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No description available.

The overall objective of this project was to investigate the role of locally derived growth factors and extracellular matrix proteins in regulating prepubertal heifer mammary development. In the first experiment, short-term treatment of heifers with GH or E increased proliferation of mammary epithelial cells. Coinciding with increased epithelial cell proliferation, IGF-I protein increased and IGFBP-3 protein decreased within mammary tissues. Thus, proliferation was associated with an apparent net increase in the biological availability of IGF-I within the mammary gland. In the second experiment, decreased mammary development and epithelial cell proliferation in response to ovariectomy coincided with decreased mammary expression of IGF-I mRNA and decreased binding of IGF-I to mammary microsomes. Taken together, these results imply an important role for locally derived IGF-I in regulating heifer mammary development. However, in contrast to our hypothesis, IGF-I mRNA did not differ between cleared or intact mammary fat pad, suggesting that expression of IGF-I mRNA is not regulated by epithelial:stromal interactions. Neither ovariectomy or epithelial:stromal interactions influenced the mRNA expression of IGFBP-3 or IGFBP-5 within mammary tissues. Ovariectomy increased the proportion of ERa positive mammary epithelial cells. In contrast, GH administration to prepubertal heifers did not influence the proportion of ERa-positive epithelial cells. Interestingly, mammary development was more severely affected in heifers ovariectomized before six weeks of age than heifers ovariectomized at three months of age, implying a critical period of ovarian stimulation during the first six weeks of age. Localization of laminin, fibronectin, and collagen in mammary parenchyma suggested specific roles for extracellular matrix proteins in regulating mammary development and ductal morphogenesis. Laminin was decreased and fibronectin was increased by ovariectomy, suggesting a possible role for interactions between the ovary and extracellular matrix proteins within the heifer mammary gland. Finally, the mitogenic capacities of mammary tissue extracts from control and ovariectomized heifers did not differ in their ability to stimulate in vitro proliferation of MAC-T cells. In conclusion, the overall results support the hypothesis that locally derived IGF-I regulates prepubertal heifer mammary development. However, ERa expression and extracellular matrix proteins also appear to be important regulators of heifer mammary development. / Ph. D.

Die Reifung der Zona reticularis der Nebennieren-Rinden (NNR) und ihrer Androgen-Sekretion vor der Pubertät unterscheidet sich bei Menschen und höheren Primaten von der NNR-Reifung anderer Species, z.B. der Nager. Die Sekretion der NNR-Androgene leitet die Pubertätsentwicklung ein. Die NNR-Androgene erlangen medizinische Bedeutung dadurch, dass sie bei Frauen zu Hirsutismus und Fertilitätsstörungen führen können. Neben diesen Symptomen stellen sie einen Risikofaktor für die Entwicklung eines Polycystischen-Ovar-Syndroms (PCOS) dar, das ungefähr 7 % der prämenopausalen Frauen betrifft. Lange Zeit war nicht bekannt, wie die Differenzierung der Zona reticularis beim Menschen reguliert wird. Sicher ist ACTH bei weitem das bedeutsamste übergeordnete Hormon für die globale adrenocorticale Differenzierung und Funktion. Weitere Faktoren sind speziell für die Androgensekretion verantwortlich, aber nicht genau definiert. Nun wurde zunächst in zellbiologischen Experimenten belegt, dass die ACTH-Wirkung durch ein Spezies-spezifisches Muster von Wachstumsfaktoren autokrin moduliert wird und so die postnatale Entwicklung der Nebenniere steuern kann. Die vorliegenden Untersuchungen an menschlichen NNR-Zellen von Kindern und Erwachsenen in Primärkultur zeigen erstmals, dass IGF-I und IGF-II differenzierte Funktionen dieser Zellen aufrecht erhalten. IGF-I und, mehr noch, IGF-II steigern die Steroid-Biosynthese und ACTH-Ansprechbarkeit, und sie fördern die Bildung von Androstendion, einem delta5-Androgen der Zona reticularis. Darüberhinaus bewirkt Insulin in physiologischen sowie in micromolaren Konzentrationen den IGFs ähnliche Änderungen der Steroidsynthese. In Querschnittsuntersuchungen an gesunden Kindern vor der Pubertät sowie Kindern mit einfacher Adipositas konnte