Reactive oxygen species (ROS) is cytotoxic to the cell and is known to contribute to secondary cell death following primary traumatic brain injury (TBI). We described in our study that PN is the main mediator for both lipid peroxidation and protein nitration, and occurred almost immediately after injury. As a downstream factor to oxidative damage, the peak of Ca2+-dependent, calpainmediated cytoskeletal proteolysis preceded that of neurodegeneration, suggesting that calpain-mediated proteolysis is the common pathway leading to neuronal cell death. The time course study clearly elucidated the interrelationship of these cellular changes following TBI, provided window of opportunity for pharmacological intervention. Furthermore, we conducted a pharmacological study to solidify our hypothesis. First of all, we tested the potency of a membrane permeable, catalytic scavenger of PN-derived free radicals, tempol for its ability to antagonize PN-induced oxidative damage. Tempol successfully inhibited PNinduced protein nitration at dosages of 30, 100 and 300mg/kg. Moreover, early single dose of 300mg/kg was administered and isolated mitochondria were examined for respiratory function and oxidative damage level. Our data showed that tempol reduced mitochondrial oxidative damage, and maintained mitochondrial function within normal limits, which suggested that tempol is efficiently permeable to mitochondrial membrane and mitochondrial oxidative damage is essential to mitochondrial dysfunction. Next, we found that calpainmediated proteolysis is reduced at early treatment with a single dose of tempol. However, the effect of tempol on calpain is short-lived possibly due to systematic elimination. In our multiple dose study, tempol showed a significant inhibitory effect on SBDPs. Consequently, we measured neuordegeneration with the de Olmos aminocupric silver staining method at 7 days post-injury and detected a significant decrease of neuronal cell death. Together, the time course study and pharmacological study strongly support the hypothesis that PN is the upstream mediator in secondary cell death in the CCI TBI mouse model. Moreover, inhibition of PN-mediated oxidative damage with the antioxidant, tempol, is able to attenuate multiple downstream injury mechanisms. However, targeting PN alone may be clinically impractical due to its limited therapeutic window. This limitation may be overcome in future studies by a combination of multiple therapeutic strategies.

<p>Detection and quantification of protein-ligand binding interactions is extremely important for understanding interactions that occur in biological systems. Since traditional techniques for characterizing these types of interactions cannot be performed in complex systems such as cell lysates, a series of energetics-based techniques that are capable of assessing protein stability and measuring ligand binding affinities have been developed to overcome some of the limitations of previous techniques. Now that the capabilities of the energetics-based techniques have been exhibited in model systems, the false-positive rates of the techniques, the range of biological questions to which the techniques can be addressed, and the use of the techniques to discover novel interactions in unknown systems remained to be shown. The Stability of Proteins from Rates of Oxidation (SPROX) technique and the Pulse Proteolysis (PP) technique were applied to a wide range of biological questions in both yeast and human cell lysates to evaluate the scope of these experimental workflows. The false-positive rate of iTRAQ-SPROX protein target discovery on orbitrap mass spectrometer systems was determined to be < 0.8 %. The iTRAQ-SPROX technique was successfully applied to the discovery of both known and novel protein-protein, protein-ATP, and protein-drug interactions, leading to the quantification of protein-ligand binding affinities in each of these studies. In the pursuit of discovering geldanamycin protein interactors, the use of iTRAQ-SPROX and SILAC-PP in combination was determined to be advantageous for confirming protein-ligand interactions since the techniques utilize different quantitation strategies that are subject to separate technical errors in quantitation. Finally, the iTRAQ-SPROX and SILAC-PP techniques were used to evaluate the interactions of manassantin A in a human cell lysate. In this work, a previously unknown protein target of manassantin A, Filamin A, was detected as a hit protein using both the iTRAQ-SPROX and SILAC-PP protocols. The work completed in this dissertation has expanded the understanding of the limitations of energetics-based techniques and shown that biological replicate analyses are essential to confirm ligand interactions with novel protein targets.</p> / Dissertation

Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...

