Downstream regulatory antagonist modulator (DREAM) is a calcium sensing protein that co-assembles with KV4 potassium channels to regulate ion currents as well as with DNA in the nucleus, where it regulates gene expression. The interaction of DREAM with A-type KV4 channels and DNA has been shown to regulate neuronal signaling, pain sensing, and memory retention. The role of DREAM in modulation of pain, onset of Alzheimer’s disease, and cardiac pacemaking has set this protein as a novel therapeutic target. Moreover, previous results have shown a Ca2+ dependent interaction between DREAM and KV4/DNA involving surface contacts at the N-terminus of DREAM. However, the mechanisms by which Ca2+ binding at the C-terminus of DREAM induces structural changes at the C- and N-terminus remain unknown. Here, we present the use of biophysics and biochemistry techniques in order to map the interactions of DREAM and numerous small synthetic ligands as well as KV channels. We further demonstrate that a highly conserved network of aromatic residues spanning the C- and N-terminus domains control protein dynamics and the pathways of signal transduction on DREAM. Using molecular dynamics simulations, site directed mutagenesis, and fluorescence spectroscopy we provide strong evidence in support of a highly dynamic mechanism of signal transduction and regulation. A set of aromatic amino acids including Trp169, Phe171, Tyr174, Phe218, Phe235, Phe219, and Phe252 are identified to form a dynamic network involved in propagation of Ca2+ induced structural changes. These amino acids form a hydrophobic network connecting the N- and C-terminus domains of DREAM and are well conserved in other neuronal calcium sensors. In addition, we show evidence in support of a mechanism in which Ca2+ signals are propagated towards the N-terminus and ultimately lead to the rearrangement of the inactive EF-hand 1. The observed structural motions provide a novel mechanism involved in control of the calcium dependent KV4 and DNA binding. Altogether, we provide the first mechanism of intramolecular and intermolecular signal transduction in a Ca2+ binding protein of the neuronal calcium sensor family.

The structural characteristics associated with the binding of beneficial metals (i.e. - Mg2+, Zn2+ and Ca2+) to natural proteins has typically received more attention than competitive binding by toxic metals (e.g. – Pb2+, Hg2+, Cd2+, La3+, etc.). In this thesis, a statistical analysis of Pb2+-binding in crystallized protein structures indicates that Pb2+ does not bind preferentially with nitrogen, as generally assumed, but binds predominantly with oxygen, and to a lesser degree, sulfur. A comparison of Ca2+ and Pb2+ indicates that Pb2+ binds with a wider range of coordination numbers, with less formal change, and with less defined structure than Ca2+. The Pb2+ ion also appears to displace Ca2+ with little conformational stress in calcium binding proteins (CaBP’s). Experimental data from the binding of metals with engineered fluorescent proteins indicate that both Pb2+ and Gd3+ will occupy grafted calcium-binding sites with greater affinity than Ca2+, and strong evidence is presented to support the hypothesis that Pb2+ and Gd3+ will bind non-specifically on the protein surface. These results suggest that toxicity is associated with two binding mechanisms: displacement of the metal cofactor which disrupts protein function, and non-specific binding which maintains higher solubility of the metal.

Os recentes avanços nas tecnologias de comunicação e computação viabilizaram o pronto acesso às informações do prontuário eletrônico do paciente (PEP). O potencial de difusão de informações clínicas resultante suscita preocupações acerca da priva-cidade do paciente e da confidencialidade de seus dados. As normas presentes na legislação dispõem que o conteúdo do prontuário deve ser sigiloso, não cabendo o acesso a ele sem a prévia autorização do paciente, salvo quando necessário para be-neficiá-lo. Este trabalho propõe o MACA, um modelo de autorização contextual para o controle de acesso baseado em papéis (CABP) que contempla requisitos de limita-ção de acesso ao PEP em ambientes abertos e distribuídos. O CABP regula o acesso dos usuários ao PEP com base nas funções (papéis) que eles exercem numa organi-zação. Uma autorização contextual usa informações ambientais disponíveis durante o acesso para decidir se um usuário tem o direito e a necessidade de acessar um re-curso do PEP. Isso confere ao MACA flexibilidade e poder expressivo para estabele-cer políticas de acesso ao PEP e políticas administrativas para o CABP que se adap-tam à diversidade ambiental e cultural das organizações de saúde. O MACA ainda permite que os componentes do PEP utilizem o CABP de forma transparente para o usuário final, tornando-o mais fácil de usar quando comparado a outros modelos de CABP. A arquitetura onde a implementação do MACA foi integrada adota o serviço de diretórios LDAP (Lightweight Directory Access Protocol), a linguagem de pro-gramação Java e os padrões CORBA Security Service e Resource Access Decision Fa-cility. Com esses padrões abertos e distribuídos, os componentes heterogêneos do PEP podem solicitar serviços de autenticação de usuário e de autorização de acesso de modo unificado e coerente a partir de múltiplas plataformas. A implementação do MACA ainda tem a vantagem de ser um software livre, de basear-se em componen-tes de software sem custos de licenciamento e de apresentar bom desempenho para as demandas de acesso estimadas. Por fim, a utilização rotineira do MACA no con-trole de acesso ao PEP do InCor-HC.FMUSP, por cerca de 2000 usuários, evidenciam a exeqüibilidade do modelo, da sua implementação e da sua aplicação prática em casos reais. / The recent advances in computing and communication technologies allowed ready access to the electronic patient record (EPR) information. High availability of clinical information raises concerns about patients privacy and data confidentiality of their data. The legal regulation mandates the confidentiality of EPR contents. Everyone has to be authorized by the patients to access their EPR, except when this access is necessary to provide care on their behalf. This work proposes MACA, a contextual authorization model for the role-based access control (RBAC) that considers the ac-cess restrictions requirements for the EPR in open and distributed environments. RBAC regulates user’s access to EPR based on organizational functions (roles). Con-textual authorizations use environmental information available at access time, like user/patient relationship, in order to decide whether a user is allowed to access an EPR resource. This gives flexibility and expressive power to MACA, allowing one to establish access policies for the EPR and administrative policies for the RBAC that considers the environmental and cultural diversity of healthcare organizations. MACA also allows EPR components to use RBAC transparently, making it more user friendly when compared with other RBAC models. The implementation of MACA architecture uses the LDAP (Lightweight Directory Access Protocol) directory server, the Java programming language and the standards CORBA Security Service and Re-source Access Decision Facility. Thus, heterogeneous EPR components can request user authentication and access authorization services in a unified and coherent way across multiple platforms. MACA implementation complies with free software pol-icy. It is based on software components without licensing costs and it offers good performance for the estimated access demand. Finally, the daily use of MACA to control the access of about 2000 users to the EPR at InCor-HC.FMUSP shows the feasibility of the model, of its implementation and the effectiveness of its practical application on real cases.

