time PCR and c-fos protein after repeated treatment of D-AMP by the Western Blotting. Results . Hyperactivity, impulsivity and attention deficit were observed in SHR. Decreased brain volume in caudate-putamen and vermis cerebelli in SHR were demarcated using MRI. Functional MRI (fMRI) and altered c-fos expression indicated plasticity changes of the prefrontal cortex (PFC) in SHR. Dopamine content was found to decrease in mesocortical and mesolimbic dopamine pathways, but increased in the striatum. Dopamine D4 receptors gene and protein expression were decreased in the PFC in SHR. We also found that the expression of the synaptosomal-associated protein 25 (SNAP-25) gene was initially lower in the PFC but higher in the striatum in SHR. However, this disparity of SNAP-25 in the PFC vanished after repeated treatment of D-AMP between SHR and WKY. Conclusions. In the present study, we demonstrated that SHR could be established as an ADHD model by completing complex assessments of face validity, construct validity and prediction validity. We suggested that the "synaptogenesis hypotheses" might contribute to the abnormal release of dopamine and dysfunction of PFC and the striatum in SEER. In conclusion, our results have provided further new information relevant to the understanding of ADHD in human via the analysis of the SHR model. / Li, Qi. / Adviser: David Yen. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 1375. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 108-125). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / School code: 1307.

Background and Purpose: The endothelium is a single-cell layer positioned at the blood-vascular wall interface, where in response to blood-borne signals and hemodynamic forces, endothelial cells act as central regulators of vascular homeostatic processes including vascular tone, growth and remodeling, inflammation and adhesion, and blood fluidity and coagulation. Agonist- or flow-stimulated endothelium-dependent vasorelaxation becomes impaired in states of cardiovascular disease (CVD) risk and has been identified as a possible biomarker of overall endothelial dysfunction leading to vascular dysregulation and disease pathogenesis. Accordingly, it is important to elucidate the mechanisms accounting for this endothelial vasomotor dysfunction. Upon stimulation, endothelial cells can synthesize and release a variety of endothelium-derived relaxing factors (EDRFs), the most prominent of which is nitric oxide (NO) derived from NO synthase (NOS). In addition, under certain CVD risk conditions including hypertension and aging, stimulated endothelial cells can become a prominent source of endothelium-derived contracting factors (EDCFs) produced in a cyclooxygenase (COX)-dependent manner. Consequently, endothelial dysfunction may be caused by under-active EDRF signaling and/or competitive over-active EDCF signaling. Much attention has been given to elucidating the mechanisms of under-active EDRF signaling and its role in causing endothelial dysfunction, wherein excess reactive oxygen species (ROS) accumulation and oxidative stress under CVD risk conditions have been recognized as major factors in reducing NO bioavailability thus causing under-active EDRF signaling and endothelial dysfunction. Less attention however, has been given to elucidating the mechanisms of over-active COX-mediated EDCF signaling and its role in causing endothelial dysfunction. Moreover, while COX-mediated EDCF signaling activity has been investigated in some segments of the vasculature, most notably the aorta, it has not been well-investigated in the common carotid artery (CCA), a highly accessible cerebral blood flow conduit particularly advantageous in exploring the roles of the endothelium in vascular pathogenesis. It was the global purpose of this thesis to gain a better understanding of the cellular-molecular mechanisms accounting for endothelial dysfunction in the CCA of animal models known to exhibit COX-mediated EDCF signaling activity, in particular essential (spontaneous) hypertension and aging. Experimental Objective and Approach: This thesis comprises three studies. Study I and Study II investigated the CCA of young-adult (16-24wk old) normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Study III investigated the CCA of Adult (25-36wks old) and Aging (60-75wks old) Sprague Dawley (SD) rats treated in vivo (or not; CON) with L-buthionine sulfoximine (BSO) to chronically deplete the cellular anti-oxidant glutathione (GSH) and increase ROS accumulation and oxidative stress. The global objective and approach across these studies was to systematically examine the relative contributions of NOS and COX signaling pathways in