Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-12T13:09:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao_MitalleMatos_PPGG.pdf: 3448234 bytes, checksum: fe598459348fe967a8f1c2e5067f7d99 (MD5) / Made available in DSpace on 2016-07-12T13:09:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao_MitalleMatos_PPGG.pdf: 3448234 bytes, checksum: fe598459348fe967a8f1c2e5067f7d99 (MD5) Previous issue date: 2015-03-02 / CNPq / Danos causados por doenças virais estão entre os principais fatores limítrofes da produtividade do feijão-caupi. Em condições de estresse os fatores de transcrição (TFs) participam ativamente das etapas iniciais do processo de detecção e sinalização, regulando a expressão de vários grupos gênicos. Neste sentido, objetivou-se caracterizar TFs da família MYB e avaliar sua expressão diferencial frente à infecção viral, bem como determinar genes de referência (RGs) para normalização dos dados em RT-qPCR sob diferentes condições de estresse e controles. Por meio de análises in silico no banco NordEST, identificamos no transcriptoma do feijão-caupi um total de 86 candidatos a TF MYB, classificados em três subfamílias. A análise dos componentes estruturais do domínio R2R3-MYB permitiu observar a conservação dos aminoácidos característicos desta classe protéica em feijão-caupi. Por sua vez, o padrão de distribuição em pseudocromossomos de Phaseolus vulgaris indicou que genes MYB sofreram duplicações em tandem e intercromossomais, contribuindo para sua expansão no feijão-caupi. A análise filogenética formou 18 subclados, apoiados pela estrutura dos motivos funcionais da região C-terminal das proteínas. Das tags SuperSAGE diferencialmente expressas sob infecção viral, três foram reguladas positivamente, indicando a participação de candidatos MYB na resposta ao estresse viral. Dos sete RGs avaliados em três conjuntos experimentais, β-tubulina, Skip16 e Act2/7 + Skip16 foram as melhores combinações para seca, salinidade e vírus, respectivamente, podendo ser recomendados como normalizadores para estudos de expressão diferencial em feijão-caupi. Neste estudo identificamos a maior família de TFs em plantas observando sua participação ativa na resposta de defesa contra estresses em feijão-caupi. / Damages caused by viral diseases are among the main factors affecting the cowpea productivity. Under stress conditions, transcription factors (TFs) actively participate in the initial stages of the detection and signaling process by regulating the expression of various gene groups. In this sense, the objective of the present work was to characterize members of the MYB TF-family and evaluate their differential expression under viral infection, also determining reference genes (RGs) for data normalization in RT-qPCR under different stress and control conditions. Using in silico approaches to analyze the NordEST databank, a total of 86 MYB TF-candidates could be identified, being classified into three subfamilies. An analysis of the structural components of the R2R3-MYB domain allowed the identification of conserved amino acid residues of this protein class in cowpea. In turn, the MYB distribution pattern in the pseudochromosomes of Phaseolus vulgaris indicated that MYB members suffered in tandem and interchromosomal duplications, contributing to their expansion in cowpea. Phylogenetic analysis formed 18 subclades, supported by structural features of motifs in the C-terminal region of the protein. Of differentially expressed SuperSAGE tags under viral infection, three were upregulated, indicating the involvement of MYB candidates in response to viral stress. Considering the seven tested RGs under three experimental conditions, β-tubulin, Skip16 and Act2/7 + Skip16 were the best combinations for drought, salinity and viruses, respectively, recommended as normalizer genes in studies of differential expression in cowpea. In the present work we identified members of the largest family of TFs in plants observing their active participation in defense against stress response in cowpea.