gezeigt werden, dass die Körperzusammensetzung mit den NNR-Androgenen zusammenhängt. Über die Mediatoren IGF-I, Insulin und Leptin wird offensichtlich der NNR der Zustand von Gewicht und Wachstum des Kindes signalisiert, auch bei pathologischer Körperzusammensetzung, wie dem Prader-Willi-Syndrom. Während die Adipositas die Androgen-Bildung steigern kann, ist sie jedoch selbst nicht der kausale Faktor einer vorzeitigen Nebennierenrindenreifung. Der Prämaturen Pubarche können in 5 - 10 % der untersuchten weiblichen Population ein nicht-klassisches AGS oder NNR-Tumoren zugrunde liegen. Bei den verbleibenden Kindern besteht eine eigentlich harmlose Reifungsbeschleunigung mit normaler Wachstumsprognose. Betrachtet man diese Kinder mit idiopatischer Prämaturer Adrenarche jedoch genauer, so finden sich zwei Untergruppen mit langfristigen Risiken: erstens zeigen Kinder mit einer sogenannte manifesten Heterozygotie für einen 21-Hydroxylase-Defekt Auffälligkeiten des Wachstums, die eine Endgrössenreduktion bewirken könnten, und zweitens wird bei Jugendlichen mit einer Überstimulierbarkeit der NNR diese "Exaggerated Adrenarche" für ein nachfolgendes PCOS verantwortlich gemacht. Schliesslich scheint es vor dem Hintergrund der sich epidemieartig ausbreitenden Zunahme des Übergewichts im Kindesalter angezeigt, den Bezug dieser NNR-Störungen zur Adipositas und der Hyperinsulinämie weiter zu klären. / The prepubertal maturation of the zona reticularis of the adrenal cortex and its androgen secretion in man and higher primates differs from other species, e.g. rodents. The secretion of adrenal androgens induces the pubertal development. The importance of adrenal androgens is derived from them being the cause for hirsutism and fertility disorders in women. In addition they represent a risk factor for the development of the polycystic ovary syndrome (PCOS), that affects about 7% of all pre-menopausal women. The regulation of the differentiation of the zona reticularis was unknown for a long time. However, ACTH is by far the most important hormone to regulate the global adrenocortical differentiation and function. In addition, other yet undefined factors are specifically responsible for the secretion of adrenal androgens. The cell-biological experiments presented here demonstrate that the effects of ACTH can be modulated in an autocrine manner by a species-specific pattern of growth factors so as to allow for the control of the postnatal development of the adrenal gland. The present investigations in human adrenocortical cells of children and adults in primary culture show for the first time that IGF-I and IGF-II maintain the differentiated function of these cells. IGF-I and to an even greater extent IGF-II enhance the biosynthesis of steroids and ACTH-responsiveness, and they promote the production of androstenedione, a delta5-androgen of the zona reticularis. Moreover, insulin, in physiological as well as in micromolar concentrations, induces changes in steroid production similar to the IGFs. In cross-sectional studies of healthy pre-pubertal children and children with simple obesity, it was shown that body composition is associated with adrenal androgens. Mediated by IGF-I, insulin and leptin, body composition apparently signals the child's state of weight and growth to the adrenals, even in patients with abnormal body composition, e.g. the Prader-Willi syndrome. While obesity may enhance androgen production, it is not the direct causal factor to induce premature adrenal maturation. In 5-10% of the female population investigated, premature pubarche is caused by non-classical adrenal hyperplasia or an adrenocortical tumour. In the remaining children, there is merely a harmless acceleration of maturation with normal growth prediction. A closer look at the children with idiopathic premature adrenarche, however, reveals two subgroups with long-term risks: First, children with a so called manifest heterozygosity of a 21-hydroxylase-defect show growth abnormalities, possibly reducing final height. Second, in adolescents with enhanced stimulation of the adrenal cortex, this 'exaggerated adrenarche' is held responsible for the subsequent development of PCOS. Finally, with regard to the rapidly spreading epidemic of overweight in children, it seems essential to study into greater depth the relationship between these adrenal dysfunctions and obesity or hyperinsulinism.