Nous étudions la migration et la protéolyse de la matrice extracellulaire dans le cancer du sein. Pour cela, nous avons mis en place deux systèmes modèles. Le premier, se base sur une lame basale reconstituée et permet d'évaluer le potentiel invasif de lignées cellulaires tumorales. Nous montrons que les cellules cancéreuses migrent différemment à travers un gel pour former des amas de taille variable directement corrélé à leur pouvoir invasif. Dans notre système, seule la migration de type mésenchymateuse est utilisée par les cellules. Ce type de mouvement est directement dépendant de protéases sécrétées par les cellules. Nous avons, donc mesurée la synthèse au niveau transcriptionnel de la classe d'enzyme majoritairement impliquée dans la dissémination tumorale, les matrice métalloprotéases (MMPs). Nous avons ainsi pu montrer que l'expression de 3 MMPs est corrélée aux capacités migratoires des cellules donc à leur potentiel invasif. Le processus physique par lequel les enzymes dégradent les matrices est très peu étudié au niveau expérimental. Le second système que nous utilisons se base sur un modèle de matrice conjonctive majoritairement composer de collagène de type I. Nous utilisons la gélatine, pour étudier la protéolyse de gels protéiques par différentes classes de protéases. A partirdes études sur la solubilisation enzymatique des gels par l'-chymotrypsine, la protéinase K et la papaïne, nous montrons qu'il existe des mécanismes de dégradation distincts. Le premier est un mécanisme anormal dont la cinétique est limitée par la diffusion de l'enzyme, le second est brownien et la cinétique est limitée par la réaction. Ce second mécanisme dépend directement d'interactions eléctrostatiques entre l'enzyme et le gel. Nous observons pour deux des enzymes que l'évolution des temps de dégradation mais également la cinétique dépendent de la concentration en protéine dans les gels. / We study the migration and proteolysis of the extracellular matrix in breast cancer. For this, we set up two model systems. The first is based on a reconstituted basement membrane and allows the evaluation of invasive potential tumor cell lines. We show that cancer cells migrate differently across the gel to form clusters of variable size directly correlates with their invasiveness. In our system, only the migration of mesenchymal type is used by the cells. This type of movement is directly dependent proteases secreted by the cells. We therefore measured the synthesis at the transcriptional level of the enzyme class mainly involved in tumor dissemination, the matrix metalloproteases (MMPs). We were able to show that the expression of 3 MMPs is correlated with migratory capacity of cells, therefore their invasive potential. The physical process by which enzymes degrade the matrix is very little studied at the experimental level. The second system we use is based on a model of connective matrix mainly composed of collagen type I. We use gelatin for the study of protein gels proteolysis by different classes of proteases. Based on the study of gels enzymatic solubilization by a- chymotrypsin, proteinase K and papain, we show that there are distinct mechanisms of degradation. The first mechanism is abnormal whose kinetic is limited by enzyme diffusion, and the second is Brownian and the kinetic is reaction limited. The second mechanism depends directly on electrostatic interactions between enzyme and gel. We observe for two enzymes that the evolution of degradation time but also the degradation kinetics depend on the concentration of protein in gels.

Differences have been shown in the steady-state accumulation and half-lives between Brassica FAD3 (BF3) and tung FAD3 (TF3) proteins expressed in yeast cells cultured at 30°C. TF3 has a greater steady-state accumulation and longer half-life than BF3. These differences are attributed to post-translational modification and have been shown to be controlled by an Nterminal element. I attempted to determine specific amino acids important for regulation, and further characterize the mechanism contributing to the differences. Through site-directed mutagenesis, it was shown that replacing lysine residues with asparagines in the BF3 and TF3 Ntermini increased protein stability, while replacing an asparagine with lysine in the TF3 Nterminus decreased its stability. Furthermore, I showed that the TF3 polyglutamic region (six consecutive glutamic acid residues) is primarily responsible for the higher steady-state amount of TF3 in comparison to BF3. This negatively charged region likely acts as an electrostatic shield protecting the protein from degradation.

