Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

O presente trabalho tem como objetivo avaliar a influência das variáveis ambientais na mortalidade de idosos (acima de 65 anos) por doenças cardiovasculares (DCV) e respiratórias (RES), nos condados de Los Angeles, Orange e Santa Bárbara na Califórnia, através de análises estatísticas, em particular de componentes principais (ACP). Para isso foram utilizados dados diários das estações meteorológicas dos condados (temperatura, pressão, umidade e velocidade do vento), de poluição do ar (NO2, CO, SO2 e O3) e da mortalidade por DCV e RES, durante o período 1975-2005. Além disso, foram calculados 7 índices de conforto térmico e estabelecidas as zonas de conforto para diferentes graus de percepção térmica a partir do índice de Temperatura Efetiva (TE). Os resultados verificaram tendências diferentes na variação temporal das DCV e RES. Enquanto as doenças RES tiveram um aumento bastante leve em todos os condados, as DCV apresentaram uma ligeira diminuição nos condados de Orange e Santa Bárbara, e uma diminuição constante e acentuada no condado de Los Angeles. Também se verificou uma variação sazonal significativa, com evidente aumento do número de óbitos de ambas as doenças durante o inverno e queda durante o verão. Em relação aos poluentes, foram observadas muitas ultrapassagens dos padrões nacionais de qualidade do ar principalmente para o O3, com 1443 vezes para o condado de Los Angeles, 771 para o Orange e 114 para Santa Bárbara. A partir dos índices de conforto térmico notou-se que o mês mais estressante devido ao calor foi setembro principalmente para os condados de Los Angeles e Santa Bárbara, e julho e agosto para o condado de Orange. Analisando a média de óbitos por sensação térmica observou-se um aumento das doenças nos extremos, principalmente quando a sensação é Muito Frio, com médias por doenças RES de 16,42, 3,31 e 0,81 e por DCV de 77,32, 15,37 e 2,92 nos condados de Los Angeles, Orange e Santa Bárbara, respectivamente. Com base na análise de correlação entre as variáveis e as doenças, observaram-se as melhores correlações no condado de Los Angeles, tanto para as doenças RES quanto DCV, e as correlações mais baixas foram observadas no condado de Santa Bárbara. Os resultados da ACP nos condados de Los Angeles e Orange verificaram o aumento da mortalidade por DCV no inverno, associadas a correlações positivas com os níveis de poluição principalmente em Los Angeles e ao estresse por frio, umidade e vento (via índice de conforto térmico) em Orange. Por outro lado, a mortalidade por doenças RES esteve mais associada às variações meteorológicas do que às concentrações de poluentes, observando-se associações positivas com o índice de calor (IC). Neste sentido, o condado de Orange apresentou maiores correlações com IC do que o condado de Los Angeles, além de uma variância maior (11,36 % contra 9,89 %). Por último, no condado de Santa Bárbara os resultados foram muito diferentes, já que tanto episódios de frio quanto de calor não mostraram impactos claros na população idosa, com respeito a ambos os grupos de doenças. O distintivo foi que os níveis de SO2 tiveram a maior associação positiva à mortalidade principalmente por DCV (0,53), embora tiver o condado as concentrações mais baixas das estudadas (média de 0,29 ppb). Em suma, os condados possuem estruturas sociais diferentes, apesar da semelhança do clima, fazendo com que os resultados sejam bastante dispares entre os mesmos. As perspectivas destes estudos pretendem colocar estes resultados para climas futuros, pois diversos episódios de ondas de calor ocorreram após o ano de 2005. / The objective of this study was to evaluate the influence of environmental variables on the mortality of elderly patients (over 65 years) due to cardiovascular diseases (CD) and respiratory diseases (RD) in the counties of Los Angeles, Orange and Santa Bárbara, California; through statistical analyzes, in particular, principal components (PCA). For this purpose, daily data of the meteorological stations of the counties (temperature, pressure, humidity and wind speed), air pollution (NO2, CO, SO2 and O3) and mortality by CD and RD were used during the period 1975-2005. In addition, seven thermal comfort indexes were calculated and comfort zones were established for different degrees of thermal perception according to the Effective Temperature index (TE). The results showed different trends in the temporal variation of CD and RD. While RD diseases had a fairly mild increase in all counties, CD showed a slight decrease in Orange and Santa Barbara counties and a steady decline in Los Angeles county. There was also a significant seasonal variation, with an evident increase in the number of deaths from both diseases during the winter and fall during the summer. In relation to the pollutants, many national standards for air quality were exceeded, mainly for the O3, with 1443 times for Los Angeles, 771 for Orange and 114 for Santa Barbara. From the thermal comfort indexes it was noted that the most stressful month due to the heat was September, mainly for the counties of Los Angeles and Santa Barbara, and July and August for the county of Orange. Analyzing the average number of deaths due to thermal sensation, there was an increase of the illnesses in extreme, especially when the sensation is Very Cold, with RD averages of 16.42, 3.31 and 0.81 and CD average of 77.32, 15.37 and 2.92 in the counties of Los Angeles, Orange and Santa Barbara, respectively. Based on correlation analysis between variables and diseases, the best correlations were found in Los Angeles for both RD and CD, and the lowest correlations were observed in Santa Barbara. The results of PAC in the counties of Los Angeles and Orange showed the increase in CD mortality in winter associated to positive correlations with the levels of pollution mainly in Los Angeles and stress due to cold, humidity and wind (via thermal comfort index) in Orange. On the other hand, mortality due to RD diseases was more associated with meteorological variations than with pollutant concentrations, with positive associations with heat index (HI). In this sense, Orange County presented higher correlations with HI than the county of Los Angeles, in addition to a greater variance (11.36% vs. 9.89%). Finally, in Santa Barbara the results were very different, since both episodes of cold and heat did not show clear impacts in the elderly population, regarding both groups of diseases. The difference was that SO2 levels had the highest positive association with CD mortality (0.53), although this county had the lowest concentrations studied (mean 0.29 ppb). In sum, counties have different social structures, despite the similarity of the climate, making the results quite different between them. The perspectives of these studies intend to place these results for future climates, since several episodes of heat waves occurred after 2005.