mediating the acetylcholine (ACh)-stimulated endothelium-dependent relaxation (EDRF) and contractile (EDCF) activities of isometrically-mounted CCA in tissue baths in vitro, with a particular focus on elucidating the mechanisms of COX-mediated EDCF signaling activity. An added objective was to examine the in vivo hemodynamic characteristics of the CCA in each animal model investigated, serving both to identify the pressure-flow environment that the CCA is exposed to in vivo and to provide assessment of potential hypertension, aging, and oxidative stress effects on large artery hemodynamics. Key Findings: Study I hemodynamic analysis confirmed a hypertensive state in young adult SHR while also exposing a reduction in mean CCA blood flow in SHR compared to WKY accompanied by a multi-faceted pressure-flow interaction across the cardiac cycle relating to flow and pressure augmentation. Study III hemodynamic analysis found that neither aging nor chronic BSO-induced GSH depletion affected CCA blood pressure or blood flow parameters in SD rats. Study I and II demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from young adult SHR, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to WKY. Examining ACh-stimulated contractile function specifically from a quiescent (non pre-contracted) state revealed that EDCF activity did exist in WKY CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in SHR CCA could not fully suppress its >2-fold augmented EDCF activity vs. WKY CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the SHR-WKY CCA model revealed that the EDCF signaling activity was completely dependent on the COX-1 (but not COX-2) isoform of COX and was almost exclusively mediated by the thromboxane-prostanoid (TP) sub-type of the prostaglandin (PG) G-protein coupled receptor family and by Rho-associated kinase (ROCK), a down-stream effector of the molecular switch RhoA. Furthermore, it was found that while exogenous ROS-stimulated CCA contractile function was similarly >2-fold augmented in SHR vs. WKY and dependent on COX-1 and TP receptor and ROCK effectors, ACh-stimulated CCA EDCF signaling activity was only minimally affected by in-bath ROS manipulating compounds. Additional biochemical and molecular analysis revealed that ACh stimulation was associated with PG over-production from an over-expressed COX-1 in SHR CCA, and with CCA plasma membrane localization and activation of RhoA. Study III demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from Aging SD rats, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to Adult SD rats. Specific examination of ACh-stimulated contractile function revealed that EDCF activity did exist in Adult CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in Aging CCA could not fully suppress its >3-fold augmented EDCF activity vs. Adult CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the Adult-Aging SD rat CCA model revealed that EDCF signaling activity was completely dependent on COX-1, but while exogenous ROS was able to elicit a COX-dependent CCA contractile response, in-bath ROS manipulating compounds were found to be without effect on ACh-stimulated CCA EDCF signaling activity. Furthermore, biochemical analysis revealed that aging was not associated with a change in tissue (liver and vascular) GSH content or ROS accumulation. Chronic in vivo BSO treatment was effective in depleting tissue GSH content and increasing ROS accumulation, to a similar extent, in both Adult and Aging SD rats. However, regardless of age, neither ACh-stimulated NO-mediated EDRF signaling activity nor COX-mediated EDCF signaling activity were affected by these BSO-induced perturbations. Conclusions and Perspective: In the CCA of animals at the early pathological stages of either essential hypertension (young adult SHR) or normotensive aging (Aging SD rats), endothelial vasomotor dysfunction can be caused solely by over-active EDCF signaling, apparently disconnected from changes in NO bioavailability or oxidative stress. While NO and ROS may act, respectively, as negative and positive modulators of the established COX-PG-TP receptor-RhoA-ROCK cell-signaling axis mediating endothelium-dependent contractile activity, these factors do not appear to be essential to the mechanism(s) underlying the development of over-active EDCF signaling. Further elucidation of the cellular-molecular causes of over-active EDCF signaling, and its patho-biological consequences, in the SHR-WKY and Adult-Aging SD rat CCA models of EDCF activity established and hemodynamically characterized in this thesis, may help to identify new or more effective targets to be used in prevention or treatment strategies to combat the pathogenesis of CVD.

