The Identification and Biochemical Properties of the Catalytic Specificity of a Serine Peptidase Secreted by Aspergillus fumigatus Fresenius

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Occupational chronic solvent encephalopathy in Finland 1995 - 2007 : incidence and diagnostic methods

The occurrence of occupational chronic solvent encephalopathy (CSE) seems to decrease, but still every year reveals new cases. To prevent CSE and early retirement of solvent-exposed workers, actions should focus on early CSE detection and diagnosis. Identifying the work tasks and solvent exposure associated with high risk for CSE is crucial. Clinical and exposure data of all the 128 cases diagnosed with CSE as an occupational disease in Finland during 1995-2007 was collected from the patient records at the Finnish Institute of Occupational Health (FIOH) in Helsinki. The data on the number of exposed workers in Finland were gathered from the Finnish Job-exposure Matrix (FINJEM) and the number of employed from the national workforce survey. We analyzed the work tasks and solvent exposure of CSE patients and the findings in brain magnetic resonance imaging (MRI), quantitative electroencephalography (QEEG), and event-related potentials (ERP). The annual number of new cases diminished from 18 to 3, and the incidence of CSE decreased from 8.6 to 1.2 / million employed per year. The highest incidence of CSE was in workers with their main exposure to aromatic hydrocarbons; during 1995-2006 the incidence decreased from 1.2 to 0.3 / 1 000 exposed workers per year. The work tasks with the highest incidence of CSE were floor layers and lacquerers, wooden surface finishers, and industrial, metal, or car painters. Among 71 CSE patients, brain MRI revealed atrophy or white matter hyperintensities or both in 38% of the cases. Atrophy which was associated with duration of exposure was most frequently located in the cerebellum and in the frontal or parietal brain areas. QEEG in a group of 47 patients revealed increased power of the theta band in the frontal brain area. In a group of 86 patients, the P300 amplitude of auditory ERP was decreased, but at individual level, all the amplitude values were classified as normal. In 11 CSE patients and 13 age-matched controls, ERP elicited by a multimodal paradigm including an auditory, a visual detection, and a recognition memory task under single and dual-task conditions corroborated the decrease of auditory P300 amplitude in CSE patients in single-task condition. In dual-task conditions, the auditory P300 component was, more often in patients than in controls, unrecognizable. Due to the paucity and non-specificity of the findings, brain MRI serves mainly for differential diagnostics in CSE. QEEG and auditory P300 are insensitive at individual level and not useful in the clinical diagnostics of CSE. A multimodal ERP paradigm may, however, provide a more sensitive method to diagnose slight cognitive disturbances such as CSE.

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

Introduction Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Methods Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90 degrees light scattering and FtsZ polymer pelleting assays. The gamma 32P-GTP synthesised by NDK from GDP and gamma 32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound P-32-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. Results NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK. Conclusion Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.

Students' choice of a master in management in Portugal: A means-end chain approach

In the increasingly competitive market of higher education introduced by the Bologna Declaration, understanding the decision-making of master in management students is at the center of institutional management and marketing efforts on its mission to attract prospective students in a less costly, more efficient manner. The means-end chain approach, applied to the choice of a Portuguese institution in which to pursue a master in management, points to the position in rankings and to the non-specificity of the program as the most important attributes. Additionally, results show that students with distinct demographic, household, or background characteristics choose in significantly different manners.

