Dissertations / Theses on the topic 'Non genomic effects'

Nandrolone and other anabolic androgenic steroids (AAS) at elevated concentration can alter the expression and function of neurotransmitter systems and contribute to neuronal cell death. This effect can explain the behavioural changes, drug dependence and neuro degeneration observed in steroid abuser. Nandrolone treatment (10-8M–10-5M) caused a time- and concentration-dependent downregulation of mu opioid receptor (MOPr) transcripts in SH-SY5Y human neuroblastoma cells. This effect was prevented by the androgen receptor (AR) antagonist hydroxyflutamide. Receptor binding assays confirmed a decrease in MOPr of approximately 40% in nandrolonetreated cells. Treatment with actinomycin D (10-5M), a transcription inhibitor, revealed that nandrolone may regulate MOPr mRNA stability. In SH-SY5Y cells transfected with a human MOPr luciferase promoter/reporter construct, nandrolone did not alter the rate of gene transcription. These results suggest that nandrolone may regulate MOPr expression through post-transcriptional mechanisms requiring the AR. Cito-toxicity assays demonstrated a time- and concentration dependent decrease of cells viability in SH-SY5Y cells exposed to steroids (10-6M–10-4M). This toxic effects is independent of activation of AR and sigma-2 receptor. An increased of caspase-3 activity was observed in cells treated with Nandrolone 10-6M for 48h. Collectively, these data support the existence of two cellular mechanisms that might explain the neurological syndromes observed in steroids abuser.

Nandrolone and other anabolic androgenic steroids (AAS) at elevated concentration can alter the expression and function of neurotransmitter systems and contribute to neuronal cell death. This effect can explain the behavioural changes, drug dependence and neuro degeneration observed in steroid abuser. Nandrolone treatment (10-8M–10-5M) caused a time- and concentration-dependent downregulation of mu opioid receptor (MOPr) transcripts in SH-SY5Y human neuroblastoma cells. This effect was prevented by the androgen receptor (AR) antagonist hydroxyflutamide. Receptor binding assays confirmed a decrease in MOPr of approximately 40% in nandrolonetreated cells. Treatment with actinomycin D (10-5M), a transcription inhibitor, revealed that nandrolone may regulate MOPr mRNA stability. In SH-SY5Y cells transfected with a human MOPr luciferase promoter/reporter construct, nandrolone did not alter the rate of gene transcription. These results suggest that nandrolone may regulate MOPr expression through post-transcriptional mechanisms requiring the AR. Cito-toxicity assays demonstrated a time- and concentration dependent decrease of cells viability in SH-SY5Y cells exposed to steroids (10-6M–10-4M). This toxic effects is independent of activation of AR and sigma-2 receptor. An increased of caspase-3 activity was observed in cells treated with Nandrolone 10-6M for 48h. Collectively, these data support the existence of two cellular mechanisms that might explain the neurological syndromes observed in steroids abuser.

Negli ultimi anni si è assistito ad un crescente aumento nell’ambiente di sostanze che sono in grado di alterare il sistema endocrino poiché agiscono come l’ormone naturale estrogeno e per questo definite distruttori endocrini (EDs). Si ipotizza che l’esposizione agli EDs durante il periodo fetale e neo-natale sia la causa dell’insorgenza di numerosi disordini dell’apparato riproduttivo maschile come sterilità, cancro del testicolo, criptorchidismo e ipospadia che vengono definite con il termine unico di sindrome del testicolo disgenico (TDS). E’ importante studiare gli effetti degli estrogeni e degli xenoestrogeni, una classe di EDs, durante il periodo fetale ed in particolare durante lo sviluppo dell’apparto riproduttivo e conoscere i meccanismi, con cui tali sostanze esplicano i loro effetti. OBIETTIVI. Verificare l'espressione dei recettori degli estrogeni (ERs) nei precursori dei gameti adulti, ovvero nelle cellule germinali di topo denominate PGCs e analizzare i pathways molecolari attivati in queste cellule dagli estrogeni e dallo xenoestrogeno lindano. Inoltre si vuole verificare la presenza di ERs funzionali nelle cellule somatiche testicolari embrionali utilizzando costrutti ERE-luc e AP1-Luc per valutare l'attività estrogenica di xenoestrogeni. RISULTATI. Lo studio condotto in questa tesi mette in evidenza l'esistenza di pathways molecolari attivabili dall’estrogeno (E2) nelle gonadi embrionali di topo, in particolare, nel testicolo, sia nelle cellule germinali primordiali che nelle cellule somatiche. Abbiamo osservato che l’E2 è in grado di attivare, attraverso il recettore degli estrogeni ERα , importanti chinasi nelle PGCs (AKT, ERK1/2 e SRC) con effetti positivi sulla crescita e proliferazione di tali cellule. Si è osservato che il lindano, al contrario dell’E2, influenza negativamente la sopravvivenza di tali cellule attraverso una azione pro-apoptotica diretta, probabilmente derivante da effetti negativi sull’attività chinasica AKT. Inoltre abbiamo descritto per la prima volta l'esistenza di un recettore dell’estrogeno funzionale nelle cellule del Leydig testicolari durante lo stadio precoce dello sviluppo e messo a punto un test in vitro che può essere utilizzato per valutare l'attività estrogenica di xenoestrogeni direttamente sulle cellule del testicolo embrionale di mammiferi. CONCLUSIONI: Questi risultati rafforzano l’ipotesi dell’origine fetale della TDS. Numerosi studi hanno messo in evidenza gli effetti diretti degli estrogeni sul sistema endocrino, sull'espressione genica e su funzioni specifiche delle cellule somatiche testicolari embrionali, in particolare sulle cellule del Leydig, tuttavia tali studi sono carenti nelle cellule germinali. E’ importante conoscere i meccanismi di azione degli xenoestrogeni sullePGCs visto che attraverso esse viene trasmesso il genoma alle generazioni successive.
In the recent years the increased presence of human made compounds that mimic the action of estrogens termed endocrine disrupters (ED) in environment and in food and the exposure to these compounds during fetal and neonatal period has been hypotized to be the cause of the raise of disorders of male reproductive function, such a decrease of sperm count, increase in the incidence of testicular cancer and cryptorchidism and hypospadias termed Testicular Dysgenesis Syndrome (TDS). For these reason, it is important to know how the estrogens and xenoestrogens, a class of ED, act during the fetal development and to know the mechanism by which these compounds exert their effects. AIMS: To study the expression of estrogen receptors (ERs) in the embryonic precursors of the adult gametes termed PGCs and to analyze the existence in such cells of intracellular molecular pathways modulable by estrogens and xenoestrogen lindane. To verify the presence of functional ER-beta in embryonic testicular somatic cells using an ERE-luc and AP1-Luc assay and to evaluate estrogenic activity of putative EDs on mammalian embryonic testis. RESULTS: The data described in this thesis highlights the existence of functional estrogen-dependent pathways in embryonic mouse gonads in particular in testis, both in germ and somatic cells. We found that E2 is able to activate via ER-beta multiple intracellular signalling in PGCs and that the xenoestrogens, lindane affect the survival in such cells through a direct pro-apoptotic action likely resulting from its adverse effect on AKT activity. Othermore, we described for the first time the existence of a functional ERα pathway in putative Leydig cells from early stage of testis development and describe an in vitro assay that can be used to evaluate estrogenic activity of compounds on mammalian embryonic testis. CONCLUSIONS: These results support the notion of the TDS origin during early stages of testis development. While data are accumulating showing direct effect of estrogens and EDs on gene expression and specific functions of somatic cells of the embryonic testes, in particular Leydig cells, such results on germ cells are lacking and further studies are needed to investigate the effects of these compounds on embryonic germ cell function including epigenetic regulation.