Un remodelage vasculaire anormal est à la base de la pathogenèse des maladies cardio-vasculaires (MCV) telles que l’athérosclérose et l’hypertension. Des dysfonctionnements au niveau de la migration, l’hypertrophie et la prolifération des cellules musculaires lisses vasculaires (CMLV) sont des évènements cellulaires qui jouent un rôle primordial dans le remodelage vasculaire. L’insulin-like growth factor 1 (IGF-1), puissant facteur mitogène, contribue au développement des MCV, notamment via l’activation des protéines MAPK et PI3-K/PKB, composantes clés impliquées dans les voies de croissance cellulaire. Ces molécules sont également impliquées dans la modulation de l’expression de nombreux facteurs de transcription, incluant le facteur Egr-1. Egr-1 est régulé à la hausse dans différents types de maladies vasculaires impliquant les voies de signalisation de croissance et de stress oxydant qui par ailleurs peuvent être déclenchées par l’IGF-1. Cependant, la question d’une possible modulation de l’expression d’Egr-1 dans les CMLV demeure inabordée; plus spécifiquement, la caractérisation de la voie de signalisation reliant l’action d’IGF-1 à l’expression d’Egr-1 reste à établir. Dans cette optique, l’objectif de cette étude a été d’examiner l’implication de MAPK, PKB et des dérivés réactifs de l’oxygène (DRO) dans l’expression d’Egr-1 induite par l’IGF-1 dans les CMLV. L’IGF-1 a induit une augmentation marquée du niveau protéique de l’Egr-1 en fonction du temps et de la concentration utilisés. Cette augmentation a été inhibée en fonction des doses d’agents pharmacologiques qui ciblent les voies de signalisation de MAPK, PKB et DRO. De plus, l’expression du facteur de transcription, Egr-1, en réponse de l’IGF-1, a été atténuée suite à un blocage pharmacologique des processus cellulaires responsables de la synthèse d’ARN et de synthèse protéique. Pour conclure, on a démontré que l’IGF-1 stimule l’expression d’Egr-1 via les voies de signalisation, impliquant ERK1/2/JNK, PI3K/PKB. On a également proposé que les DRO jouent un rôle important dans ce processus. Dans l’ensemble, nous avons suggéré un nouveau mécanisme par lequel l’IGF-1 promeut la prolifération et l’hypertrophie cellulaire, processus à la base des anomalies vasculaires. / Aberrant vascular remodelling underlies the pathogenesis of major cardiovascular diseases (CVD), such as atherosclerosis and hypertension. Abnormal growth, migration and proliferation of vascular smooth muscle cells (VSMC) are believed to play a critical role in vascular remodelling. IGF-1, potent mitogen, is believed to contribute to the development of CVD through the hyperactivation of proliferative and growth promoting pathways including mitogen-activated protein kinase (MAPK) and protein kinase B (PKB) pathways. It has also been implicated in modulating the expression of multiple transcription factors, including the early growth response protein-1 (Egr-1). Egr-1 upregulation has been observed in different models of vascular diseases implicating growth and redox signalling such as observed in response to IGF-1. However, modulation of Egr-1 expression by IGF-1 in VSMC, more specifically the signaling pathways involved in this process remain poorly characterized. Therefore, in the present studies, we investigated the implication of MAPK, PKB and ROS in IGF-1-induce Egr-1 expression in VSMC. IGF-1 induced a marked increase in Egr-1 protein level in a time and dose-dependent fashion. This increase was dose dependently inhibited by different pharmacological inhibitors targeting MAPK, PKB and reactive oxygen species (ROS) generation. Furthermore, pharmacological inhibitors of RNA and protein synthesis also attenuated IGF-1-induce response on Egr-1 expression. In conclusion, we showed that IGF-1 stimulates the expression of Egr-1 through ERK1/2/JNK and PI3K/PKB. We also propose that ROS generation plays an important role in this response. Overall, we propose a novel mechanism through which IGF-1 may exert its deleterious responses in vascular abnormalities.