alpha-melanocyte stimulating hormone (alpha-MSH) is an important regulator of food intake, metabolic rate, and inflammation. Recently, alpha-MSH was shown to influence sympathetic activity and blood pressure regulation. In the present study, we investigated the cardiovascular effects of alpha-MSH in the nucleus tractus solitarii (NTS) of spontaneously hypertensive rats (SHR). Because nitric oxide (NO) is well-known to involve in central cardiovascular regulation, we elucidated the role of NO in the cardiovascular responses induced by alpha-MSH. In urethane-anesthetized SHR, unilateral microinjection of alpha-MSH (0.3-300 pmol) into the NTS produced dose-responsive depressor and bradycardic effects. The cardiovascular effects of alpha-MSH were abrogated by the antagonist of melanocortin receptor (MC3/4-R), SHU9119. Pretreatment with precursor of nitric oxide, L-arginine, enhanced the duration of alpha-MSH-mediated hypotensive effects, whereas prior application of L-NAME, a universal inhibitor of nitric oxide synthase (NOS), significantly attenuated the effects of alpha-MSH. Prior injection with inhibitor of inducible NOS, aminoguanidine, but not inhibitor of neuronal NOS, 7-nitroindazole, attenuated the hypotensive effect of alpha-MSH. In summary, these results indicated alpha-MSH induced depressor and bradycardic effects in the NTS of SHR. Besides, the hypotensive mechanism of alpha-MSH was mediated via MC4-R and involved with iNOS activation in the NTS of SHR.

The way physicians are paid for the provision of care is a relevant aspect of health care systems. Fee-for-service (FFS) payment system has been criticized for affecting quality of care, contributing to the fragmentation of health care, and for rising costs of health care systems. Alternative payment plans (APP) have been introduced as options to the traditional FFS payment scheme. Despite the link between payment methods and behavior of physicians that has been established; there is a lack of evidence about the impact of payment systems on wellness of physicians, specifically on their perception of professional equity and daily distress of physicians. The purpose of this study was to explore the effects of APP on physicians’ perceptions of professional equity and daily distress. The following questions guided this dissertation: 1) Does professional equity perceived by physicians vary among practitioners paid by FFS, APP, or blended alternatives? 2) Is the payment method associated with daily distress of medical practitioners? and 3) Are levels of professional equity, daily distress, and career satisfaction of physicians different by gender and payment methods? In 2011, a cross-sectional study was conducted with physicians practicing in the Saskatoon Health Region (SHR), the largest health authority of Saskatchewan, Canada. Physicians completed a questionnaire evaluating their perceptions of professional equity and daily distress. Analyses of variances (ANOVA) were performed to assess differences in professional equity (overall and by its fulfillment, financial, and recognition dimensions) and daily distress among physicians paid by FFS, APP, and blended schemes. As multivariable analyses, a linear regression was used to test the interaction between specialty and payment methods on the perception of professional equity, controlling for the number of patients, gender, and age group. A mixed linear regression model was built to predict daily distress, testing demographics, workload, complexity of patients, payment method, career satisfaction, and practice profile; the random component of the model considered the influence of geographic area of practice. Also, a multivariate analysis of variance (MANOVA) was conducted to evaluate differences among professional equity, daily distress, and career satisfaction by payment method and gender. In total, 382 (48.1%) physicians participated in the study. Response bias was tested and found to be negligible (Appendix F). The ANOVA identified that physicians paid by APP perceived higher professional equity than those paid by FFS (p=0.005), as well as higher levels of income (p=0.03) and recognition (p=0.001) equity than those with FFS. In the multivariable analyses, a higher level of professional equity was predicted among family practitioners (FPs) paid by APP and blended schemes in comparison to those paid by FFS. Additionally, the payment method was a predictor of daily distress when adjusted by other factors. Lower levels of distress were found among physicians who had more than 75% of patients with complex conditions and were paid by APP compared to those paid by FFS and blended methods. The MANOVA identified that female physicians had poorer wellness indicators than male practitioners. Multiple comparisons identified higher levels of equity among male physicians paid by APP than those with FFS, although this benefit was not observed among female ones. In conclusion, physicians paid by APP perceived higher professional equity (fair economic rewards and appropriate recognition) in comparison to those paid by FFS. Particularly, FPs paid by APP perceived higher professional equity than those FPs paid by FFS. Additionally, the payment method was identified as an associated factor with distress; lower levels of daily distress were predicted among physicians paid by APP who see high proportions of patients with complex conditions. Notwithstanding, female physicians had poorer wellness indicators and the impact of APP on professional equity was only distinguished among males. A potential unequal impact of APP must be recognized between female and male physicians.

