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A proteólise limitada de certas proteínas leva à liberação de peptídeos opióides endógenos. Vários relatos apontam que peptídeos derivados da hemoglobina como hemorfinas e hemopressinas têm efeito antinociceptivo, pela atividade de modulação de receptores acoplados a proteínas G. No presente estudo, um ensaio de captura do substrato (ECS) foi combinado com a marcação isotópica e LC-MS/MS para identificar e caracterizar um novo fragmento da hemoglobina que se liga à EP24.15. O peptídeo AGH, identificado neste trabalho, inibe respostas de hipernocicepção periféricas através de receptores opióides do tipo <font face=\"Symbol\">m . A persistência do peptídeo AGH no tecido nervoso perfundido sugere relevância fisiológica. Embora o AGH seja derivado de hemoglobina e tenha atividade opióide, falta-lhe a sequência chave das hemorfinas (YPWT), indicando que ele pode pertencer a uma nova classe de peptídeos derivados da hemoglobina. Adicionalmente, o AGH modula as interações entre as proteínas 14-3-3<font face=\"Symbol\">e e EP24.15 in vitro, podendo estar relacionado com a secreção não convencional da EP24.15. / Limited proteolysis of certain proteins leads to the release of endogenous opioid peptides. Several reports have shown that hemoglobin-derived peptides such as hemorphins and hemopressins have an antinociceptive effect by modulating GPCR activity. In the present study, a substrate capture assay (SCA) was combined with isotopic labeling and LC-MS/MS to identify and characterize a new bioactive hemoglobin fragment that binds to EP24.15. AGH, a new peptide identified in this work, inhibits peripheral hyperalgesic responses through <font face=\"Symbol\">m opioid receptors (MOR). The persistence of AGH peptide in perfused nervous tissue suggests its physiological relevance. Although AGH is derived from hemoglobin and it is a peptide with opioid activity, it lacks the key sequence of hemorphins (YPWT), indicating that it is part of a new class of peptides derived from hemoglobin. Additionally, the AGH modulates interactions between 14-3-3<font face=\"Symbol\">e and EP24.15 proteins in vitro and may be related to the unconventional EP24.15 secretion.

Insulin plays a vital role in whole-body metabolism and provides a major anabolic stimulus for cellular signaling pathways, including those involved in the metabolism of glucose and protein. Consequently, insulin dysregulation (ID) is known to alter molecular signal transduction in insulin-sensitive tissues such as skeletal muscle, thereby disrupting glucose metabolism and compromising protein synthetic capacity. Our first objective was to induce ID in healthy horses by administering dexamethasone (DEX), a potent glucocorticoid, for 21 days. We evaluated the effects on insulin-stimulated muscle protein signaling components involved in the mammalian target of rapamycin (mTOR) pathway. DEX-induced ID reduced insulin-stimulated activation of downstream (rpS6, 4EBP-1) mTOR signaling and increased atrogin-1 abundance, a marker for protein breakdown (P < 0.05). Additionally, 21 days of DEX elevated plasma amino acids levels in insulin-stimulated conditions, indicative of reduced uptake or increase release into circulation (P < 0.05). The second objective was to evaluate the short-term effects of DEX treatment in healthy horses. Plasma insulin, glucose and amino acid dynamics and activation of mTOR signaling pathways following an oral sugar test (OST) or intake of a high protein meal were evaluated before and after 7 days of DEX treatment, and after 7 days of no treatment. Seven days of DEX treatment increased basal levels of glucose, insulin and several amino acids (P < 0.05). Additionally horses treated with DEX had an exacerbated insulin response to the OST and consumption of the high protein meal in comparison to control horses (P < 0.05). The majority of blood metabolites returned to basal levels after 7 days of recovery from DEX treatment, indicating these effects were transient. Short-term DEX treatment decreased overall activation of mTOR and FoxO3 but increased total FoxO3 and IRS-1 abundance (P < 0.05). Postprandial activation of rpS6 was greater in horses treated with DEX for 7 days but was lower in those horses after 7 days of recovery from treatment (P < 0.05). Postprandial activation of ULK and AMPK tended to be greater in DEX treated horses (P < 0.1). Akt phosphorylation and mysotatin abundance were lower after the OST in DEX treated horses (P < 0.05). The final objective was to evaluate whether similar changes in postprandial metabolic responses would be seen in horses with naturally occurring ID. Plasma insulin, glucose and amino acid responses following ingestion of a high protein meal were determined in mature horses with equine metabolic syndrome (EMS). Horses with EMS had higher basal plasma insulin concentrations but lower levels of aspartate, glutamate, asparagine and plasma urea nitrogen in comparison to healthy controls (P < 0.05). Consumption of a high protein meal resulted in a 9-fold greater insulin response and higher postprandial levels of various amino acids (P < 0.05). Together this research indicates that ID affects whole body protein metabolism by altering cellular signaling pathways in healthy and diseased horses.