The elderly : reminiscing, coping with loss, accepting death

This study investigated loss, death and dying, reminiscing, coping and the process of adaptation from the sUbjective perspective. A number of theories and models of death and dying were reviewed in the background literature search with the focus on reminiscing as a coping phenomenon. The format of the study was audio-taped interviews with ten sUbjects and the recording of their memories and reminiscing of life stories. The sUbjects were required to complete an initial questionnaire in a demographic data collection process. Two separate interviews consisted of a primary data collecting interview and a verification interview four to eight weeks later. An independent chart review completed the data collecting process. Data analysis was by the examination of the emerging themes in the subjects' personal narratives which revealed the sUb-categories of reminiscing, loss (including death and dying), acceptance, hope, love, despair and belief. Belief was shown to be the foundation and the base for living and reminiscing. Reminiscing was found to be a coping phenomenon, within the foundation of a belief system. Both living and reminiscing revealed the existence of a central belief or value with a great deal of importance attached to it. Whether the belief was of a spiritual nature, a value of marriage, tradition, a work ethic or belief in an abstract value such as fate,it gave support and control to the individuals' living and reminiscing process. That which caused despair or allowed acceptance indicated the sUbjects' basic belief and was identified in the story narrations. The findings were significant to health care in terms of education, increased dignity for the elderly and better understanding by society. The profiles represented an average age of 86.3 years with age showing no bearing on the life experiences associated with the emerging themes. Overwhelmingly, belief was shown to be the foundation in reminiscing. A Judeo-Christian cultural value base supported the belief in 90% of the sUbjects; however, different beliefs were clearly shown indicating that belief is central to all thinking beings, in everyday life and in reminiscing. Belief was not necessarily spiritual or a practised or verbalized religion. It was shown to be a way of understanding, a fundamental and single thread tying the individual's life and stories together. The benefits were the outcomes, in that knowledge of an individual's belief can optimize care planning for any age group, and/or setting. The strength of the study was the open question format and the feedback process of data verification. The unrestricted outcomes and non-specificity were significant in a world where dying is everybody's business.

UNDERSTANDING THE RELATIONSHIP BETWEEN ONCOLYTIC AD5 DELETED E1b55KDA LYTIC INFECTION AND P53 IN MAMMALIAN CELLS

Adenoviruses are the most commonly used in the development of oncolytic therapy. Oncolytic adenoviruses are genetically modified to selectivity replicate in and kill tumor cells. The p53 molecule is a tumor suppressor protein that responds to viral infection through the activation of apoptosis, which is inhibited by adenovirus E1B55kDa protein leading to progressive viral lytic cycle. The non-specificity of replication has limited the use of wild type adenovirus in cancer therapy. This issue was resolved by using an E1b deleted Ad that can only replicate in cells with a deficiency in the p53 protein, a common feature of most cancer cells. Although demonstrating a moderate success rate, E1b55kDa deleted Ad has not been approved as a standard therapy for all cancer types. Several studies have revealed that E1b deleted Ad replication was independent of p53 status in the cell, as the virus replicated better in some p53 deficient cancers more than others. However, this mechanism has not been investigated deeply. Therefore, the objective of this study is to understand the relationship between p53 status, levels and functional activity, and oncolytic Ad5dlE1b55kDa replication efficiency. Firstly, five transient p53 expression vectors that contain different regulatory elements were engineered and then evaluated in H1299, HEK293 and HeLa cell lines. Data indicated that vector that contains the MARs and HPRE regulatory elements achieved the highest stability of p53 expression. Secondly, we used these vectors to examine the effect of various p53 expression levels on the replication efficiency of oncolytic Ad5dlE1b55kDa. We found that the level of p53 in the cell had an insignificant effect on the oncolytic viruses’ replication. However, the functional activity of p53 had a significant effect on its replication, as Ad5dlE1b55kDa was shown to have selective activity in H1299 cells (p53-null). In contrast, a decrease in viral replication was found in HeLa cells (p53-positive). Finally, the effect of p53’s functional activity on the replication efficiency of oncolytic Ad5dlE1b55kDa was examined. Viral growth was evaluated in H1299 cells expressing number of p53 mutants. P53-R175H mutant successfully rescued viral growth by allowing the virus to exert its mechanism of selectivity. The mechanism entailed deregulating the expression of specific genes, cell cycle and apoptosis, in the p53 pathway to promote its production leading to efficient oncolytic effect. These results confirmed that oncolytic Ad5dlE1b55kDa sensitivity is mutation-type specific. Therefore, before it is applied clinically as cancer therapy for p53 deficient tumors, the type of p53 mutation must be determined for efficient antitumor effect.