L’adénovirus a été étudié dans l’optique de développer de nouveaux traitements pour différentes maladies. Les vecteurs adénoviraux (AdV) sont des outils intéressants du fait qu’ils peuvent être produits en grandes quantités (1X1012 particules par millilitre) et de par leur capacité à infecter des cellules quiescentes ou en division rapide. Les AdVs ont subi bon nombre de modifications pour leur permettre de traiter des cellules tumorales ou pour transporter des séquences génétiques exogènes essentielles pour le traitement de maladies monogéniques. Toutefois, les faibles niveaux d’expression du récepteur primaire de l’adénovirus, le CAR (récepteur à l’adénovirus et au virus coxsackie), réduit grandement l’efficacité de transduction dans plusieurs tumeurs. De plus, certains tissus normaux comme les muscles n’expriment que très peu de CAR, rendant l’utilisation des AdVs moins significative. Pour pallier à cette limitation, plusieurs modifications ont été générées sur les capsides virales. L’objectif de ces modifications était d’augmenter l’affinité des AdVs pour des récepteurs cellulaires spécifiques surexprimés dans les tumeurs et qui seraient exempts dans les tissus sains avoisinant. On peut mentionner dans les approches étudiées: l’utilisation de ligands bispécifiques, l’incorporation de peptides dans différentes régions de la fibre ou la substitution par une fibre de sérotypes différents. Notre hypothèse était que les domaines d’interaction complémentaire (K-Coil et ECoil) permettraient aux ligands de s’associer aux particules virales et d’altérer le tropisme de l’AdV. Pour ce faire, nous avons inclus un domaine d’interaction synthétique, le K-Coil,dans différentes régions de la fibre virale en plus de générer des mutations spécifiques pour abolir le tropisme naturel. Pour permettre la liaison avec les récepteurs d’intérêt dont l’EGF-R, l’IGF-IR et le CEA6, nous avons fusionné le domaine d’interaction complémentaire, le E-Coil, soit dans les ligands des récepteurs ciblés dont l’EGF et l’IGF-I, soit sur un anticorps à un seul domaine reconnaissant la protéine membranaire CEA6, l’AFAI. Suite à la construction des différents ligands de même que des différentes fibres virales modifiées, nous avons determiné tout d’abord que les différents ligands de même que les virus modifiés pouvaient être produits et que les différentes composantes pouvaient interagir ensemble. Les productions virales ont été optimisées par l’utilisation d’un nouveau protocole utilisant l’iodixanol. Ensuite, nous avons démontré que l’association des ligands avec le virus arborant une fibre modifiée pouvait entraîner une augmentation de transduction de 2 à 21 fois dans différentes lignées cellulaires. À cause de la difficulté des adénovirus à infecter les fibres musculaires occasionnée par l’absence du CAR, nous avons cherché à savoir si le changement de tropisme pourrait accroître l’infectivité des AdVs. Nous avons démontré que l’association avec le ligand bispécifique IGF-E5 permettait d’accroître la transduction autant dans les myoblastes que dans les myotubes de souris. Nous avons finalement réussi à démontrer que notre système pouvait induire une augmentation de 1,6 fois de la transduction suite à l’infection des muscles de souriceaux MDX. Ces résultats nous amènent à la conclusion que le système est fonctionnel et qu’il pourrait être évalué dans des AdVs encodant pour différents gènes thérapeutiques. / Adenoviruses have been studied as a way to develop new treatments for different diseases. Adenoviral vectors (AdV) are considered interesting tools for this propose, because they can be produced at high titers (1X1012 particles per millilitre) in laboratory and they have the capacity to infect non-dividing and dividing cells. AdV have been often modified in order to obtain the ability to kill tumour cells or to deliver exogenous genetic sequences essential to treat monogenic disease. However, weak expression of the primary adenovirus receptor, the CAR (Coxsackie and adenovirus receptor) reduces greatly the transduction efficiency of AdV for the tumour cells. Moreover, some normal tissues express low amount of CAR, like the skeletal muscle, reducing the appeal of using AdV as a gene delivery vehicle for this tissue. To address this problematic, many modifications were done on the adenoviral capsid. The goal of these modifications were to generate an AdV able to target specific cellular receptors that were expressed in tumour cells but not in normal cells. Several approaches were done to modify the tropism of AdV, such as incubation with a bispecific ligands, incorporation of peptides within the adenoviral fiber structure or substitution of the viral fiber with a different serotype fiber. The hypothesis of my project was to determine if an interaction domain fused within a ligand could bind the complementary domain incorporated on a virus and change the tropism of the AdV. The first step was to include a synthetic interaction domain, the K-Coil, within specific region of the adenoviral fiber, as well as inserting two point mutations to abolish the natural tropism. To target the EGF-R, IGF-IR and the CEA6, we fused the complementary interaction domain, the E-Coil, to the respective ligand known as the EGF and the IGF-I or to a single domain antibody (known as AFAI) that bind specifically to CEA6. The specific interaction between the E-Coil and K-Coil was used to associate the ligand with the fiber in order to retarget the AdV toward the selected receptor. We showed that the different ligands as well as the modified fibers could be produced and that both E-Coil and K-Coil expressing partners could interact together. We optimized the viral production by using an iodixanol purification protocol. More importantly, we clearly demonstrated that the ligand association with the fiber could increase the transduction efficiency between 2 to 21 fold against various tumour cells. The difficulty of adenovirus to infect muscle cells because of the lack of CAR expression brought us to evaluate the potential of our retargeted AdV to increase the transduction for the tissue. We showed that the use of IGF-E5 could increase the transduction efficiency in myoblasts as wells as in myotubes. We finally demonstrated that our retargeting system could increase the transduction efficiency for skeletal muscle by 1,6 fold in new born MDX mice. In conclusion, our results show that the retargeting system is indeed functional. This system could be assessed using vectors that express therapeutic genes.