Na busca por agonistas, antagonistas e inibidores de natureza peptídica do fator de crescimento de fibroblastos ácido humano (hFGF-1), iniciamos o presente trabalho fazendo uma análise conformacional teórica do peptídeo Ac- WFVGLKKNGSSKRGPRT-NH2 (107-123 [hFGF-1]). Em trabalho anterior, este composto havia se mostrado um agonista da atividade mitogênica da proteína capaz de inibir a ligação de 125I-hFGF-1 aos seus receptores celulares e de se ligar à resina heparina-Sepharose (Oyama et al. 1996). Os cálculos das propriedades dinâmicas deste peptídeo (I; Tabela 1) demonstraram que ele não adotava nenhuma conformação preferencial, o que poderia justificar a baixa atividade apresentada pelo mesmo (104 vêzes menor do que a da proteína nativa). Este peptídeo foi ressintetizado, purificado, caracterizado química e biologicamente, confirmando os resultados anteriores. Seu comportamento randômico foi comprovado experimentalmente através de uma análise estrutural parcial por 1H-RMN. O resultado desta análise demonstrou que este peptídeo exibe uma configuração random coil, em solução aquosa (Kiyota et al., 1996, 1999). Diante desta constatação e com base em dados descritos na literatura (Harper & Lobb 1988; Burgess et al., 1991; Pantoliano et al., 1994, Springer et al., 1994; Thompson et al., 1994; Imamura et al., 1995; Blaber et al., 1996; Ornitz et al., 1996; Zhu et al.,1991, 1995, 1997; Schwizer, 1995; Sieber & Moe, 1996; Rizo et al., 1996); desenhamos dezessete peptídeos relacionados ao Sítio 2 (97-132) do hFGF-1 mostrados na Tabela 1 (ver arquivo PDF). Todos os peptídeos foram sintetizados manualmente por síntese em fase sólida, sempre que possível purificados à homogeneidade por RP-HPLC e caracterizados quimicamente por RP-HPLC, análise de aminoácidos e espectrometria de massas. Os peptídeos cíclicos foram obtidos por: a) oxidação das sulfidrilas das cisteínas e formação de ponte dissulfeto intramolecular; b) reação entre os grupos amina e carboxila de cadeias laterais de dois diferentes resíduos de aminoácidos com formação de uma ligação lactama. Os testes de atividade mitogênica foram realizados sempre que os peptídeos eram obtidos com pureza ≥90% determinada por RP-HPLC analítica em dois sistemas diferentes de solventes. Os resultados obtidos em culturas de fibroblastos de camundongos Balb/c 3T3 mostraram que: 1) II, III, VI-IX e XIII eram inativos; 2) o cíclico IV era mitogênico (ED50 ~50 µM) ao contrário do seu análogo linear correspondente (III); 3) V, um análogo de IV que apresenta deleção de um resíduo (Asn), exibia uma atividade mitogênica menor do que a de IV; 4) X exibia uma atividade mitogênica menor do que a do I; 5) os cíclicos XII e XV exibiam atividades comparáveis a do I, enquanto que os seus análogos lineares correspondentes (XI e XIV) eram inativos; 6) XVI e XVII exibiam atividades mitogênicas também comparáveis à de I. Paralelamente a estes estudos, foi desenvolvido um modelo teórico dos domínios extracelulares DII e DIII do FGFR-1β. A partir do posicionamento gráfico das moléculas de hFGF-1 e de um hexassacarídeo de heparina junto a este modelo do receptor, foram desenhados os peptídeos Ac-170NTTDKENEVLH180-NH2 (XVIII) e Ac-194SLAGNSIGLSH204-NH2 (XIX) (Oyama et al., 1997). Estes dois peptídeos cujas seqüências primárias são, respectivamente, relacionadas àquelas dos loops DE e FG do domínio DIII, foram também sintetizados e testados neste trabalho como possíveis ligantes do sítio 2 do hFGF-1.Os testes biológicos demonstraram que o peptídeo XIX, na faixa de concentração testada, não exibia nenhuma atividade inibitória sobre as atividades mitogênicas dos FGFs -1 e -2. Por outro lado, notou-se um claro efeito inibitório de XVIII sobre a atividade mitogênica de ambas as proteínas, sendo este efeito mais significativo para o FGF-2 (Kiyota et al., 1998). Alguns dos peptídeos estudados foram submetidos a análises espectroscópicas com o objetivo de determinar suas conformações em solução. Este conhecimento forneceria subsídios para o desenho de novos peptídeos mitogênicos e, mais ainda, para determinação dos requisitos estruturais destes peptídeos e, como reflexo, do hFGF1 para expressão de suas atividades mitogênicas. Assim, foi feita uma análise parcial do peptídeo mitogênico IV e de seu análogo linear III inativo em solução aquosa empregando fluorescência e 1HRMN ( (Kiyota et al., 1998). Estes peptídeos foram analisados também por técnicas de CD e 1H-NMR (Kiyota et al., 1999). Os resultados obtidos mostraram que III e IV parece se organizarem de forma semelhante em suas porções N-terminais, em estruturas correspondentes a β-turns. Por outro lado, as conformações das porções C-terminais destes peptídeos diferiram; somente em IV, observouse a presença de uma família de confôrmeros com estruturas helicoidais nessa porção do esqueleto e que eram superponíveis. O mesmo não foi observado na porção C-terminal de III. A análise conformacional do peptídeo XVIII em solução foi também realizada empregando-se CD e 1H-RMN. Os resultados obtidos indicaram que este peptídeo tem forte tendência em assumir uma estrutura helicoidal em solução aquosa contendo 50% CD3OH. O conjunto dos resultados obtidos neste trabalho nos permitem concluir que: 1) a presença de W107 e F108 é, de fato, essencial para a expressão da atividade mitogênica de peptídeos derivados do sítio 2 do hFGF-1; 2) a presença de N106 adjacente ao core hidrofóbico constituído pelos resíduos W107F108 é importante; 3) a ciclização do peptídeo IV foi decisiva para a expressão de sua atividade, indicando que, não apenas a presença, mas o posicionamento adequado das cadeias laterais de N106, W107 e F108 são determinantes; 4) apenas uma das lisinas (K112 ou K113) é essencial para a atividade mitogênica (X, XVI e XVII); 5) o importante parece ser a manutenção do posicionamento das cadeias laterais em relação aos dos outros resíduos contidos na seqüência, uma vez que, a substituição do segmento 112KKNGS116 por uma prolina e/ou deleção de 120GPRT123 (XI e XIV) abolem a atividade mitogênica do peptídeo; 6) a ciclização que mantem a distância e as orientações relativas entre WF e KR (considerados essenciais para a atividade) em seqüência (XII e XV), leva à recuperação da atividade; 7) a atividade inibitória específica para o FGF-2 exibida pelo peptídeo XVIII parece ser um indicativo de que a alça DE do domínio