Background and Purpose: The endothelium is a single-cell layer positioned at the blood-vascular wall interface, where in response to blood-borne signals and hemodynamic forces, endothelial cells act as central regulators of vascular homeostatic processes including vascular tone, growth and remodeling, inflammation and adhesion, and blood fluidity and coagulation. Agonist- or flow-stimulated endothelium-dependent vasorelaxation becomes impaired in states of cardiovascular disease (CVD) risk and has been identified as a possible biomarker of overall endothelial dysfunction leading to vascular dysregulation and disease pathogenesis. Accordingly, it is important to elucidate the mechanisms accounting for this endothelial vasomotor dysfunction. Upon stimulation, endothelial cells can synthesize and release a variety of endothelium-derived relaxing factors (EDRFs), the most prominent of which is nitric oxide (NO) derived from NO synthase (NOS). In addition, under certain CVD risk conditions including hypertension and aging, stimulated endothelial cells can become a prominent source of endothelium-derived contracting factors (EDCFs) produced in a cyclooxygenase (COX)-dependent manner. Consequently, endothelial dysfunction may be caused by under-active EDRF signaling and/or competitive over-active EDCF signaling. Much attention has been given to elucidating the mechanisms of under-active EDRF signaling and its role in causing endothelial dysfunction, wherein excess reactive oxygen species (ROS) accumulation and oxidative stress under CVD risk conditions have been recognized as major factors in reducing NO bioavailability thus causing under-active EDRF signaling and endothelial dysfunction. Less attention however, has been given to elucidating the mechanisms of over-active COX-mediated EDCF signaling and its role in causing endothelial dysfunction. Moreover, while COX-mediated EDCF signaling activity has been investigated in some segments of the vasculature, most notably the aorta, it has not been well-investigated in the common carotid artery (CCA), a highly accessible cerebral blood flow conduit particularly advantageous in exploring the roles of the endothelium in vascular pathogenesis. It was the global purpose of this thesis to gain a better understanding of the cellular-molecular mechanisms accounting for endothelial dysfunction in the CCA of animal models known to exhibit COX-mediated EDCF signaling activity, in particular essential (spontaneous) hypertension and aging. Experimental Objective and Approach: This thesis comprises three studies. Study I and Study II investigated the CCA of young-adult (16-24wk old) normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Study III investigated the CCA of Adult (25-36wks old) and Aging (60-75wks old) Sprague Dawley (SD) rats treated in vivo (or not; CON) with L-buthionine sulfoximine (BSO) to chronically deplete the cellular anti-oxidant glutathione (GSH) and increase ROS accumulation and oxidative stress. The global objective and approach across these studies was to systematically examine the relative contributions of NOS and COX signaling pathways in mediating the acetylcholine (ACh)-stimulated endothelium-dependent relaxation (EDRF) and contractile (EDCF) activities of isometrically-mounted CCA in tissue baths in vitro, with a particular focus on elucidating the mechanisms of COX-mediated EDCF signaling activity. An added objective was to examine the in vivo hemodynamic characteristics of the CCA in each animal model investigated, serving both to identify the pressure-flow environment that the CCA is exposed to in vivo and to provide assessment of potential hypertension, aging, and oxidative stress effects on large artery hemodynamics. Key Findings: Study I hemodynamic analysis confirmed a hypertensive state in young adult SHR while also exposing a reduction in mean CCA blood flow in SHR compared to WKY accompanied by a multi-faceted pressure-flow interaction across the cardiac cycle relating to flow and pressure augmentation. Study III hemodynamic analysis found that neither aging nor chronic BSO-induced GSH depletion affected CCA blood pressure or blood flow parameters in SD rats. Study I and II demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from young adult SHR, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to WKY. Examining ACh-stimulated contractile function