Das Ziel dieser Arbeit war es einen geeigneten internen Standard für die linksventrikuläre mRNA-Quantifizierung bei kardialer Hypertrophie und Herzinsuffizienz in der Ratte zu finden. Die mRNA-Expression von GAPDH, 18SrRNA, Cyclophilin and Porphobilinogen-Desaminase (PBGD) wurde vier Wochen nach Induktion von Hypertrophie (kleiner aortokavaler Shunt) und Herzinsuffizienz (großer aortokavaler Shunt bzw. Myokardinfarkt) mit Hilfe des Ribonuklease Protektion Assay (RPA) und der TaqMan PCR bestimmt. Die linksventrikuläre ANP-mRNA-Expression war in allen untersuchten Modellen unabhängig von der angewendeten Detektionsmethode erhöht. Die mRNA-Expression der Housekeeping Gene mit Hilfe des RPA bestimmt, war in allen untersuchten Modellen im Vergleich zu den Kontrollen unverändert (GAPDH: kleiner Shunt: 105.1+-7.4, großer Shunt: 105.2+-6.8, MI: 88.4+-3.7; 18SrRNA: kleiner Shunt: 110.7+-8.2, großer Shunt: 104.4+-8.9, MI: 107.5+-12.0; Cyclophilin: kleiner Shunt: 96.4+-7.9, großer Shunt: 112.9+-4.9, MI: 95.7+-13.8; PBGD: kleiner Shunt: 81.9+-6.3, großer Shunt: 83.7+-4.7, MI: 79.8+-9.7; % Kontrolle). In der sehr sensitiven TaqMan PCR zeigte sich eine veränderte mRNA-Expression von GAPDH, PBGD und Cyclophilin, lediglich 18S wurde unverändert exprimiert (GAPDH: kleiner Shunt: 114.5+-18.7, großer Shunt: 133.6+-19.1, MI: 64.2+-6.2, p / The purpose of this study was to identify an appropriate left ventricular mRNA as internal standard in gene expression analysis in cardiac hypertrophy and heart failure in the rat. Expression levels of GAPDH, 18SrRNA, Cyclophilin and porphobilinogen desaminase (PBGD) were measured four weeks after induction of either cardiac hypertrophy (small aortocaval shunt) or heart failure (large aortocaval shunt or myocardial infarction) using Ribonuclease protection assay (RPA) and TaqMan PCR. The left ventricular expression of ANP mRNA was increased in all these experimental models independently of the used method. Using RPA the mRNA expression of all studied housekeeping genes was unchanged in all experimental models compared to controls (GAPDH: small shunt: 105.1+-7.4, large shunt: 105.2+-6.8, MI: 88.4+-3.7; 18SrRNA: small shunt: 110.7+-8.2, large shunt: 104.4+-8.9, MI: 107.5+-12.0; Cyclophilin: small shunt: 96.4+-7.9, large shunt: 112.9+-4.9, MI: 95.7+-13.8; PBGD: small shunt: 81.9+-6.3, large shunt: 83.7+-4.7, MI: 79.8+-9.7; % control). Using the TaqMan PCR as a much more sensitive method only 18SrRNA levels were unchanged whereas GAPDH, PBGD and Cyclophilin mRNA expression was regulated (GAPDH: small shunt: 114.5+-18.7, large shunt: 133.6+-19.1, MI: 64.2+-6.2, p

Os bastonetes Gram positivos irregulares (BGPIs) compõem um grupo de espécies bacterianas com ampla diversidade fenotípica e que podem estar presente no meio ambiente, na microbiota humana e de animais. A identificação acurada de BGPIs em nível de gênero e espécie empregando métodos bioquímicos convencionais é bastante limitada, sendo recomendado, portanto, o uso de técnicas moleculares. No presente estudo, foram identificadas amostras de BGPIs oriundas de espécimes clínicos de humanos, de produtos farmacêuticos e de áreas limpas através da análise de sequencias do gene 16S rRNA e de outros genes conservados (housekeeping genes). Os resultados obtidos pelo sequenciamento dos genes 16S rRNA e rpoB demonstraram C. striatum multi-resistente (MDR) como responsável por surto epidêmico em ambiente hospitalar da cidade do Rio de Janeiro. Quinze cepas de C. striatum foram isoladas em cultura pura a partir de secreção traqueal de pacientes adultos submetidos a procedimentos de entubação endotraqueal. A análise por eletroforese em gel de campo pulsado (PFGE) indicou a presença de quatro perfis moleculares, incluindo dois clones relacionados com cepas MDR (PFGE I e II). Os dados demonstram a predominância de PFGE I entre cepas MDR isoladas de unidades de terapia intensiva e enfermarias cirúrgicas. Uma potencial ligação causal entre a morte e a infecção por C. striatum MDR (PFGE tipos I e II) foi observada em cinco casos. Adicionalmente, acreditamos que este seja o primeiro estudo de identificação de espécies de Nocardia relacionadas com infecções humanas pela análise da sequencia multilocus (MLSA) no Brasil. Diferente dos dados observados na literatura (1970 a 2013) e obtidos pelos testes fenotípicos convencionais, a caracterização molecular de quatro lócus (gyrB-16S-secA1-hsp65) permitiu a