O tipo biológico influenciado pela genética Bos taurus indicus garante maior adaptação aos animais em regiões com altas temperaturas, no entanto exibe alguns atributos de carcaça e associados à palatabilidade da carne inferiores quando comparados ao tipo biológico predominantemente Bos taurus taurus. Existe a hipótese de que tais diferenças estejam associadas ao conteúdo mitocondrial na musculatura destes animais. Objetivou-se avaliar as diferenças no metabolismo oxidativo, pela medida de respiração mitocondrial em fibras permeabilizadas submetidas a duas temperaturas (38,5 e 40 °C) utilizando respirômetro de alta resolução (Oroboros Instruments), do Longissimus lumborum imediatamente após abate, entre os tipos biológicos Brahman e Angus com taxa e extensão da proteólise distintas ao longo de 14 dias de maturação. Foram investigadas as atividades enzimáticas de lactato desidrogenase e citrato sintase, pH, bem como autólise de &mu;-calpaína e degradação de proteínas miofibrilares de 18 animais das raças Brahman (6), Brangus (6) e Angus (6). A taxa de acidificação das carcaças foi diferente entre os tipos biológicos (P = 0,049) e entre os tempos postmortem (P < 0,001), sendo que as carcaças dos animais Brahman às 6h postmortem apresentaram pH tão alto quanto as carcaças de Brangus e Angus às 3h posmortem. A atividade de citrato sintase 1h postmortem também foi diferente (P = 0,086) entre os tipos biológicos, sendo que maior atividade (P = 0,075) foi identificada em Brahman comparado a Brangus. O fluxo de consumo de oxigênio mitocondrial no estado 4 (ST4_O), apresentou interação (P = 0,050) entre os tipos biológicos e temperatura, assim como a respiração mitocondrial do sistema de transporte de elétrons desacoplado da produção de ATP (P = 0,047), ambos superiores para Brahman a 38,5 °C na comparação entre tipos biológicos. Todavia, ocorreu uma queda da ST4_O, apenas para os animais Brahman, quando a temperatura foi aumentada. A maior resistência a acidificação e maior respiração mitocondrial imediatamente após o abate para o tipo biológico Brahman, foram acompanhadas pela redução (P = 0,058) da autólise completa de &mu;-calpaína às 24h postmortem. A degradação da titina após 7d de maturação foi reduzida (P = 0,056) e se manteve menor (P = 0,003) aos 14d para Brahman comparado aos outros tipos biológicos. O aparecimento da banda 150 kDa da alfa-II-espectrina às 24h postmortem também é menor (P = 0,011) para Brahman, assim como a degradação da desmina (P = 0,019) e troponina-T (P = 0,001). As duas últimas proteínas tiveram sua degradação reduzida ao longo dos 14d postmortem para este tipo biológico. Portanto, o tipo biológico Brahman tem um mecanismo muscular adaptativo que envolve maior conteúdo mitocondrial, com maior vazamento de prótons pelo sistema de transporte de elétrons e que pode estar relacionado a redução da taxa de ativação da &mu;-calpaína, redução da degradação proteica e, consequentemente, a redução da taxa e da extensão do amaciamento da carne destes animais. / Biological type influenced by Bos taurus indicus ensures higher animals adaptation to warm environment, however exhibit less desirable carcass and palatability traits when compared to predominant Bos taurus taurus biological types. There is a hypothesis that muscle mitochondrial content is associated with those differences. It was aimed to evaluate the oxidative metabolism, measuring mitochondrial respiration in permeabilized fibers sumitted to two temperatures (38.5 and 40 °C) using high resolution respirometry (Oroboros Instruments), in Longissimus lumborum imediately after slaughter between biological types Brahman and Angus with different proteolysis rate and extension during 14d aging. Were investigated lactate dehydrogenase and citrate sinthase activities, pH, as well as &mu;-calpain autolysis and protein degradation between 18 animals from breeds Brahman (6), Brangus (6) and Angus (6). The carcass pH decline is different between biological types (P = 0.049) and between time postmortem (P < 0.001), with Brahman carcasses showing pH at 6h postmortem as high as Brangus and Angus pH at 3h postmortem. Citrate sinthase activity 1h postmortem is different (P = 0,086) between biological types, with Brahman showing higher (P = 0.075) values than Brangus animals. The mitochondrial oxygen flux consumption at state 4 (ST4_O) showed interaction (P = 0.050) between biological types and temperature, as well as respiration for electron transport system uncopled from ATP production (P = 0.047), both higher for Brahman at 38.5 °C in biological types comparison. However, decreased ST4_O was observed only for Brahman animals when temperature was increased. Higher resistance to pH decline and higher mitochondrial respiration after slaughter to Brahman cattle were followed by reduced (P = 0.058) complete &mu;-calpain autolysis at 24h postmortem. Titin degradation after 7d aging was reduced (P = 0.056) and kept lower (P = 0.031) after 14d to Brahman when compared to other biological types. The 150 kDa band from alpha-IIspectrin degradation at 24h postmortem is lower (P = 0.011) to Brahman, as well as desmin degradation (P = 0.019) and troponin-T (P = 0.001). Desmin and troponin-T had its degradation reduced extended until 14d aging for this biological type. Therefore, Brahman biological type has an adaptive muscular mechanism that involves higher mitochondrial content, with higher proton leak in electron transport system and that can be related with reduced rate for &mu;-calpain activation, reduced protein degradation and, consequentely, reduced rate and extension to beef tenderization for these animals.