specifically from a quiescent (non pre-contracted) state revealed that EDCF activity did exist in WKY CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in SHR CCA could not fully suppress its >2-fold augmented EDCF activity vs. WKY CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the SHR-WKY CCA model revealed that the EDCF signaling activity was completely dependent on the COX-1 (but not COX-2) isoform of COX and was almost exclusively mediated by the thromboxane-prostanoid (TP) sub-type of the prostaglandin (PG) G-protein coupled receptor family and by Rho-associated kinase (ROCK), a down-stream effector of the molecular switch RhoA. Furthermore, it was found that while exogenous ROS-stimulated CCA contractile function was similarly >2-fold augmented in SHR vs. WKY and dependent on COX-1 and TP receptor and ROCK effectors, ACh-stimulated CCA EDCF signaling activity was only minimally affected by in-bath ROS manipulating compounds. Additional biochemical and molecular analysis revealed that ACh stimulation was associated with PG over-production from an over-expressed COX-1 in SHR CCA, and with CCA plasma membrane localization and activation of RhoA. Study III demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from Aging SD rats, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to Adult SD rats. Specific examination of ACh-stimulated contractile function revealed that EDCF activity did exist in Adult CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in Aging CCA could not fully suppress its >3-fold augmented EDCF activity vs. Adult CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the Adult-Aging SD rat CCA model revealed that EDCF signaling activity was completely dependent on COX-1, but while exogenous ROS was able to elicit a COX-dependent CCA contractile response, in-bath ROS manipulating compounds were found to be without effect on ACh-stimulated CCA EDCF signaling activity. Furthermore, biochemical analysis revealed that aging was not associated with a change in tissue (liver and vascular) GSH content or ROS accumulation. Chronic in vivo BSO treatment was effective in depleting tissue GSH content and increasing ROS accumulation, to a similar extent, in both Adult and Aging SD rats. However, regardless of age, neither ACh-stimulated NO-mediated EDRF signaling activity nor COX-mediated EDCF signaling activity were affected by these BSO-induced perturbations. Conclusions and Perspective: In the CCA of animals at the early pathological stages of either essential hypertension (young adult SHR) or normotensive aging (Aging SD rats), endothelial vasomotor dysfunction can be caused solely by over-active EDCF signaling, apparently disconnected from changes in NO bioavailability or oxidative stress. While NO and ROS may act, respectively, as negative and positive modulators of the established COX-PG-TP receptor-RhoA-ROCK cell-signaling axis mediating endothelium-dependent contractile activity, these factors do not appear to be essential to the mechanism(s) underlying the development of over-active EDCF signaling. Further elucidation of the cellular-molecular causes of over-active EDCF signaling, and its patho-biological consequences, in the SHR-WKY and Adult-Aging SD rat CCA models of EDCF activity established and hemodynamically characterized in this thesis, may help to identify new or more effective targets to be used in prevention or treatment strategies to combat the pathogenesis of CVD.

Orientador: Katashi Okoshi / Resumo: Introdução: A sobrecarga de pressão causada pela hipertensão arterial sistêmica (HAS) pode gerar mudança na arquitetura do colágeno, favorecer a fibrose, bem como o desbalanço entre a produção de espécies reativas de oxigênio (ERO) e a capacidade antioxidante. Aumento das ERO pode gerar ativação de vias sinalizadoras como a do fator nuclear kappa B (NF-kB) e das proteínas quinases ativadas por mitógenos (MAPK). Alterações dessas vias contribuem para o processo de remodelamento cardíaco causado pela HAS. O exercício físico desempenha importante papel na atenuação dos fatores de risco cardiovascular como a HAS. Dessa forma, o objetivo desse estudo foi avaliar a influência do treinamento físico sobre o remodelamento cardíaco de ratos espontaneamente hipertensos (SHR) na fase que antecede o desenvolvimento de insuficiência cardíaca. Métodos: Foram constituídos quatro grupos experimentais