MODELING HETEROTACHY IN PHYLOGENETICS

Il a été démontré que l’hétérotachie, variation du taux de substitutions au cours du temps et entre les sites, est un phénomène fréquent au sein de données réelles. Échouer à modéliser l’hétérotachie peut potentiellement causer des artéfacts phylogénétiques. Actuellement, plusieurs modèles traitent l’hétérotachie : le modèle à mélange des longueurs de branche (MLB) ainsi que diverses formes du modèle covarion. Dans ce projet, notre but est de trouver un modèle qui prenne efficacement en compte les signaux hétérotaches présents dans les données, et ainsi améliorer l’inférence phylogénétique. Pour parvenir à nos fins, deux études ont été réalisées. Dans la première, nous comparons le modèle MLB avec le modèle covarion et le modèle homogène grâce aux test AIC et BIC, ainsi que par validation croisée. A partir de nos résultats, nous pouvons conclure que le modèle MLB n’est pas nécessaire pour les sites dont les longueurs de branche diffèrent sur l’ensemble de l’arbre, car, dans les données réelles, le signaux hétérotaches qui interfèrent avec l’inférence phylogénétique sont généralement concentrés dans une zone limitée de l’arbre. Dans la seconde étude, nous relaxons l’hypothèse que le modèle covarion est homogène entre les sites, et développons un modèle à mélanges basé sur un processus de Dirichlet. Afin d’évaluer différents modèles hétérogènes, nous définissons plusieurs tests de non-conformité par échantillonnage postérieur prédictif pour étudier divers aspects de l’évolution moléculaire à partir de cartographies stochastiques. Ces tests montrent que le modèle à mélanges covarion utilisé avec une loi gamma est capable de refléter adéquatement les variations de substitutions tant à l’intérieur d’un site qu’entre les sites. Notre recherche permet de décrire de façon détaillée l’hétérotachie dans des données réelles et donne des pistes à suivre pour de futurs modèles hétérotaches. Les tests de non conformité par échantillonnage postérieur prédictif fournissent des outils de diagnostic pour évaluer les modèles en détails. De plus, nos deux études révèlent la non spécificité des modèles hétérogènes et, en conséquence, la présence d’interactions entre différents modèles hétérogènes. Nos études suggèrent fortement que les données contiennent différents caractères hétérogènes qui devraient être pris en compte simultanément dans les analyses phylogénétiques.

Uma contribuição ao estudo do stress: introversão-extroversão, dogmatismo e vulnerabilidade ao stress

Os dois tipos de stress fisiológico e psicológico constituem, a meu ver, um único fenômeno, embora, do ponto de vista didático, faça-se necessária esta divisão. Neste estudo ambos os tipos foram abordados teoricamente, mas o mesmo se propõe a analisar a influência dos fatores de personalidade - extroversão, introversão e dogmatismo- na vulnerabilidade ao stress. Os determinantes cognitivos foram considerados como mediadores desta relação. O instrumental utilizado para a verificação empírica foi composto dos seguintes testes: 16 PF de Cattell e Eber, Fator P de Toulouse-Pieron, Escala de Dogmatismo de Rokeach e o Sorting-Test, adaptado para esse estudo. Esses instrumentos foram utilizados para testar as hipóteses; o introvertido e o não-dogmático, tanto na condição isolada quanto em combinação, apresentam vulnerabilidade ao stress maior do que o extrovertido e o dogmático nas mesmas condições. A análise da regressão múltipla demonstrou que nenhuma diferença quanto à vulnerabilidade ao stress foi observada em relação aos tipos extrovertido, introvertido, dogmátlco e não-dogmático, quer isoladamente, quer nas combinações extrovertido-dogmático e introvertido-não-dogmático. É provável que algumas circunstâncias possam explicar esse fenômeno. Em primeiro lugar é possível que a vulnerabilidade ao stress esteja mais relacionada com traço de personalidade do que com tipo. Em segundo lugar, pode ser que o instrumento utilizado para medir extroversão-introversão não forneça uma medida válida: uma vez que o conceito se baseia nas primeiras formulações da teoria da ativação. Poderíamos, ainda, acrescentar uma outra relação na influência dos fatores de personalidade na vulnerabilidade ao stress . Esta relação pode ser estudada sob os dois aspectos do stress: a especificidade e a não-especificidade. Assim sendo, poder-se-ia conjecturar que tanto os fatores de personalidade quanto os determinantes cognitivos estariam mais relacionados com a especificidade do que com a não-especificidade. Esta última estaria mais vinculada aos aspectos fisiológicos. Esta última proposição poderia servir de um esquema para uma diferente análise teórica e empírica da influência dos fatores de personalidade na vulnerabilidade ao stress, enfatizando mais a especificidade do que a não-especificidade.

