Resumo: A formação da musculatura esquelética nos membros de frango é um processo complexo, dividido em várias etapas, desde a formação do somito, progenitor da musculatura, até o músculo propriamente dito. O presente trabalho teve o objetivo de caracterizar o perfil de expressão de genes associados aos fatores miogênicos MyoD, MRF4 e Miogenina (responsáveis pela formação e manutenção da musculatura) e Miostatina. Utilizaram-se 40 animais de duas linhagens (20 de corte e 20 de postura), cujos tecidos das coxas e sobrecoxas foram coletados em duas idades (21 e 42 dias póseclosão). A técnica de PCR quantitativa foi empregada nas análises da expressão gênica e os dados obtidos foram analisados com a utilização do programa REST (Ferramenta para análise de expressão relativa - Relative Expression Software Tool) que se baseia em quantificação relativa. Devido à divergência fenotípica no padrão de crescimento das linhagens estudadas, esperava-se encontrar diferenças na expressão de miostatina, as quais não foram confirmadas. Por outro lado, foram verificadas diferenças para MRF4 nos músculos Gastrocnêmio (coxa) e Biceps (sobrecoxa) em ambas as idades, e 9 para Miogenina, em Gastrocnêmio, aos 42 dias; com maior expressão na linhagem CC. A caracterização dos genes estudados foi parcial, abrindo a possibilidade para que outros genes possam ser selecionados com intuito de compreender o desenvolvimento muscular em frangos. / Abstract: The development of the chicken limb muscles is a complex process, divided into several stages from the initial somite formation to the muscle itself. This study aimed to characterize expression profile of genes associated to myogenic factors MyoD, MRF4 and Miogenin (responsible for muscle formation and maintenance) and Miostatin. It was used 40 animals from two lines (20 broiler and 20 layer), whose thigh and drumstick tissues were collected at two ages (21 and 42 days post-hatch). Gene expression employed techniques of quantitative PCR and data were analyzed by REST program, which is based on a relative quantification. Due to phenotypic divergence in the growth pattern of the strains studied, it was expected to find differences in the expression of miostatin that were not confirmed. On the other hand, differences were found for MRF4, with highest expression in CC line. The characterization of genes studied was partial, opening the possibility that other genes may be selected in order to understand the muscle development in chickens. / Orientador: Patrícia Pasquali Parise-Maltempi / Coorientador: Helena Javiel Alves / Banca: Millor Fernandes do Rosário / Banca: Anderson Luis Alves / Mestre

Made available in DSpace on 2014-06-11T19:22:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-14Bitstream added on 2014-06-13T19:49:26Z : No. of bitstreams: 1 marchesin_ml_me_rcla.pdf: 512126 bytes, checksum: f1af03206b1ca9a151524c3b6960cca3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A formação da musculatura esquelética nos membros de frango é um processo complexo, dividido em várias etapas, desde a formação do somito, progenitor da musculatura, até o músculo propriamente dito. O presente trabalho teve o objetivo de caracterizar o perfil de expressão de genes associados aos fatores miogênicos MyoD, MRF4 e Miogenina (responsáveis pela formação e manutenção da musculatura) e Miostatina. Utilizaram-se 40 animais de duas linhagens (20 de corte e 20 de postura), cujos tecidos das coxas e sobrecoxas foram coletados em duas idades (21 e 42 dias póseclosão). A técnica de PCR quantitativa foi empregada nas análises da expressão gênica e os dados obtidos foram analisados com a utilização do programa REST (Ferramenta para análise de expressão relativa - Relative Expression Software Tool) que se baseia em quantificação relativa. Devido à divergência fenotípica no padrão de crescimento das linhagens estudadas, esperava-se encontrar diferenças na expressão de miostatina, as quais não foram confirmadas. Por outro lado, foram verificadas diferenças para MRF4 nos músculos Gastrocnêmio (coxa) e Biceps (sobrecoxa) em ambas as idades, e 9 para Miogenina, em Gastrocnêmio, aos 42 dias; com maior expressão na linhagem CC. A caracterização dos genes estudados foi parcial, abrindo a possibilidade para que outros genes possam ser selecionados com intuito de compreender o desenvolvimento muscular em frangos. / The development of the chicken limb muscles is a complex process, divided into several stages from the initial somite formation to the muscle itself. This study aimed to characterize expression profile of genes associated to myogenic factors MyoD, MRF4 and Miogenin (responsible for muscle formation and maintenance) and Miostatin. It was used 40 animals from two lines (20 broiler and 20 layer), whose thigh and drumstick tissues were collected at two ages (21 and 42 days post-hatch). Gene expression employed techniques of quantitative PCR and data were analyzed by REST program, which is based on a relative quantification. Due to phenotypic divergence in the growth pattern of the strains studied, it was expected to find differences in the expression of miostatin that were not confirmed. On the other hand, differences were found for MRF4, with highest expression in CC line. The characterization of genes studied was partial, opening the possibility that other genes may be selected in order to understand the muscle development in chickens.

