Os problemas da poluição atmosférica atingem todos os grandes centros urbanos, em particular as megacidades com população maior do que 10 milhões de habitantes. Emissões veiculares e industriais destacam-se entre as principais responsáveis pelas altas concentrações de poluentes do ar nesses centros urbanos. Os diferentes componentes da poluição atmosférica, a dose e o tempo de exposição, podem levar a diversos impactos na saúde humana. Esse estudo teve por objetivo avaliar a genotoxicidade da poluição atmosférica (PM2,5 e NO2) e sua correlação com modificações no perfil de metilação das citocinas IL-10 e TNF-alfa, em trabalhadores da cidade de São Paulo/SP ocupacionalmente expostos. Participaram deste estudo 57 indivíduos do gênero masculino, com idades variando entre 28 e 66 anos de idade, trabalhadores em turnos diários de atividades externas na cidade de São Paulo e, portanto, ocupacionalmente expostos à poluição atmosférica. Foram recrutadas 3 categorias de profissionais: 1) controladores de tráfego (n=18); 2) taxistas (n=21) e profissionais do Instituto Florestal (n=18). Esses trabalhadores foram divididos em dois grupos em função dos locais de trabalho e exposição: 1) grupo área urbana (AU), composto pelos controladores de tráfego e taxistas e 2) grupo área periurbana (APU), composto pelos profissionais do Instituto Florestal. Amostradores individuais de poluição atmosférica foram utilizados para a coleta dos poluentes PM2,5 e NO2. A análise da genotoxicidade foi realizada pelo teste de micronúcleos nas células epiteliais da mucosa oral e em linfócitos do sangue periférico. O perfil de metilação das citocinas IL-10 e TNFalfa foi realizado por sequenciamento de nova geração. Nossos resultados mostraram uma diferença na concentração do PM2,5 entre os grupos (AU=32,92?g.m-3, APU=25,77ug.m-3; p=0,0311). Não foi encontrada diferença na concentração de NO2 entre os grupos. Foram encontradas diferenças nas frequências de micronúcleos, tanto em mucosa oral (AU=2,78%, APU=1,16%; p < 0,0001) quanto em linfócitos periféricos (AU=1,51%, APU=0,73%; p < 0,0001). Também foi encontrada diferença na metilação média do gene IL-10 entre os grupos (AU=25%, APU=30%; p=0,0120). Não foi encontrada diferença na metilação média do gene TNF-alfa entre os grupos. Concluímos que os trabalhadores da área urbana da cidade estão expostos a maiores concentrações de PM2,5, possuem maiores frequências de micronúcleos tanto em células da mucosa oral quanto em linfócitos periféricos e apresentam um perfil de hipometilação do gene IL-10 em comparação com os trabalhadores da área periurbana da cidade / The problems of air pollution affect all major urban centers, particularly megacities with populations greater than 10 million. Vehicular and industrial emissions stand out among the main responsible for the high concentrations of air pollutants in these urban centers. The different components of air pollution, the dose and time of exposure, can lead to different impacts on human health. This study aimed to evaluate the genotoxicity of air pollution (PM2,5 and NO2) and its correlation with changes in the methylation profile of the cytokines IL-10 and TNF-alpha in workers of São Paulo/SP occupationally exposed. The study included 57 male individuals, with age range between 28 and 66 years old, workers in daily shifts of outdoor activities in the São Paulo city and therefore occupationally exposed to air pollution. Were recruited professional of three categories: 1) traffic controllers (n=18); 2) taxi drivers (n=21) and professionals from the Forestry Institute (n=18). These workers were divided into two groups according to workplaces and exposure: 1) urban area group (UA), composed of traffic controllers and taxi drivers and 2) peri-urban area group (PUA), composed of professionals from the Forestry Institute. Individual samplers of air pollution were used for the collection of PM2,5 and NO2 pollutants. The analysis was performed by genotoxicity micronucleus test in the buccal mucosa epithelial cells and in peripheral blood lymphocytes. The methylation profile of the cytokines IL-10 and TNF-alpha was done by next generation sequencing. Our results showed a difference in PM2,5 concentration between the groups (UA=32,92ug.m-3, PUA=25,77ug.m-3; p=0,0311). No difference was found in NO2 concentrations between groups. Differences were found in the frequency of micronuclei in both buccal mucosa (UA=2,78%, PUA=1,16%; p < 0,0001) and in peripheral lymphocytes (UA=1,51%, PUA=0,73%; p < 0.0001). Difference was also found in average methylation of the IL-10 gene between the groups (UA=25%, PUA=30%; p=0,0120). There was no difference in the average methylation of TNF-alpha gene between the groups. We conclude that the workers of the urban area of the city are exposed to higher concentrations of PM2,5, have higher frequencies of micronuclei in both the buccal mucosa cells and in peripheral lymphocytes and have a hypomethylation profile of IL-10 gene in comparison with workers the peri-urban area of the city