de ratos: normotensos Wistar (W) sedentários (W-SED, n=27); W exercitados (W-EX, n=31); SHR sedentários (SHR-SED, n=27); e SHR exercitados (SHR-EX, n=32). A partir de 13 meses de idade, os animais dos grupos exercitados foram submetidos a protocolo de exercício em esteira, cinco dias por semana, durante quatro meses. A avaliação estrutural e funcional in vivo do coração foi realizada por ecocardiograma. A função miocárdica in vitro foi avaliada em preparações de músculo papilar isolado do ventrículo esquerdo (VE). Amostras de tecido do VE foram obtidas para análises bioquímicas, histológicas e mo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: The pressure overload caused by systemic arterial hypertension (SAH) may change the collagen architecture, induce fibrosis, as well as imbalance between the reactive oxygen species (ROS) production and antioxidant capacity. Increased ROS leads to activation of signaling pathways such as nuclear factor kappa B (NF-kB) and mitogen-activated protein kinases (MAPK). Alterations in these pathways contribute to cardiac remodeling process induced by SAH. Physical exercise plays an important role in mitigating cardiovascular risk factors such as hypertension. Therefore, the aim of this study was to evaluate the influence of physical training, started before clinical evidence of heart failure, on cardiac remodeling in spontaneously hypertensive rats (SHR). Methods: Four experimental groups were used: sedentary (W-SED n=27) and trained (W-EX, n=31) normotensive Wistar rats, and sedentary (SHR-SED, n=27) and exercised (SHR-EX, n=32) hypertensive rats. Rats of the exercise groups underwent a protocol of treadmill exercise five days a week, for four months; exercise started at 13 months of age. Echocardiogram was performed to evaluate in vivo cardiac structures and function. In vitro myocardial function was analyzed in left ventricular (LV) papillary muscle preparations. LV tissue samples were obtained for biochemical, histological, and molecular analysis. Total myocardial collagen was assessed by histology and hydroxyproline quantification. Cardiomyocyte size was measured i... (Complete abstract click electronic access below) / Doutor

O Transtorno de Déficit de Atenção e Hiperatividade (TDAH) é um dos transtornos neuropsiquiátricos mais prevalentes da infância caracterizado pelos sintomas de desatenção, hiperatividade e impulsividade. O TDAH é uma desordem neurocomportamental heterogênea e fenotipicamente complexa e sua etiologia ainda não foi completamente esclarecida, mas sabe-se que a interação de fatores ambientais e genéticos e o acúmulo de seus efeitos possivelmente aumenta a vulnerabilidade ao transtorno. Nesse estudo, foram investigados o imunoconteúdo de proteínas sinápticas e do desenvolvimento a partir de neurônios da região do córtex pré-frontal de animais SHR, um dos modelos animais mais validados para o estudo do TDAH. Também foi realizada uma análise morfomética do padrão de desenvolvimento dessas células ao longo de diferentes dias in vitro e o papel do BDNF, fator neurotrófico crucial para a sobrevivência e maturação das sinapses, no desenvolvimento dos neurônios SHR. A análise do imunoconteúdo da SNAP-25 mostrou aumento nos níveis dessa proteína no 2º DIV e diminuição no 5º DIV nos neurônios SHR em relação ao controle WKY, sem alterações entre as cepas nos outros dias analisados. Em relação aos níveis de sinaptofisina nos neurônios SHR, foi observado aumento somente no 5º DIV. A análise do proBDNF mostrou diminuição nos neurônios SHR no 5º DIV e aumento no 8º DIV. A imunodetecção do CREB mostrou que os neurônios SHR apresentam níveis diminuídos dessa proteína somente no 1º DIV. O receptor TrkB também apresentou alterações no seu imunoconteúdo, com aumento no 2º DIV e diminuição no 5º DIV nos neurônios SHR. O imunoconteúdo do BDNF e do TrkB fosforilado não apresentaram alterações entre as linhagens nos dias analisados. Além disso, foi realizada uma análise morfométrica de diferentes parâmetros de desenvolvimento dos neurônios ao longo de diferentes dias in vitro por meio da marcação da proteína da região somatodendrítica MAP-2. Foi observada diminuição no comprimento total dos neuritos dos neurônios SHR no 5º DIV em relação aos neurônios WKY. Também foi verificado redução no número de raízes no 2º DIV e redução no número de pontos de ramificação no 5º DIV nos neurônios SHR. As