Identification of residues and domains of Raf important for function in vivo and in vitro

Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. We engineered these mutations into the homologous residues of vertebrate Raf-1 and analyzed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf-1 activation loop, and mutation of this residue in Raf-1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype.

Fas ligand and tumour counter-attack in colorectal cancer stratified according to microsatellite instability status

Expression of membrane-bound Fas ligand (FasL) by colorectal cancer cells may allow the development of an immune-privileged site by eliminating incoming tumour-infiltrating lymphocytes (TILs) in a Fas-mediated counter-attack. Sporadic colorectal cancer can be subdivided into three groups based on the level of DNA microsatellite instability (NISI). High-level NISI (NISI-High) is characterized by the presence of TILs and a favourable prognosis, while microsatellite-stable (MSS) cancers are TIL-deficient and low-level MSI (MSI-Low) is associated with an intermediate TIL density. The purpose of this study was to establish the relationship between MSI status and FasL expression in primary colorectal adenocarcinoma. Using immunohistochemistry and a selected series of 101 cancers previously classified as 31 MSI-High, 30 NISI-Low, and 40 MISS, the present study sought to confirm the hypothesis that increased TIL density in MSI-High cancers is associated with low or absent membrane-bound FasL expression, while increased FasL in MSS cancers allows the killing of host TILs. TUNEL/CD3 double staining was also used to determine whether MSS cancers contain higher numbers of apoptotic TILs in vivo than MSI-High or MSI-Low cancers. Contrary to the initial hypothesis, it was found that MSI-High cancers were associated with higher FasL expression (p = 0.04) and a stronger intensity of FasL staining (p = 0.007). In addition, mucinous carcinomas were independently characterized by increased FasL expression (p = 0.03) and staining intensity (p = 0.0005). Higher FasL expression and staining intensity did not correlate with reduced TIL density or increased numbers of apoptotic TILs. However, consistent with the hypothesis that curtailment of the host anti-tumour immune response contributes to the poor prognosis in MSS cancers, it was found that apoptotic TILs were most abundant in MSS carcinomas and metastatic Dukes' stage C or D tumours (p = 0.004; p = 0.046 respectively). This study therefore suggests that MSS colorectal cancers are killing incoming TILs in an effective tumour counter-attack, but apparently not via membrane-bound FasL. Copyright (C) 2003 John Wiley Sons, Ltd.

Effects of glucose-insulin-potassium infusion on chronic ischaemic left ventricular dysfunction

Background: Glucose-insulin-potassium (GIK) infusion improves cardiac function and outcome during acute ischaemia. Objective: To determine whether GIK infusion benefits patients with chronic ischaemic left ventricular dysfunction, and if so whether this is related to the presence and nature of viable myocardium. Methods: 30 patients with chronic ischaemic left ventricular dysfunction had dobutamine echocardiography and were given a four hour infusion of GIK. Segmental responses were quantified by improvement in wall motion score index (WMSI) and peak systolic velocity using tissue Doppler. Global responses were assessed by left ventricular volume and ejection fraction, measured using a three dimensional reconstruction. Myocardial perfusion was determined in 15 patients using contrast echocardiography. Results: WMSI (mean (SD)) improved with dobutamine (from 1.8 (0.4) to 1.6 (0.4), p < 0.001) and with GIK (from 1.8 (0.4) to 1.7 (0.4) p < 0.001); there was a similar increment for both. Improvement in wall motion score with GIK was observed in 55% of the 62 segments classed as viable by dobutamine echocardiography, and in 5% of 162 classed as non-viable. There was an increment in peak systolic velocity after both doputamine echocardiography (from 2.5 (1.8) to 3.2 (2.2) cm/s, p < 0.01) and GIK (from 3.0 (1.6) to 3.5 (17) cm/s, p < 0.001). The GlK effects were not mediated by changes in pulse, mean arterial pressure, lactate, or catecholamines, nor did they correlate with myocardial perfusion. End systolic volume improved after GlK (p = 0.03), but only in 25 patients who had viable myocardium on dobutom ne echocardiography. Conclusions: In patients with viable myocardium and chronic left ventricular dysfunction, GlK improves wall motion score, myocardial velocity, and end systolic volume, independent of effects on haemodynamics or catecholamines. The response to GlK is observed in areas of normal and abnormal perfusion assessed by contrast echocardiography.