Myogenic differentiation of muscle cells in culture is characterized by changes in morphology and in pattern of gene expression. When the myoblasts in culture are induced to diffrentiate either by cell to cell contact or by serum-starvation, a vast array of muscle tissue-specific genes including α-actin, are activated and is accompanied by a concomitant down-regulation of non-muscle genes such as, β- and γ-actins, tubulins etc.. The coordinate activation of muscle-specific genes is suggested to be mediated by cis-acting regulatory sequences in the muscle gene-promoters and muscle-tissue-specific DNA-binding proteins belonging to the MyoD class of regulators. But the mechanism behind the repression of non-muscle gene expression during differentiation has not yet been well understood. To date, no consensus has been achieved on the mechanism governing the down-regulation of β- actin gene, and no information is available on the regulation of -y-actin gene during muscle differentiation. The results from the present study showed that during differentiation of BC^3H1 cells the β- and γ-actins genes were down-regulated to ≈25% of their initial levels in undifferentiated cells. Measurement of half-life during differentiation indicated that the half-lives of both β- and γ-actins decreased to ≈25% of their original levels in myoblasts. These results suggest that changes in mRNA stability play an important role in the down-regulation of non-muscle actin genes. Second messengers and oncogenes are known to block the differentiation program of muscle cells. In the present study cAMP and ElA were observed to inhibit the down-regulation of β- and γ-actin genes in BC^3H1 cells. In both cAMP-and E1A- treated cells the β- and γ-actin mRNAs were found to have a higher half-life than the untreated differentiated BC^3H1 cells. This observation also suggests that mRNA stability might play an important role in the regulation of β- and γ-actin gene expression. The muscle-specific α-actin is activated by cell-cell contact and serum-starvation. Results in the present study suggested that cAMP was able to inhibit the activation of α-actin expression mediated by serum-starvation while it had no significant effect on the signal mediated by cell-cell contact. It is hypothesized that the two signals mediating a-actin activation might follow different intracellular signalling pathways. The effects of cAMP and E1A on the expression of muscle-specific and non-muscle actins could be a direct primary event or might be an indirect secondary event, mediated by other intracellular factors such as myogenin. The results showed that cAMP did not block the transcription of the myogenin while secondary evidences suggested that cAMP might negatively-regulate myogenin at a point downstream of transcription. E1A was observed to block the expression of myogenin gene suggesting that E1A might be mediating its effect through myogenin. Because the muscle-specific(α) and non-muscle(β- and γ-) isoforms were expressed both in the presence and in the absence of myogenin, myogenin's role in the regulation of actin genes is unclear. / Thesis / Master of Science (MS)

To determine a possible role for NCX in myogenically active smooth muscle arterioles, studies were conducted by manipulation of extracellular Na+ levels and inhibition of the exchanger. Western blotting was performed for the identification of the NCX protein. Real-time PCR was performed to demonstrate the level of expression of mRNA, for the NCX isoforms. Antisense oligonucleotides against NCX mRNA were introduced in an isolated cremaster arteriole followed by functional studies after 24 hours. Level of expression of NCX was determined by western blotting. The data are consistent with the presence of NCX1 in the cremaster arterioles.

In isolated skeletal muscle arterioles, increasing transmural pressure causes an increase in constriction. This active myogenic response varies with age and fiber type. Increased transmural pressure activates both Ca2+-activated (KCa) potassium channels and voltage-dependent (Kv) potassium channels; these channels have a role in the negativefeedback pathways that modulate depolarization and myogenic constriction. We tested the hypothesis that increased KCa channel and Kv channel activity contribute to reduced myogenic responsiveness in skeletal muscle arterioles of aged rats. 1A arterioles were isolated from soleus, an oxidative muscle, and superficial gastrocnemius, a glycolytic muscle, of young (4 mos) and aged (24 mos) Fischer 344 rats. Myogenic responses were assessed by increasing intraluminal pressure (0-140 cm H2O) in increments of 20cm H2O. Vasoconstrictor response were determined in response to increasing concentrations of the KCa channel blocker, charybdotoxin (CTX; 10-10 to 10-7 M) and the Kv channel blocker, 4-Aminopyridine (4-AP; 10-5 to 10-2 M). To determine the role of potassium channels in modulating the myogenic response, cannulated arterioles from soleus and gastrocnemius were incubated with CTX (50 nM) and 4-AP (5mM) for 15 minutes prior to evaluation of the myogenic response. Increased Kv channel activity contributes to reduced myogenic constriction in soleus and gastrocnemius muscle arterioles from aged rats. In soleus muscle arterioles, KCa channel activity opposes myogenic tone in young but not old rats. In gastrocnemius muscle arterioles, treatment with CTX did not eliminate age-related differences in the myogenic response, and the KCa channel contribution to myogenic tone was, in fact, greater arterioles from young as compared to old rats. Kv channels contribute to greater myogenic constriction in soleus arterioles, KCa channels appear to be more active in gastrocnemius muscle arterioles as compared to soleus muscle arterioles. Therefore Kv and KCa channels are tonically active in skeletal muscle arterioles, contributing to a hyperpolarizing force that opposes myogenic constriction. Furthermore, increased Kv channel activity contributes to the age-related reduction of myogenic constriction in soleus and gastrocnemius muscle arterioles.