Cette thèse explore et développe des méthodes pour exploiter les effets génétiques de dominance ou/et d'épistasie sur des modèles de sélection génomique dans les schémas de sélection utilisant le croisement chez le porc et le maïs. Le Chapitre 2 a consisté à estimer et à exploiter la variance de dominance intra-race à travers des stratégies de planification des accouplements pour maximiser le progrès génétique global de l’âge à 100 kg (AGE), de l’épaisseur de lard dorsal (BD) et du poids moyen des porcelets par portée (APWL), dans une population porcine de race Landrace française. La maximisation de la valeur génétique totale au lieu de la seule partie additive dans la planification des accouplements a donné à la descendance un avantage moyen de -0,79 jour, -0,04 mm et 11,3 g pour AGE, BD et APWL, respectivement. En contrepartie, le gain génétique additif attendu a légèrement été réduit (1,8% pour AGE par exemple). Ces résultats indiquent que la planification des accouplements peut améliorer les performances des descendants sans compromettre considérablement le progrès génétique. Dans le Chapitre 3, l'efficacité de la planification des accouplements et de l’évaluation génomique, en tenant compte des effets additifs et de dominance, pour améliorer les performances des individus croisés (CB) a été étudiée par simulation dans un croisement à deux voies chez le porc. Les effets de l’utilisation de différentes sources d'informations dans l'évaluation génétique (uniquement des données de race pure (PB) ou des données PB et CB), de plusieurs valeurs d'héritabilité au sens étroit et large, et de plusieurs stratégies d’accouplement pour produire les animaux CB (accouplements au hasard, minimisant la consanguinité future ou maximisant la valeur génétique totale attendue des animaux CB) ont été évaluées. La sélection des animaux PB sur leurs performances en PB a donné un gain génétique de 0,2 écart-type génétique par génération pour le caractère « performance en croisement ». Ce gain a été doublé lorsque les animaux PB étaient sélectionnés sur leur performance en croisés. Les stratégies d’accouplement a entraîné une légère augmentation des performances des animaux CB. Lorsque la corrélation génétique entre les performances exprimées chez les animaux PB et CB est faible, la sélection des animaux PB pour leur performance en croisés en utilisant les informations CB est une stratégie plus efficace pour exploiter l'hétérosis et augmenter les performances des animaux CB au niveau commercial. Dans le Chapitre 4, la théorie des modèles d'évaluation génétique chez des hybrides à partir du croisement de lignées pures (comme pour le maïs) a été revue dans un contexte génomique. La covariance entre les hybrides due aux effets de substitution additifs, à la dominance et à l’épistasie a été dérivée analytiquement. En utilisant les marqueurs SNP, il est possible de décomposer l’aptitude spécifique à la combinaison (SCA) en dominance et épistasie intergroupes, et de décomposer les aptitudes générales à la combinaison (GCA) en effets additifs intra-lignée et une épistasie additive par additive intra-ligne. Un jeu de données publiques sur des hybrides Dent × Flint a été analysé. Le modèle proposé a été comparé à d'autres modèles génomiques en termes d'estimations des composantes de variance et de capacité prédictive, y compris un modèle supposant un effet commun des gènes des lignées pures. L'étude confirme que la majeure partie de la variabilité observée chez les hybrides est expliquée par la GCA et que les variances dues à la dominance et à l'épistasie sont moins importants et du même ordre de grandeur. Les modèles basés sur la définition d’effets différents (comme traditionnellement considérés chez le maïs), ou commun aux origines (comme considérés intra-race en sélection animale) ont abouti à des capacités prédictives similaires pour les hybrides
This thesis explores and develops methodology to exploit dominance or/and epistasis genetic effects on genomic selection models in pig and maize crossbreeding schemes. The Chapter 2 consisted of estimating and exploiting within-breed dominance variance through mate allocation strategies to maximize the overall genetic merit of the traits age at 100 Kg (AGE), backfat depth (BD) and average piglet weight per litter (APWL), in a French Landrace pig population. Maximizing total genetic values instead of breeding values in matings gave to the progeny an average advantage of 0.79 days, 0.04 mm, and 11.3 g for AGE, BD and APWL, respectively, but slightly reduced the expected additive genetic gain (e.g. 1.8 % for AGE). These results indicate that genomic mate allocation can improve the performance of the offspring without dramatically compromising the additive genetic gain. In Chapter 3, the effectiveness of mate allocation strategies and genomic evaluations, accounting for additive and dominance effects, to improve crossbred (CB) performance were investigated by simulation in a two-way pig crossbreeding scheme. Effects of the sources of information used in the genetic evaluation (only purebred (PB) data or PB and CB data), of several narrow and broad-sense heritability values, and of several options for mate allocation to produce the CB (mating at random, minimizing expected future inbreeding, or maximizing the expected total genetic value of crossbred animals) were evaluated. Selecting PB animals for PB performance yielded a genetic gain of 0.2 genetic standard deviations of the trait “CB performance” per generation, whereas selecting PB animals for CB performance doubled the genetic response. Mate allocation strategy resulted in a slight increase of the CB performance. When the genetic correlation between PB and CB is low, selecting PB animals for CB performance using CB information is a more efficient strategy to exploit heterosis and increase performance at the CB commercial level. In Chapter 4, the theory of hybrid genetic evaluation models from single-cross of pure lines (as in maize) was revisited in a genomic context. Covariance between hybrids due to additive substitution effects and dominance and epistatic deviations were analytically derived. Using SNP genotypes, it is possible to split specific combining ability (SCA) into dominance and across-groups epistasis, and to split general combining ability (GCA) into within-line additive effects and within-line additive by additive epistasis. A publicly available maize data set of Dent × Flint hybrids was analyzed. The proposed model was compared to other genomic models in terms of variance components estimation and predictive ability, including a model assuming a common effect of genes across origins. The study confirms that most variation in hybrids is accounted for by GCA, and that variances due to dominance and epistasis are small and have similar magnitudes. Models based on defining effects either differently (as it is traditionally done in maize) or identically across origins (as it is done in single breeds in livestock) resulted in similar predictive abilities for hybrids