DIII do receptor pode estar envolvido na ligação a esta proteína. Estas conclusões são relevantes e essenciais para: 1) o entendimento dos requisitos estruturais para a atividade mitogênica dos peptídeos estudados e, como reflexo, do hFGF-1, 2) o desenho de novos agonistas, antagonistas ou inibidores do sistema FGF. / In our search for small potent agonists or inhibitors related to hFGF-1(97-132), we first investigated the preferred conformation in solution of Ac -WFVGLKKNGSSKRGPRT-NH2 (I), by 1H-NMR. This compound has been described as a weak agonist of the mitogenic activity of this growth factor able to inhibit the binding of 125I-hFGF-1 to their cellular receptors, and to heparin-Sepharose columns (Oyama et al., 1996). We found that this peptide is in a random coil configuration, which could explain its low activity (104 times less potent than the native protein). On the basis on these results and on several data available in the literature (Harper & Lobb 1988; Burgess et al., 1991; Pantoliano et al., 1994, Springer et al., 1994; Thompson et al., 1994; Imamura et al., 1995; Ornitz et al., 1996; Blaber et al., 1996; Zhu et al., 1991, 1995; 1997; Schwizer, 1995; Sieber & Moe, 1996; Rizo et al., 1996), we designed seventeen peptides related to the Site 2 (97-132)[hFGF-1] listed on the Table 1: some were linear and some were cyclic. They were synthesized manually using the solid phase method, purified by RP-HPLC, and chemically characterized by RP-HPLC, amino acid analysis and mass spectrometry. Conformational constraints of certain peptides were achieved by cyclization. Intramolecular dissulfide bonds were formed by the oxidation of the thiol groups of two cysteins residues with air oxigen and/or K2Fe(CN)6. Lactama bonds were formed between the functional side chain group of acidic and basic residue. The synthetic peptides were tested in of their ability to inducing mitogenic response on Balb/c 3T3, A-31 clone fibroblasts cultures. The results obtained were the following: 1) peptides II, III, VI-IX were essentially inactive on Balb/c 3T3 fibroblasts in the range of concentrations used (up to 200 µM), 2) in the same range of concentration, peptide IV showed an ED50 60µM (similar to that found for peptide I) while its correspondent linear analog (III) was inactive; 3) V, analog of IV, that has Asn deleted, exibihited mitogenic activity lower than IV; 4) X showed a mitogenic activity on Balb/c fibroblasts lower than I, 5) cyclic peptides XII and XV showed mitogenic activities similar to that of I, while their correspondent linear (XI and XIV) and cyclic (XIII) analogs were inactive; 6) XVI and XVII showed mitogenic activities similar to that found for I. In parallel, two peptides [Ac-170NTTDKENEVLH180-NH2 (XVIII) and Ac-194SLAGNSIGLSH204-NH2 (XIX)], derived from DIII FGFR-1β and designed as putative ligands of Site2 hFGF-1, were synthesized and tested. In the range of concentration used (up to 200 µM), XIX was inactive and exhibited no inhibitory effect on FGF-1 and FGF-2 mitogenic activities. Nevertheless, XVIII inhibited the mitogenic activity of both proteins, being this effect clearly more significant for the FGF-2 (Kiyota et al., 1998). Some of synthetic peptides have been spectroscopically analyzed in order to disclose the structural features that characterize the active (Kiyota et al., 1999). A detailed analysis was undertaken with peptides III and IV using circular dichroism (CD) and 1H-NMR. Although the similarities in their primary sequences, III has shown inactive when tested on Balb/c 3T3 fibroblasts culture while IV was mitogenic with ED50 values around 50 µM. I was also not capable of inhibiting the binding of hFGF-1 to its cellular receptors, I was inactive while II inhibits it with ID50 values of about 30 µM. Circular dichroism study showed that while at increasing SDS concentrations the spectra of III suggested the presence of an equilibrium among partially structured states, those of IV indicated that this peptide exists in unordered extended conformation, folds into a β-conformation and, finally, assumes a helix rich structure. 1H-NMR analysis revealed the existence of a well defined γ-turn encompassing residues 4-6 that nicely fits with that present in the same portion of the crystallized hFGF-1. Superposition of the final structures of III and IV over the entire sequence revealed that only the C-terminal portion of III has the tendency to fold into a regular structure. Together these data indicate that the turn existence in IV allowed it to acquire the structural determinants for the expression of mitogenicity probably through a more appropriate arrangement of residues 8-10. More importantly, they demonstrate that we have found a short agonist of hFGF-1 able to structurally mimic its corresponding stretch. Conformational analysis of XVIII in solution was undertaken also by using CD e 1HRMN. The results obtained indicated that it has a strong tendency to assume helicoidal configuration in aqueous solution containing 50% CD3OH. Altogether these data led us to conclude that: 1) the presence of Asn106 adjacent to hydrophobic core constituted by W107F108 is essential for the mitogenic activity of IV; 2) conformation constraint by cyclization was efficient for the correct N106, W107 and F108 side chains orientation in peptide IV for an effective cellular receptors binding; 3) peptides related to hFGF-1 (114-123) seem to be promising mitogenic agonists; 4) the only one lysine between L126 and N129 is enough for the mitogenic activity expression (X, XVI and XVII); 5) deletions of residues, replacement of deleted fragment by Pro followed by restriction of peptide conformation might keep the frame of the residues considered essential for the mitogenic activity along the peptides backbones; 6)The inhibitory effect on the FGF-2 mitogenic activity observed in peptide XVIII was indicative that the loop DE of DIII FGFRs seems to be involved in the binding of this protein.These conclusions are very relevant in terms of the knowledge of the structural requirements for the mitogenic activity of studied peptides and, as a reflex, of the hFGF-1. Furthermore, they constitute additional guidelines for designing new constrained peptides derived from this segment of FGF-1, which may result in more potent agonists, antagonists or inhibitors of such important target.