No description available.

Renal autoregulation is unique and critically important in maintaining homeostasis in the body via control of renal blood flow and filtration. The myogenic reflex responds directly to pressure variation and is present throughout the vasculature in varying degrees, while the tubuloglomerular feedback (TGF) mechanism adjusts microvascular resistance and glomerular filtration rate (GFR) to maintain distal tubular NaCl delivery. No simple models are available which allow the independent contributions of the myogenic and TGF responses to be compared and which include control over multiple metabolic and physiological parameters. Independently developed mathematical models of myogenic autoregulation and TGF control of GFR have been combined to produce a comprehensive model for the rat kidney which is responsive to multiple small step changes in mean arterial pressure. The system encompasses every level of the renal vasculature and the tubular system of the nephrons while simultaneously incorporating the modulatory effects of changes in viscosity and shear stress-induced nitric oxide (NO) production. The vasculature of the rat kidney has previously been divided via a Strahler ordering scheme using morphological data derived from micro-CT imaging. This data, combined with an extensive literature review of the relevant experimental data, led to the development of order-specific parameter sets for each of the eleven vascular levels. The model of the myogenic response depends primarily on circumferential wall tension, corresponding to a distally dominant resistance distribution with the highest contributions localized to the afferent arterioles and interlobular arteries. The constrictive response is tempered by the vasodilatory influence of flow-induced NO. Experimental comparison with data from groups that inhibited the TGF mechanism showed that the model was able to accurately reproduce the characteristics of renal myogenic autoregulation. This myogenic model was coupled with a system of equations that represented both spatial and temporal changes in concentration of the filtrate in the tubular system of the nephrons and the corresponding resistance changes of the afferent arteriole via the TGF mechanism. Computer simulation results of the system response to pressure perturbations were examined, as well as the interaction between mechanisms and the modulatory influences of metabolic and hemodynamic factors on the steady state and transient characteristics of whole-organ renal autoregulation. The responses of the model were consistent with experimental observations and showed that the frequency of the myogenic reflex was approximately 0.4 Hz while that of TGF was 0.06 Hz, corresponding to a 2-3 sec response time for myogenic contraction and 16.7 sec for TGF. Within the autoregulatory range step increases in pressure induced damped oscillations in tubular flow, macula densa NaCl concentration, arteriolar diameter, and renal blood flow. The model demonstrated that these oscillations were triggered by TGF and confined to vessels less than 100 micrometer in diameter. The pressure response in larger vessels remained important in characterizing total autoregulatory efficacy. Examination of the steady-state and transient characteristics of the model results demonstrates the necessity of considering the whole organ response in studies of renal autoregulation. A comprehensive model of autoregulation also allows for the examination of pathological states, such as the altered NO production in hypertension or the excess tubular reabsorption of water seen in diabetes. The model was able to reproduce experimental results when simulating diseased states, enabling the analysis of impaired autoregulation as well as the identification of key factors affecting the autoregulatory response.

The zebrafish (Danio rerio) has been used as a model for studying vertebrate development in the cardiovascular system. In order to monitor heart contraction and cytosolic calcium oscillations, fish were either embedded in methylcellulose or anesthetized with tricaine. Using high-resolution differential interference contrast and calcium imaging microscopy, we here show that dopamine and verapamil alter calcium signaling and muscle contraction in anesthetized zebrafish, but not in embedded zebrafish. In anesthetized fish, dopamine increases the amplitude of cytosolic calcium oscillation with a subsequent increase in heart contraction, whereas verapamil decreases the frequency of calcium oscillation and heart rate. Interestingly, verapamil also increases myocardial contraction. Our data further indicate that verapamil can increase myocardial calcium sensitivity in anesthetized fish. Taken together, our data reinforce in vivo cardiac responses to dopamine and verapamil. Furthermore, effects of dopamine and verapamil on myocardial calcium and contraction are greater in anesthetized than embedded fish. We suggest that while the zebrafish is an excellent model for a cardiovascular imaging study, the cardio-pharmacological profiles are very different between anesthetized and embedded fish.

No description available.

No description available.