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
A predição do mérito genético dos indivíduos é um dos maiores desafios no melhoremento de plantas e animais. A predição é difícil por que as características importantes possuem natureza complexa, onde alguns caracteres possuem poucos genes de efeito maior, enquanto que outros são controlados por um elevado número de genes de efeito pequeno, além disso, efeitos não-additivos como dominância e epistasia podem ser importantes para o controle da variação genética. Para obter altas acurácias na predição é importante usar o modelo que corresponde com a arquitetura genética da característica e adicionalmente a adequada partição das várias fontes de variação genética (aditiva, dominancia e epistasia) é desejada para várias aplicações como capacidade geral e específica de combinação. No capítulo 1 foi revisado os aspectos gerais da predição genômica (GP), a aplicação dessa abordagem com diferentes propósitos em características com distintas arquiteturas genéticas e no final alguns modelos estatísticos aplicado na GP. No capítulo 2 foi avaliado modelos de regressão genômica (WGR) aditivos e aditivo-dominante com diferentes prioris, essas são premissas sobre a presença ou não de marcas com efeito maior. Adicionalmente no capítulo 3 foi avaliado a inclusão da informação oriunda do pedigree na predição genômica, usando os modelos BayesA aditivo e aditivo-dominante e também com o RKHS, que teoricamente pode predizer os efeitos aditivo e não aditivos confundidos. Esses modelos foram aplicados na altura de árvores (HT) aos 6 anos de idade, diâmetro na altura do peito (DBH) e resistência a ferrugem, mesurados em 923 indivíduos de pinos oriundos de uma população estruturada em 71 irmãos completos e genotipados com 4722 marcadores genéticos. Também foram simulados 6 características com distintas arquiteturas genéticas (poligenica e oligogênica com três leveis de dominância) para esses estudos. As populações simuladas usadas nessas características foram derivadas a partir de um programa de melhoramento padrão de pinos. No capítulo 2 para as caracteríticas oligogenica simuladas e para resistência a ferrugem o BayesA e BayesB forneceram as melhores acurácias para predição genotípica, porem as diferentes priores usadas em WGR produziram resultados similares para HT e para característica poligênicas simuladas. Contudo a inclusão da dominância nos modelos WGR aumentaram a acurácia apenas para características simuladas com elevado efeito de dominância e para HT. Quando o BayesB foi ajustado em uma geração para predizer na geração seguinte, a inclusão da dominância aumentou as acurácias apenas para características oligogenicas simuladas com elevada dominancia. Independente do modelo adotado, a acurácia da predição genotípica total decresceu com o aumento dos efeitos de dominancia nas características simuladas. Então esses resultados refletem que a predição da dominancia foi complexa quando comparado com a predição dos efeitos aditivos, e para a aplicações posteriores dos efeitos de dominância, algumas propriedades genéticas da população devem ser avaliadas como MAF e número de meios irmãos e irmãos completos. No capítulo 3, a inclusão do informação oriunda do pedigree no modelo genômico, não produziu acurácias mais elevadas quando comparado com os modelos que usaram apenas informações de marcadores, e ambos modelos foram substancialmente mais acurados que o modelo baseado apenas em informação de pedigree. Em HT, DBH e características poligênicas simuladas com efeitos aditivos e dominantes, os modelos baseados em RKHS mostraram acurácias ligeiramente superiores que o BayesA para predição genotípica total, enquanto que o BayesA foi a melhor opção para resistência a ferrugem e características oligogenicas. Para a predição dos valores de melhoramento o BayesA aditivo foi o melhor modelo.
The prediction of individual genetic merit is one of most important challenges in plant and animal breeding. Prediction is difficult because the important traits have a complex nature, where some traits have few genes with major effects, while others are controlled by a large number of genes with small effects. Non-additive effects such as dominance and epistasis can also be important for controlling the genetic variation. In order to achieve higher accuracies in the prediction, it is important to use the model that matches the genetic architecture of trait. The proper partition of the various sources of genetic variation (additive, dominance and epistasis) is desired for several applications, such as exploring the overall and specific combination ability. In Chapter 1, the general remarks of genomic prediction (GP) are reviewed, with the application of this approach with different proposals in distinct genetic architecture traits, together with some statistic models applied in GP. In Chapter 2, the additive and additive-dominance whole-genomic-regression (WGR) models are evaluated with different priors, together with assumptions regarding the presence or not of markers with major effects. Chapter 3 evaluates the inclusion of pedigree information in genomic prediction with additive- and additive-dominance BayesA and also with RKHS model that can theoretically predict confused additive and non- additive effects. These models were applied in tree height (HT), diameter at breast height (DBH) and rust resistance in 923 loblolly pine individuals at 6 years of age from a structured population of 71 full-sib families genotyped with 4722 genetic markers. Six traits were also simulated with distinct genetic architectures (polygenic and oligogenic traits with three dominance levels) for these studies. The simulated population for these traits was derived from a standard pine breeding program. In the oligogenic simulated traits and rust resistance in chapter 2, BayesA and BayesB provided greater accuracies for genotypic prediction; however, the different priors of WGR yielded similar results for HT and simulated polygenic traits. Therefore, the inclusion of dominance effects in WGR increases the accuracy only for simulated traits with high dominance effects and HT. When BayesB was fitted in one generation for predicting the next generation, the dominance inclusion increased the accuracies only for the oligogenic simulated trait with high dominance. Regardless of the model adopted, the accuracy of whole genotypic prediction decreased with the increase of dominance effects in simulated traits. Thus, these results reflect that dominance prediction is complex when compared to additive prediction, and for downstream applications of dominance effects, some genetic properties of the population should be evaluated, such as MAF and the number of half and full-sibs. In chapter 3, the inclusion of pedigree information in genomic model did not yield higher accuracies than models based in only marker information, and both models were substantially more accurate than models basedonly on pedigree. In HT, DBH and in polygenic traits simulated with additive-dominance effects, the RKHS-based models showed slightly higher accuracies than BayesA for whole genotypic prediction, while BayesA-based models were the best option for rust resistance and oligogenic simulated traits. For the prediction of breeding values, the BayesA additive was the best model.

The neurosteroids constitute a group of powerful hormones synthesized and acting in the central nervous system. They participate in a number of important central processes, such as memory and learning, mood and neuroprotection. Their effects emerge from rapid interactions with membrane bound receptors, such as the N-methyl-D-aspartate (NMDA) receptor, the gamma-amino-butyric acid receptor and the sigma 1 receptor. The mechanisms of action are separate from classical genomic interactions.

The aims of this thesis were to identify and characterize the molecular mechanisms underlying the effects of nanomolar concentrations of neurosteroids at the NMDA receptor.

The results show that the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate 3α5βS) differently modulate the NMDA receptor, changing the kinetics for the NMDA receptor antagonist ifenprodil, through unique and separate targets at the NR2B subunit. The effects that appear to be temperature independent were further confirmed in a calcium imagining functional assay. A second functional study demonstrated that PS and 3α5βS affect glutamate-stimulated neurite outgrowth in NG108-15 cells.

Misuse of anabolic androgenic steroids (AAS) has powerful effects on emotional states. Since neurosteroids regulate processes involved in mood it can be hypothesised that AAS can interact with the action of neurosteroids in the brain. However, chronic administration of the AAS nandrolone decanoate did not alter the allosteric effects of PS or 3α5βS at the NMDA receptor, but changed the affinity for PS, 3α5βS and dehydroepiandrosterone sulfate to the sigma 1 receptor. The results also showed that the neurosteroids displace ³H-ifenprodil from the sigma 1 and 2 receptors without directly sharing the binding site for ³H-ifenprodil at the sigma 1 receptor. The decreased affinity for the neurosteroids at the sigma 1 receptor may be involved in the depressive symptoms associated with AAS misuse.

The NMDA receptor system is deeply involved in neurodegeneration and the NMDA receptor antagonist ifenprodil exert neuroprotective actions. The findings that neurosteroids interact with ifenprodil at the NMDA receptor may be an opportunity to obtain synergistic effects in neuroprotective treatment.

Thiazolidinediones such as rosiglitazone are used in the treatment of Type-2 Diabetes, and are ligands for peroxisome proliferator-activated receptor-gamma (PPARγ), a ligand-activated transcription factor that regulates expression of genes involved in glucose and lipid metabolism. However, rosiglitazone is known to exert PPARγ-independent effects alongside its classical receptor-dependent effects. This study investigated the PPARγ-independent effects of rosiglitazone on intracellular calcium (Ca2+i) signalling in cultured monocytic and vascular smooth muscle cells Rosiglitazone rapidly (5-30min) inhibited the Ca2+ sequestration activity of the ER-resident Ca2+ pump enzyme SERCA2b in a dose-dependent manner (IC50~2μM). 10μM Rosiglitazone triggered rapid increases in [Ca2+]i; however, restoration to basal levels occurred within 72h. Consequently, cell viability was not adversely affected by rosiglitazone treatment. Initiation of the unfolded protein response (UPR) was identified as the mechanism underpinning rosiglitazone's Ca2+ homeostatic restorative properties. Rosiglitazone induced alternate splicing of the UPR transcription factor XBP-1, which led to increased mRNA and protein expression of SERCA2b (shown via bioinformatics analysis to be a UPR target gene), and increased ER Ca2+-ATPase activity in rosiglitazone-treated cells. In tissue (rabbit aortic ring) samples, 10μM rosiglitazone induced transient (within 1h) non-significant losses in vasorelaxatory sensitivity to sodium nitroprusside, but extended treatment (4-24h) increased sensitivity beyond that of vehicle-treated samples. Thus, at cell and tissue levels, this data suggests that rosiglitazone initially causes increased [Ca2+]i due to inhibition of SERCA2b, but extended incubation induces - via upregulation of UPR target genes - the restoration of Ca2+ homeostasis, and possibly even improvements in function in some contexts. Clearly, the data presented in this in vitro study must be treated tentatively, and more research is needed before these potentially beneficial effects can be firmly identified as being clinically significant. Nevertheless, the data obtained here may constitute preliminary evidence that, alongside its PPARy-dependent effects, rosiglitazone may exert functional improvements on the vasculature.