The principle objective of this investigation was to develop an understanding of the biochemical and physiological response of free-ranging adult polar bears (<i>Ursus maritimus</i>) to prolonged fasting. A body condition index was developed from two measures, total body mass and straight-line body length, and was used as a covariate in the analyses of all other data. Protein and amino acid catabolism and urea synthesis were significantly lower in fasting bears when compared to feeding bears, and in fat bears when compared to lean bears. The inference from these results is that the energy metabolism in both states (fasting and fat) is one in which lipid is the predominant fuel for energy and nitrogen is conserved. Nutritional state (feeding versus fasting) had no significant effect on the plasma concentrations of non-esterified fatty acid, glycerol, and ketone bodies, or on the plasma ratio of acyl-carnitine to free carnitine. Furthermore, acetoacetate concentration was below the level of detection (<196 [mu]mol/L) in all bears, and â-hydroxybutyrate concentration never exceeded 291 [mu]mol/L. These results suggest polar bears are able to regulate closely the synthesis, release, and use of lipid metabolites without significant alteration in their plasma concentrations. Fasting polar bears showed no evidence of essential fatty acid (EFA) deficiency; the proportions of the diet-derived EFA linoleic (18:2[omega]6) and á-linolenic (18:3[omega]3) acids in the plasma and adipose tissue of fasting polar bears were greater than that in feeding polar bears. Plasma triiodothyronine concentrations and rectal temperatures were lower in fasting bears captured during summer-fall than in feeding bears, which suggests metabolic rates were decreased during fasting to conserve body fuels. Liver glycogen concentrations were found to be higher in fasting polar bears than in feeding bears. Furthermore, the results from intravenous administration of glucose (glucose tolerance test) to polar bears indicated the rates of insulin secretion and clearance in polar bears were slow relative to rates reported for other mammals. The inference from these results is that polar bears are not as dependent on glucose for energy as are other mammals and, as a consequence, are more lax in regulating their body glucose stores.

The ability to evade apoptosis is an acquired characteristic associated with many normal and pathophysiological processes. TMEM 85 represents a novel transmembrane domain containing human protein isolated in our previous screen for Bax suppressors, but whose function is currently unknown. Using viability and growth assays, we confirmed that TMEM 85 is anti-apoptotic. Four unique human cDNA sequences containing regions distinct from and of perfect identity to our cDNA were present in the database. Analysis of TMEM 85 suggests that it consists of five exons, alternatively spliced to produce at least four different mRNA's and proteins (TMEM 85v1-v4). RT-PCR analysis using RNA isolated from mice and humane tissues show that all transcripts are expressed. Yeast contain an orthologue of the human TMEM 85v1 protein, YGL213C. Surprisingly, the viability assay indicated that mutants lacking YGL231c do not show a hyper-responsive apoptotic phenotype, however its overexpression shows that it is nevertheless anti-apoptotic. Using a yeast strain expressing chromosomally TAP-tagged YGL231c, we found no up-regulation of the endogenous gene due to stress. The deletion mutant is also known to expresses a synthetically lethal phenotype in the presence of alpha-synuclein. While expression of alpha-synuclein caused significant death in both the wild type and deletion mutants, TMEM 85v2 was unable to exhibit a protective role. These findings demonstrate the complexity of the TMEM 85 gene and its anti-apoptotic function in both yeast and human.

