Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.

The Zn(Scys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles. The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A. The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins. Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxin-encoding gene from Clostridium pasteurianum. The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar. The most significant differences are increased lengths of the M-S bonds in Zn-rubredoxin (average length, 2.345 A) as compared with Fe-rubredoxin (average length, 2.262 A). An increase of the CA-CB-SG-M dihedral angles involving Cys-6 and Cys-39, the first cysteines of each of the Cys-Xaa-Xaa-Cys metal binding motifs, has been observed. Another consequence of the replacement of iron by zinc is that the region around residues 36-46 undergoes larger displacements than the remainder of the polypeptide chain. Despite these changes, the main features of the FeS4 site, namely a local 2-fold symmetry and the characteristic network of N-H...S hydrogen bonds, are conserved in the ZnS4 site. The Zn-substituted rubredoxin provides the first precise structure of a Zn(Scys)4 unit in a protein. The nearly identical fold of rubredoxin around iron or zinc suggests that at least in some of the sites where the metal has mainly a structural role-e.g., zinc fingers-the choice of the relevant metal may be directed by its cellular availability and mobilization processes rather than by its chemical nature.

A joint hazard and time scaling model to compare survival curves.

To provide a more general method for comparing survival experience, we propose a model that independently scales both hazard and time dimensions. To test the curve shape similarity of two time-dependent hazards, h1(t) and h2(t), we apply the proposed hazard relationship, h12(tKt)/ h1(t) = Kh, to h1. This relationship doubly scales h1 by the constant hazard and time scale factors, Kh and Kt, producing a transformed hazard, h12, with the same underlying curve shape as h1. We optimize the match of h12 to h2 by adjusting Kh and Kt. The corresponding survival relationship S12(tKt) = [S1(t)]KtKh transforms S1 into a new curve S12 of the same underlying shape that can be matched to the original S2. We apply this model to the curves for regional and local breast cancer contained in the National Cancer Institute's End Results Registry (1950-1973). Scaling the original regional curves, h1 and S1 with Kt = 1.769 and Kh = 0.263 produces transformed curves h12 and S12 that display congruence with the respective local curves, h2 and S2. This similarity of curve shapes suggests the application of the more complete curve shapes for regional disease as templates to predict the long-term survival pattern for local disease. By extension, this similarity raises the possibility of scaling early data for clinical trial curves according to templates of registry or previous trial curves, projecting long-term outcomes and reducing costs. The proposed model includes as special cases the widely used proportional hazards (Kt = 1) and accelerated life (KtKh = 1) models.

Amygdala activity at encoding correlated with long-term, free recall of emotional information.

Positron emission tomography of cerebral glucose metabolism in adult human subjects was used to investigate amygdaloid complex (AC) activity associated with the storage of long-term memory for emotionally arousing events. Subjects viewed two videos (one in each of two separate positron emission tomography sessions, separated by 3-7 days) consisting either of 12 emotionally arousing film clips ("E" film session) or of 12 relatively emotionally neutral film clips ("N" film session), and rated their emotional reaction to each film clip immediately after viewing it. Three weeks after the second session, memory for the videos was assessed in a free recall test. As expected, the subjects' average emotional reaction to the E films was higher than that for the N films. In addition, the subjects recalled significantly more E films than N films. Glucose metabolic rate of the right AC while viewing the E films was highly correlated with the number of E films recalled. AC activity was not significantly correlated with the number of N films recalled. The findings support the view derived from both animal and human investigations that the AC is selectively involved with the formation of enhanced long-term memory associated with emotionally arousing events.

Learning and recall of form discriminations during reversible cooling deactivation of ventral-posterior suprasylvian cortex in the cat.

Extrastriate visual cortex of the ventral-posterior suprasylvian gyrus (vPS cortex) of freely behaving cats was reversibly deactivated with cooling to determine its role in performance on a battery of simple or masked two-dimensional pattern discriminations, and three-dimensional object discriminations. Deactivation of vPS cortex by cooling profoundly impaired the ability of the cats to recall the difference between all previously learned pattern and object discriminations. However, the cats' ability to learn or relearn pattern and object discriminations while vPS was deactivated depended upon the nature of the pattern or object and the cats' prior level of exposure to them. During cooling of vPS cortex, the cats could neither learn the novel object discriminations nor relearn a highly familiar masked or partially occluded pattern discrimination, although they could relearn both the highly familiar object and simple pattern discriminations. These cooling-induced deficits resemble those induced by cooling of the topologically equivalent inferotemporal cortex of monkeys and provides evidence that the equivalent regions contribute to visual processing in similar ways.