Tout d'abord, nous avons démontré l'origine périphérique de la corticostérone après l'administration d'un stress aigu. Pour cela, nous avons utilisé un modèle de souris déficient en transporteur de corticostérone : Corticosterone binding-globulin (Cbg-/-). Ensuite, nous avons déterminé si les effets rapides du stress sur le rappel mnésique dépendaient de mécanismes non-génomiques. Nous avons précisé si ces effets étaient médiés par les récepteurs aux minéralocorticoïdes (MR) ou aux glucocorticoïdes de l'hippocampe. Dans ce but dans un premier temps, nous avons injecté un complexe macromoléculaire de corticostérone (Cort-3CMO-BSA) qui ne franchit pas la membrane cellulaire pour évaluer l'implication de mécanismes membranaires. Dans un deuxième temps nous avons administré dans l'hippocampe dorsal (HD) ou ventral (HV), 15 minutes avant le stress, l'antagoniste MR (RU 28318) et l'antagoniste GR (RU 38486) et nous avons évalué les performances mnésiques à 15, 60, 105 et 120 minutes après le stress. En effet ces délais ont été choisis selon l'apparition de pics de corticostérone induit par le stress, mesurés par microdialyse, dans l'HD et l'HV.Les principaux résultats obtenus sont : i) les souris Cbg -/- ne présentent pas de déficit mnésique 15 min après l'administration d'un stress aigu, contrairement aux souris contrôles qui ont un déficit mnésique important; ii) De même, l'administration de métyrapone (un inhibiteur de synthèse de la corticostérone) prévient des effets rapides du stress sur la mémoire; iii) Nous avons démontré que les effets rapides délétères sont médiés par des récepteurs membranaires, puisque l'injection de Cort-3CMO-BSA dans l'HD produit des effets similaires au stress aigu. De plus, l'effet de l'injection du complexe Cort-3CMO-BSA n'est pas bloqué par l'injection systémique d'anisomycine (un inhibiteur de synthèse protéique) nous avons montré que les récepteurs membranaires aux glucocorticoïdes de type MR sont responsables des effets cognitifs rapides du stress et de la cort-3CMO-BSA sur le rappel mnésique ;iv) Dans l'HD, l'injection du RU 28318 bloquait les effets délétères du stress quand les performances mnésiques étaient évaluées 15 min après le stress, mais non aux délais plus longs. Au contraire, le RU 38486 prévenait les déficits mnésiques quand les performances étaient évaluées à 60 mais non à 105 min après le stress. Dans l'HV, le schéma opposé est observé puisque l'injection du RU 38486 est dénuée d'effet quand il est injecté à 60 min après le stress mais il bloque les déficits mnésiques induits 105 min après le stress. L'implication des récepteurs MR et GR et l'efficacité de leur antagoniste semble dépendant de l'évolution de la concentration de corticostérone au cours du temps dans l'HD et l'HV.Pour conclure, notre étude a mis en évidence que le stress aigu diminue le rappel mnésique hipocampo-dépendant par l'intermédiaire d'un mécanisme de "switch" impliquant les récepteurs MR puis GR de l'HD à des délais plus courts et ensuite seulement les récepteurs GR de l'HV à des délais plus long.

The use of molecular markers to predict non-tested materials in field trials has been extensively employed in breeding programs. The genomic prediction of single crosses is a promising approach in maize breeding programs as it reduces selection cycle and permits the selection of promising crosses. Accounting for non-additive effects on genomic prediction can increase prediction accuracy of models depending on the traits genetic architecture. Genomic prediction was first developed for single environments andrecently extended to exploit the genotype by environment interactions for prediction of non-evaluated individuals. The employment of multi-environment genomic models is advantageous in several aspects and has enabled significant higher prediction accuracies than single environment models. However, only a small number of studies regarding the inclusion of non-additive effects in these models are reported. Moreover, the genotype by environment interaction complexity can largely impact the prediction accuracyof these models. Thus, the objectives were to i)evaluate the contribution of additive and non-additive (dominance and epistasis) effects for the prediction of agronomical traits with different genetic architecture in tropical maize single-crosses grown under two nitrogen regimes (ideal and stressing), and ii)verify the impact of the genotype by environment interaction complexity, and the inclusion of dominance deviations, on the prediction accuracy of hybrids grain yield using a multi-environment prediction model. For this, we used phenotypic and genotypic data of 906 single-crosses evaluated during two years, at two locations, under two nitrogen regimes, totaling eight contrasting environments (combination of year x locations x nitrogen regimes). The traits considered in the study were grain yield, ear, and plant height. The results regarding the inclusion of additive and non-additive effects (dominance and epistasis) in genomic prediction models suggest that non-additive effects play an important role instressing conditions, having a high, medium and low contribution for phenotypic expression of grain yield, plant height, and ear height, respectively. The inclusion of dominance deviations in multi-environment prediction model increases the prediction accuracy. Furthermore, a linear relationship between genotype by environment complexity and prediction accuracywas found.
O uso de marcadores moleculares para a predição do fénotipo de materiais não testados em campo tem sido amplamente utilizado em programas de melhoramento genético de plantas. A predição genômica de hibridos simples é uma ferramenta promissora no melhoramento genético do milho, pois além da redução do tempo necessário para cada ciclo de seleção, ela pode ser utilizada para a identificação de cruzamentos promissores. Dependendo da característica em estudo, a inclusão de efeitos não aditivos em modelos de predição genômica pode aumentar significativamente sua acurácia de predição. Além disso, estes modelos foram inicialmente propostos para a predição de materiais em apenas um único ambiente. Atualmente, foram expandidos para considerarem os efeitos da interação genótipos por ambiente. O uso de tais modelos têm se mostrado vantajoso em vários aspectos, um deles é o considerável aumento da acurácia de predição de novos materiais. Contudo, ainda são escassos estudos envolvendoa inclusão de efeitos não aditivos nesses modelos. Ademais, fatores como a complexidade da interação genótipo por ambiente pode influenciar de maneira significativa a acurácia preditiva de modelos considerando múltiplos ambientes. Portanto, os objetivos foram: i)avaliar a contribuição de efeitos aditivos e não aditivos (dominância e epistasia) para a predição de caracteres agronômicos com diferentes arquiteturas genéticas em cruzamentos simples de milho tropical cultivados sob dois níveis de disponibilidade de nitrogênio (ideal e estressado), e ii)verificar o impacto da complexidade da interação genótipo por ambiente, e da inclusão de desvios de dominância na acurácia de predição de modelos multi-ambientes para a predição da produtividade grãos de híbridos simples de milho. Para isto, foram utilizados os dados fenótipicos e genotípicos de 906 híbridos simples de milho avaliados durante dois anos, em dois locais, sob dois níveis de adubação nitrogenada, totalizando oito ambientes distintos (combinação ano xlocal x nivel de adubação nitrogenada). Os caracteres estudados foram produtividade de grãos, altura de espiga, e plantas. Os resultados acerca da inclusão de efeitos aditivos e não aditivos (dominancia e epistasia) sugerem que, efeitos não aditivos são mais importantes sob condições de estresse, contribuem de maneira significativa para produtividade grãos, de modo intermediário para altura de plantas e possuem pouca importância para altura de espiga. A inclusão de desvios de dominância em modelos de predição multi-ambientes aumentou de forma significativa a acurácia de predição. Além disto, observou-se uma relação linear entre complexidade da interação genótipos por ambientes e acurácia preditiva do modelo.

Qualitativ unterschiedliche genomische und nichtgenomische Mechanismen vermitteln die starken anti-inflammatorischen und immunmdulatorischen Eigenschaften der Glucocorticoide (GC). Der genomisch vermittelte Mechanismus ist bereits gut untersucht und dokumentiert, während der nichtgenomisch vermittelte Mechanismen noch einen Gegenstand vielseitiger Untersuchungen darstellt. Wir haben uns daher die Frage gestellt, ob Beclometason und Clobetasol besonders geeignet für die topische Applikation sind, weil sie sich in ihrem Wirkungsspektrum von systemisch zu applizierenden GC wie Dexamethason unterscheiden. Wir verglichen dazu die Effekte auf den Sauerstoffverbrauch mittels der Clark-Elektrode (nichtspezifisch nichtgenomischer Mechanismus), auf die IL-6-Synthese mittels ELISA (genomischer Mechanismus) und auf die Apoptose mittels Durchflusszytometrie (nichtgenomischer und genomischer Mechanismus) in ruhenden und stimulierten humanen PBMC. Dabei zeigten Beclometason und Clobetasol in sehr niedrigen Konzentrationen (10-10, 10-8 M) einen stärkeren Effekt auf den Sauerstoffverbrauch, waren aber in hohen Konzentrationen (10-5, 10-4 M) weniger potent im Vergleich zu Dexamethason. Auch hinsichtlich ihrer genomischen Potenz waren die topischen GC in einer Konzentration von 10-10 M und 10-8 M effektiver als Dexamethason, in höheren Konzentrationen unterschieden sie sich aber nicht. Alle drei GC induzierten Apoptose konzentrationsabhängig und unterschieden sich nicht in Konzentrationen zwischen 10-8 M und 10-5 M. In einer Konzentration von 10-4 M war die Induktion von Apoptose durch die topischen GC in PBMC und Jurkat-T-Zellen aber signifikant stärker im Vergleich zu Dexamethason. Diese Ergebnisse zeigen, dass sich topische und systemische GC in ihrer genomischen und nichtgenomischen Potenz signifikant unterscheiden. Es ist daher davon auszugehen, dass nichtgenomische Effekte eine deutlichere klinische Relevanz besitzen als bisher angenommen.
Several different genomic and non-genomic mechanisms mediate the important anti-inflammatory and immunomodulatory effects of glucocorticoids (GCs). The genomic effects are the most important while the clinical relevance of non-genomic actions is still a matter of debate. We therefore investigated whether beclomethasone and clobetasol are particularly suitable for topical application because they differ in their spectrum of activity from systemically administered GCs such as dexamethasone. We compared effects on oxygen consumption as measured with a Clark electrode (nonspecific non-genomic glucocorticoid effects), on interleukin-6 synthesis by means of ELISA (genomic effects) and on apoptosis using flow cytometry (non-genomic and genomic effects) in quiescent and mitogen-stimulated PBMCs. Beclomethasone and clobetasol had stronger effects on the oxygen consumption of quiescent and stimulated cells at lower concentrations (10-10, 10-8 M) but were less potent at higher concentrations (10-5, 10-4 M) in comparison with dexamethasone. Also in terms of genomic potency, topical GCs were more effective than dexamethasone at 10-10 M and 10-8 M but gave similar results at higher concentrations. The ability of all three GCs to induce apoptosis was found to be concentration-dependent and similar at concentrations between 10-8 and 10-5 M but, compared with 10-4 M dexamethasone, 10-4 M beclomethasone or clobetasol was significantly more effective at inducing apoptosis in both PBMCs and Jurkat T cells. These results show that systemic and topical GCs differ significantly in their ability to induce genomic and non-genomic effects. This suggests that non-genomic effects are more therapeutically relevant in certain clinical conditions than currently assumed.