O preparo e produção de material didático para cursos on-line demanda o trabalho de uma equipe multidisciplinar (com técnicos e especialistas de diferentes áreas) junto aos professores-autores. A organização e o processo de produção são complexos e específicos para cada projeto e instituição de ensino. Nesta pesquisa, o objetivo é analisar como ocorreu o processo colaborativo de produção de material didático no curso semipresencial de Licenciatura em Ciências da USP/UNIVESP (no período de 2013). Este estudo é definido como pesquisa qualitativa, identificada como estudo de caso. Para alcançar os objetivos previstos foi realizada coleta de dados documentais e entrevistas com profissionais que participaram do processo de criação. Para análise dos dados obtidos foi realizado inicialmente o levantamento de posicionamentos teóricos ligados aos principais termos que permeiam o estudo. Por meio de análise de conteúdo foram estabelecidas as relações entre a teoria estudada e os dados coletados. Ao final, conclui-se que, por se tratar de um trabalho em equipe, o conteúdo (texto) original precisa passar por alterações e adequações ao longo de todo o processo até se tornar material didático do curso (em seus diferentes formatos). Vale destacar que esse processo de produção é acompanhado, discutido e validado pelo professor em todas as suas etapas. Como em toda equipe multidisciplinar, a comunicação e as inter-relações entre os profissionais são constantes e significativas para o processo. É nesse contexto de trabalho em equipe (junto ao professorautor) que ocorrem momentos de cocriação didática, resultado de um trabalho colaborativo e de criação. Verificou-se que esse processo de atuação das equipes na produção de material didático tem momentos intercalados de trabalho individualizado e colaborativo. Apesar desse fluxo estar desenhado de forma linear, com etapas predefinidas, é nas inter-relações e trocas de conhecimentos, sugestões e adequações do conteúdo que ocorrem momentos de cocriação. Trata-se de um processo híbrido: linear e de cocriação. Linear em sua estrutura e forma de trabalho e de cocriação como parte natural das relações humanas e da produção coletiva de conhecimento. / The preparation and the production of didactic materials for on-line courses demand a multidisciplinary teamwork (technicians and specialists from different areas) along with teacherauthors. The organization and the process of production are complex and specific for each project and teaching institution. The objective of this research is to analyze how the collaborative process of the didactic material produced in the blended learning course of the Licentiate Degree in Science from USP/UNIVESP (conducted over a certain period of time. Qualitative approach has been adopted for this study, more specifically, a case study research. In order to accomplish our goals, we have conducted data collection by using review of documents as well as interviews with the professionals that participated in the creation process. For the purpose of analyzing the collected data, we have gathered diverse theoretical approaches related to the main topics of this study. By having the content analyzed, a relationship between the studied theory and the data collected has been established. In conclusion, due to the fact that it is the product of a teamwork, the original content (text) needs to go through some changes and adaptations during the whole process until the final version of the didactic material (in all its different formats) is completed. It is important to emphasize that every step during the process of production is attended, discussed and validated by the teacher-author. As in any multidisciplinary teamwork, the communication and the interrelationships are constant and meaningful to the process. It is in this context of teamwork (including the teacher-author) that the didactic co-creation occurs, being the result of collaborative and creation work. It has been verified that the groups worked in intercalated moments throughout the production of the material: as individuals or in collaboration. Despite the fact that there are predefined steps which are supposed to flow in a linear way, co-creation takes place during the interrelationships, the exchange of knowledge and suggestions and the adaptation of the content. It turns out to be a hybrid process: linear and co-creative. Linear in its structure and its way of working and co-creative as a natural part of human relationships and as a collective production of knowledge.

Il est maintenant clairement établi que la vitamine B12 (cobalamine) joue un rôle important dans la formation des globules rouges, et joue un rôle essentiel dans l'efficacité du fonctionnement du système nerveux, comme les fonctions cognitives. La carence en vitamine B12 est répandue dans le monde et provoque une anémie mégaloblastique et des déficits neurologiques. La carence en vitamine B12 et l'hyperactivité glucocorticoïde (GC) contribuent à la détérioration de la plasticité et des fonctions de l'hippocampe. Pour comprendre comment la carence en cobalamine dans le système nerveux central génère des déficits neurologiques fonctionnels, nous avons utilisé un modèle de souris génétiquement modifiées, dont le gène codant le récepteur de la transcobalamine (souris CD320 KO) est invalidé exclusivement dans le cerveau. Nos analyses comportementales indiquent des déficits dans l'apprentissage visuo-spatial hippocampo-dépendant chez les souris KO. Les résultats ont montré qu'une dose journalière physiologique d'hydroxycortisone (8 mg / kg / jour I.P.) a un effet positif dans la restauration dans les performances d'apprentissage, chez les souris KO par rapport aux contrôles. Conformément aux déficits comportementaux, ce modèle knock-out montre une diminution de l'expression de protéines clés impliquées dans la plasticité cérébrale. Les résultats de western blot des extraits d'hippocampe ont révélé que les souris KO femelles montrent une diminution de l'expression des récepteurs des glucocorticoïdes (GR) et du coactivateur Proliférateur de péroxysome 1 (PGC-1), la protéine de réponse de croissance précoce -1 (EGR-1), Synapsines (Syn I, II), protéines clés connues pour être impliquées dans l'activité synaptique. L'étude du modèle CD320 KO pourrait permettre de mieux comprendre les effets de la carence en vitamine B12 observés chez l'homme, afin d’identifier des réponses potentielles aux différents troubles neurologiques associés ; notamment peut être un traitement palliatif basé sur les corticoïdes / It is now clearly established that vitamin B12 (or cobalamin), plays an important role in the formation of red blood cells, as well as it has a vital role in the efficient functioning of the nervous system such as cognitive functions. Vitamin B12 deficiency is widespread worldwide and causes megaloblastic anemia and neurological deficits. Vitamin B12 deficiency and the hyperactivity of the glucocorticoid (GC) contribute to the deterioration of hippocampal plasticity and functions. To understand whether cobalamin