Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative syndrome of unknown etiology that most commonly affects people in middle and high age. The hallmark of ALS is a progressive and simultaneous loss of upper and lower motor neurons in the central nervous system that leads to a progressive muscle atrophy, paralysis and death usually by respiratory failure. ALS is not a pure motor neuronal syndrome; it extends beyond the motor system and affects extramotor areas of the brain as well. The majority of the patients suffer from a sporadic ALS disease (SALS) while in at least ten percent the disease appears in a familial form (FALS). Mutations in the gene encoding the antioxidant enzyme superoxide dismutase-1 (SOD1) are the most common cause of FALS. More than 165 SOD1 mutations have been described, and these confer the enzyme a cytotoxic gain of function. Evidence suggests that the toxicity results from structural instability which makes the mutated enzyme prone to misfold and form aggregates in the spinal cord and brain motor neurons. Recent studies indicate that the wild-type human SOD1 protein (wt-hSOD1) has the propensity to develop neurotoxic features. The aim of the present study was to investigate if wt-hSOD1 is involved in the pathogenesis of SALS and FALS patients lacking SOD1 mutations and to evaluate the neurotoxic effect of misfolded wt-hSOD1 protein in vivo by generating a transgenic wt-hSOD1 mice model. We produced specific SOD1-peptide-generated antibodies that could discriminate between the misfolded and native form of the enzyme and optimized a staining protocol for detection of misfolded wt-hSOD1 by immunohistochemistry and confocal microscopy of brain and spinal cord tissue. We discovered that aggregates of misfolded wt-hSOD1 were constitutively present in the cytoplasm of motor neurons in all investigated SALS patients and in FALS patients lacking SOD1 gene mutations. Interestingly, the misfolded wt-hSOD1 aggregates were also found in some motor neuron nuclei and in the nuclei of the surrounding glial cells, mainly astrocytes but also microglia and oligodendrocytes, indicating that misfolded wt-hSOD1 protein aggregates may exert intranuclear toxicity. We compared our findings to FALS with SOD1 mutations by investigating brain and spinal cord tissue from patients homozygous for the D90A SOD1 mutation, a common SOD1 mutation that encodes a stable SOD1 protein with a wild-type-like enzyme activity. We observed a similar morphology with a profound loss of motor neurons and aggregates of misfolded SOD1 in the remaining motor neuron. Interestingly, we found gliosis and microvacuolar degeneration in the superficial lamina of the frontal and temporal lobe, indicating a possible frontotemporal lobar dementia in addition to the ALS disorder. Our morphological and biochemical findings were tested in vivo by generating homozygous transgenic mice that over expressed wt-hSOD1. These mice developed a fatal ALS-like disease, mimicking the one seen in mice expressing mutated hSOD1. The wt-hSOD1 mice showed a slower weight gain compared to non-transgenic mice and developed a progressive ALS-like hind-leg paresis. Aggregates of misfolded wt-hSOD1 were found in the brain and spinal cord neurons similar to those in humans accompanied by a loss of 41 % of motor neurons compared to non-transgenic litter mates. In conclusion, we found misfolded wt-hSOD1 aggregates in the cytoplasm and nuclei of motor neurons and glial cells in all patients suffering from ALS syndrome. Notable is the fact that misfolded wt-hSOD1 aggregates were also detected in FALS patients lacking SOD1 mutations indicating a role for SOD1 even when other genetic mutations are present. The neurotoxicity of misfolded wt-hSOD1 protein was confirmed in vivo by wt-hSOD1 transgenic mice that developed a fatal ALS-like disease. Taken together, our results support the notion that misfolded wt-hSOD1 could be generally involved and play a decisive role in the pathogenesis of all forms of ALS.