On the induced electric field gradients in the human body for magnetic stimulation by gradient coils in MRI

Prior theoretical studies indicate that the negative spatial derivative of the electric field induced by magnetic stimulation may he one of the main factors contributing to depolarization of the nerve fiber. This paper studies this parameter for peripheral nerve stimulation (PNS) induced by time.-varying gradient fields during MRI scans. The numerical calculations are based on an efficient, quasi-static, finite-difference scheme and an anatomically realistic human, full-body model. Whole-body cylindrical and planar gradient sets in MRI systems and various input signals have been explored. The spatial distributions of the induced electric field and their gradients are calculated and attempts are made to correlate these areas with reported experimental stimulation data. The induced electrical field pattern is similar for both the planar coils and cylindrical coils. This study provides some insight into the spatial characteristics of the induced field gradients for PNS in MRI, which may be used to further evaluate the sites where magnetic stimulation is likely to occur and to optimize gradient coil design.

Heat resistance of Bacillus spores when adhered to stainless steel and its relationship to spore hydrophobicity

Twenty-one strains of Bacillus (10 B. stearothermophilus, 3 B. cereus, and 8 B. licheniformis strains) were assayed for spore surface hydrophobicity on the basis of three measures: contact angle measurement (CAM), microbial adhesion to hydrocarbons (MATH), and hydrophobic interaction chromatography (HIC). On the basis of the spore surface characteristics obtained from these assays, along with data on the heat resistance of these spores in water, eight strains of Bacillus (three B. stearothermophilus, three B. cereus, and two B. licheniformis strains) either suspended in water or adhering to stainless steel were exposed to sublethal heat treatments at 90 to 110degreesC to determine heat resistance (D-value). Significant increases in heat resistance (ranging from 3 to 400%) were observed for the eight strains adhering to stainless steel. No significant correlation was found between these heat resistance increases and spore surface characteristics as determined by the three hydrophobicity assays. There was a significant positive correlation between the hydrophobicity data obtained by the MATH assay and those obtained by the HIC assay, but these data did not correlate with those obtained by the CAM assay.

Evaluating a model of parental influence on youth physical activity

Objective: To test a conceptual model linking parental physical activity orientations, parental support for physical activity, and children's self-efficacy perceptions with physical activity participation. Participants and Setting: The sample consisted of 380 students in grades 7 through 12 (mean age, 14.0 +/- 1.6 years) and their parents. Data collection took place during the fall of 1996. Main Outcome Measures: Parents completed a questionnaire assessing their physical activity habits, enjoyment of physical activity, beliefs regarding the importance of physical activity, and supportive behaviors for their child's physical activity. Students completed a 46-item inventory assessing physical activity during the previous 7 days and a 5-item physical activity self-efficacy scale. The model was tested via observed variable path analysis using structural equation modeling techniques (AMOS 4.0). Results: An initial model, in which parent physical activity orientations predicted child physical activity via parental support and child self-efficacy, did not provide an acceptable fit to the data. Inclusion of a direct path from parental support to child physical activity and deletion of a nonsignificant path from parental physical activity to child physical activity significantly improved model fit. Standardized path coefficients for the revised model ranged from 0.17 to 0.24, and all were significant at the p < 0.0001 level. Conclusions: Parental support was an important correlate of youth physical activity, acting directly or indirectly through its influence on self-efficacy. Physical activity interventions targeted at youth should include and evaluate the efficacy of individual-level and community-level strategies to increase parents' capacity to provide instrumental and motivational support for their children's physical activity.

Correlação entre a depuração plasmática de creatinina utilizando urina coletada durante 24 horas e 12 horas

Introdução: A concentração da creatinina no plasma é usada para avaliar a função renal, mas a depuração da creatinina plasmática (DCP) constitui método mais sensível para essa finalidade. Objetivo: Correlacionar a DCP em coleta urinária de 12 horas noturna com a de 24 horas. Métodos: Noventa e cinco voluntários (34-64 anos) coletaram urina durante 24 horas em dois frascos: diurno (das 7h às 19h) e noturno (das 19h às 7h do dia seguinte). A coleta de sangue se deu em jejum para medidas bioquímicas. A correlação entre as variáveis foi feita pelo teste Pearson (r) e a concordância de medidas, pelo teste de Bland-Altman. Resultados: Urinas de quatro indivíduos foram recusadas por erro de coleta. Na amostra final (n = 91; 42 homens), havia 23 hipertensos e cinco diabéticos. A DCP (mL/min/1,73 m2) foi menor no período noturno em mulheres (77,8 ± 22,7 versus 88,4 ± 23,6; p < 0,05) e similar em homens (91,2 ± 22,9 versus 97,3 ± 30,9; p > 0,05). As correlações entre a DCP na urina de 12 horas noturna ou diurna e a de 24 horas foram fortes (r = 0,85 e 0,83, respectivamente). Em 85 e 83 dos 91 indivíduos, a medida da DCP noturna e diurna, respectivamente, foi concordante com a de 24 horas. Conclusão: A urina de 12 horas, sobretudo quando coletada à noite, fornece valores de DCP similares àqueles obtidos em coleta de 24 horas. Como essa coleta é mais fácil de ser feita em pacientes ambulatoriais à noite, esse período deveria ser preferido para a medida da filtração glomerular.