alterações observadas em proteínas que são relacionadas aos processos de sinapses e de desenvolvimento neuronal podem auxiliar na compreenssão das diferenças encontradas no padrão de desenvolvimento dos neurônios SHR. Essas modificações a nível proteico podem estar alterando o crescimento e o padrão de arborização dendrítica e implicar em modificações na funcionalidade dessas células importantes para a melhor compreensão das bases neurobiológicas do TDAH. / Attention deficit hyperactivity disorder (ADHD) is one of the most common neuropsychiatric disorders of childhood characterized by symptoms of inattention, hyperactivity and impulsivity. ADHD is a heterogeneous and phenotypically complex neurobehavioral disorder with unknown etiology, but the interaction between environmental and genetic factors have been described to increase the vulnerability to the disorder. In this study, we investigated the immunocontent of synaptic and development proteins of prefrontal cortex neurons from one of the most validated animal models for the study of ADHD (SHR). We also performed a morphometric analysis along development of these cells at different days in vitro and the role of a neurotrofic factor (BDNF) in neuronal outgrowth. SNAP-25 immunocontent was increased at 2 DIV and decreased at 5 DIV in SHR neurons. Synaptophysin levels show increases only at 5 DIV in SHR neurons. The levels of proBDNF were decreased at 5 DIV and increased at 8 DIV in SHR neurons. CREB immunodetection showed that SHR neurons present decreased levels only at 1 DIV. The TrkB receptor also presented changes in immunocontent, with increase at 2 DIV and decrease at 5 DIV in the SHR neurons. Morphometric analysis during neuronal development by immunostaining with MAP-2 somatodendritic protein show decrease in total length at 5 DIV in SHR neurons in relation to WKY neurons. Besides that, SHR neurons exhibit reduction in number of roots at 2 DIV and number of branch points at 5 DIV. Changes in proteins related to synaptic processes and neuronal during development can help to understand differences found in the pattern of development of the neurons SHR. These changes at protein level may be altering neuronal outgrowth and dendritic arborization and possible involve modifications in functionality of these cells important for better understanding the neurobiological bases of ADHD.

The literature reports beneficial effects of aminoguanidine and its derivatives on the cardiovascular system. This work utilized strategies of Medicinal Chemistry, such as molecular hybridization and bio-ossification, for the synthesis of 14 aromatic aminoguanidine derivatives (DAGs), in order to investigate effective, safe and less effective adverse therapeutic alternatives for the treatment of hypertension. After pharmacological screening with the 14 DAGs, in order to select the most efficient derivative with respect to its cytotoxic activity and vasorelaxant effect, the work follows with the characterization of the cardiovascular effects induced by LQM01 in Spontaneously Hypertensive Rats (SHR), Through experimental approaches in vitro and in vivo, using the method of preparations with superior mesenteric artery isolated from rat and direct and indirect blood pressure measurement, respectively. The LQM01 derivative showed very low cytotoxic activity in MTT assays with reduced cell growth only at high concentrations. The derivative 01 also promoted a vasorelaxant effect similar to the Guanabenzo antihypertensive drug, a positive control, in rings pre-contracted with FEN, independently of endothelium. The observed effect showed a significant deviation of the response curve from LQM01 to the right, in precontracted FEN rings, in the presence of the 5 mM TEA blocker, a non-selective blocker of the K+ channels of the 4-AP blocker, a blocker (1mM) and TEA 1mM, a selective BKCa blocker, thus suggesting the participation of KV, BKCa in the vasorelaxation effect induced by LQM01. As in the depolarizing solution of KCl 80mM vasorelaxation was reduced, suggesting the inhibition of Ca2+ influx through the VOCCs, such hypothesis is justified by the attenuation of the CaCl2-induced contractions in the depolarizing solution nominally without Ca+2. In non-anesthetized SHR rats, i.v. in bolus administration of LQM01 induced dose-dependent hypotension with a more significant hypotensive effect at doses of 5 and 