The development of peptides as therapeutic agents has been hampered by their poor enzymatic stability and bioavailability. Many strategies, such as chemical modification, synthesis of peptidomimetics and formulation, have been employed to overcome these issues. For central nervous system (CNS) active peptides, the blood brain barrier is an added hurdle. Nasal delivery is believed to provide a direct access to the brain via the olfactory nerve, which would bypass the blood brain barrier. This route of administration, however, is dependant on the size and physico-chemical properties of the administered drug. For these reasons, three CNS active peptides were chosen as models. Leu-enkephalin, endomorphin-1 and a-conotoxin MII are three peptides that differ in their size, amino acid sequence and conformation. Using chemical modifications to improve their stability and ability to cross biological membranes, in vitro assessments of derivatives of these peptides were performed and in vivo nasal delivery was attempted on the most promising candidates. The chemical modifications consisted in the addition of lipids and/or sugars to the N- or C-terminus of the peptides. Assessment of the in vivo bioavailability after nasal administration, however, proved to be challenging. The initial method chosen for this purpose was the use of tritiated acetic anhydride which would radiolabel the peptide via acetylation at the N-terminus of the peptide derivatives. Consequently, in vitro stability and permeability of each acetylated derivatives was also studied. Acetylation of the lipidic derivatives, which formed an amide bond, proved to be beneficial for the stability of the lipidic peptides. In contrast, acetylation of the Nterminus sugar derivatives, which formed an ester bond at one or several positions of the sugar, was an unstable modification. Thus, an extraction method for the tested peptides from rat tissues was developed, and LC-MS/MS analyses were conducted to measure the level of peptide in the olfactory bulbs, brain and blood. Leu-enkephalin derivatives were all amide derivatives at the C-terminus of the peptide. The most successful Leu-enkephalinamide derivatives were C8-LeuEnk (2), C12- LeuEnk (3) and Lac-LeuEnk (8), which are the Leu-enphelinamide peptide modified with a C8 lipoamino acid, a C12 lipoamino acid and a lactose moiety respectively. They all exhibited improved permeability across Caco-2 monolayers and stability in Caco-2 cell homogenate and/or plasma. Problems of solubility encountered with C12-LeuEnk (3), however, hampered its testing in vivo after nasal administration. C8-LeuEnk (2) and Lac-LeuEnk (8) were administered intranasally to male Sprague-Dawley rats. Both peptides were found in the olfactory bulbs after 10 minutes administration (2: 49.2 ± 15.6 nM; 8: 40.6 ± 14.6 nM) while blood concentration remained low, showing that the peptide reached the olfactory bulbs directly from the nasal cavity via the olfactory nerve. Brain concentrations were 13.5 ± 10.1 nM for C8-LeuEnk (2) and 13.6 ± 6.9 nM for Lac-LeuEnk (8). These two peptides brain concentrations seemed to be high enough to exhibit analgesic effect when compared to their binding affinity in vitro. This was not statistically significant, however, due to the high standard deviations observed (Kiμ C8-LeuEnk (2) = 7.74 ± 1.15 nM; Kiμ Lac-LeuEnk (8) = 6.69 ± 1.81 nM). Endomorphin-1 was only modified at the N-terminus as previous results have shown that the activity of the peptide is strongly decreased by C-terminus modifications. The most successful modification, regarding permeability across Caco-2 monolayers and water solubility, was shown to be the addition of a lactose moiety to the N-terminus of the peptide. Lac-Endo1 (16) exhibited a permeability of 1.91 ± 0.76 x 10-6 cm/s and was soluble at the concentration used for in vivo nasal administration (2 mg/Kg, 50 μL administration). After 10 minutes administration, Lac-Endo1 (16) was found in the olfactory bulbs (418 ± 410 nM), in the brain (4.01 ± 4.61 nM) and in the blood (1.58 ± 1.85 nM). The large standard deviations observed reflect the difficulties encountered with the extraction process of this peptide. A direct transport for the nasal cavity to the olfactory bulb was observed as illustrated by the low blood concentrations. Brain concentrations, however, were too low to expect a strong analgesic effect from this compound after nasal administration (Kiμ Lac-Endo1 (16) = 11.3 ± 1.2 nM). a-Conotoxin MII is a 16 amino acid long peptide containing two disulfide bonds. The formation of these two disulfide bonds leads to low yields in the synthesis of the derivatives of this peptide. Addition of a lipidic moiety to the peptide did not seem to improve its permeability through biological membranes. This modification resulted in highly lipophilic peptides with dissolution issues in water based media such as those used in the permeability experiments. The most successful a-conotoxin MII derivative was GS-Ctx (25) which exhibited a permeability of 4.22 ± 0.53 x 10-7 cm/s across Caco-2 monolayers. This permeability, however, was too low to consider in vivo administration. In conclusion, we successfully synthesised a series of derivatives of Leu-enkephalin, endomorphin-1 and a-conotoxin MII and screened them through Caco-2 monolayers for permeability and Caco-2 cell homogenates and human plasma for stability. Three derivatives (C8-LeuEnk (2), Lac-LeuEnk (8) and Lac-Endo1 (16)) were intranasally administered and found in the olfactory bulbs 10 minutes after administration. The low blood concentrations observed show that a direct transport from the nasal cavity to the brain occurs. Thus, nasal administration could be an option for delivering to the brain low molecular weight peptides exhibiting increased stability and permeability in vitro.