identificação das espécies N. nova, N. cyriacigeorgica, N. asiatica e N. exalbida/gamkensis relacionadas com quadros de nocardiose em humanos. Cepas de N. nova isoladas de diferentes materiais clínicos de um único paciente apresentaram padrões de susceptibilidade antimicrobianos idênticos e dois perfis PFGE, indicando a possibilidade de quadros de co-infecção por N. nova em humanos. Em outra etapa da investigação, amostras de BGPIs obtidos de ambientes de salas limpas que não puderam ser identificadas por critérios convencionais foram submetidas a análise da sequência do gene 16S rRNA e caracterizadas 95,83% em nível de gênero e 35,42% em espécies. Para gêneros mais encontrados no estudo, foram analisados os genes rpoB e recA de dezessete cepas de Microbacterium e utilizado o MLSA para a identificação de sete cepas identificadas como Streptomyces. Os ensaios permitiram a identificação de três cepas de Microbacterium e de uma única amostra de Streptomyces ao nível de espécie. A análise da sequencia do gene rpoB também se mostrou eficaz na identificação de espécies de cepas de Corynebacterium. Finalmente, para as cepas ambientais pertencentes à classe Actinobacteria os dados morfológicos, bioquímicos e genotípicos permitiram documentar a cepa 3117BRRJ como representante de uma nova espécie do gênero Nocardioides, para o qual o nome Nocardioides brasiliensis sp. nov. e as cepas 3712BRRJ e 3371BRRJ como representante de um novo gênero e espécie para o qual o nome Guaraldella brasiliensis nov. foi proposto. / Irregular Gram positive rods (coryneform bacteria; IGPRs) comprise a group of species that has a wide phenotypic diversity and makes the conventional identification limited and that may be present in the environment, humans and animals hosts. In order to provide further accurate identification of these microorganisms in a genus and species terms it is recommended the use of molecular methods. In this study, we analyzed the 16S rRNA gene sequences and other conserved genes (housekeeping) in order to elucidate the identification of clinical isolates, pharmaceutical and clean room areas. We have documented a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. C. striatum was mostly isolated in pure culture from tracheal aspirates of adults undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE I, mostly isolated from patients of intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE I and II) infection was observed in five cases. We also performed the identification of Nocardia species of human infections by multilocus sequence analysis (MLSA) and characterized their antimicrobial and phenotypic profiles. An overview from 1970 to 2013 of the case reports on Nocardia species related to human infections, except mycetomas, in Brazil, was also accomplished. Molecular characterization by four-locus (gyrB-16S-secA1-hsp65) has provided the species identification N. nova, N. cyriacigeorgica, N. asiatica and N. exalbida/gamkensis. N. nova strains isolated from different clinical specimens of one patient showed identical antimicrobial susceptibility patterns. PFGE analysis performed to determine the genetic relatedness of these N. nova strains two distinct profiles, which were designated A and B. This is the first report on identification of Nocardia species by MLSA in Brazil. The IGPRs obtained in clean room environments that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis that allowed the identification of 95.83% at genus level and of 35.42% at the species level. The most common genus found in the clean room environments were Microbacterium and Streptomyces. The analysis of rpoB and recA genes sequence of seventeen Microbacterium strains contributed to the identification of three strains at species level. MLSA of seven Streptomyces strains identified a single sample at the species level. Moreover, the rpoB gene sequence analysis was effective in identifying Corynebacterium strains at species level. A new genus and species were found among clean room environmental strains belonging to the Actinobacteria class. Based on the morphological, genotypic and biochemical data presented in this study the 3117BRRJ strain represent a novel specie of the genus Nocardioides, for which the name Nocardioides brasiliensis sp. nov. was proposed and the 3712BRRJ and 3371BRRJ represent a new genus and species for which the name Guaraldella brasiliensis nov. were proposed.