Le récepteur à l'IGF1 (IGF-1R) ainsi que ses voies de régulation sous-jacentes ont été largement étudié pour leur importance au cours du développement et leur rôle mitotique sur divers types cellulaires. A l'échelle de la peau, l'IGF-1R contribue à l'homéostasie épidermique et est souvent associé au compartiment basal pour son effet pro-prolifératif. Très peu d'études ont montré son implication au niveau de la différenciation épidermique et celles-ci présentent des résultats contradictoires. Au cours du vieillissement, la peau s'amincit et la barrière épidermique présente des défauts de perméabilité. Parallèlement, l'activité de l'IGF- 1R, maximale à l'adolescence, diminue avec l'âge. Cette étude a contribué à éclaircir le rôle de l'IGF-1R sur la différenciation épidermique et à montrer un lien entre vieillissement, perte en qualité de la peau et diminution d'activité de l'IGF-1R. L'élaboration de modèles 2D et 3D mimant le vieillissement par diminution d'activité de l'IGF-1R, nous a permis de confirmer le rôle mitotique de l'IGF-1R sur les kératinocytes et les progéniteurs et de démontrer son effet régulateur sur la différenciation épidermique par augmentation ou diminution de ces marqueurs. L'IGF-1R renforce l'adhésion cellulaire sur différentes matrices, impliquant de possibles interactions avec les intégrines α6 et β1. Ces résultats ont été corrélés aux observations de biopsies de peaux jeunes et âgées. Nous avons aussi montré que l'IGF-1R conférait un état sénescent aux cellules soumises à de fortes doses d'H2O2. Ces travaux montrent ainsi que l'IGF-1R est nécessaire pour le processus de différenciation épidermique et pour en assurer sa protection face au stress oxydant / Insulin-like growth factor 1 receptor (IGF-1R) and its signaling pathway have been widely studied for their growth promoting role on many cell types and their implication in development. On skin, the IGF-1R function has been associated to basal proliferation and contributes to epidermal morphogenesis, but very little is known about its involvement on keratinocytes differentiation and the few studies existing depict contradictory results. IGF-1R activity is maximal during teenage and tend to decrease during aging. Aged skin depicts major thinning and defects in permeability of skin barrier. Our work consisted in clarifying IGF-1R role on epidermal differentiation process and emphasized a correlation between aging, loss of skin quality and IGF-1R activity. By building 2D and 3D aging like models with low IGF-1R activity, we confirmed IGF-1R mitogenic role on both basal and progenitor-like keratinocytes. We demonstrated that IGF-1R activity regulated keratinocytes differentiation by either enhancing or slowing down differentiation markers deposition. More importantly, we highlighted the importance of IGF-1R activity for keratinocytes adhesion on both laminin-332 and collagen I/IV coatings, implying possible interactions with α6 and β1 integrins. This relationship was further correlated on skin biopsies of young and aged donors. In a parallel study, we showed that IGF-1R could induce cell senescence under acute H2O2 stress. Taken together, IGF-1R is necessary for the epidermal differentiation process and protects epidermis from acute oxidative stress induced damages

O estudo teve como objetivo avaliar as respostas hormonais, fisiológicas e comportamentais de vacas Holandesas frente a situações de conforto ou estresse térmico ambiental. O experimento desenvolvido entre os verões de 2007 e 2008 no Campus Administrativo da Universidade de São Paulo (USP), Pirassununga, SP, utilizou 28 fêmeas de 1ª a 3ª lactações com produção média de 20 kg/dia divididas em dois grupos experimentais após a parição, com e sem disponibilidade de climatização em galpão do tipo free-stall. Os parâmetros ambientais foram avaliados através do cálculo do índice de temperatura e umidade (ITU). As colheitas de dados fisiológicos (temperatura retal, temperatura de superfície corporal do dorso e base da cauda, frequência respiratória), hormonais (cortisol e IGF-I), comportamentais e de produção e qualidade do leite foram realizadas em cinco condições climáticas no ano (outono, inverno, primavera, verão seco e verão chuvoso) caracterizadas pela temperatura, umidade relativa e radiação solar. No verão os animais foram submetidos ao Teste de Capacidade Termolítica (CT) e a um estudo comparativo de um período de sete dias sob estresse calórico em câmara climática e desafio com aplicação de ACTH. No experimento 1 o teste de capacidade termolítica foi validado. A CT foi igual para animais em lactação ou secos (P>0,05), e maior para vacas mantidas sob sistema de climatização ao longo do ano (P<0,01). Houve influência