The development of peptides as therapeutic agents has been hampered by their poor enzymatic stability and bioavailability. Many strategies, such as chemical modification, synthesis of peptidomimetics and formulation, have been employed to overcome these issues. For central nervous system (CNS) active peptides, the blood brain barrier is an added hurdle. Nasal delivery is believed to provide a direct access to the brain via the olfactory nerve, which would bypass the blood brain barrier. This route of administration, however, is dependant on the size and physico-chemical properties of the administered drug. For these reasons, three CNS active peptides were chosen as models. Leu-enkephalin, endomorphin-1 and a-conotoxin MII are three peptides that differ in their size, amino acid sequence and conformation. Using chemical modifications to improve their stability and ability to cross biological membranes, in vitro assessments of derivatives of these peptides were performed and in vivo nasal delivery was attempted on the most promising candidates. The chemical modifications consisted in the addition of lipids and/or sugars to the N- or C-terminus of the peptides. Assessment of the in vivo bioavailability after nasal administration, however, proved to be challenging. The initial method chosen for this purpose was the use of tritiated acetic anhydride which would radiolabel the peptide via acetylation at the N-terminus of the peptide derivatives. Consequently, in vitro stability and permeability of each acetylated derivatives was also studied. Acetylation of the lipidic derivatives, which formed an amide bond, proved to be beneficial for the stability of the lipidic peptides. In contrast, acetylation of the Nterminus sugar derivatives, which formed an ester bond at one or several positions of the sugar, was an unstable modification. Thus, an extraction method for the tested peptides from rat tissues was developed, and LC-MS/MS analyses were conducted to measure the level of peptide in the olfactory bulbs, brain and blood. Leu-enkephalin derivatives were all amide derivatives at the C-terminus of the peptide. The most successful Leu-enkephalinamide derivatives were C8-LeuEnk (2), C12- LeuEnk (3) and Lac-LeuEnk (8), which are the Leu-enphelinamide peptide modified with a C8 lipoamino acid, a C12 lipoamino acid and a lactose moiety respectively. They all exhibited improved permeability across Caco-2 monolayers and stability in Caco-2 cell homogenate and/or plasma. Problems of solubility encountered with C12-LeuEnk (3), however, hampered its testing in vivo after nasal administration. C8-LeuEnk (2) and Lac-LeuEnk (8) were administered intranasally to male Sprague-Dawley rats. Both peptides were found in the olfactory bulbs after 10 minutes administration (2: 49.2 ± 15.6 nM; 8: 40.6 ± 14.6 nM) while blood concentration remained low, showing that the peptide reached the olfactory bulbs directly from the nasal cavity via the olfactory nerve. Brain concentrations were 13.5 ± 10.1 nM for C8-LeuEnk (2) and 13.6 ± 6.9 nM for Lac-LeuEnk (8). These two peptides brain concentrations seemed to be high enough to exhibit analgesic effect when compared to their binding affinity in vitro. This was not statistically significant, however, due to the high standard deviations observed (Kiμ C8-LeuEnk (2) = 7.74 ± 1.15 nM; Kiμ Lac-LeuEnk (8) = 6.69 ± 1.81 nM). Endomorphin-1 was only modified at the N-terminus as previous results have shown that the activity of the peptide is strongly decreased by C-terminus modifications. The most successful modification, regarding permeability across Caco-2 monolayers and water solubility, was shown to be the addition of a lactose moiety to the N-terminus of the peptide. Lac-Endo1 (16) exhibited a permeability of 1.91 ± 0.76 x 10-6 cm/s and was soluble at the concentration used for in vivo nasal administration (2 mg/Kg, 50 μL administration). After 10 minutes administration, Lac-Endo1 (16) was found in the olfactory bulbs (418 ± 410 nM), in the brain (4.01 ± 4.61 nM) and in the blood (1.58 ± 1.85 nM). The large standard deviations observed reflect the difficulties encountered with the extraction process of this peptide. A direct transport for the nasal cavity to the olfactory bulb was observed as illustrated by the low blood concentrations. Brain concentrations, however, were too low to expect a strong analgesic effect from this compound after nasal administration (Kiμ Lac-Endo1 (16) = 11.3 ± 1.2 nM). a-Conotoxin MII is a 16 amino acid long peptide containing two disulfide bonds. The formation of these two disulfide bonds leads to low yields in the synthesis of the derivatives of this peptide. Addition of a lipidic moiety to the peptide did not seem to improve its permeability through biological membranes. This modification resulted in highly lipophilic peptides with dissolution issues in water based media such as those used in the permeability experiments. The most successful a-conotoxin MII derivative was GS-Ctx (25) which exhibited a permeability of 4.22 ± 0.53 x 10-7 cm/s across Caco-2 monolayers. This permeability, however, was too low to consider in vivo administration. In conclusion, we successfully synthesised a series of derivatives of Leu-enkephalin, endomorphin-1 and a-conotoxin MII and screened them through Caco-2 monolayers for permeability and Caco-2 cell homogenates and human plasma for stability. Three derivatives (C8-LeuEnk (2), Lac-LeuEnk (8) and Lac-Endo1 (16)) were intranasally administered and found in the olfactory bulbs 10 minutes after administration. The low blood concentrations observed show that a direct transport from the nasal cavity to the brain occurs. Thus, nasal administration could be an option for delivering to the brain low molecular weight peptides exhibiting increased stability and permeability in vitro.

Genetically based Central Nervous System (CNS) disorders remain a largely unresolved issue in the world today. Our genome is the source of our greatest strengths and weaknesses. For this reason, intelligent modification of the genome's DNA is a profoundly beneficial goal in the maximization of the overall health of the human race. Potential benefit in this field is currently limited in both effectiveness and safety in regards to the delivery of therapeutic genes into the nucleus, which is protected by many evolution-based barriers. Evolution has also favored the development of highly specialized and infectiously effective viruses capable of overcoming such boundaries. By neutering the naturally occurring and pathologically benign Adeno-Associated Virus (AAV) we have transformed what was once a virus, into a "pure" vector, taking full advantage of evolution's diligent enhancement of these genetic hijackers without introducing unacceptable danger to patients. We utilized the logically engineered, castrated form of AAV serotype 9 (recombinant AAV9/rAAV9) to act as a vehicle for two reporter genes, Enhanced Green Fluorescent Protein (EGFP) and Firefly Luciferase (Fluc) with the goal of assessing and improving the efficiency of vector transduction in murine CNS. We found that rAAV9, when infused into the intrathecal space of mice is capable of extensive and intensive transduction of both neurons and astrocytes throughout the entire length of the SC as well as the hind regions of the brain (brainstem and cerebellum). We also found that efficiency of transduction was best in our highest dose groups, 1E+12 genome copies (GC) in Experiment 1 and 2E+12 GC in Experiment 2, both of which received rAAV9 particles via the two commercially available (100μL and the 200μL) ALZET® Osmotic Pump designs. Administering dosage higher than this directly into the intrathecal space was limited by the size of the pump reservoir and rAAV9 production titer. We are currently attempting to achieve a more complete CNS transduction by performing another experiment in which we place the pump cannula directly into the intracerebroventricular (ICV) space of the lateral ventricles. Our findings reveal that infusion of rAAV9 by intrathecal placed pump cannula is more effective than any other method tested in this study int the transduction of neurons and glial cells of adult&ndashmouse CNS. By elucidating a mode of delivery that maximizes the robustness of transduction efficiency, our results are a critical building block in designing a cure for the array of genetic-based diseases of the CNS, which are currently untreatable

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Saúde Coletiva, 2014. / Submitted by Ana Cristina Barbosa da Silva (annabds@hotmail.com) on 2015-02-20T15:58:21Z No. of bitstreams: 1 2014_CarolinaDantasRochaXavierdeLucena_Parcial.pdf: 239046 bytes, checksum: 24343f3229bd87027b7fa4c2be8aad64 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2016-03-22T18:48:33Z (GMT) No. of bitstreams: 1 2014_CarolinaDantasRochaXavierdeLucena_Parcial.pdf: 239046 bytes, checksum: 24343f3229bd87027b7fa4c2be8aad64 (MD5) / Made available in DSpace on 2016-03-22T18:48:33Z (GMT). No. of bitstreams: 1 2014_CarolinaDantasRochaXavierdeLucena_Parcial.pdf: 239046 bytes, checksum: 24343f3229bd87027b7fa4c2be8aad64 (MD5) / Em 2011, o Ministério da Saúde publicou uma portaria tornando o preenchimento do Cartão Nacional de Saúde (CNS) obrigatório em 2012. O objetivo deste trabalho é descrever o perfil epidemiológico das Autorizações de Internações Hospitalares (AIH) eletivas de 2012 e analisar descritivamente se a obrigatoriedade do CNS permite uma qualidade da informação do Sistema de Informação Hospitalar (SIH). Trata-se de um estudo de natureza descritiva de delineamento transversal, desenvolvido com incorporação de método quantitativo de pesquisa, em que foram analisadas as AIH eletivas aprovadas de 2012. Para análise dos dados, utilizou-se o software Statistical Package for the Social Sciences (SPSS), através do qual se realizaram análises descritivas de distribuições de frequências e desenvolveu-se um software através do Java para que fosse possível a análise através do CNS de duplicidades de AIH para uma mesma internação. De acordo com este estudo, 54% das AIH eletivas no Brasil em 2012 foram causadas por Doenças do aparelho digestivo, geniturinário, circulatório e neoplasias. Observou-se que mais de 50 mil AIH iniciaram no ano de 2008, resultando em um tempo de permanência de 1.460 a 1.827 dias. Mais de 180 mil foram informadas com motivo de apresentação PERMANÊNCIA, portanto, nestes casos foram geradas mais de uma AIH para uma mesma internação, ocorrendo superestimação do número de internações eletivas no país já que se utiliza o dado de número de internação como sendo o nº de AIH. Conclui-se com este estudo a necessidade de implementação de regras no SIH que permita o registro fidedigno e real de uma internação, qualificando a informação, sendo imprescindível para a epidemiologia, planejamento e gestão. Faz-se necessário ainda, tornar o CNS uma fonte mais segura e fidedigna já que foram encontradas situações de duplicação e erros. Contudo, a implantação da obrigatoriedade do CNS nas AIH no ano de 2012 foi positiva para a qualificação da informação já que permitiu análises que podem ser utilizadas para a tomada de decisão dos gestores dos sistemas estudados. / In 2011 the Brazilian Ministry of Health published a directive that included a filling of the National Health Card (CNS) in 2012. The goal of this study is to describe the epidemiological profile of elective Authorize Hospital Admittance (AIH) and descriptively analyze the requirement of CNS enables quality of information from the Hospital Information System (SIH). This is a descriptive cross-sectional study, developed with the incorporation of the quantitative research method, where we analyzed the AIH approved in 2012. The analysis was performed by SPSS software, through which it conducted descriptive analyzes of frequency distributions and developed a software using Java to make possible the analysis of CNS through the duplicates of AIH for the same hospitalization. According to this study, 54% of elective AIH in Brazil in 2012 were caused by the digestive, genitourinary, circulatory diseases and neoplasms appliance. It was observed that more than 50 000 AIH began in 2008, resulting in a residence time 1460-1827 days. Over 180000 were informed on the occasion of presentation STAY therefore these cases were generated over an AIH for the same hospitalization, occurring overestimation of the number of elective admissions in the country since it uses the given number of hospitalization as the number AIH. The conclusion to this study the need to implement rules in SIH enabling the trusted and true record of a hospital, qualifying information, and essential for epidemiology, planning and management. It is still necessary to make the CNS safer and reliable source since duplication and errors were found. However, the implementation of mandatory CNS in AIH in 2012 was positive for the classification of the information as allowed analyzes that can be used for decision making of managers of the systems studied.

Os receptores para mineralocorticoides (MR) e glicocorticoides (GR) representam importantes sítios de ligação para os glicocorticoides. Enquanto a ativação crônica e excessiva de GR leva à atrofia do hipocampo, a ativação do MR é neuroprotetora. Considerando as alterações no giro denteado (GD), decorrentes do envelhecimento, e a possível participação do MR e GR neste processo, o objetivo deste trabalho foi correlacionar o desbalanço de tais receptores com a degeneração neuronal nesta região do hipocampo de cães idosos. Para isso, utilizaram-se cortes histológicos do hipocampo de 6 cadáveres caninos jovens/adultos (até 8 anos de idade) e 12 idosos (idade igual ou superior a 10 anos), de ambos os sexos e de qualquer raça, os quais foram submetidos à: coloração de Nissl, para a identificação de células nervosas; imuno-histoquímica, para o estudo da expressão do MR e GR; e marcação fluorescente (fluoro-jade B), para detecção de neurônios em degeneração. A camada polimórfica do GD de cães idosos apresentou redução (em 16%) na contagem de neurônios e maior número de neurônios em degeneração. Em conjunto com estas alterações celulares, a menor marcação/expressão do MR e a menor relação MR:GR foram correlacionadas com a degeneração neuronal na camada polimórfica do GD de cães idosos. Além disso, cadelas idosas apresentaram menor densidade celular na camada granular, quando comparadas aos machos idosos, sugerindo uma ação diferencial dos esteroides sexuais nas alterações do GD. Os resultados indicam que o desbalanço na relação MR:GR pode interferir na sobrevivência neuronal no GD de cães idosos. / The mineralocorticoid (MR) and glucocorticoid (GR) receptors bind the glucocorticoid hormones. The chronic and excessive GR activation leads to hippocampus atrophy. By contrast, MR activation is neuroprotective. Considering the aging changes in the dentate gyrus (DG) and the possible participation of MR and GR in this process, the objective of this study was to correlate the unbalance of these receptors with the neuronal degeneration in this hippocampal region of aged dogs. For that purpose, cadaveric histologic sections of hippocampus of 6 young/adult dogs (until 8 years old) and 12 aged dogs (more than 10 years old), of both sex and any breed, were included. The Nissl staining and imunochemistry were performed to identify nerve cells and to study the MR and GR expression, respectively. Moreover, fluorescent labeling (fluoro- Jade B) was used to detect degenerating neurons. The polimorfic layer of the DG of aged dogs showed reduction (of 16%) on the neurons counting and more degenerating neurons. Together with this cells changes, the less MR expression and MR:GR relation were correlated with the neuronal degeneration in the polimorfic layer of DG of aged dogs. Besides that, aged females presented lower cell density in the granular layer, when compared to aged males, suggesting a differential sex steroid action on changes in the GD. Our results indicate that unbalance on the MR:GR relation may interfere with neuronal survival in the DG of aged dogs.