Pesquisa de anticorpos anti-PGL-I em pacientes infectados pelo HIV em área endêmica de hanseníase

Esse estudo investiga a infecção subclínica de Mycobacterium leprae em pacientes infectados e não infectados pelo HIV, através da dosagem de anticorpos anti-PGL-I, e avalia se existe uma possível correlação dos resultados sorológicos encontrados com o estado de imunossupressão dos pacientes infectados pelo HIV. Foi realizado um estudo transversal analítico em 350 pacientes infectados pelo HIV e em 350 pacientes não infectados pelo HIV para detecção de anticorpos IgM anti-PGL-I em região endêmica para hanseníase. Avaliou-se uma possível correlação do estado de imunossupressão dos pacientes infectados pelo HIV (contagem de linfócitos CD4+, carga viral e uso ou não de terapia antirretroviral) com a soropositividade para PGLI. Dentre os pacientes infectados pelo HIV, 6% (21/350) apresentaram sorologia positiva para PGL-I e dos indivíduos não infectados pelo HIV, 29,1% (102/350) tinham PGL-I positivo. O grupo controle apresentou cerca de cinco vezes mais indivíduos com anticorpos anti-PGL-I do que o grupo infectado pelo HIV. Não houve diferença estatisticamente significativa na correlação do estado de imunossupressão do paciente com o resultado da sorologia anti-PGL-I. Houve uma menor produção de anticorpos anti-PGL-I em indivíduos infectados pelo HIV, o que pode indicar uma baixa taxa de infecção subclínica por M. leprae, ou uma baixa produtividade específica desses anticorpos, ou ambas as hipóteses. A desregulação de linfócitos B em indivíduos infectados pelo HIV pode ser a causa da baixa produção de anticorpos anti-PGL-I. Não houve correlação do estado de imunossupressão do paciente com o resultado da sorologia anti-PGL-I.

Estudo sobre acetilação da isoniazida em pacientes com tuberculose pulmonar e da sua implicação na redução ou eliminação da carga bacilar no escarro

A N-acetiltransferase 2 é a principal enzima responsável pelo metabolismo e inativação da isoniazida no organismo humano. Mutações no gene NAT2 levam a 3 perfis genotípicos de acetilação que alteram os níveis séricos do fármaco: acetiladores lentos, intermediários e rápidos, o que pode alterar o desfecho terapêutico. O objetivo do estudo foi investigar se os diferentes perfis podem influenciar no tempo de negativação da cultura de escarro, e se existe correlação entre carga bacilar e gravidade da doença com tempo de conversão da cultura. A população de estudo foi composta por 62 pacientes, que tiveram seus DNAs sequenciados para identificação de mutações no gene NAT2 e seus perfis de acetilação determinados. A análise genotípica detectou 10 SNPs, sendo as mutações 341 T>C (39,65%) e 481 C>T (38,71%) as mais frequentes. A determinação das variantes alélicas identificou NAT2*5B (29,03%), NAT2*6A (23,39%) e NAT2*4 (24,19%) como os alelos mais frequentes e NAT2*5B/*5B como o genótipo mais frequente (20,4%). Dentre os 62 pacientes, foi possível correlacionar tempo de negativação da cultura e perfil de acetilação entre 43 deles, os quais 58,3% e 55,6% tiveram o genótipo lento com maior frequência no mês 1 e mês 3, respectivamente. Por meio de dados microbiológicos, a carga bacilar e a gravidade da doença também foram comparadas com o tempo de negativação, indicando que os pacientes com doença moderada ou avançada (76,7%) e aqueles com carga bacilar alta (60,4%), não tiveram associação estatística com o tempo de conversão da cultura. Por último, curvas de crescimento de isolados de M. tuberculosis de pacientes foram construídas para verificar possíveis diferenças na duração da fase lag entre os isolados, porém não foi observada diferença estatística entre elas. Com base nos resultados encontrados, verifica-se que não existe associação entre o perfil de acetilação do paciente, a carga bacilar, a gravidade da doença e o tempo de negativação da cultura de escarro

Fadiga e comportamento tensão-deformação cíclico da liga de alumínio 7050-T7451

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Tecnologia, Departamento de Engenharia Mecânica, 2016.