Diferentes métodos de diagnóstico molecular na diferenciação das espécies de Sarcocystis spp

The Sarcocystis genus includes obligatory two-host life cycle protozoan parasites. It is the most numerous of the six genera of the Sarcocystidae family. The infection caused by parasites of this genus is a zoonotic and cosmopolitan disease known as sarcosistosis or sarcosporidiosis. The sarcositosis though frequently asymptomatic in its definitive hosts can be fatal in its intermediate hosts. The usual diagnoses of sarcosistosis takes place through a histological demonstration of schizonts in blood vessels and organs, and the presence of cysts in muscle tissue by necropsy or biopsy, this second method still more common and based on morphological features of the sarcocyst. However, these methods can be inadequate to a precise identification of the infector species once that, besides the genus being of numerous species, these often present similar morphological features. Another factor that makes the diagnostic more difficult is the non specificity of some Sacocystis species to their hosts. Consequently, molecular diagnostic methods have been used in order to identify the infector species and the parasite specific biological cycles, demonstrating also new species and coevolutive aspects between parasite and host. Among the most employed molecular techniques the Polimerase Chain Reaction (PCR), the nested-PCR and the Restriction Fragment Length Polymorphism (RFLP) stands out

Correlation between Hoff test performance, body composition and aerobic and anaerobic fitness in professional soccer players

Purpose The purpose of the study was to investigate a possible association between the distance covered in the Hoff test with parameters of maximal oxygen uptake (V_O2MAX), anaerobic threshold, anaerobic fitness, and body composition of professional adult soccer players. Methods Twenty-five professional soccer players (20 ± 3 years) participated in the study. On different days the athletes performed: a graded incremental exercise test in a laboratory to measure V_O2MAX; a specific soccer field test called the Hoff test; a running anaerobic sprint test (RAST); an incremental test on an oval circuit to determine the velocity relative to anaerobic threshold (VAnT) and an estimation of body composition. Results The average V_O2MAX corresponded to 4.1 ± 0.1 L min-1 (54.1 ± 1.2 mL kg-1 min-1 ). The average distance covered during the Hoff test was 1,442.4 ± 30.0 m. The distance covered during the Hoff test showed significant correlations with absolute and expressed in an appropriated scale V_O2MAX (r = 0.44, p = 0.02; r = 0.42, p = 0.02, respectively) while no significant differences were found with body composition, VAnT and RAST variables. Conclusions The present study demonstrated that the distance covered during the Hoff test has weak correlation with V_O2MAX determined in treadmill running, and no correlation with VAnT, body composition and RAST outcomes, probably due to the non-specificity of the proposed tests when associated with the Hoff test.

A Case Study on Using the Via Christi Breastfeeding Assessment Tool in a Clinical Setting

Aims- The goal of this study is to obtain a subjective point of view of the use, benefits, and shortcomings of the Via Christi breastfeeding Assessment Tool in a clinical setting. Methods- This case study is based on a single yet detailed interview with a lactation consultant who has utilized the Via Christi Breastfeeding Tool in a clinical setting. Findings- Advantages of the Via Christi Breastfeeding Assessment Tool include that the tool is practical and concrete, encompassing both a subjective dimension and the basic elements of breastfeeding. Shortcomings of the tool include its non-specificity in focus on the feeding technique only and excluding other factors involved with the birth and mother- baby couplet. Conclusion- The Via Christi Breastfeeding Tool serves as a good initial screening of breastfeeding, which in result leads to the recommendation of further and more extensive study of a specific mother-baby couplet breastfeeding measures if needed.