The role of podocytes in glomerular permselectivity is tightly associated with their intricate morphology, featuring interdigitating foot processes from adjacent cells. The actin cytoskeleton is an integral component of podocyte foot processes and is regulated by a number of proteins expressed in podocytes. Rho-family of small GTPases are known key regulators of the actin cytoskeleton. This study, investigated the role of Rac1 in podocytes, using conditionally immortalized mouse podocytes (MPs). We studied Rho-GTPase activities and morphology/cytoskeleton of differentiating mouse podocytes stably expressing nephrin. We also studied the impact of transfection of various Rho-GTPase mutants and IQGAP1 mutants. We demonstrated that nephrin expression potentiates and sustains Rac1 activity during the differentiation process. We showed that Rac1 contributes to process formation in differentiating MPs and may have a similar role in vivo when podocytes are recovering from injuries.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2004. / Härtill 4 uppsatser.

Orientador: Maria Cristina Gomes Marcondes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T10:54:20Z (GMT). No. of bitstreams: 1 Salomao_EmilianneMiguel_M.pdf: 747643 bytes, checksum: a4d8091683306c9beadb732986faade9 (MD5) Previous issue date: 2005 / Resumo : A intensa mobilização de substratos dos tecidos da carcaça do hospedeiro, em função do crescimento neoplásico, promove no organismo o estado caracterizado como caquexia. Presente na maioria dos pacientes com câncer, a caquexia promove intensa perda involuntária de peso decorrente, preferencialmente, da depleção da proteína muscular em função do aumento da degradação e/ou da diminuição da síntese protéica, culminando na redução da qualidade e expectativa de vida. Trabalhos têm mostrado que o desenvolvimento do câncer dá-se de forma mais agressiva e severa quanto mais jovem for o paciente. Considerando-se que: a) os aminoácidos de cadeia ramificada, principalmente a leucina, participam ativamente na sinalização celular, estimulando a síntese como também inibindo a degradação protéica; b) a glutamina, aminoácido não essencial, participa indiretamente no aumento da síntese protéica muscular esquelética, como também da diminuição do catabolismo protéico; c) o exercício físico exerce grande estimulação da síntese protéica; temos por principal interesse, neste trabalho, minimizar as alterações metabólicas do tecido hospedeiro frente ao crescimento neoplásico. Assim, associando-se a suplementação nutricional de leucina e/ou glutamina e exercício físico aeróbio, de intensidade leve a moderada, ao crescimento do carcinossarcoma de Walker 256, avaliamos a composição corpórea, o metabolismo protéico do músculo gastrocnêmio, o estresse oxidativo e o perfil sérico (proteína, albumina, globulina, glicose, lípedes totais, colesterol total, colesterol HDL, triacilgliceróis e lactato) em ratos Wistar em pleno desenvolvimento corporal. Nossa tentativa foi, com a suplementação nutricional aliada ao exercício, prevenir ou atenuar o estado caquético do animal conseqüente a evolução tumoral. Os resultados mostraram que os grupos inoculados com tumor apresentaram redução do peso corpóreo e da ingestão alimentar no final dos experimentos. Verificamos alterações na composição corpórea química, tais como, elevado teor de água corpórea, redução do teor de gordura total e diminuição do nitrogênio colageno da carcaça nos ratos implantados com tumor de Walker 256. Houve, ainda, diminuição da síntese e aumento da degradação no músculo gastrocnêmio nos animais implantados com tumor. Além disso, observou-se aumento do conteúdo de malondialdeído (MDA), em relação ao conteúdo de antioxidantes como GST, nos músculos gastrocnêmio e cardíaco indicando aumento do estresse oxidativo associado à evolução tumoral. Observamos também, redução dos teores séricos de proteína, albumina, glicose, colesterol e HDL nesses animais portadores de tumor. A associação suplementação nutricional/exercício promoveu melhora no estado caquético dos animais, visto que provocou: ligeira melhora da eficiência alimentar; preservação de proteína total da carcaça, manutenção da incorporação de fenilalanina e menor degradação da proteína muscular no grupo suplementado com leucina, além de preservar a proteína total sérica, manutenção da glicemia, colesterol total sérico e reduzir o lactato sérico, principalmente no grupo suplementado com leucina. Esses dados sugerem que o efeito benéfico da associação entre suplementação nutricional e exercício ao crescimento neoplásico foi pouco pronunciado, provavelmente, ao fato do crescimento acelerado do tumor superar as adaptações ao programa de treinamento físico / Abstract: The intense nutrients mobilisation of the host carcass tissue, in function