Made available in DSpace on 2017-05-12T14:41:56Z (GMT). No. of bitstreams: 1 ROBERTO_FLORENTINO_SILVA.pdf: 2985671 bytes, checksum: a9f521c8ecec873a7cb17c8154221dcf (MD5) Previous issue date: 2013-06-14 / This dissertation seeks to analyze the socio-spatial dynamics of prostitution in the city of Francisco Beltrão, Paraná, in its earliest manifestations, in specific moments, and at present, at another time, and alterations in the forms, functions, structures and processes of this activity, here understood as an economic activity. Part of the overall scale of the sex industry in a globalized world, of international trafficking of people, and the forms of organization of this activity in the city of São Paulo, to give a more precise cut of this activity on the scale of Francisco Beltrão. It attempts to analyze the explosive mix of sex, money and prejudices just as a socioeconomic activity in the light of the categories of Economic Geography. / Este trabalho busca analisar a dinâmica sócio-espacial da prostituição no município de Francisco Beltrão, Paraná, em suas primeiras manifestações, num momento, e na atualidade, em outro momento, e as modificações ocorridas nas formas, funções, estruturas e processos dessa atividade, aqui entendida como atividade econômica. Parte da escala geral da indústria do sexo no mundo globalizado, do tráfico internacional de pessoas, das formas de organização dessa atividade na cidade de São Paulo, para dar um recorte mais preciso dessa atividade na escala do Município. Tenta analisar a explosiva mistura de sexo, dinheiro e preconceitos apenas como uma atividade sócio-econômica, à luz das categorias da Geografia Econômica.

Os problemas da poluição atmosférica atingem todos os grandes centros urbanos, em particular as megacidades com população maior do que 10 milhões de habitantes. Emissões veiculares e industriais destacam-se entre as principais responsáveis pelas altas concentrações de poluentes do ar nesses centros urbanos. Os diferentes componentes da poluição atmosférica, a dose e o tempo de exposição, podem levar a diversos impactos na saúde humana. Esse estudo teve por objetivo avaliar a genotoxicidade da poluição atmosférica (PM2,5 e NO2) e sua correlação com modificações no perfil de metilação das citocinas IL-10 e TNF-alfa, em trabalhadores da cidade de São Paulo/SP ocupacionalmente expostos. Participaram deste estudo 57 indivíduos do gênero masculino, com idades variando entre 28 e 66 anos de idade, trabalhadores em turnos diários de atividades externas na cidade de São Paulo e, portanto, ocupacionalmente expostos à poluição atmosférica. Foram recrutadas 3 categorias de profissionais: 1) controladores de tráfego (n=18); 2) taxistas (n=21) e profissionais do Instituto Florestal (n=18). Esses trabalhadores foram divididos em dois grupos em função dos locais de trabalho e exposição: 1) grupo área urbana (AU), composto pelos controladores de tráfego e taxistas e 2) grupo área periurbana (APU), composto pelos profissionais do Instituto Florestal. Amostradores individuais de poluição atmosférica foram utilizados para a coleta dos poluentes PM2,5 e NO2. A análise da genotoxicidade foi realizada pelo teste de micronúcleos nas células epiteliais da mucosa oral e em linfócitos do sangue periférico. O perfil de metilação das citocinas IL-10 e TNFalfa foi realizado por sequenciamento de nova geração. Nossos resultados mostraram uma diferença na concentração do PM2,5 entre os grupos (AU=32,92?g.m-3, APU=25,77ug.m-3; p=0,0311). Não foi encontrada diferença na concentração de NO2 entre os grupos. Foram encontradas diferenças nas frequências de micronúcleos, tanto em mucosa oral (AU=2,78%, APU=1,16%; p < 0,0001) quanto em linfócitos periféricos (AU=1,51%, APU=0,73%; p < 0,0001). Também foi encontrada diferença na metilação média do gene IL-10 entre os grupos (AU=25%, APU=30%; p=0,0120). Não foi encontrada diferença na metilação média do gene TNF-alfa entre os grupos. Concluímos que os trabalhadores da área urbana da cidade estão expostos a maiores concentrações de PM2,5, possuem maiores frequências de micronúcleos tanto em células da mucosa oral quanto em linfócitos periféricos e apresentam um perfil de hipometilação do gene IL-10 em comparação com os trabalhadores da área periurbana da cidade / The problems of air pollution affect all major urban centers, particularly megacities with populations greater than 10 million. Vehicular and industrial emissions stand out among the main responsible for the high concentrations of air pollutants in these urban centers. The different components of air pollution, the dose and time of exposure, can lead to different impacts on human health. This study aimed to evaluate the genotoxicity of air pollution (PM2,5 and NO2) and its correlation with changes in the methylation profile of the cytokines IL-10 and TNF-alpha in workers of São Paulo/SP occupationally exposed. The study included 57 male individuals, with age range between 28 and 66 years old, workers in daily shifts of outdoor activities in the São Paulo city and therefore occupationally exposed to air pollution. Were recruited professional of three categories: 1) traffic controllers (n=18); 2) taxi drivers (n=21) and professionals from the Forestry Institute (n=18). These workers were divided into two groups according to workplaces and exposure: 1) urban area group (UA), composed of traffic controllers and taxi drivers and 2) peri-urban area group (PUA), composed of professionals from the Forestry Institute. Individual samplers of air pollution were used for the collection of PM2,5 and NO2 pollutants. The analysis was performed by genotoxicity micronucleus test in the buccal mucosa epithelial cells and in peripheral blood lymphocytes. The methylation profile of the cytokines IL-10 and TNF-alpha was done by next generation sequencing. Our results showed a difference in PM2,5 concentration between the groups (UA=32,92ug.m-3, PUA=25,77ug.m-3; p=0,0311). No difference was found in NO2 concentrations between groups. Differences were found in the frequency of micronuclei in both buccal mucosa (UA=2,78%, PUA=1,16%; p < 0,0001) and in peripheral lymphocytes (UA=1,51%, PUA=0,73%; p < 0.0001). Difference was also found in average methylation of the IL-10 gene between the groups (UA=25%, PUA=30%; p=0,0120). There was no difference in the average methylation of TNF-alpha gene between the groups. We conclude that the workers of the urban area of the city are exposed to higher concentrations of PM2,5, have higher frequencies of micronuclei in both the buccal mucosa cells and in peripheral lymphocytes and have a hypomethylation profile of IL-10 gene in comparison with workers the peri-urban area of the city