Factors affecting the nodulation and growth of tropical woody legume seedlings

This study surveys the occurrence of nodulation in woody legume species in Panamá and Costa Rica, describes nodule and root characteristics, and researches host-bacteria specificity, nodulation potential of soils, and the effects of light, added nitrogen, and rhizobia and VA mycorrhizal fungi inoculation on seedling growth. I examined 83 species in 37 genera and found 80% to be nodulated. Percent nodulated species in the Caesalpinioideae, Mimosoideae, and Papilionoideae was 17, 95, and 86, respectively, with no correlation between nodule morphology and tribal classification. Nodules formed mainly at root branch points which supports epidermal breaks as an important rhizobia infection route. More non-nodulated than nodulated species had root hairs. Several species emitted volatile sulfur-containing compounds, including the toxic compound ethylmercaptan, from roots, germinating seeds, and other tissues. These emissions may have an allelopathic action against pathogens, predators, or other plants. In contrast to the general non-specificity of most legumes for rhizobia, Mimosa pigra L. was highly specific and only nodulated in flooded soils. This species' specificity, combined with a limited occurrence of its root nodule bacteria may limit its natural distribution, but its spread as an invasive weed is facilitated when fill material from rivers is deposited in other areas. ^ An experimental light level of 1.5% of full sun completely inhibited seedling nodulation, as do similar naturally low levels in forest understory. In the forest, trees and seedlings were not nodulated. in some soils with suspected high N content. For six experimental species, added N progressively increased seedling growth while decreasing nodule biomass; at the highest level of added N nodulation was completely suppressed. Species and individuals showed variation in nodule biomass at high N applications which may indicate an opportunity for genetic selection for optimal N acquisition. Rhizobia inoculation had a small positive effect on seedling shoot growth, but VA mycorrhiza inoculation overwhelmingly increased seedling size, biomass, and leaf mineral concentration. In lowland tropical forest, VA mycorrhizal colonization appears indispensable for legume nodulation because of the fungus' ability to supply P in deficient soils. This requirement makes the legume-rhizobia-mycorrhiza association obligately tripartite. ^

Erythropoietin receptor expression in non-small cell lung carcinoma: a question of antibody specificity

Immunohistochemical studies on formalin-fixed, paraffin-embedded (FFPE) tissue utilizing polyclonal antibodies form the cornerstone of many reports claiming to demonstrate erythropoietin receptor (EPOR) expression in malignant tissue. Recently, Elliott et al. (Blood 2006;107:1892-1895) reported that the antibodies commonly used to detect EPOR expression also detect non-EPOR proteins, and that their binding to EPOR was severely abrogated by two synthetic peptides based on the sequence of heat shock protein (HSP) 70, HSP70-2, and HSP70-5. We have investigated the specificity of the C20 antibody for detecting EPOR expression in non-small cell lung carcinoma (NSCLC) utilizing tissue microarrays. A total of 34 cases were available for study. Antibody absorbed with peptide resulted in marked suppression of cytoplasmic staining compared with nonabsorbed antibody. Four tumors that initially showed a membranous pattern of staining retained this pattern with absorbed antibody. Positive membranous immunoreactivity was also observed in 6 of 30 tumors that originally showed a predominantly cytoplasmic pattern of staining. Using the C20 antibody for Western blots, we detected three main bands, at 100, 66, and 59 kDa. Preincubation with either peptide caused abolition of the 66-kDa band, which contains non-EPOR sequences including heat shock peptides. These results call into question the significance of previous immunohistochemical studies of EPOR expression in malignancy and emphasize the need for more specific anti-EPOR antibodies to define the true extent of EPOR expression in neoplastic tissue