da exposição ao sol sobre todas as variáveis fisiológicas (P<0,01). Os níveis plasmáticos de cortisol foram maiores antes da exposição ao sol e depois do repouso por 1 hora à sombra para vacas em lactação (P=0,03) e para as sem disponibilidade de climatização (P=0,03). O IGF-I foi maior nas vacas secas em final de gestação (P<0,01). No experimento 2 a temperatura retal não teve influência da climatização, com os dois grupos apresentando valores abaixo de 38,56 ºC ao longo do ano (P=0,11). Observou-se uma tendência de alta (P<0,01) nas concentrações plasmáticas de cortisol entre outono e inverno, começando o decréscimo até o início do verão seco e um novo aumento durante o verão chuvoso, e um comportamento inverso para o IGF-I. Temperatura retal mostrou uma correlação moderada e positiva (P<0,01) com a temperatura superficial (0,46) e frequência respiratória (0,35). A temperatura do ar e ITU apresentaram correlações positivas de moderada à alta com as temperaturas retal, da base da cauda e superficial, e também com a frequência respiratória (P<0,01). No experimento 3 as vacas passaram a maior parte do dia na sombra em pé (84,2 %) independente da estação do ano. O ambiente climatizado proporcionou maior frequência de alimentação e produção de leite durante o verão (P<0,05), assim como teor de gordura 17,9 % maior (P<0,01). No experimento 4 os animais foram submetidos ao estresse pontual causado pelo uso do ACTH e ao estresse calórico prolongado em câmara climática. Tanto a administração de ACTH quanto a exposição ao calor prolongado em câmara climática aumentaram os níveis de cortisol plasmático. Durante o estresse calórico houve diminuição do IGF-I e produção leiteira e aumento das variáveis fisiológicas ligadas a termorregulação. / The study aimed to evaluate the hormonal, physiological and behavioral responses of Holstein cows in situations of a comfort or heat environment. The experiment was conducted between the summers of 2007 and 2008 in the University of São Paulo (USP), Pirassununga, SP, and used 28 females from 1st to 3rd lactations with average production of 20 kg/day divided into two experimental groups after birth, with and without an evaporative cooling system in a free-stall. Environmental parameters were evaluated by temperature and humidity index (THI). Collection of physiological data (rectal temperature, body surface, internal base of tail, respiratory rate), hormonal (cortisol and IGF-I), behavioral and production and milk quality were conducted in five climatic conditions (autumn, winter, spring, dry summer and rainy summer) characterized by air temperature, relative humidity and solar radiation. In summer the animals were subjected to Thermolysis Capacity Test (CT) and a comparative study of a period of seven days under heat stress in climatic chamber and challenged with ACTH administration. In experiment 1 the Thermolysis Capacity Test was validated. The CT was the same for dry or lactating animals (P>0.05) and higher for cows kept in evaporative cooling system throughout the year (P<0.01). The results showed influence of sun exposure on all physiological variables (P<0.01). Plasma levels of cortisol were higher before sun exposure and after the one hour rest under shade for lactating cows (P=0.03) and for no cooled animals (P=0.03). IGF-I was higher in dry cows in late gestation (P<0.01). In experiment 2 the evaporative cooling system did not show influence on rectal temperature, with both groups having values below 38.56 ºC throughout the year (P=0.11). It was observed an upward trend (P<0.01) in plasma cortisol concentrations between autumn and winter, starting the decline until the beginning of dry summer and a further increase during rainy summer, and an opposite pattern for IGF- I. Rectal temperature showed a moderate and positive correlation (P<0.01) with the body surface temperature (0.46) and respiratory rate (0.35). The air temperature and THI showed moderate to high positive correlations with rectal temperatures, and the internal base of tail, and also with the respiratory rate (P<0.01). In experiment 3 cows spent most of the day standing in the shade (84.2%) regardless of season. Cooled cows had a higher feeding frequency and milk production during summer (P<0.05) and fat content 17.9% higher (P<0.01). In experiment 4, cows were subjected to the short stress caused by the ACTH administration and prolonged heat stress in climatic chamber. Both the administration of ACTH as prolonged exposure to heat in climatic chamber increased the levels of plasma cortisol. During heat stress a decrease in plasma IGF-I and milk production was observed, and an increase in physiological variables related to thermoregulation.