Ziel: Gewinnung neuer Erkenntnisse auf dem Gebiet der Glucocorticoidforschung. Die Arbeit gliedert sich in zwei Teile: 1. Klinische Studie zu Wirkungen und Nebenwirkungen einer niedrig bis mittelhoch dosierten Methylprednisolon(MP)-Therapie bei Patienten mit entzündlich-rheumatischen Erkrankungen. 2. Durchflusszytometrische Untersuchungen mit humanen PBMC mit dem Ziel, membranständige Glucocorticoidrezeptoren (mGCR) nachzuweisen. Methodik: 1. In einer klinischen Studie wurden zwei Patientengruppen mit jeweils 20 Patienten miteinander verglichen. Alle Patienten hatten entzündlich-rheumatische Erkrankungen und bekamen eine MP-Therapie über mindestens ein Jahr. Die Dosierungen in der ersten Gruppen entsprachen einer low-dose GC-Therapie, die Patienten in der zweiten Gruppe bekamen eine medium-dose GC-Therapie. Erwünschte, unerwünschte Wirkungen sowie die Lebensqualität der Patienten wurden erhoben. 2. Humane PBMC wurden durchflusszytometrisch untersucht. Es wurden konventionelle Färbemethoden sowie eine hoch-sensitive Liposomenfärbung zur Detektion spezifischer membranständiger Antigene angewandt. Ergebnisse: 1. In den meisten Fällen waren die relativ niedrigen Dosierungen von MP geeignet, die Krankheitsaktivität der entzündlich-rheumatischen Erkrankung wirksam zu kontrollieren. Einzelne Exazerbationen waren allerdings zu verzeichnen. Bei den meisten unerwünschten Wirkungen zeigten sich keine Unterschiede zwischen den Dosisgruppen. Osteoporosetypische Rückenschmerzen traten signifikant höher in der oberen Dosisgruppe auf (p=0,04), bei dem erhöhten Augeninnendruck zeigte sich eine Tendenz (p=0,1). Häufige Nebenwirkungen auch bei niedrigen Dosierungen waren: Unterblutungen der Haut und Pergamenthaut (76,2 % bzw. 73,8 % aller Patienten) bzw. eine Cushing-Symptomatik (61,9 % aller Patienten). 2. Mit der Liposomen-Färbetechnik ließen sich erstmals mGCR auf humanen PBMC systematisch nachweisen. Bis zu 5 % der B-Lymphozyten und bis zu 7 % der Monozyten exprimierten mGCR bei Gesunden. Stimulationen des Immunsystems durch Impfungen oder eine aktive rheumatoide Arthritis führten zu einer deutlichen Erhöhung des Anteils mGCR-positiver Monozyten auf über 20 %. Schlussfolgerungen: 1. Niedrig bis mittelhoch dosierte Therapien mit MP können effektiv die Aktivität von entzündlich-rheumatischen Erkrankungen kontrollieren. Die unerwünschten Effekte sind vermutlich dosisabhängig, für die meisten ist jedoch nicht relevant, ob mit einer low-dose oder einer medium-dose Therapie behandelt wird. 2. mGCR werden auf humanen PBMC unter physiologischen Bedingungen exprimiert. Unter bestimmten immunologischen Bedingungen werden sie hochreguliert. Herkunft und Funktion der Rezeptoren müssen noch genauer geklärt werden.
Purpose: Gaining new knowledge in glucocorticoid research. The dissertation consists of two parts: 1. Clinical study on effects and side-effects of a low-dose / medium-dose therapy with methylprednisolone (MP) in patients with inflammatory rheumatic diseases. 2. Flowcytometric investigation of human PBMC in order to detect membrane-bound glucocorticoid-receptors (mGCR). Methods: 1. In a clinical study two groups of patients - 20 patients each - were compared. All patients had inflammatory rheumatic diseases and recieved MP-therapy for at least one year. The first group recieved a low-dose GC-therapy, the second group a medium-dose GC-therapy. 2. Human PBMC were examined. We used conventional and high-sensitive liposome staining technique for the detection of specific membrane-bound antigens. Results: 1. In most cases rather low dosages of MP were able to control the disease activitiy of inflammatory rheumatic diseaeses. However, we observed disease exacerbation in some cases. Most side-effects showed the same characteristics in both groups. There was a significant higher appearance of typical osteoporotic back pain in the higher dosage group (p=0,04) and a tendency to higher intraophtalmic pressure in this group (p=0,1). Common side effects with even low dosages were: skin hematoma and thin skin (76,2 % and 73,8 % respective) and a Cushing-Syndrome (61,9 % of all patients). 2. With the liposome staining technique we showed for the first time systematically mGCR on human PBMC. Up to 5 % of B-lymphocytes and 7 % of monocytes presented mGCR in healthy blood donors. Stimulation of the immunological system by vaccination or in case of an active rheumatoid arthritis led to a marked increase of mGCR-positive monocytes to more than 20 %. Conclusions: 1. Low-dose and medium-dose methylprednisolone therapy can effectivly control the activity of inflammatory rheumatic diseases. The side effects are probably dose-dependent. However, for most side effects it doesn''t matter if patients are treated with a low-dose or a medium-dose therapy. 2. mGCR are expressed on human PBMC under physiological conditions and are up-regulated under certain immunological conditions. The function of these receptors has to be examined more profoundly.

Human complex diseases, like Diabetes and Cancer, affect many people worldwide today. Despite existing knowledge, many of these diseases are still not preventable. Complex diseases are known to be caused by a combination of genetic factors, as well as environmental and life style factors. The scope of this investigation covered the genomics of Type 1 Diabetes (T1D). There are 49 human genomic regions that are known to carry markers (disease-associated single nucleotide mutations) for T1D, and these were extensively studied in this research. The aim was to find out in how far this disease may be caused by problems in gene regulation rather than in gene coding. For this, the genetic factors associated with T1D, including the single point mutations and susceptibility regions, were characterised on the basis of their genomic attributes. Furthermore, mutations that occur in binding sites for transcription factors were analysed for change in the conspicuousness of their binding region, caused by allele substitution. This is called SNP (Single nucleotide polymorphism) sensitivity. From this study, it was found that the markers for T1D are mostly non-coding SNPs that occur in introns and non-coding gene transcripts, these are structures known to be involved in gene regulatory activity. It was also discovered that the T1D susceptibility regions contain an abundance of intronic, non-coding transcript and regulatory nucleotides, and that they can be split into three distinct groups on the basis of their structural and functional genomic contents. Finally, using an algorithm designed for this study, thirty-seven SNPs that change the representation of their surrounding region were identified. These regulatory mutations are non-associated T1D-SNPs that are mostly characterised by Cytosine to Thymine (C-T) transition mutations. They were found to be closer in average distance to the disease-associated SNPs than other SNPs in binding sites, and also to occur frequently in the binding motifs for the USF (Upstream stimulatory factor) protein family which is linked to problems in Type 2 diabetes.

Le virus y de la pomme de terre est le membre type des potyvirus. Son genome est constitue d'un arn simple brin d'environ 10 kb de polarite (+), pourvu d'une proteine virale vpg liee de maniere covalente en 5 et d'une sequence poly(a) en 3. Les extremites 5 et 3 non codantes ont un role dans la traduction et la replication du genome viral. L'effet de ces sequences sur la traductibilite de messagers chimeriques electropores dans des protoplastes de plantes a ete etudie. L'extremite 5 non codante du pvy a un effet activateur de la traduction, comparable a celui de la region omega du tmv. L'extremite 3 non codante du pvy a un effet comparable a une sequence poly(a). Il semblerait que la region 5 non codante du pvy permette l'initiation interne de la traduction sur un messager bicistronique. La possibilite d'interferer avec la multiplication virale grace a des arn defectifs interferents ayant conserve les extremites 5 et 3 non codantes est envisagee. Quelques resultats preliminaires sont presentes

Le dioxyde d’azote (NO2), en tant que second polluant atmosphérique le plus meurtrier en Europe est un des plus préoccupants pour la santé humaine selon l’Agence Européenne de l’Environnement. Il est notamment connu pour être responsable de maladies cardiovasculaires, respiratoires ainsi que pour contribuer au vieillissement cutané et au développement de la dermatite atopique. Des facteurs endogènes à l’hôte tels que le microbiote cutané interviennent également dans cette pathologie. En effet, de nombreuses pathologies cutanées sont corrélées à un déséquilibre (dysbiose) du microbiote bactérien, un acteur essentiel du maintien de l’homéostasie de la peau. Or, il est fortement soupçonné que l’effet des polluants sur la peau implique des mécanismes d’action directe mais également un mécanisme d’action indirecte lié à l’altération du microbiote cutané par le polluant. En conséquence, il est pertinent d’aborder l’effet du NO2 gazeux (gNO2) sur le microbiote cutané bactérien. Cette thèse a donc pour objectif d’évaluer l’impact physiologique, morphologique et moléculaire du NO2 sur des souches bactériennes commensales d’espèces représentatives du microbiote cutané (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus capitis, Pseudomonas fluorescens, Corynebacterium tuberculostearicum). Selon l’espèce, des réponses différentes au stress nitrosant généré par le gNO2 ont ainsi été mises en évidence ainsi qu’une tolérance plus importante au gNO2 pour certaines d’entre elles. Ces travaux suggèrent par conséquent que le NO2 pourrait contribuer à la formation d’un état dysbiotique du microbiote cutané et participer à l’action indirect du polluant sur la peau
Nitrogen dioxide (NO2), as the second most deadly air pollutant in Europe, is one of the most of concern for human health according to the European Environment Agency. It is notably known to be responsible for cardiovascular and respiratory diseases and also contributes to skin aging and atopic dermatitis. Host endogenous factors such as the cutaneous microbiota are also involved in this pathology, which is common in urban and suburban areas. Indeed, many skin pathologies are correlated to an imbalance (dysbiosis) of the bacterial microbiota, an essential player in the preservation of skin homeostasis. However, it is strongly presumed that the effect of pollutants on the skin involves direct mechanisms of action but also an indirect mechanism linked to the alteration of the cutaneous microbiota by the pollutant. Consequently, it is relevant to address the effect of gaseous NO2 (gNO2) on the cutaneous microbiota. This thesis aims to assess the physiological, morphological and molecular impact of gNO2 on commensal bacterial strains of representative species of the cutaneous microbiota (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus capitis, Pseudomonas fluorescens, Corynebacterium tuberculostearicum). Depending on the species, different responses to gNO2-generated nitrosative stress were thus highlighted as well as a higher tolerance to gNO2 for some of them. This work therefore suggests that gNO2 could contribute to the formation of a dysbiotic state of the cutaneous microbiota and participate in the pollutant indirect action on the skin

Chan Hoi Yun.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 131-144).
Abstracts in English and Chinese.
DECLARATION --- p.i
ACKNOWLEDGMENTS --- p.ii
ABBREVIATIONS --- p.iii
ABSTRACT IN ENGLISH --- p.v
ABSTRACT IN CHINESE --- p.viii
CONTENTS --- p.xi
Chapter Chapter 1 --- Introduction
Chapter 1.1. --- Steroid Hormones --- p.1
Chapter 1.1.1. --- Synthesis of estrogens and progesterone --- p.1
Chapter 1.2. --- Cellular Mechanisms of Female Steroid Hormones --- p.5
Chapter 1.2.1. --- Genomic actions of female steroid hormones --- p.5
Chapter 1.2.2. --- Non-genomic actions of female steroid hormones --- p.7
Chapter 1.2.3. --- Estrogen antagonists --- p.7
Chapter 1.2.3.1. --- Classification of estrogen antagonists --- p.7
Chapter 1.2.3.2. --- Mechanisms of estrogen antagonists --- p.9
Chapter 1.3. --- Chronic (genomic) Effects of 17β-Estradiol and Progesterone --- p.10
Chapter 1.3.1. --- Effects of lipid metabolism --- p.10
Chapter 1.3.2. --- Effects on cell proliferation --- p.11
Chapter 1.3.3. --- Effects on endothelial cells --- p.12
Chapter 1.4. --- Acute Effects of 17β-Estradiol and Progesterone --- p.13
Chapter 1.4.1. --- Role of endothelium in 17β-estradiol or progesterone Relaxation --- p.13
Chapter 1.4.2. --- Involvement of plasma membrane estrogen receptors --- p.14
Chapter 1.4.3. --- Role of Ca2+ and K+ channel in estrogen relaxation --- p.14
Chapter 1.4.4. --- Interaction with vasoconstrictors --- p.15
Chapter 1.4.5. --- Interaction with endothelium-dependent dilators --- p.16
Chapter 1.4.6. --- Interaction with adrenergic response --- p.17
Chapter 1.5. --- Clinical Studies --- p.19
Chapter 1.6. --- Therapeutic Values of Estrogen and Progesterone --- p.20
Chapter 1.7. --- Objectives of the Present Study --- p.22
Chapter Chapter 2 --- Method and Materials
Chapter 2.1. --- Tissue Preparation --- p.25
Chapter 2.1.1. --- "Preparation of the rat aorta, mesenteric artery and carotid Artery" --- p.25
Chapter 2.1.2. --- Removal of the functional endothelium --- p.26
Chapter 2.2. --- Organ Bath Set-up --- p.26
Chapter 2.3. --- Force Measurement --- p.28
Chapter 2.3.1. --- Vascular action of 17β-estradiol and progesterone --- p.29
Chapter 2.3.1.1. --- Role of endothelium/nitric oxide in 17β-estradiol- or progesterone-induced relaxation --- p.29
Chapter 2.3.1.2. --- Role of inducible nitric oxide in progesterone-induced relaxation --- p.30
Chapter 2.3.1.3. --- Effect of estrogen receptor inhibitor on 17β-estradiol- induced relaxation --- p.30
Chapter 2.3.1.4. --- Interaction between progesterone and 17β-estradiol --- p.31
Chapter 2.3.1.5. --- Effect of 17β-estradiol on protein kinase C-mediated contraction --- p.31
Chapter 2.3.1.6. --- Synergistic interaction between β-adrenoceptor agonists and 17β-estradiol --- p.32
Chapter 2.4. --- Porcine Coronary Artery Experiments --- p.33
Chapter 2.4.1. --- Vessel preparation --- p.33
Chapter 2.4.2. --- Force measurement --- p.33
Chapter 2.4.3. --- Experimental protocol --- p.34
Chapter 2.4.3.1. --- Effect of physiological level of 17β-estradiol on β- adrenoceptor agonist-induced relaxation --- p.34
Chapter 2.4.3.2. --- Effect of physiological level of 17β-estradiol on phosphodiesterase inhibitor-induced relaxation --- p.34
Chapter 2.5. --- Ovariectomy --- p.35
Chapter 2.5.1. --- Method of ovariectomy --- p.35
Chapter 2.5.2. --- Preparation of blood vessels --- p.36
Chapter 2.5.3. --- Experimental protocols --- p.38
Chapter 2.5.3.1. --- Effect of ovariectomy on contractility of rat carotid arteries --- p.38
Chapter 2.5.3.2. --- Effect of ovariectomy on relaxation of rat carotid arteries --- p.38
Chapter 2.6. --- Chemicals and Solutions --- p.39
Chapter 2.7. --- Statistical Analysis --- p.42
Chapter Chapter 3 --- Results
Chapter 3.1. --- Role of Endothelium/Nitric Oxide in 17β-Estradiol- and Progesterone-induced Relaxations --- p.43
Chapter 3.1.1. --- Relaxant response of 17β-estradiol --- p.43
Chapter 3.1.2. --- Effects of inhibitors of nitric oxide activity on 17β- estradiol-induced relaxation --- p.46
Chapter 3.1.3. --- Relaxant response of progesterone --- p.46
Chapter 3.1.4. --- Effects of inhibitors of nitric oxide activity on progesterone-induced relaxation --- p.50
Chapter 3.2. --- Effect of Estrogen Receptor Inhibitor on 17β-Estradiol- induced Relaxation --- p.56
Chapter 3.3. --- Interaction between Progesterone and 17β-Estradiol --- p.56
Chapter 3.4. --- Effect of Female Sex Steroid Hormones on Protein Kinase C-mediated Contraction --- p.59
Chapter 3.4.1. --- Effect of 17β-estradiol on phorbol ester-induced contraction --- p.59
Chapter 3.4.2. --- Effect of progesterone on phorbol ester-induced contraction --- p.59
Chapter 3.5. --- Effects of β-adrenoceptor Agonists on 17β-Estradiol- induced Relaxations --- p.62
Chapter 3.5.1. --- Effect of isoproterenol on 17β-estradiol-induced relaxation --- p.62
Chapter 3.5.2. --- Role of endothelium/nitric oxide on the isoproterenol potentiation of 17β-estradiol-induced relaxation --- p.63
Chapter 3.5.3. --- Role of cyclic AMP on isoproterenol-enhancement of 17β- estradiol-induced relaxation --- p.69
Chapter 3.5.4. --- Effects of β-adrenoceptor antagonists --- p.69
Chapter 3.6. --- Effects of Physiological Concentration of 17β-EstradioI onβ-adrenoceptor Agonists-induced Relaxationsin Porcine Coronary Artery --- p.77
Chapter 3.6.1. --- Effect of 17β-estradiol on isoproterenol-induced relaxations --- p.77
Chapter 3.6.2. --- Effect of 17β-estradiol on fenoterol-induced relaxations --- p.11
Chapter 3.6.3. --- Effect of 17β-estradiol on dobutamine-induced relaxations --- p.81
Chapter 3.6.4. --- Effect of 17β-estradiol on IBMX-induced relaxation --- p.86
Chapter 3.7. --- Effect of Ovariectomy on the Vascualr Reactivity --- p.88
Chapter 3.7.1. --- Effect of ovariectomy on the contractile activity of rat carotid artery --- p.88
Chapter 3.7.1.1. --- Effect of ovariectomy on phenylephrine-induced contraction --- p.88
Chapter 3.7.1.2. --- Effect of ovariectomy on U46619-induced contraction --- p.96
Chapter 3.7.1.3. --- Effect of ovariectomy on high K+- induced contraction --- p.102
Chapter 3.7.1.4. --- Effect of ovariectomy on acetylcholine-induced relaxation --- p.106
Chapter Chapter 4 --- Discussions
Chapter 4.1. --- Role of Endothelium/Nitric oxide in 17β-Estradiol- and Progesterone-induced Relaxations --- p.110
Chapter 4.2. --- Effect of Estrogen Receptor Inhibitor on 17β-Estradiol- induced Relaxation --- p.113
Chapter 4.3. --- Interaction between Progesterone and 17β-Estradiol --- p.114
Chapter 4.4. --- Effects of Female Sex Steroid Hormones on Protein Kinase C-mediated Contraction --- p.115
Chapter 4.5. --- Effects of β-Adrenoceptor Agonists on 17β-Estradiol- induced Relaxations --- p.116
Chapter 4.6. --- Effects of 17β-Estradiol on β-Adrenoceptor Agonists- induced Relaxations in Porcine Coronary Artery --- p.121
Chapter 4.7. --- Effect of Ovariectomy on the Vascular Reactivity --- p.125
Chapter 4.8. --- Conclusions --- p.129
References --- p.131
Publications --- p.145

Thesis (M.Sc.)--York University, 2006. Graduate Programme in Kinesiology and Health Science.
Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR29625

First, the theory of MSG is extended to aperiodic heterogeneous solid structures. Integral constraints are introduced to decompose the displacements and strains of the heterogeneous material into a fluctuating part and a macroscopic part, of which the macroscopic part represents the responses of the homogenized material. One advantage of this theory is that boundary conditions are not required. Consequently, it is capable of handling micro-structures of arbitrary shapes. In addition, periodic constraints can be incorporated into this theory as needed to model periodic or partially periodic materials such as textile composites. In this study, the newly developed method is employed to investigate the finite thickness effect of textile composites.

Indiana University-Purdue University Indianapolis (IUPUI)
Differences in voluntary consumption of ethanol have been negatively correlated with differences in initial sensitivity and tolerance to ethanol’s pharmacological effects. From this perspective, both adolescent and adult alcohol-nonpreferring (NP) rats would be expected to be initially more sensitive to the sedative and hypothermic effects of ethanol and fail to acquire tolerance to those effects than preferring (P) rats. The first objective of this experiment was to assess alcohol-induced hypothermia and locomotor sedation in adolescent and adult P and NP rats over five consecutive daily administrations (saline, 1.5 g/kg, or 3.0 g/kg ethanol 17%v/v), testing the hypothesis that the P rats would acquire tolerance to the hypothermic response whereas the NP rats would not show changes across days. In addition, it was hypothesized that there would be age-related differences in initial sensitivity to ethanol, evident by adolescent rats displaying less ethanol-induced hypothermia and locomotor sedation than adult rats on Day 1. The second objective was to determine if conditioning was occurring between the administration environment and the hypothermic response and locomotor sedation elicited by ethanol exposure, via a sixth injection of saline. Female rats were surgically implanted with intraperitoneal Mini Mitter telemetry probes on postnatal day 25 or 85 and experimental manipulations began five days later. Data were collected every minute; temperature data were then converted to change from baseline scores and locomotor data were totaled for each session. On Day 1, maximum temperature reduction elicited by the 3.0 g/kg dose was greater in the NP rats than the P rats, regardless of age. That dose also produced greater levels of locomotor sedation in the adult rats compared to the adolescent rats, regardless of line. The 1.5 g/kg dose of ethanol produced a greater hypothermic response in adult rats compared to adolescent rats, locomotor activity was reduced equally across the groups. With repeated administrations, NP adult rats displayed sensitization to the hypothermic response elicited from the 3.0 g/kg dose; in contrast, tolerance to the hypothermic response was found within the 1.5 g/kg dose for the adolescent P, adult P, and the adult NP rats. Repeated saline administrations also resulted in tolerance to the hypothermic response associated with administration in the adult NP and adolescent P rats. On the Day 6 saline administrations, adult rats which had previously been exposed to the 3.0 g/kg dose, maintained their baseline body temperatures better than both of the other exposure groups. Adolescent rats failed to show any signs of conditioning when administered saline on Day 6. Contrary to prediction the P rats failed to acquire tolerance to the 3.0 g/kg dose for either measure; and the line difference in ethanol-induce hypothermia was due to sensitization of the hypothermic response in adult NP rats. These results also provide further support that adolescent rats are less sensitive to the initial aversive effects of ethanol at the 1.5 g/kg dose for ethanol-induced hypothermia and the 3.0 g/kg dose for locomotor activity. The current experiment provides evidence that initial sensitivity as well as the acquisition of tolerance to ethanol-induced hypothermia may be behavioral phenotypes correlated with selection for high and low alcohol drinking preference.