O objetivo do presente estudo foi avaliar as características físico-químicas, microbiológicas, índices de proteólise, lipólise e viscosidade do iogurte natural batido, elaborado a partir de leite integral contendo três níveis de células somáticas (CS): <400.000 células/mL, 400.000-800.000 células/mL e >800.000 células/mL. Cada tipo de leite foi obtido da ordenha de animais previamente selecionados de acordo com o nível de CS e a composição do leite. Para a fabricação do iogurte, o leite foi padronizado quanto ao teor de sólidos totais (ST) e transferido para tanque multi-uso, no qual foi submetido à pasteurização (90ºC, 15 minutos), seguida da adição da cultura starter, incubação (42ºC, aprox. 3 horas) e envase do produto. O iogurte foi mantido em câmara fria a 5ºC, sendo que os parâmetros de qualidade foram avaliados mediante a colheita de amostras nos dias 1, 10, 20 e 30 após a fabricação. A seqüência de elaboração foi repetida seis vezes, no período de março a agosto/2003. As análises efetuadas no produto incluíram: pH, acidez, percentuais de gordura, ST, sólidos não-gordurosos (SNG), nitrogênios total (NT), não caseinoso (NNC) e não protéico (NNP), ácidos graxos livres (AGL), viscosidade aparente, contagem de bactérias láticas e coliformes a 30º e 45ºC. Não foram constatadas diferenças (P > 0,05) entre os parâmetros físico-químicos e microbiológicos obtidos no leite e no iogurte. Os índices de proteólise dos iogurtes, estimados através da relação NT – NNC / NT – NNP, mantiveram-se constantes, não apresentando diferenças entre os tratamentos (P > 0,05). A viscosidade do iogurte produzido com leite contendo mais de 800.000 células/mL foi maior (P < 0,05) em relação aos outros tratamentos nos dias 10, 20 e 30 após a fabricação, observando-se uma correlação positiva (P < 0,05) com os níveis de CS no 10º e 20º dia de armazenamento. A concentração de AGL foi maior (P < 0,05) no iogurte de alta contagem de CS no 1º e 30º dia de armazenamento, sendo observada uma correlação positiva (P < 0,05) com o nível de CS nos mesmos dias. Os resultados indicam que o aumento dos níveis de CS no leite não apresenta efeitos sobre a proteólise do iogurte, porém origina um aumento na viscosidade e no grau de lipólise do produto durante o armazenamento por 30 dias. / The aim of the present study was to evaluate physical, chemical and microbiological characteristics, as well as proteolysis, lipolysis and viscosity of plain stirred yoghurt produced from whole milk with somatic cell counts (SCC) at levels of < 400,000 cells/mL, 400,000-800,000 cells/mL and > 800,000 cells/mL. Each milk treatment was obtained from selected cows, according to its SSC status and milk composition. Yoghurts were produced after standardisation of milk total solids (TS), followed by pasteurisation (90ºC, 15 minutes), addition of starter culture, incubation (42ºC, approx. 3 hours) and packaging. Yoghurts were stored at 5ºC, and quality evaluation was conducted in samples collected on days 1, 10, 20 and 30 after production. Manufacturing procedures were repeated for six times, from March to August/2003. Yoghurt analyses included: pH, acidity, fat, protein, TS, solids non-fat (SNF), total nitrogen (TN), non-casein nitrogen (NCN) and non-protein nitrogen (NPN), free fatty acids (FFA), apparent viscosity, lactic bacteria counts and coliforms at 30 and 45ºC. There were no differences (P > 0.05) in physical, chemical and microbiological parameters of milk and yoghurt among treatments. Proteolysis, as estimated by TN – NCN / TN – NPN relation, was constant for all treatment yoghurts (P > 0.05). Viscosity of high SCC yoghurt (> 800,00 cells/mL) increased (P < 0.05) on 10, 20 and 30 days storage, and a positive correlation (P < 0.05) with SCC was observed on days 20 and 30. FFA content was higher (P < 0.05) on days 1 and 30 of storage, and besides there was a positive correlation (P < 0.05) between SCC and FFA levels on the same days of storage. Results indicate that high SCC milk do not affect proteolysis of yoghurt, although it increases viscosity and lypolisis during storage for 30 days.