of the neoplastic growth, leads to the host cachexia. Present in most of the patients with cancer, the cachexia is the state characterized by the involuntary weight loss that overtakes the protein muscle depletion increasing the degradation and/or decreasing the protein synthesis process in the muscle. In these cases, there is reduction of the quality and the life expectancy. On the other hand, some studies have shown that the development of the cancer is more aggressive and severe in younger patients. The branched-chain amino acids (BCAA), especially leucine, actively participate on cell signalling, stimulating the synthesis as well as inhibiting the protein degradation. The glutamine, a non essential amino acid, indirectly activates the muscle protein synthesis, as well as inhibits the protein catabolism. Additionally, the physical exercise stimulates the protein synthesis. Knowing these facts, our main interests, were to minimize the metabolic alterations in tumor-bearing host. The nutritional supplementation of leucine and/or glutamine and moderate aerobic exercise associated to the Walker 256 tumor growth could avoid the muscular depletion, preserve the protein body mass and the energetic source, possibly preventing the cachetic state of the animal. For this purpose, we evaluated the body chemical composition, the protein metabolism, measuring the muscle protein synthesis and catabolism, as well as the oxidative stress and biochemical blood profile in young tumor-bearing rats. The results showed reduced body weight and food intake in tumorbearing rats. The tumor effects changed the chemical body composition, showing high body water content, reduced total body fat and low carcass nitrogen in Walker tumor bearing rats. Protein synthesis was reduced and proteolysis was increased in young tumor-bearing rats. Additionally, the malondyhaldeide content increased in skeletal and cardiac muscle suggesting high oxidative stress associated to the tumor development. We observed low blood protein, albumin, globulin, glucose and cholesterol in tumor-bearing rats. The improvement of the cachexia was less pronounced in tumor-bearing animal which received supplemented diet associated to physical exercise. In these groups, we verified that food efficiency was slightly increased in comparison to the non-exercised tumour-bearing group. The carcass protein was maintained as well as the phenylalanine incorporation in muscle and less tyrosine was released from skeletal muscle when tumour-bearing groups were fed leucine rich diet. In these groups, the total blood protein, glycemia, total blood cholesterol and reduced blood lactate were preserved, mainly in exercised group. Those data suggest that the beneficial effect of the association among amino acids rich diet, exercise and tumor growth was not so expressive, probably due to the exponential tumor growth overcame the physical training program / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientador: Jaime Amaya-Farfan / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-14T12:35:54Z (GMT). No. of bitstreams: 1 Zaffani_VivianeCostaSilva_M.pdf: 1237742 bytes, checksum: 013e4cb87532470b0d0ebff8e7ade481 (MD5) Previous issue date: 2009 / Resumo: A ocorrência de desvios no metabolismo protéico durante o exercício depende tanto da intensidade, duração e freqüência do exercício, como também da ingestão alimentar, especialmente da qualidade da dieta consumida. Neste contexto, proteína do soro do leite (PSL) destaca-se pelo seu alto valor nutritivo, devido tanto à composição de aminoácidos, quanto à rápida digestão, além de outras funcionalidades relacionadas com a saúde. O objetivo deste estudo foi avaliar em ratos os efeitos da ingestão da proteína do soro do leite, na sua forma intacta e hidrolisada (~12,5% de hidrólise), em associação à atividade física de endurance, sobre os níveis séricos de aminoácidos, evolução ponderal, conteúdo protéico em gastrocnêmio e sóleo, conteúdo de DNA no gastrocnêmio, níveis séricos de IGF1, síntese e degradação protéica no grastrocnêmio e síntese no sóleo. Ratos Wistar foram distribuídos em 6 grupos, de acordo com a proteína consumida (12%): caseína (CAS), isolado protéico do soro do leite (IPSL) ou hidrolisado protéico do soro do leite (HPSL)) e submetidos a um protocolo de atividade física (sedentários (S) e treinados (T)). Os ratos treinados correram em esteira, durante 9 semanas, e foram sacrificados após 48 horas de repouso e 12 horas de jejum. As três dietas utilizadas apresentaram conteúdos semelhantes de aminoácidos totais, mas as dietas IPSL e HPSL destacaram-se apresentando maiores valores absolutos de leucina, isoleucina, lisina, treonina, cistina, alanina e ácido aspártico, em relação a CAS. No geral, os níveis séricos de aminoácidos indispensáveis foram