Submitted by Rosemary Tourinho Pereira (rosemary.pereira@uniceub.br) on 2013-08-01T14:31:40Z No. of bitstreams: 1 Tese_MariliaQueirozDiasJacome_2013.pdf: 2173866 bytes, checksum: 32b85f22d0df7e610695e6c4f0f9ec51 (MD5) / Made available in DSpace on 2013-08-01T14:31:40Z (GMT). No. of bitstreams: 1 Tese_MariliaQueirozDiasJacome_2013.pdf: 2173866 bytes, checksum: 32b85f22d0df7e610695e6c4f0f9ec51 (MD5) Previous issue date: 2013-05 / Made available in DSpace on 2013-10-10T13:09:10Z (GMT). No. of bitstreams: 3 Tese_MariliaQueirozDiasJacome_2013.pdf.txt: 421905 bytes, checksum: 10fed67cea141b2d87e7fdb61dbdd5c3 (MD5) license.txt: 362 bytes, checksum: b9e0d9c10d77debde1a5ea963f0e67b4 (MD5) Tese_MariliaQueirozDiasJacome_2013.pdf: 2173866 bytes, checksum: 32b85f22d0df7e610695e6c4f0f9ec51 (MD5) Previous issue date: 2013-05 / Saber como membros de Comitês de Ética em Pesquisas (CEPs) articulam regulamentação e avaliação pode contribuir para problematizar o processo de revisão ética, vislumbrando diminuir diferenças no trabalho dos comitês. Este estudo tem o objetivo de analisar a organização e funcionamento de CEPs por coordenadores e membros e suas percepções acerca do processo de avaliação ética. Os dados foram coletados com a aplicação de questionários enviados para 645 comitês de ética. Os participantes da pesquisa são coordenadores e demais membros. Os questionários para coordenadores visavam caracterizar o perfil dos comitês e sua operacionalização, enquanto para os membros investigou-se como concebem a avaliação ética. As respostas foram categorizadas por frequência e média das respostas e passaram por teste estatístico. Responderam os questionários aplicados on-line 129 coordenadores e 295 membros. O estudo possibilitou caracterizar os comitês como localizados em instituições de ensino superior, com nove anos ou mais de funcionamento, maioria de membros no primeiro mandato e formação predominante na área de ciências biológicas e da saúde, com mestrado e doutorado, sem especialização nas áreas de bioética e ética em pesquisa. A capacitação dos membros se dá, principalmente, pela leitura das regulamentações e por orientações do coordenador e do secretário do CEP. / JÁCOME, Marília de Queiroz Dias. Análise dos comitês de ética em pesquisa no Brasil: percepção de seus coordenadores e membros. Tese (Doutorado em Bioética). Programa de Pós-Graduação em Bioética, Faculdade de Ciências da Saúde. Bioética: Universidade de Brasília, 2013. Orientação: Tereza Cristina Cavalcanti Ferreira de Araujo.