Während der letzten Dekade neurobiologischer Forschung wurde deutlich, dass inflammatorische Vorgänge in einem Netwerk nicht-neuronaler und neuronale Zellen wesentlich zur Entstehung und zur Progredienz einiger wichtiger neurodegenerativer Erkrankungen beitragen. Therapeutische Ansätze sollten daher auch auf die Protektion initial überlebender Neurone vor dieser sekundären inflammatorischen Schädigung zielen. Ein wesentlicher Bestandteil dieser sekundären Schädigung besteht aus der Migration von Makrophagen und Mikrogliazellen in die Regionen neuronaler Schädigung, wo sie grosse Mengen an toxischen Zytokinen und Sauerstoffradikalen freisetzen. In einer Makrophagen-ähnlichen Zelllinie, sowie in phagozytierenden Mikrogliazellen wurde eine nukleäres proteolytisches System identifiziert, dass in der Lage war, oxidativ geschädigte Kernproteine zu erkennen und abzubauen. Im Gegensatz zu dem bisherigen Konzept relativer Langlebigkeit der Histonproteine, wurde diese nach oxidativer Schädigung innerhalb von Minuten abgebaut und vom Chromatin entfernt. Dieser schnelle Abbau war von der nicht-kovalenten Interaktion der automodifizierten Poly(ADP-Ribose)-Polymerase-1 (PARP-1) mit dem 20S Proteasom abhängig. Die PARP-1 wurde somit als ein Signalmolekül zwischen dem Chromatinschaden und der Einleitung einer protektiven Zellantwort charakterisiert, die Mikrogliazellen das Überleben ihres eigenen Aktivierungszustandes ermöglicht. Es zeigte sich, dass dieses PARP-Proteasom-System in Abhängigkeit vom Differenzierungsgrad Makrophagen-ähnlicher Zellen abhängig ist und auch funktionell in die Chemotherapieresistenz humaner Leukämiezellen involviert ist. Darüberhinaus regulierte die PARP-1 auch die Expression des Integrins CD11a durch Interaktion mit dem translozierten NF-kappaB und HMG-I(Y) und dadurch die Migration von Mikrogliazellen zum Ort der neuronalen Schädigung. Diese Ergebnisse machem die PARP-1 zu einem potentiellen Ziel therapeutischer Interventionen zur Verhinderung der destruktiven Migration von Mikrogliazellen, womit eine Protektion initial überlebender Neurone vor weiterer inflammatorischer Schädigung erreicht werden könnte. / During the last decade of neurobiological research, it became clear that inflammatory pathways in the CNS, involving a network of non-neuronal and neuronal cells, are contributing mainly to the onset and progress of several major neurodegenerative diseases. Therapeutic approaches must therefore focus on the protection of initially surviving neurons from this secondary inflammatory damage. One major component of secondary neuronal damage is the migration of macrophages and microglia cells towards the sites of injury where they produce large amounts of toxic cytokines and oxygen radicals. In a macrophage-like cell line and in phagocytosing microglial cells a nuclear proteolytic system was identified, which was able to recognize and degrade oxidatively-damaged nuclear proteins, in particular histones. In contrast to the previous concept of relatively long-living histone proteins, they are rapidly degraded and removed from the chromatin within minutes after oxidative damage. This rapid degradation was dependent on the non-covalent interaction of the 20S proteasome with the automodified poly(ADP-ribose)-polymerase-1 (PARP-1). Therefore, the PARP-1 has been identified as a signal molecule between the detection of a chromatin-damage and a protective cellular response, which enables microglial cells to survive their own activation state. The regulation of this PARP-proteasome-system depends on the differentiation state of macrophage-like cells and is also functionally involved in the chemotherapy-resistance of human leukemia cells. Moreover, PARP-1 regulates the expression of the integrin CD11a by interaction with the translocated NF-kappaB and HMG-I(Y) and therefore microglia migration towards the sites of neuronal injury. These findings renders the PARP as potential target for therapeutic interventions to inhibit destructive microglial migration and therefore to protect initially surviving neurons from inflammatory damage.