A Distrofia Muscular de Duchenne (DMD) é uma doença recessiva ligada ao cromossomo ?X?, causada pela ausência da proteína distrofina que ocorre em vários tecidos, sendo caracterizada por uma severa disfunção da musculatura esquelética ocasionando morte prematura do paciente. Embora controverso, alguns autores reportaram que o GH (hormônio do crescimento) estaria implicado no desenvolvimento da doença e poderia ser utilizado no tratamento da mesma. Desta forma, neste estudo a dosagem sérica de IGF-I (Fator I de crescimento Similar à insulina) um peptídeo GH-dependente que regula as ações do hormônio do crescimento, foi realizada no intuito de verificar se existe correlação ou não entre o desenvolvimento da doença e a concentração sérica de IGF-I. As variações nos níveis deste hormônio foram demonstradas com o decorrer da idade sendo que, nos três primeiros meses todos os animais apresentaram comportamento semelhante com aumento dos níveis de IGF-I, porém no quarto mês os animais Distróficos benignos apresentaram redução média de 34% deste hormônio, enquanto ocorreu aumento de 1% no animal não Distrófico. Ainda, as células granulares adenohipofisárias relacionadas à produção do hormônio de crescimento em cães Golden Retriever com Distrofia Muscular apresentam-se maiores do que nos cães Golden Retriever não distróficos quanto aos seguintes parâmetros (eixo longo, área seccional e volume celular). À microscopia eletrônica de transmissão observamos que as células estudadas apresentam grânulos elétron-densos de parede dupla e distribuídos por todo o citosol. Estes grânulos apresentaram-se maiores nos animais Distróficos quando comparados aos animais não Distróficos. / The Duchenne Muscular Dystrophy (DMD) is a X linked recessive disease, caused by the absence of dystrophin which is found in a variety of tissues and characterized by a severe disfunction of the skeletal musculature that results in a premature death of the patient. Theoretically, the growth hormone (GH) is considered to be associated to the development of Muscular Dystrophy and that could be used in its treatment. Hence in this study, a IGF-I (Insulin like growth factor-I) seric dosage was performed to verify whether or not there might be a between the evolution of the disease and IGF´s seric concentration. IGF-I is a GH-peptide dependent that regulates the GH actions during the growth. Changes in IGF-I levels were recorded during the dog?s post-natal development. On the first trimester, all animals presented similar IGF-I levels, although in the fourth month, a stark 34% decrease was observed in the dystrophic animals whereas a 1% increase was seen in the healthy dog. Furthermore, the GRMD´s granule-containing cells were larger when compared to the healthy animals. The following parameters in this comparison: long axis, cross-sectional area and cell volume. The ultrastructural study showed electron-dense granules composed by a double membrane and homogeneously distributed through the cell. These granules were larger in the dystrophic animals then in healthy dogs.