Morphometry of testis and seminiferous tubules of the adult crab-eating fox (Cerdocyon thous, Linnaeus, 1766)

Body and testicular biometric parameters are very important for establishing reproductive patterns and, consequently, the development of protocols for assisted reproduction in different species. A direct correlation between the testis weight and the sperm population was observed in other studied species, because the testis size primarily reflects the total volume of the seminiferous tubule, its main component. The objective of this study was to determine the testicular volume parameters and correlate data from morphometry of testis and seminiferous tubules with body mass in six adult crab-eating foxes. The mean body weight of the crab-eating foxes in this study was 6.53 kg, with approximately 0.068% allocated to the testicular mass and 0.042% specifically to seminiferous tubules, which represented 87.5% of the testicular parenchyma. The albuginea comprised 12.5% of the testicular mass. The mean diameter of seminiferous tubules was 236 µm, and the mean thickness of the seminiferous epithelium was 62.9 µm. Values of tubular parameters indicate a sperm productivity close to those observed in previously studied carnivores.

An empirical assessment of customer satisfaction and quality of servisse: Comparing Servqual and Servperf

This paper assesses the validity and reliability of two instruments measuring quality of service, the SERVPERF and SERVQUAL scales, replicated in a novel cultural settings, a Portuguese energy company. To provide insights and strategies for managerial intervention, a relation between customers’ satisfaction and quality of service is established. The empirical study suggests a superior convergent and predictive validity of SERVPERF scale to measure quality of service in this settings when comparing to SERVQUAL. The main differences of this study with previous ones, are that this one resorts on a confirmatory factor analysis, the validation of the instruments is performed by using the same measures suggested by their creators and extends the line of research to a novel cultural settings, a Portuguese energy company. Concerning the relationship between service quality and customers’ satisfaction, all of the quality of service attributes correlate almost equally to the satisfaction ones, with a lower weight concerning tangibles.

An empirical assessment of customer satisfaction and quality of service: comparing SERVQUAL and SERVPERF

This paper assesses the validity and reliability of two instruments measuring quality of service, the SERVPERF and SERVQUAL scales, replicated in a novel cultural settings, a Portuguese energy company. To provide insights and strategies for managerial intervention, a relation between customers’ satisfaction and quality of service is established. The empirical study suggests a superior convergent and predictive validity of SERVPERF scale to measure quality of service in this settings when comparing to SERVQUAL. The main differences of this study with previous ones, are that this one resorts on a confirmatory factor analysis, the validation of the instruments is performed by using the same measures suggested by their creators and extends the line of research to a novel cultural settings, a Portuguese energy company. Concerning the relationship between service quality and customers’ satisfaction, all of the quality of service attributes correlate almost equally to the satisfaction ones, with a lower weight concerning tangibles.

Prevalence of antibodies to the BK and JC papovaviruses in isolated populations

A total of 173 sera from isolated Brazilian Indian populations, 39 from the Diauarun area, and 68 from the Alto Xingú area, respectively in the North and the South of the Xingú National Park and 66 Kren-Akorore Indians, were examined for hemagglutination - inhibiting (HI) antibodies against BK and JC viruses. The global percentages of positive sera (> 1:40) were 5.2% for BK virus and 1.7% for JC virus. The distribution of positive sera according to the population groups showed one individual to be positive for BK virus in the Diauarun Indians and none of the sera contained HI antibody to JC virus; in the Alto Xingú Indians, 4 were positive for BK virus and 3 others were positive for JC virus; as regards Kren-Akorore Indians none of the sera contained antibody to JC virus, and only 4 were BK positive. Due to the limited number of observations it was neither possible to determine the time of occurrence of seroconversion nor correlate the positivity rates for both viruses in the different tribes with the respective "contact" with the white population.

Estudo da função sistólica do ventrículo direito por ecocardiografia transtorácica: relatório de projecto

Mestrado em Tecnologia de Diagnóstico e Intervenção Cardiovascular. Área de especialização - Ultrassonografia Cardiovascular