Lui, Ching Wu Lake. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 213-225). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 論文摘要 --- p.iv / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Background of the research --- p.2 / Chapter 1.2 --- Research Site --- p.7 / Chapter 1.3 --- Objective and significance --- p.12 / Chapter 1.4 --- Research Questions --- p.15 / Chapter 1.5 --- Map of the thesis --- p.16 / Chapter Chapter 2: --- Literature Review --- p.19 / Chapter 2.1 --- Prevalent Theoretical Explanation on Household Division of Labor --- p.20 / Chapter 2.2 --- Theoretical Framework of this research --- p.29 / Chapter Chapter 3: --- Research Methodology --- p.39 / Chapter 3.1 --- Sample --- p.39 / Chapter 3.2 --- Recruitment of Couples and Data Collection --- p.42 / Chapter 3.3 --- Issues in the Interviews --- p.44 / Chapter 3.4 --- Data Analysis --- p.48 / Chapter Chapter 4: --- Does Spousal Difference in Economic Resources vary Housework Distribution? --- p.52 / Chapter 4.1 --- Nature of housework --- p.52 / Chapter 4.2 --- Who does what and more? --- p.57 / Chapter 4.3 --- Earning and Housework Division --- p.70 / Chapter Chapter 5: --- Gender Ideology and Household Division of Labor --- p.73 / Chapter 5.1 --- Overview of Couples´ة Gender Ideology --- p.73 / Chapter 5.2 --- Couples with Traditional Gender Ideology and Household Division of Labor --- p.75 / Chapter 5.3 --- Sparks of Egalitarianism in Couples --- p.94 / Chapter Chapter 6: --- Gender Strategies of Couples in Housework Division --- p.102 / Chapter 6.1 --- Husbands´ة strategies --- p.103 / Chapter 6.2 --- Women´ةs compliance --- p.109 / Chapter 6.3 --- Women´ةs resistance --- p.117 / Chapter Chapter 7: --- "Children, In-laws and Doing Gender of Couples" --- p.125 / Chapter 7.1 --- How do children escalate the doing gender of husbands and wives? --- p.125 / Chapter 7.2 --- How do parent-in-laws and parents escalate the doing gender of husbands and wives? --- p.144 / Chapter Chapter 8: --- Undoing gender: Willing househusbands and Women's liberation? --- p.165 / Chapter 8.1 --- The Context of Undoing Gender --- p.165 / Chapter 8.2 --- Comparing Househusbands and Housewives: Is gender still relevant? --- p.167 / Chapter 8.3 --- Experimenting a Househusband's life: How to undo gender? --- p.177 / Chapter Chapter 9: --- Conclusion --- p.196 / Chapter 9.1 --- Key Findings --- p.196 / Chapter 9.2 --- Implications of the study --- p.201 / Appendix 1: Labor Force Participation Rates by Sex --- p.204 / "Appendix 2: Labor Force Participation Rates at Specific Ages by Sex, 1991," --- p.205 / Appendix 3: Household division of labor of married couples in 1987，1993 and 1995 --- p.206 / Appendix 4: Relative Share of Housework of Economically Active Men and Women --- p.207 / Appendix 5: Interview Schedule --- p.208 / Bibliography --- p.213

Os bastonetes Gram positivos irregulares (BGPIs) compõem um grupo de espécies bacterianas com ampla diversidade fenotípica e que podem estar presente no meio ambiente, na microbiota humana e de animais. A identificação acurada de BGPIs em nível de gênero e espécie empregando métodos bioquímicos convencionais é bastante limitada, sendo recomendado, portanto, o uso de técnicas moleculares. No presente estudo, foram identificadas amostras de BGPIs oriundas de espécimes clínicos de humanos, de produtos farmacêuticos e de áreas limpas através da análise de sequencias do gene 16S rRNA e de outros genes conservados (housekeeping genes). Os resultados obtidos pelo sequenciamento dos genes 16S rRNA e rpoB demonstraram C. striatum multi-resistente (MDR) como responsável por surto epidêmico em ambiente hospitalar da cidade do Rio de Janeiro. Quinze cepas de C. striatum foram isoladas em cultura pura a partir de secreção traqueal de pacientes adultos submetidos a procedimentos de entubação endotraqueal. A análise por eletroforese em gel de campo pulsado (PFGE) indicou a presença de