deficiency in the central nervous system produced functional neurologic deficits, we used a transcobalamin receptor / CD320 knockout mouse that lacks the receptor for the cellular uptake of cobalamin in the brain. Our behavioral analyses indicate deficits in hippocampo-dependant visuo-spatial learning capacities in KO mice. However, a daily physiological dose of hydroxycortisone (8 mg/kg/day I.P.) has a positive effect in improving learning performances in KO mice compared to controls. Consistent with the behavioral deficits, the knockout mouse shows impaired expression of key proteins implicated in synaptic plasticity. The results of western blot analyses of hippocampus extracts revealed that the knockout female mice showed decreased expressions of glucocorticoids receptors (GR), Peroxisome proliferator-activated receptor coactivator 1 (PGC-1), early growth response protein -1 (EGR-1), Synapsines (Syn I, II), Protein arginine N-methyltransferase -1 (PRMT-1), key proteins known to be implicated in the synaptic activity. The study of the CD320 knock out model could help to better understand the effects of vitamin B12 deficiency observed in humans, in order to identify potential responses to various neurological associated disorders; with a putative palliative treatment approach using corticoids

Der Begriff von Orthos Logos ist zentral für die ethische Lehre Aristoteles’. In der Literatur ist jedoch umstritten, was der Inhalt von OL ist. Das genaue Wesen von OL liegt immer noch im Dunkel. Ziel meiner Dissertation ist es, den Beitrag von OL für Aristoteles’ Ethik zu erforschen. Dabei soll vor allem erläutert werden, was OL ist bzw. was OL leisten kann. Auf dieser Untersuchung basierend versuche ich, eine ausführliche bzw. in sich konsistente Interpretation der wichtigen Bestandteile der Nicomachischen Ethik zu liefern. Ich werde dafür argumentieren, dass OL als der praktische Syllogismus selbst aufzufassen ist, der die moralischen Subjekte darüber informiert, was sie tun sollen bzw. weswegen sie gerade dies und nicht etwas anderes tun sollen. Sofern ein hinsichtlich der Moralität noch lernender Mensch den Syllogismus nicht vollständig begreift, ist es allerdings möglich, dass OL diesem Menschen etwas anderes zu sein scheint. / The notion of the orthos logos (abbr. OL) is vital and decisive for Aristotle’s ethical project. The question of what OL really means is a vigorously debated issue. But what the OL exactly is still remains ambiguous and obscure. The purpose of my dissertation is to inquire into the philosophical contribution of the OL in Aristotle’s Ethics. To fulfil this goal, it is essentially to determine what the OL is and what the OL can do. Through this inquiry I seek to present a comprehensive and consistent reading of important parts of Aristotle’s Nicomachean Ethics. I will argue that the OL is the practical syllogism per se, which could tell moral people what should they do and why should they do this or that. But it could also appear to be something else for moral learners, since they are not capable of fully understanding the syllogism yet.

O presente trabalho tem como objetivo avaliar a influência das variáveis ambientais na mortalidade de idosos (acima de 65 anos) por doenças cardiovasculares (DCV) e respiratórias (RES), nos condados de Los Angeles, Orange e Santa Bárbara na Califórnia, através de análises estatísticas, em particular de componentes principais (ACP). Para isso foram utilizados dados diários das estações meteorológicas dos condados (temperatura, pressão, umidade e velocidade do vento), de poluição do ar (NO2, CO, SO2 e O3) e da mortalidade por DCV e RES, durante o período 1975-2005. Além disso, foram calculados 7 índices de conforto térmico e estabelecidas as zonas de conforto para diferentes graus de percepção térmica a partir do índice de Temperatura Efetiva (TE). Os resultados verificaram tendências diferentes na variação temporal das DCV e RES. Enquanto as doenças RES tiveram um aumento bastante leve em todos os condados, as DCV apresentaram uma ligeira diminuição nos condados de Orange e Santa Bárbara, e uma diminuição constante e acentuada no condado de Los Angeles. Também se verificou uma variação sazonal significativa, com evidente aumento do número de óbitos de ambas as doenças durante o inverno e queda durante o verão. Em relação aos poluentes, foram observadas muitas ultrapassagens dos padrões nacionais de qualidade do ar principalmente para o O3, com 1443 vezes para o condado de Los Angeles, 771 para o Orange e 114 para Santa Bárbara. A partir dos índices de conforto térmico notou-se que o mês mais estressante devido ao calor foi setembro principalmente para os condados de Los Angeles e Santa Bárbara, e julho e agosto para o condado de Orange. Analisando a média de óbitos por sensação térmica observou-se um aumento das doenças nos extremos, principalmente quando a sensação é Muito Frio, com médias por doenças RES de 16,42, 3,31 e 0,81 e por DCV de 77,32, 15,37 e 2,92 nos condados de Los Angeles, Orange e Santa Bárbara, respectivamente. Com base na análise de correlação entre as variáveis e as doenças, observaram-se as melhores correlações no condado de Los Angeles, tanto para as doenças RES quanto DCV, e as correlações mais baixas foram observadas no condado de Santa Bárbara. Os resultados da ACP nos condados de Los Angeles e Orange verificaram o aumento da mortalidade por DCV no inverno, associadas a correlações positivas com os níveis de poluição principalmente em Los Angeles e ao estresse por frio, umidade e vento (via índice de conforto térmico) em Orange. Por outro lado, a mortalidade por doenças RES esteve mais associada às variações meteorológicas do que às concentrações de poluentes, observando-se associações positivas com o índice de calor (IC). Neste sentido, o condado de Orange apresentou maiores correlações com IC do que o condado de Los Angeles, além de uma variância maior (11,36 % contra 9,89 %). Por último, no condado de Santa Bárbara os resultados foram muito diferentes, já que tanto episódios de frio quanto de calor não mostraram impactos claros na população idosa, com respeito a ambos os grupos de doenças. O distintivo foi que os níveis de SO2 tiveram a maior associação positiva à mortalidade principalmente por DCV (0,53), embora tiver o condado as concentrações mais baixas das estudadas (média de 0,29 ppb). Em suma, os condados possuem estruturas sociais diferentes, apesar da semelhança do clima, fazendo com que os resultados sejam bastante dispares entre os mesmos. As perspectivas destes estudos pretendem colocar estes resultados para climas futuros, pois diversos episódios de ondas de calor ocorreram após o ano de 2005. / The objective of this study was to evaluate the influence of environmental variables on the mortality of elderly patients (over 65 years) due to cardiovascular diseases (CD) and respiratory diseases (RD) in the counties of Los Angeles, Orange and Santa Bárbara, California; through statistical analyzes, in particular, principal components (PCA). For this purpose, daily data of the