10 mg/kg, accompanied by an intense bradycardic effect. However, after CNS depression in the group of animals anesthetized with sodium thiopental, both the hypotensive and bradycardic responses induced by the derivative were attenuated or virtually abolished, respectively. In addition, the LMQ01 derivative was able to promote antihypertensive activity, at doses of 5 and 10 mg/kg, by its orogastric administration from the second hour, but at the fourth and sixth hour only at the highest dose, without Significant changes in HR at both doses. Conclusion: The data suggest that LQM01 promotes vasodilator effect through channel stimulation for K+, Kv and BKCa, besides inhibiting Ca2+ influx, possibly through blocking VOCCs, as well as promoting hypotensive, bradycardic and anti- Hypertensive, which may be due to a direct action of the substance on the MLV, inducing vasorelaxation and consequent reduction of peripheral vascular resistance, contributing to its hypotensive action, but may also have an inhibitory influence on the neural control system that acts on the cardiovascular system. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Literatura relata efeitos benéficos da aminoguanidína e seus derivados sobre o sistemacardiovascular. Este trabalho utilizou-se de estratégias da Química Medicinal, como hibridação molecular e bioisosterismo, para síntese de 14 derivados aminoguanidínicos (DAGs) aromáticos, com o propósito de pesquisar alternativas terapêuticas eficazes, seguras e com menos efeitos adversos para o tratamento da hipertensão. Após triagem farmacológica com os 14 DAGs, a fim de selecionar o derivado com melhor eficácia, no que se refere a sua atividade citotóxica e efeito vasorelaxante, o trabalho segue com a caracterização dos efeitos cardiovasculares induzidos por LQM01 em Ratos Espontaneamente Hipertensos (SHR), através de abordagens experimentais in vitro e in vivo, utilizando o método de preparações com artéria mesentérica superior isolada de rato e medida direta e indireta pressão arterial, respectivamente. O derivado LQM01 apresentou em ensaios de MTT, atividade citotóxica muito baixa, com redução do crescimento celular apenas em altas concentrações. O derivado 01 também promoveu efeito vasorelaxante semelhante ao fármaco anti-hipertensivo Guanabenzo, controle positivo, em anéis pré-contraídos com FEN, de maneira independente de endotélio. O efeito observado apresentou um desvio significativo da curva-concentração resposta de LQM01 para direita, em anéis pré-contraídos com FEN, na presença do bloqueador TEA 5 mM, um bloqueador não-seletivo dos canais de K+ do bloqueador 4-AP, um bloqueador seletivo dos KV (1mM) e TEA 1mM, bloqueador seletivo dos BKCa sugerindo assim a participação dos KV , BKCa no efeito vasorelaxante induzido por LQM01. Assim como, em solução despolarizante de KCl80mM o vasorelaxamento mostrou-se reduzido, sugerindo a inibição do influxo de Ca2+ através dos VOCCs, tal hipótese é justificada pela atenuação das contrações induzidas por CaCl2 em solução despolarizante nominalmente sem Ca+2. Em ratos SHR não-anestesiados, a administração i.v. in bolus de LQM01 induziu hipotensão, de maneira independente de dose, com efeito hipotensor mais significativo nas doses de 5 e 10 mg/kg, acompanhado de efeito bradicárdico intenso. No entanto, após depressão do SNC no grupo de animais anestesiados com tiopental sódico, tanto a resposta hipotensora quanto a bradicárdica induzida pelo derivado foi atenuada ou praticamente abolida, respectivamente. Além disto, o derivado LMQ01 foi capaz de promover atividade anti-hipertensiva, nas doses de 5 e 10 mg/kg, a por meio de sua administração orogástrica a partir da segunda hora, porém na quarta e sexta hora apenas na maior dose, sem promover alterações significativas sobre a FC, em ambas as doses. Em conclusão: Os dados sugerem que o LQM01 promove efeito vasodilatador através da estimulação de canais para K+, Kv e BKCa, além de inibir o influxo de Ca2+, possivelmente através do bloqueio dos VOCCs, bem como, promover efeito hipotensor, bradicárdico e anti-hipertensivo, que pode ser devido a uma ação direta da substância sobre a MLV, induzindo vasorelaxamento e consequente redução da resistência vascular periférica, contribuindo para sua ação hipotensora, como também pode ter influência inibitória sobre o sistema de controle neural que atua sobre o sistema cardiovascular.