Les conflits armés ne sont plus ce qu’ils étaient. D’aucuns annoncent la disparition pure et simple de ceux-ci au profit de formes inédites de violence marqués par la multiplicité des acteurs, par la multiplicité des motifs et par la généralisation de nouvelles stratégies et tactiques. Ainsi, le droit international classique opposant la situation de paix à la situation de conflit armé devient peu efficace. Le droit des droits de l’homme étant conçu pour les temps de paix alors que le droit humanitaire est aménagé pour les temps de guerre, la situation de violence reste en deçà du lex lata.Pourtant, entre ces deux situations, s’interpose des situations de violence dont le régime juridique existant n’est pas suffisamment adapté ou du moins n’est pas assez efficace pour protéger les droits des personnes affectées par les situations de violence interne. L’inadaptation des organes de contrôle du DIDH, jonchée de clauses dérogatoires, de nature plutôt réactive et lente, est complétée par des mécanismes de la responsabilité pénale individuelle peu efficace.Cette thèse ambitionne de poser les balises de la domestication de la violence par le droit, dans la perspective de la protection d’un seuil inaltérable « d’humanité ». De la violence à basse intensité à la violence à haute intensité, notre recherche prend au sérieux ces situations de violence et réfléchit, à travers une approche pragmatique et normative, sur le droit désirable et mécanismes susceptibles de réguler la zone « grise » des situations de violence. S’appuyant sur la diversité des actes de violence répertoriés dans la région des Grands Lacs, cette recherche permet de constater que ces violences ne sont pas soumises à une indétermination fondamentale. Le droit humanitaire est tantôt relayé, tantôt suppléé et tantôt supplanté par les nouveaux mécanismes régulateurs des situations de violence. / The armed conflicts are no longer, what they used to be. It is usually stated that armed conflicts have disappeared and have been replaced by other new forms of violence involving multiplicity of actors, multiplicity of motives and overlapping strategies and tactics. In fact, the classical international law makes a clear distinction between the peaceful and armed conflicts situations. The human rights law was conceived to deal with peaceful situations while humanitarian law deals with times of war. The inadequate monitoring organs of the international human rights law which comprises derogatory clauses supplemented by individual liability criminal mechanisms not effective is the gape.However, between these two situations, there is others situations of violence upon which the existing legal system is not adapted or at least, is not effective to protect the rights of internal violence victims.This thesis intends to point out the regulation if violence by means of law in order to guarantee a permanent doorway for the protection of humanity. From less intensive violence to more intensive violence, this study gives due consideration to both situations of violence and through a normative and pragmatic approach, it gives an insight into the desirable law and appropriate mechanisms which can regulate the « grey » matter resulting from violence. Basing on the variety of charges of violence committed in the Great Lakes Region, this research work shows that these types of violence are not subject to a fundamental indefinite law. The humanitarian law is relayed, replaced or supplanted by new mechanisms regulating the situations of violence.