INTRODUÇÂO: A sepse representa aproximadamente 25% dos pacientes internados em Unidades de Terapia Intensiva (UTI) e com taxas de mortalidade de 20 a 40%. Além disso, os pacientes sépticos podem apresentar aguda e tardiamente disfunções de órgãos e alterações da musculatura esquelética comprometendo a recuperação na UTI e, posteriormente, a sua saúde relacionada à qualidade de vida. Atualmente, a ultrassonografia tem demonstrado ser uma ferramenta capaz de avaliar a degradação da musculatura esquelética em pacientes críticos. Em pacientes sépticos não se estudou a relação de degradação muscular com testes clínicos de força muscular e aferições bioquímicas. OBJETIVOS: Quantificar a área de secção transversa do reto femoral e a força muscular a beira leito em pacientes que desenvolveram sepse grave e choque séptico. Avaliar associação entre a área de secção transversa do reto femoral e a força muscular em pacientes sépticos. MÉTODOS: Coorte prospectiva que avaliou 37 pacientes na UTI com sepse grave ou choque séptico. A medida da área de secção transversa do reto femoral foi realizada através da ultrassonografia no dia seguinte ao início da sepse e acompanhada durante a internação. Os pacientes foram submetidos a testes clínicos de força muscular (escala do Medical Research Council e a força de preensão palmar) à medida que pudessem compreender comandos verbais. Amostras de sangue foram coletadas para análise de enzimas e hormônio após a admissão no estudo e durante a internação. RESULTADOS: Houve um declínio da área de secção transversa do reto femoral de 5,1 (4,4-5,9)cm2 no 2° dia de UTI para 4,4 (3,6-5,0)cm2 e 4,3 (3,7-5,0)cm2 na alta da UTI e na alta hospitalar, respectivamente (p<0,05). Por outro lado, os testes clínicos de força apresentaram um aumento na escala do Medical Research Council de 48,0 (36,0-56,0) pontos para 60,0 (48,0-60,0) pontos na alta da UTI, este aumento foi mantido até a alta hospitalar em 60,0 (52,0-60,0) pontos (p < 0,05). Em relação à força de preenssão palmar os pacientes apresentavam média de 42,1±21,9 % do predito no 1° dia de avaliação e este valor aumentou para 65,9±20,3 % do predito no dia da alta hospitalar (p < 0,05). Houve uma associação da área de secção transversa do reto femoral e das avaliações clínicas de força muscular durante a permanência na UTI. Aumentos no escore de lesão orgânica (SOFA) no 3° dia e ser do sexo masculino apresentaram-se como fatores independentes para a degradação muscular, assim como, o SOFA do 3° dia com a fraqueza muscular. CONCLUSÃO: O estudo demonstrou que a sepse pode levar a uma degradação muscular em apenas alguns dias de UTI, associada há uma recuperação incompleta da força muscular ao longo da internação. Além disso, testes clínicos de força muscular se associaram com a degradação muscular durante a internação hospitalar / INTRODUCTION: Sepsis represents 25% of patients in the intensive care unit (ICU) with mortality rate of 20 to 40%. In addition, septic patients can present early or lately organ dysfunction and skeletal muscles alterations that reduce patient recovery and compromises health-related to quality of life. Currently, ultrasound has been shown to be a tool capable of evaluating skeletal muscle degradation in critical patients. There are no studies in septic patients about the relation of muscle degradation with clinical tests and blood biochemistry analysis. OBJECTIVES: To quantify the rectus femoris cross-sectional area and muscle strength at the bedside in patients who developed severe sepsis and septic shock. To assess the association between the rectus femoris cross-sectional area and muscle strength in septic patients. METHODS: Prospective cohort who evaluated 37 patients in the intensive care unit with severe sepsis or septic shock. The measurement of rectus femoris cross-sectional area was performed by ultrasonography on the day after the onset of sepsis and followed up during hospitalization. Patients underwent clinical tests of muscle strength (Medical Research Council scale and handgrip strength) as they could understand verbal commands. Blood samples were collected for enzyme and hormone analysis after admission to the study and during hospitalization. RESULTS: There was a decline in rectus femoris cross-sectional area from 5.1 (4.4-5.9) cm2 on the 2nd day of ICU to 4.4 (3.6-5.0) cm2 and 4.3 (3.7-5.0) cm2 at ICU discharge and at hospital discharge, respectively (p < 0.05). In contrast, strength tests showed an increase in the scale of the Medical Research Council from 48.0 (36.0-56.0) to 60.0 (48.0-60.0) points in ICU discharge, this increase was maintained until hospital discharge reaching 60.0 (52.0-60.0) points (p < 0.05). In relation to the handgrip strength, patients presented a mean of 42.1 ± 21.9% of predicted on the 1st day of evaluation and this value increased to 65.9 ± 20.3% of predicted on the day of hospital discharge ( p < 0.05). There was an association between the rectus femoris cross-sectional area and clinical assessments of muscle strength during ICU stay. Increases in the organic lesion score (SOFA) on the 3rd day and being male presented as independent factors for muscle degradation, as well as the SOFA of the 3rd day with muscle weakness. CONCLUSION: The study demonstrated that sepsis can lead to muscle degradation in only a few days of ICU, associated with an incomplete recovery of muscle strength throughout hospitalization. In addition, clinical trials of muscle strength were associated with muscle degradation during hospital stay