meteorological stations of the counties (temperature, pressure, humidity and wind speed), air pollution (NO2, CO, SO2 and O3) and mortality by CD and RD were used during the period 1975-2005. In addition, seven thermal comfort indexes were calculated and comfort zones were established for different degrees of thermal perception according to the Effective Temperature index (TE). The results showed different trends in the temporal variation of CD and RD. While RD diseases had a fairly mild increase in all counties, CD showed a slight decrease in Orange and Santa Barbara counties and a steady decline in Los Angeles county. There was also a significant seasonal variation, with an evident increase in the number of deaths from both diseases during the winter and fall during the summer. In relation to the pollutants, many national standards for air quality were exceeded, mainly for the O3, with 1443 times for Los Angeles, 771 for Orange and 114 for Santa Barbara. From the thermal comfort indexes it was noted that the most stressful month due to the heat was September, mainly for the counties of Los Angeles and Santa Barbara, and July and August for the county of Orange. Analyzing the average number of deaths due to thermal sensation, there was an increase of the illnesses in extreme, especially when the sensation is Very Cold, with RD averages of 16.42, 3.31 and 0.81 and CD average of 77.32, 15.37 and 2.92 in the counties of Los Angeles, Orange and Santa Barbara, respectively. Based on correlation analysis between variables and diseases, the best correlations were found in Los Angeles for both RD and CD, and the lowest correlations were observed in Santa Barbara. The results of PAC in the counties of Los Angeles and Orange showed the increase in CD mortality in winter associated to positive correlations with the levels of pollution mainly in Los Angeles and stress due to cold, humidity and wind (via thermal comfort index) in Orange. On the other hand, mortality due to RD diseases was more associated with meteorological variations than with pollutant concentrations, with positive associations with heat index (HI). In this sense, Orange County presented higher correlations with HI than the county of Los Angeles, in addition to a greater variance (11.36% vs. 9.89%). Finally, in Santa Barbara the results were very different, since both episodes of cold and heat did not show clear impacts in the elderly population, regarding both groups of diseases. The difference was that SO2 levels had the highest positive association with CD mortality (0.53), although this county had the lowest concentrations studied (mean 0.29 ppb). In sum, counties have different social structures, despite the similarity of the climate, making the results quite different between them. The perspectives of these studies intend to place these results for future climates, since several episodes of heat waves occurred after 2005.

Medieval cartularies have been the focus of many studies in the past few decades. Rather than simply repositories for charter texts, cartularies are now regarded by those who study them as carefully curated collections of texts whose contents and arrangement reflect the immediate concerns and archival environment of the communities that created them. One feature of cartularies which has not received attention is the ‘growth’ of their manuscripts beyond the initial phase of creation. This growth refers not only to the addition of fresh gatherings but also to the piecemeal addition of texts into the available spaces, often in a haphazard order and by many scribes working across a number of decades. ‘Manuscript growth’ is not an uncommon feature of cartularies from the central middle ages, particularly from the thirteenth century onwards. As a phenomenon, however, it has not been recognised or studied, for the good reason that it is difficult to discuss haphazard manuscript growth in a systematic way. This thesis offers a new methodology which engages with multi-scribe contributions to ‘active’ cartularies. It takes a holistic approach which integrates the textual and ‘physical’ evidence of cartularies, and embraces all forms of scribal activity. By studying the growth of cartulary manuscripts, we can gain significant insights into the contemporary use and perception of these valuable objects. This thesis therefore takes a fresh look at the ‘genre’ of medieval cartularies through the eyes of the manuscript evidence itself, and what this can reveal about its medieval scribes and readers. Two manuscripts are taken as the basis of this study: the older cartulary of Glasgow Cathedral (AUL SCA MS JB 1/3) and the older cartulary of Lindores Abbey (in private ownership in Caprington Castle). Chapter 1 introduces the field of cartulary studies, with reference to new work in this area (particularly in relation to cartularies in France and England). Central questions in this field are introduced, such as the definition of a cartulary, their creation and function. It also discusses approaches to analysing complex codices and multi-scribe activity within other manuscript genres. In Chapter 2, a new methodology will be introduced for analysing manuscript growth. This involves rethinking our approach to some familiar elements of manuscripts: their codicology, binding history, the scribes, as well as the challenge of dating the various contributions to the cartularies. New concepts and terminology will be introduced (such as ‘relative dating’ and ‘series’) that have been developed in response to these two complex cartularies. By applying this new methodology, the creation and subsequent growth of each manuscript can be examined in detail in Chapter 3 (for Glasgow Cathedral’s cartulary) and Chapter 4 (for Lindores Abbey’s). It is shown that the contemporary experience of these two cartularies was as a collection of simultaneously ‘active’ units (either unbound or in temporary bindings), offering new scribes a choice of where to place their material. Chapter 5 draws together the analysis, and focuses on the initial creation of the cartularies, the nature of their growth by piecemeal additions, and the reasons for this growth. This reveals two communities that took an active approach to reading and extending their cartularies, treating these manuscripts as a shared space. The vexed question of ‘repeated’ texts within cartularies is reconsidered in this light. The analysis allows us to develop a deeper understanding of the cartularies’ function and the role of their scribes as primarily readers, whose interactions with the manuscript were responsive and dynamic. The institutional setting is also discussed. The thesis concludes by considering the implications of this study for our understanding of the function and typology of cartularies, their relationship to archives of single-sheet documents, and as sources for institutional identity, as well as the potential of the methodology to act as a starting point for studying scribal interactions and scribes as readers in other manuscript genres with multi-scribe growth.