semelhantes para os grupos IS e HS, em comparação com os ratos controle sedentários (CS), enquanto o grupo HT apresentou o maior nível destes aminoácidos, em relação ao CT. A evolução ponderal foi semelhante para todos os grupos de ratos até o final da oitava semana de treinamento. Na nona semana, os grupos treinados apresentaram peso significativamente menor que o CS. Não houve diferença estatística para o peso, conteúdo protéico dos músculos gastrocnêmio e sóleo, níveis séricos de IGF1 e taxas de degradação protéica muscular do gastrocnêmio, entre todos os grupos experimentais. O conteúdo e concentração de DNA no gastrocnêmio foi significativamente menor em ambos os grupos que consumiam a HPSL (HS e HT), independente da atividade física, comparado aos grupos que consumiam as proteínas intactas (CS, IS, CT e IT). As taxas de síntese protéica nos músculos gastrocnêmio e sóleo também foram menores para o grupo HT, comparado aos sedentários (CS, IS e HS), mas sem mostrar diferença com os grupos CT e IT. Os ratos do grupo HT destacaram-se por apresentar diminuição da demanda por nova síntese protéica, e da necessidade de utilização de aminoácidos do pool sérico diminuindo, consequentemente, a necessidade de aumentar a quantidade de DNA celular no músculo gastrocnêmio e ainda assim, manteve o peso, a concentração e o conteúdo protéico muscular sem diferença em relação aos demais grupos. Estes resultados, considerados em conjunto, sugerem que o consumo da proteína hidrolisada do soro do leite pode contribuir para a preservação da massa muscular no gastrocnêmio, quando associado à atividade física de endurance. / Abstract: Physical exercise promotes protein metabolic alterations depending not only on its intensity, duration and frequency, but also on food intake and especially on the quality of the diet. In this context, the milk whey proteins (PSL) stand out because of their high quality, meeting both amino-acid profile and digestibility requirements, besides other functional properties. The aim of this study was to assess the effects of milk whey protein intake in rats, in both the intact and hydrolyzed forms (~12,5% of hydrolysis), associated with physical activity of endurance, on serum amino acids levels, body weight, protein content in both the gastrocnemius and soleus muscles, total DNA content in the gastrocnemius, serum IGF1 levels, protein degradation rate in the gastrocnemius, and of protein synthesis in the soleus and gastrocnemius. Male Wistar rats were divided into six groups as follows: protein consumed (12%), casein - CAS, milk whey protein isolate - IPSL, or milk whey protein hydrolyzate -HPSL) and physical activity protocol (sedentary, S, and trained, T). The trained rats were exercised on the treadmill during nine weeks and sacrificed following 48 hours of rest; the last 12 hours being fasted. The three diets tested produced similar contents of total amino acids, although the IPSL and HPSL diets stood out because of the higher absolute values of leucine, isoleucine, lysine, threonine, cysteine, alanine and aspartate than those of CAS. As a whole, the serum indispensable amino acid levels were similar when comparing both IS and HS groups with the control group (CS). However, the HT group showed higher levels of these amino acids than the CT group. No difference in body weight evolution was apparent among the groups until the end of the eighth week of training. Nevertheless, on the ninth week the trained groups showed significantly lower weights than group CS. There were no significant differences, among all groups studied, in the weight, the content and concentration of both gastrocnemius and soleus muscles, and serum IGF1 levels, as well as the degradation rate of proteins in the gastrocnemius muscle. The content and concentration of DNA in the gastrocnemius were significantly lower in both groups fed HPSL (HS and HT), regardless of physical activity, than in the groups fed intact protein (CS, IS, CT and IT). The rate of protein synthesis in both gastrocnemius and soleus muscles were also lower in the HT group than those found in the CS, IS and HS groups. However, there was no difference when compared to those of the IT and CT groups. Summarizing, the HT group stood out because of its lower demand for new protein synthesis and amino acid utilization from the serum pool, consequently decreasing the need for higher amount of cellular DNA in the gastrocnemius muscle. Even so, this group kept the same muscle mass, protein content and concentration, as those of the other groups. These results suggest that the consumption of hydrolyzed milk whey protein may contribute to the preservation of the gastrocnemius muscle when associated with physical activity of endurance. / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição