As funções terapêuticas da atividade: um estudo comparativo em terapia ocupacional

O propósito deste estudo foi verificar as alterações ocorridas em relação a assistência em terapia ocupacional na Unidade Psiquiátrica do Hospital São Paulo, vinculada ao Departamento de Psiquiatria e Psicologia Médica da Universidade Federal de São Paulo, através da análise da aplicação de um instrumento terapêutico da terapia ocupacional, a atividade. Este estudo baseou-se em pesquisa retrospectiva nos prontuários de pacientes internadas na Unidade Psiquiátrica. A pesquisa se propôs sistematizar o registro de procedimentos de terapeutas ocupacionais e de outros profissionais em relação a atividade, no sentido de dar uma contribuição universalização de alguns aspectos do discurso da Terapia Ocupacional, sistematizando-os em categorias. Pretendeu-se verificar a aplicabilidade do instrumento terapêutico pesquisado, através da comparação dos procedimentos encontrados em dois períodos. Os procedimentos metodológicos constaram de: a.- definição dos períodos a serem pesquisados: de 1/1/1988 a 31/12/1988 e de 1/1/1994 a 31/12/1994; b- coleta de dados: foram levantados em 257 prontuários de pacientes internadas na Unidade Psiquiátrica nos períodos acima definidos, os dados sócio demográficos e todos os registros encontrados nos prontuários, sobre uso de atividade e sobre terapia Ocupacional: c- categorização dos dados sobre atividade e terapia ocupacional segundo referências empíricas e funções da atividade e posteriormente em categorias empíricas em terapia ocupacional; d- análise dos dados: através do cotejamento do material encontrados nos dois períodos em relação As categorias criadas e A literatura. A análise dos dados demonstrou que não houve alterações no perfil sócio demográfico da clientela, não se alterando também a pouca atenção da equipe frente a dados relativos a origem, ocupação e perfil econômico. Em relação a idade média das pacientes e tempo de internação a distribuição dos mesmos difere, os indices mais altos encontrados relacionam-se ao segundo período. Quanto ao instrumento terapêutico, a atividade, o estudo comparativo, permitiu identificar modificações em relação a compreensão e uso do mesmo. No primeiro período, o recurso terapêutico, atividade, relaciona-se com a proposta de ambientoterapia, isto 6, toda a utilização da atividade insere-se num movimento mais amplo, grupal, com a participação efetiva de toda a equipe, voltado à experiências sociabilizantes, com raras abordagens especificas de terapeutas ocupacionais. No segundo período a utilização da atividade enquanto instrumento terapêutico, aproxima-se dos recursos teóricos e práticos da terapia ocupacional psicodinâmica, o que permitiu enfoques mais individualizados, introduziu a questão do processo terapêutico, a utilização deste recurso como uma aborgagem eletiva, ampliou o contato das terapeutas ocupacionais com a clientela em grupos específicos de terapia ocupacional e no ambulatório de egressos. Perdeu-se neste segundo período alguns procedimentos relativos a proposta da ambientoterapia e a uniformidade da equipe frente a esta questão. Percebeu-se que, a aplicabilidade deste recurso terapêutico, nos dois períodos, baseia-se nas possíveis funções da atividade, tomadas sempre como recursos estratégicos. Nos dois períodos foi possível verificar que a aplicabilidade deste instrumento terapêutico não se relacionou a categorias diagnósticas, podendo num sentido mais amplo, ter indicação a todos as pacientes.The purpose of this study was to verify the changes occurred within the Occupational Therapy assistance in the Psychiatric Unit of Hospital São Paulo, linked to the Psychiatry and Medical Psychology Department of Universidade Federal de Sao Paulo through the analysis of the use a therapeutic means of Occupational Therapy named the activity. The study was based on retrospective research of inpatients'clinical charts. The research purpose was to sistematyze the procedures of occupational therapists and of other professionals concerning the activity in order to contribute to the generalization of certains aspects of occupational therapy practices by arranging them into categories.Our intention was to verify the applicability of the therapeutic means under analysis through the comparison of procedures found in two distinct periods. The experimental methods consisted of: a. Definition of periods to be researched: from 01/01/1988 to 31/12/1988 and from 01/01/1994 to 31/12/1994. b.Data collection: Data were collected from 257 inpatineclinical charts during the above defined periods, specifically social demographics data and all the information found in charts concerning use of the activity and occupational therapy. c. data categorization about the activity and occupational therapy according to empirical references and functions of the activity and subsequently to empirical categories in occupational therapy d.data analyses: through comparison of the material found in both periods, through created categories and in reference to the existing literature. Data analyses showed that were no changes in patients social demographics profile, as well as in team members'poor charting of data relative to the source, occupation and economical profile of impatient. Concerning the age of the patients and hospitalization period, their distribution differed with higher levels found in the second period. As for the use of the therapeutic instrument, the activity, the comparison allowed the identification of due to its application.changes. In the first period, the activity relates to the millieu therapy proposal, that is, all the activity application inserts itself in a broader movement, with the effective participation of all the members focused on socialization with rare specif interventions of occupational therapists. In the secon period, the use of the activity as a therapeutic instrument follows the theoretical and practical resource of the psychodinamic occupational therapy; that allowed more specific approaches and introduced the subject of the therapeutic process in occupational therapy. The use of this resource as a choice approach enlarged the contact of occupational therapists with patients in Occupational Therapy groups and in the out patient follow-up treatment. During this second period some procedures relating to the millieu therapy project and the unity of the team concerning this subject were lost. We have noticed that the applicability of this therapeutic resource, the activity, during both periods is based on the possible functions of the activity, always taken as strategis resources. It was possible to verify, in both periods, that the applicability of this therapeutic instrument is not related to diagnostic categories, and it that can be beneficial to all the patients.BV UNIFESP: Teses e dissertaçõe