O aumento da expectativa de vida da população mundial tem se associado com uma maior prevalência de doenças neurodegenerativas. A Doença de Alzheimer (DA) é a doença neurodegenerativa mais comum e a principal causa de demência em indivíduos com mais de 60 anos, sendo caracterizada por um declínio progressivo na memória e função mental dos pacientes. Esses sintomas são acompanhados por alterações histopatológicas no cérebro desses indivíduos, incluindo a presença de uma grande quantidade de placas senis, formadas pela deposição do peptídeo beta-amiloide (Aβ), e de emaranhados neurofibrilares formados pela hiperfosforilação da proteína Tau. Estudos indicam que a deposição do Aβ é uma das principais responsáveis pelo desenvolvimento da DA, causando dano neuronal através da ativação de várias vias pró-apoptóticas e dando origem aos sintomas de demência típicos dessa doença. Até o momento, não existem tratamentos eficazes para o combate à DA, de forma que a maior parte das intervenções farmacológicas é destinada apenas ao tratamento de alguns de seus sintomas. A proteína translocadora (TSPO) se localiza em pontos de contato entre as membranas mitocondriais interna e externa e está relacionada com o transporte de colesterol para o interior da mitocôndria e com a regulação da esteroidogênese e da apoptose. Estudos mostram que ligantes da TSPO apresentam efeitos neuroprotetores em diferentes modelos experimentais de lesão cerebral e doenças neurodegenerativas. Especificamente em relação à DA, um estudo indicou que o 4’-clorodiazepam (4’-CD), um ligante da TSPO, apresenta efeitos neuroprotetores em um modelo animal dessa doença, sendo um possível candidato para o seu tratamento. Dessa forma, o objetivo desse estudo foi verificar o efeito neuroprotetor do 4’-CD em diferentes modelos in vitro de toxicidade induzida pelo Aβ, além de seus efeitos sobre o desenvolvimento de neurônios hipocampais. Inicialmente, demonstramos que o 4’-CD reduziu a morte celular de células SH-SY5Y expostas a um modelo de toxicidade induzida pela administração de Aβ. Esses efeitos estiveram associados com a redução da expressão da proteína pró-apoptótica Bax e com um aumento da expressão da survivina, uma proteína anti-apoptótica. A expressão das proteínas Bcl-xl e procaspase-3, por outro lado, não foi alterada pelos tratamentos. Posteriormente, estudamos os efeitos neuroprotetores do 4’-CD contra a toxicidade induzida pela administração do Aβ em culturas organotípicas de hipocampo. Nesses experimentos, foi demonstrado que o 4’-CD reduz a morte celular de culturas organotípicas de hipocampo expostas ao Aβ através de um aumento na expressão da enzima SOD, sem alterar, no entanto, a expressão das proteínas Akt e procaspase-3. Por fim, foi avaliado o efeito do 4’-CD sobre o desenvolvimento de culturas primárias de neurônios hipocampais de camundongos machos e fêmeas. Foi observado que as culturas de neurônios hipocampais das fêmeas apresentaram um desenvolvimento mais rápido do que as dos machos. O 4’-CD acelerou a maturação e aumentou a ramificação neurítica dos neurônios hipocampais dos machos, mas não exerceu qualquer efeito sobre os neurônios das fêmeas. Em suma, foi observado que o 4’-CD apresenta efeitos neuroprotetores contra o Aβ em células SH-SY5Y e em culturas organotípicas do hipocampo, apresentando-se como um fármaco em potencial para o tratamento da DA. Além disso, foi observado que o 4’-CD exerceu um efeito dependente do sexo sobre o desenvolvimento de culturas primárias de neurônios hipocampais, estimulando o desenvolvimento e a ramificação neurítica de neurônios hipocampais de machos, mas não de fêmeas. / The increase in life expectancy of the world population has been associated with a higher prevalence of neurodegenerative diseases. The Alzheimer’s Disease (AD) is the most common neurodegenerative disorder and the main cause of dementia among people over 60 years, being characterized by a progressive decline in the memory and mental function of the patients. These symptoms are associated with histopathological changes in the brain of these patients, including the presence of senile plaques, formed by the deposition of amyloid-beta (Aβ), and neurofibrillary tangles, which are related to the hyperphosphorylation of Tau protein. Studies indicate that Aβ deposition is a major contributor to AD progression, promoting neuronal damage through the activation of different pro-apoptotic pathways and giving rise to the typical dementia symptoms of this disease. To date, there are no effective treatments for AD, so that most of the pharmacological intervention is intended for the treatment of some of its symptoms. The translocator protein (TSPO) is located in contact sites between the outer and the inner mitochondrial membranes and is involved in the cholesterol transport into the mitochondria and in the regulation of steroidogenesis and apoptosis. Studies show that TSPO ligands present neuroprotective effects in different experimental models of brain injury and neurodegenerative diseases. Specifically regarding AD, a study indicated that 4’-chlorodiazepam (4’-CD), a TSPO ligand, is neuroprotective in an animal model of this disease, being a possible candidate for its treatment. Therefore, the aim of this study was to evaluate the neuroprotective effect of 4’-CD in different experimental models of Aβ- induced neurotoxicity in vitro, as well as its effects on the development of hipocampal neurons. First, it was demonstrated that 4’-CD decreased the cell death of SH-SY5Y cells exposed to the Aβ. This effect was associated with the inhibition of the Aβ-induced upregulation of Bax, a pro-apoptotic protein, and downregulation of survivin, a prosurvival protein. On the other hand, the expression of Bcl-xl and procaspase-3 was not change by the treatments. After, it was studied the neuroprotective effects of 4’-CD against Aβ in organotypic hipocampal cultures. In these experiments, it was shown that 4’-CD decreases the cell death of organotypic hippocampal slices exposed to the Aβ by increasing the protein expression of SOD, but without changing the expression of Akt and procaspase-3. Finally, due to the importance of the processes of neuronal development and maturation in the regeneration of CNS after injury, it was evaluated the effect of 4’-CD on the development of primary hippocampal neurons of male and female mice. It was observed that female primary hippocampal neurons presented an increased rate of development than male neurons. 4’-CD stimulated the development and increased the neuritic branching of male but not from female neurons. In summary, it was observed that 4’-CD presented a neuroprotective effect against Aβ in SH-SY5Y cells and in rat organotypical hippocampal slices, presenting itself as a promising agent for the treatment of AD. Also, it was observed that 4’-CD modulates the development of hippocampal neurons in a sex-dependent manner, stimulating the development of male but not from female cells.