Agressões nos períodos críticos do crescimento do sistema nervoso podem modificar os eventos de desenvolvimento. Entre os vários fatores nocivos está a anóxia. O organismo do neonato tem suprimento de energia anaeróbica relativamente rica, foi observado que a acidose ocorre com menor facilidade, propiciam sobrevivência. A proteína de astrócitos, S100<font face=\"Symbol\">b, exerce efeitos parácrinos e autócrinos em neurônios e glia. Sua estimulação promove sobrevivência e proteção neuronal, atuando como fator trófico e neurotrófico. Modelo animal de anóxia neonatal desenvolvido em nosso laboratório, nos revelou ativação neural pela expressão de Fos e alterações comportamentais, o que nos instigou a explorar os efeitos da anóxia nas células da glia no Hipocampo e Núcleo do Trato Solitário. Para sua exposição à anoxia, durante 25 minutos, foi utilizada câmara, saturada com nitrogênio gasoso 100%. Grupos P2 e P7 nas condições: Basal (B), sem estimulo; Sham (S) como controle experimental e Anóxia (A) com falta de oxigênio, foram analisados por S100<font face=\"Symbol\">b-IR com técnicas ABC/DAB e Western blot. Observamos significante diferença de S100<font face=\"Symbol\">b-IR no núcleo do trato solitário, somente no grupo P2 A 2 h em relação ao grupo P2 S 2 h. A reatividade glial de S100<font face=\"Symbol\">b na formação hipocampal (CA1, CA3+CA2 e DG), apresentou diferença significante no grupo anoxia de acordo com o estágio de maturação do animal. A técnica por Western blot em toda a formação hipocampal, apresentou aumento de S100<font face=\"Symbol\">b no grupo A em ambos P2 e P7, a avaliação de um todo foi diferente daquela de áreas especificas. / Attacks to the nervous system at critical growth periods can modify developmental events. Among the various harmful factors at is anoxia. The high anaerobic energy supply to the newborn and a less easily acidosis occurrence provides survival. The astrocyte S100<font face=\"Symbol\">b protein exerts paracrine and autocrine effects on neurons and glia. Its stimulation promotes neuronal survival and protection, as a trophic and neurotrophic factor. An animal model of neonatal anoxia improved in our lab revealed neural activation by Fos expression and behavioral changes, which prompted us to explore the anoxia effects on glial cells in the Hypocampus and Nucleus of Solitary Tract. For their exposure to anoxia, a chamber, saturated with 100% nitrogen gas, for 25 minutes were used. Groups with P2 and P7, conditions: Baseline, without stimulation; Sham as the experimental control, and Anoxia with lack of oxygen, were evaluated by S100<font face=\"Symbol\">b-IR by ABC/DAB and Western blot techniques. The nucleus of solitary tract, significant different S100<font face=\"Symbol\">b-IR observed, only in the P2 A 2 h compared to P2 S 2 h. The glial S100<font face=\"Symbol\">b-IR at the hippocampal formation (CA1, CA2 + CA3 and DG) presented significant difference in the anoxic group according to the maturational stage of the animal. Western blot technique of the entire hippocampal formation, showed increase of S100<font face=\"Symbol\">b at the group A at both P2 and P7, the whole evaluation was different from of that of specific areas.

Une étape critique au cours du développement de médicaments est la mesure ou la prédiction des concentrations du médicament dans un tissu cible, qui peuvent ensuite être liées à des mesures de leur efficacité ou leur toxicité. Les concentrations de médicaments ne pouvant être mesurées dans le cerveau humain, ils doivent être simulés ou prédits en utilisant des approches alternatives de modélisation. L'objectif de cette thèse est de développer in silico des approches de prédiction combinant à la fois des données précliniques in vitro et in vivo dans un modèle physiologique structuré, avec une stratégie translationnelle afin de permettre la simulation de concentrations totales et libres des médicaments dans le cerveau humain. Des modèles pharmacocinétiques physiologiques (PBPK) ont été développés dans cette thèse et évalués pour des médicaments de référence déjà sur le marché, et pour un médicament en cours de développement clinique dans l'industrie pharmaceutique. Ces modèles ont été développés à partir de stratégies de type « Bottom-up » sur la base de données in vitro pour la prédiction de la distribution des médicaments dans le cerveau et comparées à des méthodes de type «top-down » en utilisant des données in vivo. Cette thèse est une thèse sur article construite à partir de 5 articles scientifiques qui sont soit publiés soit en cours de soumission. Le premier article est une revue de la littérature publiée dans le Journal de l'AAPS. Cette revue discute les modèles PBPK disponibles actuellement et a permis d’élaborer des hypothèses de travail dans cette thèse afin de proposer des améliorations de ces modèles. Le deuxième article un article de recherche original publié dans Molecular Pharmaceutics. Cet article vise à démontrer l'élaboration d'une approche cohérente de modélisation pharmacocinétique chez le rat qui peut s’adapter en fonction de la quantité et de la qualité des données obtenues in vivo au cours du développement des médicaments. Un arbre de décision a été construit pour faciliter le paramétrage et la structure appropriée du modèle en fonction des données disponibles. Le troisième article est un article de recherche original publié dans Journal