The Significance of the Location of Mutations for the Native-State Dynamics of Human Lysozyme

The conversion of human lysozyme into amyloid fibrils is associated with a rare but fatal hereditary form of nonneuropathic systemic amyloidosis. The accumulation of large amounts of aggregated protein is thought to be initiated by the formation of transient intermediate species of disease-related lysozyme variants, essentially due to the loss of global cooperativity under physiologically relevant conditions. Interestingly, all five naturally occurring, amyloidogenic, single-point mutations are located in the β-domain of lysozyme, the region that is predominantly unfolded during the formation of the transient intermediate species. Given the lack of known naturally occurring, amyloidogenic, single-point mutations in the α-domain, we chose three specific mutations to address the effects that location may have on native-state dynamics, as studied by hydrogen-deuterium (HD) exchange experiments analyzed by NMR spectroscopy, and mass spectrometry. We compared the effect of a destabilizing α-domain mutation (I23A) with that of the well-characterized I59T β-domain variant. We also investigated the effect of a mutation that has minor effects on native-state stability at the domain interface (I56V) and compared it with that of a variant with similar stability within the C-helix (I89V). We show that when variants have similar reduced native-state stabilities, the location of the mutation (I23A versus I59T) is crucial to the native-state dynamics, with the α-domain mutation having a significantly lower ability to populate transient intermediate species under physiologically relevant conditions. Interestingly, the mutation at the interface (I56V) has a greater effect in facilitating the formation of transient intermediate species at elevated temperatures compared with the variants containing α-domain mutations, even though this mutation results in only minor changes to the native-state stability of lysozyme. These findings reveal that the location of specific mutations is an important factor in determining the native-state dynamical properties of human lysozyme in the context of its propensity to populate the aggregation-prone transient intermediate species associated with pathogenic amyloid formation.This research was supported by the Biotechnology and Biological Sciences Research Council (BB/E019927/1 to C.M.D., C.V.R., and J.R.K.), the Medical Research Council (E.D.G. and C.M.D.), the Belgian Program of Interuniversity Attraction Poles administered by the Federal Office for Scientific Technical and Cultural Affairs (PAI numbers P6/19 and P7144 to C.M.D. and M.D.), the European Union’s Sixth Framework Program (LSHM-CT-2006-037525 to C.M.D. and M.D.), and Programme grants from the Wellcome Trust and the Leverhulme Trust (C.M.D.). It was also supported by a Korean Government Scholarship for Overseas Studies (M.A.), the Winston Churchill Foundation (C.L.H.), and Boerhinger Ingleheim funds (A.D.). The NMR facility at the Department of Chemistry, University of Cambridge, is supported in part by an EPSRC Core Capability grant (EP/K039520/1)

Flow cytometric measurement of the cellular propagation of TDP-43 aggregation

Amyotrophic lateral sclerosis is a devastating neuromuscular degenerative disease characterized by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology including TAR DNA-binding protein of 43 kDa (TDP-43) aggregates. Previous work suggests that TDP-43 can move between cells. Here we used a novel flow cytometry technique (FloIT) to analyze TDP-43 inclusions and propagation. When cells were transfected to express either mutant G294A TDP-43 fused to GFP or wild type TDP-43fused to tomato red and then co-cultured, flow cytometry detected intact cells containing both fusion proteins and using FloIT detected an increase in the numbers of inclusions in lysates from cells expressing wild type TDP-43-tomato. Furthermore, in this same model, FloIT analyses detected inclusions containing both fusion proteins. These results imply the transfer of TDP-43 fusion proteins between cells and that this process can increase aggregation of wild-type TDP-43 by a mechanism involving co-aggregation with G294A TDP-43.This work was supported by the NHMRC under Grant 1084144 and 1095215

Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris.

We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general

Fluorescence Quantum Yield of Thioflavin T in Rigid Isotropic Solution and Incorporated into the Amyloid Fibrils

In this work, the fluorescence of thioflavin T (ThT) was studied in a wide range of viscosity and temperature. It was shown that ThT fluorescence quantum yield varies from 0.0001 in water at room temperature to 0.28 in rigid isotropic solution (T/η→0). The deviation of the fluorescence quantum yield from unity in rigid isotropic solution suggests that fluorescence quantum yield depends not only on the ultra-fast oscillation of ThT fragments relative to each other in an excited state as was suggested earlier, but also depends on the molecular configuration in the ground state. This means that the fluorescence quantum yield of the dye incorporated into amyloid fibrils must depend on its conformation, which, in turn, depends on the ThT environment. Therefore, the fluorescence quantum yield of ThT incorporated into amyloid fibrils can differ from that in the rigid isotropic solution. In particular, the fluorescence quantum yield of ThT incorporated into insulin fibrils was determined to be 0.43. Consequently, the ThT fluorescence quantum yield could be used to characterize the peculiarities of the fibrillar structure, which opens some new possibilities in the ThT use for structural characterization of the amyloid fibrils

Aspergillus as a multi-purpose cell factory: current status and perspectives

Aspergilli have a long history in biotechnology as expression platforms for the production of food ingredients, pharmaceuticals and enzymes. The achievements made during the last years, however, have the potential to revolutionize Aspergillus biotechnology and to assure Aspergillus a dominant place among microbial cell factories. This mini-review will highlight most recent breakthroughs in fundamental and applied Aspergillus research with a focus on new molecular tools, techniques and products. New trends and concepts related to Aspergillus genomics and systems biology will be discussed as well as the challenges that have to be met to integrate omics data with metabolic engineering attempts