Since 1976, Syria has suffered two separate but in nature similar rebellions which was different in terms of spreading, impact and size. The existing literature fails to compare these rebellions and answer the question on how two similar uprising can produce different outcomes. The objective of this study is to answer that question and produce a foundation that is usable when studying future uprisings in the Middle East and Syria. This is a qualitative research that uses multiple case studies that are compared in order to fulfil the objective of this study. The research if a desk study and uses an abductive approach. Two different theoretical concepts have been used in order to understand the dynamics that shaped the two rebellions. These are Frank H. Zimmerman's theory on why insurgencies fail and Frederick D. Miller's model of movement decline. The authors pinpoint different factors that makes a uprising prone to failure or success and these factors has been used as a lens when analysing the findings of this essay. The findings of this essay suggest that key differences that made an impact on the conflicts where the amount of local support, external support and the role of social media. In order to fully understand the dynamics that shapes modern rebellions, further research on the role of globalisation and social media is conflict must be conducted.

本研究從台灣學生在國際評量的結果談起，在亮麗成績的背後可以發現儘管學生有極為優異的數學與科學表現，但卻不喜歡學習或對其評價甚低，而且在學習上相當缺乏自信心，學生間的學習落差也持續擴大。在逐步的歸結中本研究排除了謙虛的文化因素使然，而是升學所造成繁重的課業壓力、冗長的在學時間與普遍的課後補習等額外壓抑(surplus-repression)所造成，它們不僅造成學生學習上的異化，更影響了學生的身心健康發展。這些可算是目前我們所須面對的教育困境之一。 技術理性社會(the society of technological rationality)會將升學視為很合算的經濟投資，而國家則將教育視為推動社會與經濟發展的一個關鍵性因素。在這樣的社會中，不僅依最有效率的方式來開發資源，更將人視為極度有待開發的「人力資源」(human resources)。這套企業化、技術化與科學性的語言與思考模式，藉由提升效能與改善生產力來取得群眾的支持與擁護；更將教育人員、知識份子和廣大經濟秩序的價值系統予以銜接。如此應運而生的技術理性教育方式，一方面協助生產並維持現存主宰經濟、政治與文化所需的知識類型，另一方面更進而合理化這套經濟和文化權力分配的模式。 在此境況之下的教育，逐漸淪為一種企業經營，講究的是管理、效益與成本，如將將企業管理的模式移植到教育行政之中，為的是以最高效率來達成既定的政策與目標；所孕育出的開發課程觀，其課程設計與發展所追求的是提升社會效率，使得課程(curricula)的意義不再是有待理解的文本或內容，而是達成既定目標的課程技術；在教學上則不思原有諸多方法的改進，而是將資訊科技視為是提升教學效率的最佳利器，使得教學成為教學工程學，而這些既是教育的困境也是教育的異化。 技術理性能成為思維的主流模式，不僅得力於其能大大提升生產力、改善民眾生活與增加社會財富，進而獲得民眾的認同與取得合法性而已，同時也得力於數學與科學為其合理性的撐腰，對此的瞭解與批判主要應歸功於Marcuse與Husserl的貢獻。此外，作為技術批判先行者的Heidegger，則對算計性思維(calculative thinking)展開追根究柢的哲學性反思，就過度張揚的主體性、對象化的世界、技術成為時代的形上學(metaphysics)也有發人深省與獨到之處。藉由前述等學者的論述，本研究展開嘗試性地探索與追問，希望有助於技術理性框架性教育的鬆解。 關鍵詞：技術理性、額外壓抑、算計性思維、框架的教育 / This study aims to analyze what lies behind Taiwanese students’ brilliant performances on international assessment. Though our students surpass in math and science, they don’t seem to enjoy learning or have enough confidence in their own learning. On the contrary, Taiwanese students show very low interest in learning. The gap between students’ academic performances among one another has been widening. Throughout the process of deduction, this study excludes the Chinese cultural element of being humble. It is believed that surplus-repression, like the heavy study load caused by entrance exams and long school hours coupled with the common phenomenon of going to cram schools after school, leads to diverse results of students’ learning as well as immense influences on both their mental and physical development. These are parts of the educational predicaments we have to face them now. The society of technological rationality considers advanced education as a good investment while the government treats education as a key component to promoting economic development. In this kind of society, the resources are used in the most efficient ways. Human beings, known as human resources, are considered potential resources waiting to be developed. This enterprise and technology driven, and scientifically thinking pattern, has gained support and popularity from the public by improving efficiency and productivity, so as to cast a link among educationalists, intellectuals, and the value system of the tremendous economic order. This generated education of technological rationality, in one way, assists production and maintains the knowledge genre that the present dominant economy, politics, and culture need. On the other hand, the education rationalizes the pattern that the economic and cultural power dominates. Under this circumstance, education has gradually become a managing business; emphasizing on management, benefits, and expense. To implement the enterprise managing pattern into education, administration is to carry out designated policy and reach goals with the best efficiency. What the thus-gestated curricula viewpoints is trying to pursue is the elevation of social efficiency with its curricula arrangement. Hence, curricula would no longer mean context or content waiting to be comprehended, but the strategies to attain appointed objectives. In teaching, the focus would switch to make the most of information technology, thus making it the greatest tool to boost teaching efficiency and to transfer teaching into teaching engineering instead of adopting traditional teaching methods to improve teaching. These’re not only the educational predicaments but also the alienations of education. The reason technological rationality has become the main stream in thinking is because it not only advances productivity excessively, shapes up public life, accumulates public wealth, and earns the public’s identity to get legitimacy, but it wins support for its rationality from math and science. Marcuse and Husserl should be honored for their devotion to figuring it out thoroughly. Nonetheless, Heidegger, the pioneer to conduct technological criticism, has initiated philosophical reflections over calculative thinking to go into whys and wherefores. He has got thought-provoking and unique opinions toward over-exaggerating subjectivity, objectized world, and technology becoming the era’s metaphysics. Based on the statements given by the previous scholars, this thesis is attempting to launch some explorations and queries, in hope of assisting the relief to the enframing education through technological rationality. keywords: technological rationality, surplus-repression, calculative thinking, enframing education