L’estradiol (E2) est une hormone femelle qui joue un rôle essentiel, à la fois dans la régulation et dans la détermination de certaines conditions physiologiques in vivo, telle que la différenciation et la prolifération cellulaire. Lorsque l’E2 est donné en supplément, par exemple dans le cas de thérapie hormonale, deux effets sont observés, un effet génomique et un effet non-génomique, de par son interaction avec les récepteurs à œstrogène du noyau ou de la membrane cellulaire, respectivement. L’effet non-génomique est plus difficile à étudier biologiquement parce que l’effet se produit sur une échelle de temps extrêmement courte et à cause de la nature hydrophobe de l’E2 qui réduit sa biodisponibilité et donc son accessibilité aux cellules cibles. C’est pourquoi il est nécessaire de développer des systèmes d’administration de l’E2 qui permettent de n’étudier que l’effet non-génomique de l’œstrogène. Une des stratégies employée consiste à greffer l’E2 à des macromolécules hydrophiles, comme de l’albumine de sérum bovin (BSA) ou des dendrimères de type poly(amido)amine, permettant de maintenir l’interaction de l’E2 avec les récepteurs d’œstrogène de la membrane cellulaire et d’éviter la pénétration de l’E2 dans le noyau des cellules. Toutefois, ces systèmes macromolécules-E2 sont critiquables car ils sont peu stables et l’E2 peut se retrouver sous forme libre, ce qui affecte sa localisation cellulaire. L’objectif de cette thèse est donc de développer de nouvelles plateformes fonctionnalisées avec de l’E2 en utilisant les approches de synthèses ascendantes et descendantes. Le but de ces plateformes est de permettre d’étudier le mécanisme de l’effet non-génomique de l’E2, ainsi que d’explorer des applications potentielles dans le domaine biomédical. L’approche ascendante est basée sur un ligand d’E2 activé, l’acide 17,α-éthinylestradiol-benzoïque, attaché de façon covalente à un polymère de chitosan avec des substitutions de phosphorylcholine (CH-PC-E2). L’estradiol est sous forme de pro-drogue attachée au polymère qui s’auto-assembler pour former un film. L’effet biologique de la composition chimique du film de chitosan-phosphorylcholine a été étudié sur des cellules endothéliales. Les films de compositions chimiques différentes ont préalablement été caractérisés de façon physicochimique. La topographie de la surface, la charge de surface, ainsi que la rhéologie des différents films contenant 15, 25, ou 40% molaires de phosphorylcholine, ont été étudiés par microscopie à force atomique (AFM), potentiel zêta, résonance plasmonique de surface et par microbalance à cristal de quartz avec dissipation (QCM-D). Les résultats de QCM-D ont montré que plus la part molaire en phosphorylcholine est grande moins il y a de fibrinogène qui s’adsorbe sur le film de CH-PC. Des cellules humaines de veine ombilicale (HUVECs) cultivées sur des films de CH-PC25 et de CH-PC40 forment des amas cellulaire appelés sphéroïdes au bout de 4 jours, alors que ce n’est pas le cas lorsque ces cellules sont cultivées sur des films de CH-PC15. L’attachement de l’estradiol au polymère a été caractérisé par plusieurs techniques, telles que la résonance magnétique nucléaire de proton (1H NMR), la spectroscopie infrarouge avec transformée de Fourier à réfraction totale atténuée (FTIR-ATR) et la spectroscopie UV-visible. La nature hydrogel des films (sa capacité à retenir l’eau) ainsi que l’interaction des films avec des récepteurs à E2, ont été étudiés par la QCM-D. Des études d’imagerie cellulaires utilisant du diacétate de diaminofluoresceine-FM ont révélé que les films hydrogels de CH-PC-E2 stimulent la production d’oxyde nitrique par les cellules endothéliales, qui joue un rôle protecteur pour le système cardiovasculaire. L’ensemble de ces études met en valeur les rôles différents et les applications potentielles qu’ont les films de type CH-PC-E2 et CH-PC dans le cadre de la médecine cardiovasculaire régénérative. L’approche descendante est basée sur l’attachement de façon covalente d’E2 sur des ilots d’or de 2 μm disposés en rangées et espacés par 12 μm sur un substrat en verre. Les ilots ont été préparés par photolithographie. La surface du verre a quant à elle été modifiée à l’aide d’un tripeptide cyclique, le cRGD, favorisant l’adhésion cellulaire. L’attachement d’E2 sur les surfaces d’or a été suivi et confirmé par les techniques de SPR et de QCM-D. Des études d’ELISA ont montré une augmentation significative du niveau de phosphorylation de la kinase ERK (marqueur important de l’effet non-génomique) après 1 heure d’exposition des cellules endothéliales aux motifs alternant l’E2 et le cRGD. Par contre lorsque des cellules cancéreuses sont déposées sur les surfaces présentant des motifs d’E2, ces cellules ne croissent pas, ce qui suggère que l’E2 n’exerce pas d’effet génomique. Les résultats de l’approche descendante montrent le potentiel des surfaces présentant des motifs d’E2 pour l’étude des effets non-génomiques de l’E2 dans un modèle in vitro.
Estradiol (E2) is an essential female hormone in the regulation and determination of various physiological conditions in vivo, such as cell proliferation and differentiation. When supplementing exogenous E2 as a clinical strategy for hormone therapy, it generates genomic and non-genomic effect simultaneously via binding to the estrogen receptors in the cell nucleus or membrane site. Compared to the genomic effect, it is quite difficult to monitor the E2-induced non-genomic biological behavior because this effect occurs in extremely transient time scale and the bioavailability and accessibility of E2 to target cells is very low due to the hydrophobic nature of E2. As a result, it is indispensable to develop E2 delivery systems to specifically understand estrogenic non-genomic nature. One of strategies is to graft E2 to the hydrophilic macromolecules, e.g. bovine serum albumin (BSA) or poly(amido)amine dendrimer, to maintain E2 interacting with membrane estrogen receptors instead of penetrating into the cell nucleus. However, the instability of those E2-macromolecules systems, either containing free E2 leaching or discrepancies of cellular localizations, led to controversies. Herein, the objective of present thesis is to develop novel E2-functionlized platforms by the principle of bottom-up and top-down approaches for understanding the mechanism of estrogenic non-genomic effect, and further, to explore their potential applications in the biomedicine. As a bottom-up approach, an activated E2 ligand, 17α-ethinylestradiol-benzoic acid was covalently conjugated onto a phosphorylcholine substituted chitosan polymer (CH-PC-E2) as a prodrug strategy for the fabrication of self-assembled films. Through a series of combined physicochemical and cellular investigations, the relationship between various chemical compositions of chitosan-phosphorylcholine (CH-PC) films and cellular responses was also evaluated. Based on atomic force microscopy (AFM) examination, zeta-potential measurements, surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D) measurements, surface topography, charge, and rheology of CH-PC films with 15, 25, and 40 mol% PC contents were characterized. Moreover, QCM-D measurements indicated that the amount of fibrinogen adsorbed on CH-PC films decreased significantly with increasing PC content. Finally, it was also showed that human umbilical vein endothelial cells (HUVECs) form spheroids on CH-PC25 and CH-PC40 films, but not on CH-PC15 films cultured over 4 days. In addition, the CH-PC-E2 polymer conjugates were prepared and characterized by several techniques, such as 1H nuclear magnetic resonance (1H NMR), Fourier transformed infrared-attenuated total refraction (FTIR-ATR) and UV/Vis spectra measurements. The hydrogel nature of CH-PC-E2 film as well as its interactions to estrogen receptors was further extensively investigated by QCM-D study. In the cellular study, CH-PC-E2 hydrogel films can significantly stimulate the production of nitric oxide, a protective molecule in the cardiovascular system, in the endothelial cells by a diaminofluorescein-FM diacetate imaging study. The studies above demonstrated the different roles and potential applications of CH-PC-E2 and CH-PC surfaces in the cardiovascular regenerative medicine. As a top-down approach, micropatterned substrates were used for E2 functionalization, which were prepared by photolithography via aligning ~ 2 μm in diameter gold arrays onto a glass substrate. After that, a cell adhesive peptide, cyclic RGD was introduced to the glass surface in order to induce the attachment of cells. Meanwhile, estradiol was covalently immobilized on the gold surface and the process was monitored and validated by combining SPR and QCM-D studies. In the micropatterned substrate-coupled cell ELISA study, a phosphorylation level of extracellular signal-regulated kinase (ERK), which is an important non-genomic marker, was significantly elevated by this E2-functionalized micropatterned surface after 1 hour incubation. Furthermore, E2-functionalized micropatterned substrate didn’t proliferate cancer cells indicating the absence of genomic effect stimulation. Based on these results, our E2-functionalized micropatterned substrates can function as an in vitro model for the elucidation of estrogenic non-genomic behaviors.