A arginina é um aminoácido condicionalmente essencial que participa de inúmeras reações metabólicas no organismo como, por exemplo, o ciclo da uréia, a síntese de creatina e a geração de óxido nítrico (NO). Além dessas funções a arginina é associada, com a sensibilidade à insulina, a secreção de GH e mais recentemente com a síntese protéica muscular. O objetivo deste trabalho foi investigar o efeito da suplementação via oral crônica de L-arginina sobre a síntese protéica muscular, pela via da mTOR, a fim de contribuir com as novas discussões científicas acerca deste aminoácido de ampla atuação. Métodos: Foram utilizados ratos wistar machos adultos com cerca de 200g de peso corporal divididos em quatro grupos de quatorze animais denominados na seguinte forma: Arginina Treinado (AT), Arginina Sedentário (AS), Dieta-Controle Treinado (CT) e Dieta-Controle Sedentário (CS). Ambas as dietas foram elaboradas com base das recomendações da AIN-93, sendo que a dieta enriquecida com arginina recebeu acréscimo de 2% deste aminoácido e a dieta controle recebeu um mix de aminoácidos não essenciais para garantir que ambas fossem isonitrogenadas e isocalóricas e as proporções de aminoácidos presente nas rações foi conferida por aminograma. O treinamento dos animais consistiu em exercício anaeróbio com sessões que eram compostas de 4 séries de 10 saltos com um minuto de descanso entre estas em tanque de água. Os saltos eram desempenhados com carga de 50% do peso corporal acoplado ao tórax dos animais na freqüência de 5 dias por semana por 6 semanas. A evolução da massa corporal dos animais bem como o consumo de ração foram avaliadas três vezes por semana e estimada uma média semanal. Foram realizados testes de tolerância oral à glicose (OGTT) e tolerância à insulina (ITT) no início e ao final do experimento em todos os animais para avaliar alterações na sensibilidade à insulina. Após 72hs da última sessão de treinamento os animais foram anestesiados para infusão de insulina, coleta dos músculos gastrocnêmio e plantar, fígado, sangue e eutanasiados conforme protocolo aprovado pelo CEA-USP. As análises bioquímicas foram determinações séricas de insulina, GH, IGF-1 e a proteína transportadora de IGF-1 (IGFBP-3), glicose plasmática, uréia e creatinina séricas, IGF-1 muscular e hepático por kits comerciais de tecnologia multiplex Luminex e aminograma sérico por cromatografia. As análises moleculares foram realizadas para as proteínas chaves envolvidas na via de síntese protéica muscular em sua forma total e fosforilada, sendo estas: IRS-1, Akt, mTOR, 4E-BP1 e p70S6K determinadas por método de western blotting. Resultados: Não foram encontradas diferenças estatisticamente significativas nos parâmetros avaliados com exceção da creatinina que se mostrou mais elevada nos grupos suplementados com arginina. A suplementação de arginina, nas concentrações administradas, bem como o exercício de alta intensidade pelo período determinado não foram capazes de alterar a expressão das proteínas envolvidas na regulação de síntese protéica muscular de ratos nem a sensibilidade celular à insulina. Conclusão: não houve aumento da síntese protéica muscular com a suplementação de arginina, nestas condições experimentais. / The arginine is an amino acid conditionally essential that participates in innumerous metabolic reactions in the body like, for instance, the urea cycle, the synthesis of creatine and production of nitric oxide (NO). Besides those functions the arginine is associated, with the insulin sensitivity, GH secretion and most recently with muscle protein synthesis. The aim of this work was to investigate the effect of L-arginine chronic oral supplementation on the muscle protein synthesis, through mTOR pathway, in order to contribute with new scientific discussions about this broad action amino acid. Methods: Wistar male adult rats were used with about 200g body weight distribute into four groups of fourteen animals named this way: Trained Arginine (TA), sedentary Arginine (SA), Trained Diet-Control (TC) and Sedentary Diet-control (SC). Both diets were elaborated based on the AIN-93 recommendations, considering that the enriched diet with arginine was added 2% of this amino acid and the control diet received a mix of non-essential amino acid in order to ensure that both were isonitrogenous and isocaloric and the proportions of present amino acids in the rations have been checked through aminogram. The animals training consisted of anaerobic exercise with sections composed by four jump series, with one minute rest among these in a PVC cube water. The jumps were performed with a load of 50% of their body weight attached in the animal\'s trunk, five days a week over six weeks. The animals\' body weight evolution as well as the food intake were evaluated three times a week in order to figure a weekly average. Oral glucose test tolerance (OGTT) and insulin test tolerance (ITT) have been done in the beginning and in the end of the experiment in all animals to evaluate insulin sensitive changes. The animals were anesthetized to insulin infusion, gastrocnemic and plantaris muscles, liver and blood collects 72 hrs after the last training section and afterwards sacrificed according to CEA-USP approved protocol. The biochemical analysis were blood determination of insulin, GH, IGF-1 and its binding protein 3 (IGFBP-3), glucose, urea and creatinine, and muscle and liver IGF-1 through commercial kits of multiplex Luminex technology and seric aminogram through chromatography. The molecular analysis were performed for the key proteins of the muscle protein synthesis pathway in its total and phosphorylated form: IRS-1, Akt-1, mTOR, 4E-BP1 and p70S6K determined by western blotting method. Results: Significant statistical differences were not found to all evaluated biomarkers in this experiment except for creatinine which was more elevated in groups supplemented with arginine. The arginine supplementation, in these given doses, as well as the high-intense exercise, failed in stimulate both the expression of the proteins involved in the muscle protein synthesis regulation and the insulin sensitivity in the rats in this condition. Conclusion: There hasn\'t been any increase in the muscle protein synthesis with arginine supplementation, in these experimental conditions.