of Pharmaceutical Sciences. Cette étude porte sur le développement d'un modèle PBPK pour la prédiction de la pénétration cérébrale des médicaments, dans lequel son transfert à travers la BHE a été traité de façon mécanistique en séparant les paramètres régissant la quantité (perméabilité) à travers la BHE de sa liaison dans le tissu cérébral. Une stratégie de type vitro - vivo en fonction de la perméabilité des médicaments à travers les monocouches cellulaires in vitro a été proposé afin d'extrapoler la composante de transport actif du composé à l’aide de facteurs d’extrapolation (RAF). Deux autres articles sont en cours d’écriture ou soumis. Ces articles viennent compléter les approches de PBPK pour les médicaments du SNC décrites dans les deux autres articles originaux. Une dernière partie de la thèse constitue la discussion qui met très clairement en évidence l'importance du choix d’une approche de modélisation appropriée ou mieux encore la combinaison des approches fondées sur les connaissances physiologiques, les données expérimentales et les applications prévues dans le développement du médicament. L'avantage du paramétrage mécanistique dans ces modèles PBPK est qu’il améliore leur prédictivité et la simulation de différences inter-espèces. Cette thèse a considérablement contribué à démontrer la nécessité d’associer des données in vitro à des données in vivo dans la structuration des modèles PBPK qui se révèlent alors comme des outils précieux pour la prédiction de la pharmacocinétique cérébrale chez l'homme. / A critical step during drug development is the measurement or the prediction of drug concentrations in the target tissue, which can then be linked to measures of drug efficacy or toxicity. Drug concentrations cannot be directly measured in the human brain, and must be simulated or predicted using alternative modeling approaches. The objective of this thesis is to develop in silico approaches to predict BBB penetration of drugs, combining in vitro and in vivo preclinical data in a physiologically structured model, with a translational strategy to allow the simulation of total and free drug concentrations in the human brain. Physiologically based pharmacokinetic (PBPK) models were developed and evaluated for reference molecules already on the market, as well as for a drug currently under clinical development within the pharmaceutical industry. These models were developed based on both ‘bottom-up’ (model parameter values predicted from in vitro data) and ‘top-down’ (model parameters estimated from in vivo data) strategies. This thesis is comprised of 5 scientific papers which are either published or submitted to peer-reviewed journals. The first article is a review of the literature, published in the AAPS journal. This review discusses the currently published PBPK models available for the mechanistic prediction of BBB penetration of drugs, and proposes a strategy for in vitro-in vivo (IVIVE) extrapolation. The second article is an original research article published in Molecular Pharmaceutics. This article aims to show the development of a coherent pharmacokinetic modeling approach in the rat which can be adapted based on the quantity and quality of data obtained in vivo during the development of new drugs. A decision tree was constructed to enable the appropriate parameterization and model structure based on the available data. The third article is an original research article published in Journal of Pharmaceutical Sciences. This article was based on the development of a PBPK model for the mechanistic prediction of BBB penetration of drugs, in which the active and passive components of permeability were considered separately, as well as the intra-brain tissue binding parameters. An in vitro-in vivo strategy was proposed to extrapolate the active transport component using a relative activity factor (RAF) to account for in vitro-in vivo differences in transporter activity and/or abundance. Two additional articles are either submitted or under preparation. These articles extend the PBPK approaches described in the previous two published original research articles. The final part of this thesis consists of a discussion which emphasizes clearly the importance of the appropriate choice of modeling approach, or even better, a combination of approaches based on physiological knowledge, experimental data and knowledge gathered during the course of drug development. The advantage of mechanistic parameterization of PBPK models is the improved ability for inter-species extrapolation for the subsequent simulation of free or total drug concentrations within the human brain. This thesis has considerably contributed to this rapidly evolving field of CNS drug research and development, showing the importance of combining in vitro and in vivo data within a physiologically based model structure, thus providing a valuable tool for the quantitative prediction of the penetration of drugs in the human brain.

The purpose of the present study was to evaluate the reliability of parallel-forms of the Comprehensive Neuropsychological Screening Device (CNS). Forty-five subjects ranging in age from 16 to 69 were administered Form A and Form B of the CNS at two week intervals. Results indicated that the CNS has adequate test-retest reliability. The results suggest the applicability of using the CNS as a screening device for brain dysfunction.