A proteólise é provavelmente o fenômeno mais importante que ocorre durante a maturação da maioria dos tipos de queijos e influencia fortemente suas características de aroma, sabor e textura. Neste trabalho estudou-se a aplicação de diferentes técnicas analíticas para extração e determinação dos compostos nitrogenados liberados durante a maturação do queijo Prato, compostos esses indicadores da extensão e da profundidade da maturação. Complementando esses estudos, utilizou-se métodos mais específicos como eletroforese em uréia-PAGE e RP-HPLC para avaliação dos peptídeos e aminoácidos formados. Os resultados mostraram que as frações nitrogenadas estudadas e os métodos utilizados na avaliação direta da proteólise se mostraram adequados para uso em análises de rotina. A determinação espectrofotométrica da tirosina e triptofano comprovou ser uma metodologia adequada para avaliação da intensidade de maturação, podendo ser usada como um método rápido alternativo ao de Kjeldahl. Os grupamentos amínicos livres analisados com TNBS ou ninidrina-cádmio também são técnicas mais rápidas e de fácil execução, além de fornecer informações que refletem melhor as degradações das proteínas durante a maturação. O acompanhamento da proteólise do queijo Prato ao longo da maturação indicou alto grau de associação entre os resultados obtidos em todos os métodos utilizados, sugerindo que todos podem ser empregados na sua avaliação e monitoramento. A avaliação instrumental da textura demonstrou a correlação dos atributos adesividade, elasticidade e coesividade com os outros índices usados na avaliação da proteólise, evidenciando que as alterações percebidas pelos consumidores podem ser medidas por parâmetros objetivos. A análise eletroforética demonstrou o aumento na concentração de peptídeos ao longo da maturação, sendo evidente o desdobramento gradual da &#945;s1-caseína formando a fração &#945;s1-I (f24-199) e da &#946;-caseína, formando as frações &#947;1, &#947;2 e &#947;3. A análise por RP-HPLC também demonstrou um aumento no número de picos durante a maturação, com maior variação naqueles eluídos nas regiões intermediária e final da separação. / Proteolysis is probably the most important biochemical event which occurs during the ripening of most cheese varieties, with a major impact on flavour and texture. This work is about the application of different analytical techniques for extraction and determination of the nitrogen fractions liberated during the maturation of Prato cheese. These compounds are indicators of the extension and depth of maturation. Complementing these studies, more specific methods such as urea-PAGE electrophoresis and RP-HPLC were applied. The results showed that the nitrogen fractions studied and the methodology used in direct evaluation of the proteolysis are of potential for use in routine applications. The spectrophotometric determination of tyrosine and tryptophane confirmed that this procedure is good for estimating the extent of cheese ripening. Although they are considered gross indices, they could be used as an alternative fast method for Kjeldahl. Determinations of total free aminic groups by reaction with TNBS or ninhydrincadmium are also fast and easy. Proteolysis of Prato cheese during the maturation showed a high degree of correlation with the results obtained in all tested methods, suggesting that all of them can be used for its evaluation and monitoring. Instrumental measurement of texture indicated correlation of the attributes adhesiveness, elasticity and cohesiveness with the other indices used for proteolysis evaluation, meaning that the alterations noticed by the consumers can be measured by objective parameters. Electrophoretical analysis demonstrated an increase in peptides concentration as ripening progressed, revealing a gradual breakdown of &#945;s1-casein to &#945;s1-I (f24-199). There was a degradation of &#946;-casein with a concomitant increase in concentration of &#947;-caseins. The RP-HPLC analysis also showed an increase in the number of peaks, with large variation in those eluted in the middle and final portion of the chromatogram.