Wong, Yun Sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 142-162). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.vi / Acknowledgement --- p.ix / Statement of Work --- p.x / Table of Contents --- p.xi / List of Tables --- p.xiv / List of Figures --- p.xvi / Glossary of Terms and Abbreviations --- p.xviii / Chapter Section I: --- Introduction --- p.1 / Chapter Chapter 1: --- General Overview of Asthma --- p.2 / Chapter 1.1 --- Asthma definition and its phenotype --- p.2 / Chapter 1.2 --- Asthma epidemiology and its prevalence in past decades --- p.4 / Chapter 1.3 --- Hygiene hypothesis and asthma development --- p.8 / Chapter 1.4 --- Asthma pathogenesis and innate immunity --- p.12 / Chapter 1.5 --- The environmental factors and genetic makeup in relation with asthma --- p.17 / Chapter Chapter 2: --- Study Plan and Obj ective --- p.21 / Chapter Section II: --- Literature Review --- p.24 / Chapter Chapter 3: --- Indoor Environmental factors of Asthma --- p.25 / Chapter 3.1 --- Overview of the indoor environmental factors --- p.25 / Chapter 3.2 --- House dust endotoxin --- p.27 / Chapter 3.2.1 --- Determinants of endotoxin exposure in home environment --- p.21 / Chapter 3.2.2 --- Protective role of endotoxin in allergy and asthma development --- p.29 / Chapter 3.2.3 --- Deleterious effect of endotoxin exposure in asthma: the dark side --- p.31 / Chapter 3.3 --- Allergen --- p.34 / Chapter 3.3.1 --- Allergens: an update --- p.34 / Chapter 3.3.2 --- Determinants of allergens in home environment --- p.35 / Chapter 3.3.3 --- Allergens avoidance: environmental intervention --- p.36 / Chapter 3.4 --- Nitrogen dioxide --- p.40 / Chapter 3.4.1 --- Determinants of indoor nitrogen dioxide and its relation with gas cooking --- p.40 / Chapter 3.4.2 --- The adverse effects of nitrogen dioxide on respiratory symptoms --- p.41 / Chapter 3.4.3 --- Reactive nitrogen species and nitrosative stress in asthma --- p.42 / Chapter Chapter 4: --- CD14 Single Nucleotide Polymorphisms and Asthma --- p.45 / Chapter 4.1 --- Overview of CD14 receptor --- p.45 / Chapter 4.2 --- Action of CD14 receptor in endotoxin response --- p.47 / Chapter 4.3 --- Relation of CD14 with asthma --- p.48 / Chapter 4.3.1 --- Associations between CD14 polymorphisms and asthma phenotypes --- p.48 / Chapter 4.3.2 --- Endotoxin switch concept: from gene to gene - environment --- p.52 / Chapter Section III: --- Study Core --- p.55 / Chapter Chapter 5: --- Methodology in indoor environment investigation and its result --- p.57 / Chapter 5.1 --- Study Population --- p.57 / Chapter 5.2 --- Home Visiting Protocol --- p.60 / Chapter 5.2.1 --- The International Study of Asthma and Allergies in Childhood (ISAAC) --- p.60 / Chapter 5.2.2 --- ISAAC questionnaire --- p.61 / Chapter 5.2.3 --- House dust collection procedures --- p.62 / Chapter 5.2.4 --- Indoor nitrogen dioxide measurements --- p.65 / Chapter 5.2.4.1 --- Ogawa passive sampler --- p.65 / Chapter 5.2.4.2 --- Preparation and measurement procedures --- p.66 / Chapter 5.2.4.3 --- Indoor nitrogen dioxide quantification --- p.67 / Chapter 5.3 --- House dust extraction --- p.69 / Chapter 5.4 --- House dust endotoxin measurement --- p.70 / Chapter 5.5 --- Allergen measurement --- p.72 / Chapter 5.6 --- Statistical Analysis --- p.75 / Chapter 5.7 --- Results --- p.77 / Chapter 5.7.1 --- Demographic data and subjects characteristics --- p.77 / Chapter 5.7.2 --- "Dust weight, endotoxin and allergen levels and their determinants in household" --- p.82 / Chapter 5.7.3 --- Indoor NO〕2levels and its determinant in household --- p.95 / Chapter 5.7.4 --- Associations between indoor environmental factors and respiratory health --- p.96 / Chapter 5.7.4.1 --- Clinical symptoms --- p.96 / Chapter 5.7.4.2 --- Exhaled NO levels --- p.101 / Chapter 5.7.4.3 --- Spirometric indices --- p.103 / Chapter Chapter 6： --- Methodology in genotyping CD14 polymorphisms and its result --- p.105 / Chapter 6.1 --- Study population --- p.105 / Chapter 6.2 --- Serum Total and allergen-specific IgE measurement --- p.106 / Chapter 6.3 --- CD14 Genotyping s --- p.107 / Chapter 6.3.1 --- Genotyping promoter SNPs ofCD14/-159 and -1359 --- p.107 / Chapter 6.3.2 --- Genotyping promoter SNP of CD14/-1619 --- p.109 / Chapter 6.3.3 --- Validation of genotyping by sequencing --- p.111 / Chapter 6.4 --- Statistical Analysis --- p.112 / Chapter 6.5 --- Results --- p.113 / Chapter 6.5.1 --- Subjects characteristics and clinical features. --- p.113 / Chapter 6.5.2 --- Associations between CD14 SNPs and asthma phenotypes --- p.114 / Chapter Chapter 7: --- Discussion --- p.120 / Chapter 7.1 --- Influence of indoor factors on asthmatic children --- p.120 / Chapter 7.2 --- CD14 polymorphisms in modifying asthma phenotypes --- p.135 / Chapter Chapter 8: --- Conclusion and Further Works --- p.138 / References --- p.141 / Appendix 1 Questionnaire / Appendix 2 Publications