<p>Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. </p><p>The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development.</p><p>In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease.</p><p>In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. </p><p>In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.</p>

The subject of this study is the policing of the drug problem during the period between the nationalization of the police force in 1965 and the introduction of imprisonment as a penalty for drug consumption in 1993. The influence of four key factors are discussed: legislation, organization, the conception of and the attention paid to the drug problem. Qualitative and quantitative data from police archives, the prosecutor general, parliamentary publications, a public TV newsroom and various statistics from other sources are combined. The analysis shows four important stages in the process towards a substantially increased drug control: By the end of the 1960s, all data indicate increased attention to the drug problem. The penal law on narcotics was passed in 1968. It laid the foundation for subsequent policing activities. The police was also given increased possibilities to use telephone-tapping to combat serious crimes. By the end of the 1970s, the creation of a special narcotics police organization at the medium level of police hierarchy institutionalized, emphasized and assigned manpower to combat serious crimes. Since the beginning of the 1980s new target groups have been added. The "street dealer" is a new direct target. With the strengthening of the preventive role of the police new indirect target groups emerged: the police also emphasized their work against recruitment of new drug users. The main objective of the new penal law on narcotics of 1968 was to help drug addicts recover and merge back in society again. Addiction was seen as a disease. Drug dealing by the addicts was reprehensible but not a priority matter to the police. Over time, this attitude changed and in 1989 the police saw distinct actions against street dealers as the most important element in the repressive policy.

Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development. In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease. In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.

Huntington’s disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which translocates, and accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients and is consistently observed in all animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression and an understanding of the effects of mHtt may lead to strategies to slow the progression of the disease. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of these models using two N-mHtt-affected promoters in in vitro transcription assays and in two cell models of HD using techniques including overexpression of known N-mHtt-interacting transcription factors, chromatin immunoprecipitation, promoter deletion and mutation analyses and in vitro promoter binding assays. Based on our results and those in the literature, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at affected promoters. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as the observation that some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.