quatro perfis moleculares, incluindo dois clones relacionados com cepas MDR (PFGE I e II). Os dados demonstram a predominância de PFGE I entre cepas MDR isoladas de unidades de terapia intensiva e enfermarias cirúrgicas. Uma potencial ligação causal entre a morte e a infecção por C. striatum MDR (PFGE tipos I e II) foi observada em cinco casos. Adicionalmente, acreditamos que este seja o primeiro estudo de identificação de espécies de Nocardia relacionadas com infecções humanas pela análise da sequencia multilocus (MLSA) no Brasil. Diferente dos dados observados na literatura (1970 a 2013) e obtidos pelos testes fenotípicos convencionais, a caracterização molecular de quatro lócus (gyrB-16S-secA1-hsp65) permitiu a identificação das espécies N. nova, N. cyriacigeorgica, N. asiatica e N. exalbida/gamkensis relacionadas com quadros de nocardiose em humanos. Cepas de N. nova isoladas de diferentes materiais clínicos de um único paciente apresentaram padrões de susceptibilidade antimicrobianos idênticos e dois perfis PFGE, indicando a possibilidade de quadros de co-infecção por N. nova em humanos. Em outra etapa da investigação, amostras de BGPIs obtidos de ambientes de salas limpas que não puderam ser identificadas por critérios convencionais foram submetidas a análise da sequência do gene 16S rRNA e caracterizadas 95,83% em nível de gênero e 35,42% em espécies. Para gêneros mais encontrados no estudo, foram analisados os genes rpoB e recA de dezessete cepas de Microbacterium e utilizado o MLSA para a identificação de sete cepas identificadas como Streptomyces. Os ensaios permitiram a identificação de três cepas de Microbacterium e de uma única amostra de Streptomyces ao nível de espécie. A análise da sequencia do gene rpoB também se mostrou eficaz na identificação de espécies de cepas de Corynebacterium. Finalmente, para as cepas ambientais pertencentes à classe Actinobacteria os dados morfológicos, bioquímicos e genotípicos permitiram documentar a cepa 3117BRRJ como representante de uma nova espécie do gênero Nocardioides, para o qual o nome Nocardioides brasiliensis sp. nov. e as cepas 3712BRRJ e 3371BRRJ como representante de um novo gênero e espécie para o qual o nome Guaraldella brasiliensis nov. foi proposto. / Irregular Gram positive rods (coryneform bacteria; IGPRs) comprise a group of species that has a wide phenotypic diversity and makes the conventional identification limited and that may be present in the environment, humans and animals hosts. In order to provide further accurate identification of these microorganisms in a genus and species terms it is recommended the use of molecular methods. In this study, we analyzed the 16S rRNA gene sequences and other conserved genes (housekeeping) in order to elucidate the identification of clinical isolates, pharmaceutical and clean room areas. We have documented a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. C. striatum was mostly isolated in pure culture from tracheal aspirates of adults undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE I, mostly isolated from patients of intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE I and II) infection was observed in five cases. We also performed the identification of Nocardia species of human infections by multilocus sequence analysis (MLSA) and characterized their antimicrobial and phenotypic profiles. An overview from 1970 to 2013 of the case reports on Nocardia species related to human infections, except mycetomas, in Brazil, was also accomplished. Molecular characterization by four-locus (gyrB-16S-secA1-hsp65) has provided the species identification N. nova, N. cyriacigeorgica, N. asiatica and N. exalbida/gamkensis. N. nova strains isolated from different clinical specimens of one patient showed identical antimicrobial susceptibility patterns. PFGE analysis performed to determine the genetic relatedness of these N. nova strains two distinct profiles, which