Os recentes avanços nas tecnologias de comunicação e computação viabilizaram o pronto acesso às informações do prontuário eletrônico do paciente (PEP). O potencial de difusão de informações clínicas resultante suscita preocupações acerca da priva-cidade do paciente e da confidencialidade de seus dados. As normas presentes na legislação dispõem que o conteúdo do prontuário deve ser sigiloso, não cabendo o acesso a ele sem a prévia autorização do paciente, salvo quando necessário para be-neficiá-lo. Este trabalho propõe o MACA, um modelo de autorização contextual para o controle de acesso baseado em papéis (CABP) que contempla requisitos de limita-ção de acesso ao PEP em ambientes abertos e distribuídos. O CABP regula o acesso dos usuários ao PEP com base nas funções (papéis) que eles exercem numa organi-zação. Uma autorização contextual usa informações ambientais disponíveis durante o acesso para decidir se um usuário tem o direito e a necessidade de acessar um re-curso do PEP. Isso confere ao MACA flexibilidade e poder expressivo para estabele-cer políticas de acesso ao PEP e políticas administrativas para o CABP que se adap-tam à diversidade ambiental e cultural das organizações de saúde. O MACA ainda permite que os componentes do PEP utilizem o CABP de forma transparente para o usuário final, tornando-o mais fácil de usar quando comparado a outros modelos de CABP. A arquitetura onde a implementação do MACA foi integrada adota o serviço de diretórios LDAP (Lightweight Directory Access Protocol), a linguagem de pro-gramação Java e os padrões CORBA Security Service e Resource Access Decision Fa-cility. Com esses padrões abertos e distribuídos, os componentes heterogêneos do PEP podem solicitar serviços de autenticação de usuário e de autorização de acesso de modo unificado e coerente a partir de múltiplas plataformas. A implementação do MACA ainda tem a vantagem de ser um software livre, de basear-se em componen-tes de software sem custos de licenciamento e de apresentar bom desempenho para as demandas de acesso estimadas. Por fim, a utilização rotineira do MACA no con-trole de acesso ao PEP do InCor-HC.FMUSP, por cerca de 2000 usuários, evidenciam a exeqüibilidade do modelo, da sua implementação e da sua aplicação prática em casos reais. / The recent advances in computing and communication technologies allowed ready access to the electronic patient record (EPR) information. High availability of clinical information raises concerns about patients privacy and data confidentiality of their data. The legal regulation mandates the confidentiality of EPR contents. Everyone has to be authorized by the patients to access their EPR, except when this access is necessary to provide care on their behalf. This work proposes MACA, a contextual authorization model for the role-based access control (RBAC) that considers the ac-cess restrictions requirements for the EPR in open and distributed environments. RBAC regulates user’s access to EPR based on organizational functions (roles). Con-textual authorizations use environmental information available at access time, like user/patient relationship, in order to decide whether a user is allowed to access an EPR resource. This gives flexibility and expressive power to MACA, allowing one to establish access policies for the EPR and administrative policies for the RBAC that considers the environmental and cultural diversity of healthcare organizations. MACA also allows EPR components to use RBAC transparently, making it more user friendly when compared with other RBAC models. The implementation of MACA architecture uses the LDAP (Lightweight Directory Access Protocol) directory server, the Java programming language and the standards CORBA Security Service and Re-source Access Decision Facility. Thus, heterogeneous EPR components can request user authentication and access authorization services in a unified and coherent way across multiple platforms. MACA implementation complies with free software pol-icy. It is based on software components without licensing costs and it offers good performance for the estimated access demand. Finally, the daily use of MACA to control the access of about 2000 users to the EPR at InCor-HC.FMUSP shows the feasibility of the model, of its implementation and the effectiveness of its practical application on real cases.