Determination of absorption curves, dissolution profiles and establishment of in vitro-in vivo correlation by in silico methods using GastroPlusTM and DDDPlusTM

The use of computer programs to predict drug absorption in humans and to simulate dissolution profiles has become a valuable tool in the pharmaceutical area. The objective of this study was to use in silico methods through software GastroPlusTM and DDDPlusTM to simulate drug absorption curves and dissolution profiles, and to establish in vitro-in vivo correlations (IVIVCs). The work presented herein is divided into five chapters and includes the drugs ketoprofen, pyrimethamine, metronidazole, fluconazole, carvedilol and doxazosin. In Chapter 1, simulated plasma curves for ketoprofen matrix tablets are presented and IVIVC was established. The use of simulated intrinsic dissolution tests for pyrimethamine and metronidazole as a tool for biopharmaceutics classification is detailed in Chapter 2. In Chapter 3, simulation of plasma curves for fluconazole capsules with different dissolution profiles is demonstrated as a tool for biowaiver. IVIVC studies were also conducted for carvedilol immediate-release tablets from dissolution profiles in Chapter 4. Chapter 5 covers the application of simulated dissolution tests for development of doxazosin extended-release formulations. Simulation of plasma curves and IVIVC using the software GastroPlusTM as well as intrinsic dissolution tests and dissolution profiles using the software DDDPlusTM proved to be a tool of wide application in predicting biopharmaceutical characteristics of drugs and formulations, allowing the reduction of time and costs of experimental laboratory work.

Análise da dinâmica e quantificação metabólica de imagens de medicina nuclear na modalidade PET/CT.

A presença da Medicina Nuclear como modalidade de obtenção de imagens médicas é um dos principais procedimentos utilizados hoje nos centros de saúde, tendo como grande vantagem a capacidade de analisar o comportamento metabólico do paciente, traduzindo-se em diagnósticos precoces. Entretanto, sabe-se que a quantificação em Medicina Nuclear é dificultada por diversos fatores, entre os quais estão a correção de atenuação, espalhamento, algoritmos de reconstrução e modelos assumidos. Neste contexto, o principal objetivo deste projeto foi melhorar a acurácia e a precisão na análise de imagens de PET/CT via processos realísticos e bem controlados. Para esse fim, foi proposta a elaboração de uma estrutura modular, a qual está composta por um conjunto de passos consecutivamente interligados começando com a simulação de phantoms antropomórficos 3D para posteriormente gerar as projeções realísticas PET/CT usando a plataforma GATE (com simulação de Monte Carlo), em seguida é aplicada uma etapa de reconstrução de imagens 3D, na sequência as imagens são filtradas (por meio do filtro de Anscombe/Wiener para a redução de ruído Poisson caraterístico deste tipo de imagens) e, segmentadas (baseados na teoria Fuzzy Connectedness). Uma vez definida a região de interesse (ROI) foram produzidas as Curvas de Atividade de Entrada e Resultante requeridas no processo de análise da dinâmica de compartimentos com o qual foi obtida a quantificação do metabolismo do órgão ou estrutura de estudo. Finalmente, de uma maneira semelhante imagens PET/CT reais fornecidas pelo Instituto do Coração (InCor) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) foram analisadas. Portanto, concluiu-se que a etapa de filtragem tridimensional usando o filtro Anscombe/Wiener foi relevante e de alto impacto no processo de quantificação metabólica e em outras etapas importantes do projeto em geral.

The Role of Passage Topic Knowledge in Typical and Poor Comprehenders' Recall

This dissertation examines the role of topic knowledge (TK) in comprehension among typical readers and those with Specifically Poor Comprehension (SPC), i.e., those who demonstrate deficits in understanding what they read despite adequate decoding. Previous studies of poor comprehension have focused on weaknesses in specific skills, such as word decoding and inferencing ability, but this dissertation examined a different factor: whether deficits in availability and use of TK underlie poor comprehension. It is well known that TK tends to facilitate comprehension among typical readers, but its interaction with working memory and word decoding is unclear, particularly among participants with deficits in these skills. Across several passages, we found that SPCs do in fact have less TK to assist their interpretation of a text. However, we found no evidence that deficits in working memory or word decoding ability make it difficult for children to benefit from their TK when they have it. Instead, children across the skill spectrum are able to draw upon TK to assist their interpretation of a passage. Because TK is difficult to assess and studies vary in methodology, another goal of this dissertation was to compare two methods for measuring it. Both approaches score responses to a concept question to assess TK, but in the first, a human rater assigns a score whereas in the second, a computer algorithm, Latent Semantic Analysis (LSA; Landauer & Dumais, 1997) assigns a score. We found similar results across both methods of assessing TK, suggesting that a continuous measure is not appreciably more sensitive to variations in knowledge than discrete human ratings. This study contributes to our understanding of how best to measure TK, the factors that moderate its relationship with recall, and its role in poor comprehension. The findings suggest that teaching practices that focus on expanding TK are likely to improve comprehension across readers with a variety of abilities.