were designated A and B. This is the first report on identification of Nocardia species by MLSA in Brazil. The IGPRs obtained in clean room environments that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis that allowed the identification of 95.83% at genus level and of 35.42% at the species level. The most common genus found in the clean room environments were Microbacterium and Streptomyces. The analysis of rpoB and recA genes sequence of seventeen Microbacterium strains contributed to the identification of three strains at species level. MLSA of seven Streptomyces strains identified a single sample at the species level. Moreover, the rpoB gene sequence analysis was effective in identifying Corynebacterium strains at species level. A new genus and species were found among clean room environmental strains belonging to the Actinobacteria class. Based on the morphological, genotypic and biochemical data presented in this study the 3117BRRJ strain represent a novel specie of the genus Nocardioides, for which the name Nocardioides brasiliensis sp. nov. was proposed and the 3712BRRJ and 3371BRRJ represent a new genus and species for which the name Guaraldella brasiliensis nov. were proposed.

The purpose of this study is to determine to what extent the homemaking responsibilities some fourth-grade children assume affect the way in which they perform their school responsibilities.

Les chimiokines sont des protéines appartenant à la famille des cytokines. A l'origine, ces molécules ont été étudiées pour leur implication dans la régulation du trafic leucocytaire. Depuis une dizaine d'années, de nombreuses études ont cependant démontré que les chimiokines sont aussi impliquées dans les différentes étapes de la cancérogénèse : inhibition de l'immunité anti-tumorale, rôle de facteur de croissance des cellules tumorales, régulation de l'angiogénèse et particulièrement induction de la migration des métastases. En effet, les chimiokines sont capables de guider les cellules métastatiques de la tumeur primaire vers leur lieu d'implantation. Ce guidage passe par la liaison de la chimiokine à son récepteur, ce qui conduit à l'activation de différentes cascades signalétiques intracellulaires qui aboutiront in fine à la migration et le guidage des cellules le long du gradient de chimiokine, et ce jusqu'au lieu d'implantation où une tumeur secondaire pourra alors se former.C'est la compréhension du rôle des chimiokines dans la migration des métastases qui a conduit à l'étude de l'expression de leurs récepteurs dans le cancer. Après avoir étudié dans un premier temps les outils nécessaires à l'obtention de résultats fiables notamment en PCR quantitative, le profil d'expression de différents récepteurs aux chimiokines a été analysé chez des patients atteints d'un cancer colorectal. Les résultats montrent que seules les expressions des récepteurs CCR10 et CXCR4 diminuent chez les patients ayant développé des métastases. De plus cette baisse est corrélée à un mauvais pronostique. Cependant, des études in vitro démontrent que les lignées coliques métastatiques surexpriment ces récepteurs, et que la migration de celles-ci est sensible à un gradient de CCL27, le ligand du récepteur CCR10.Ces résultats a priori paradoxaux prennent tout leur sens lorsqu'on rapproche le concept métastatique du concept de cellule souche. Dans les deux cas, ces types cellulaires aux capacités phénotypiques communes (migration, survie, prolifération) peuvent donner une population cellulaire hétérogène. La mise au point d'une méthode permettant de faire passer les cellules tumorales (prolifératives) en cellules métastatiques (migratoires) et vice versa nous a conduit à un concept syncrétique dans lequel les notions de cellules tumorales, de métastases et de cellules souches se confondent. Dans celui-ci, les cellules tumorales, qu'elles soient ou non issues de cellules souches saines, sont capables de changer réversiblement de phénotype pour former des métastases (équivalent de la transition épithélio-mésenchymateuse). La notion de réversibilité est ici essentielle car elle permet d'expliquer comment une cellule métastatique, une fois implantée, peut s'activer et redonner une population cellulaire tumorale proliférative. Cela implique aussi que les métastases, capables de rester plusieurs mois en dormance, partagent les mêmes mécanismes que les cellules souches.Enfin, l'étude en parallèle de nouvelles voies de régulation de l'angiogénèse - qui fournit aux métastases le moyen de quitter la tumeur primaire - faisant intervenir le récepteur à la thrombine PAR1 d'une part et la chimiokine CXCL4, plus communément appelée PF4 d'autre part, pourrait aboutir à l'élaboration de nouvelles molécules à visée thérapeutique. / Chimiokines are proteins belonging to the cytokines' family. In the beginning, chemokines were studied for their implication in the leucocytic trafic regulation. Since ten years, many studies however show that chemokines are also implied in all of carcinogenesis steps: antitumor immunity inhibition, role of growth factor of tumoral cells, angiogenesis regulation and especially metastases migration induction. Indeed, chemokines are able to guide metastatic cells from the primary tumour towards their secondary site thanks to chemokine / chemokine receptors interactions which lead to metastases migration along a chemokine gradient until their secondary site. Then, metastases activation will lead to the development of a secondary tumour.It is the understanding of chemokines' role in metastases migration that led to the study of their receptors expression in cancer. After having initially studied the tools that were necessary to obtain reliable results in particular in quantitative PCR, expression profile of several chemokine receptors was established on patients developing a colorectal cancer. Results show that only CCR10 and CXCR4 expressions significantly decrease in patients who develop metastases. Moreover this decrease is correlated with poor prognostic. However, in vitro studies show that metastatic colon cell lines overexpress the both receptors, and that migration of those cells is sensitive to a CCL27 gradient, the CCR10 ligand.These paradoxical results may however be easily explained if the metastatic concept is compared to the stem cell one. In both cases, these cellular types share phenotypical properties (migration, survival, proliferation) and are able to give a heterogeneous cellular population. The development of a protocol that turns proliferative tumoral cells into migratory metastatic ones (and vice versa) led us to a syncretic concept in which tumoral cells, metastases and stem cells notions are merging. In this concept, tumoral cells (which result or not from healthy stem cells), are able to reversibly change their phenotype to form metastases (‘epithelial-mesenchymal transition' equivalent). Reversibility is actually an essential concept because it may explain how a metastatic cell, once established, can be activated again and become a proliferative tumoral cell. That also implies that metastases, able to remain several months in dormancy, share the same mechanisms as stem cells ones.Lastly, the study of new angiogenesis regulation pathways - which provides to metastases the means of leaving the primary tumour - involving on the one hand PAR1 thrombin receptor, and on the other hand chemokine CXCL4 (also called PF4), may lead to the development of new therapeutic molecules.

Autoportante is a collection of stories of human architectures designed for public engagement, a study of the sense of responsibility people feel in appreciating, using and mantaining public and private structures that provide us with support. Hence, the word autoportante is defined by me as “one’s own automatic ability to care for support structures in society”. My research question being: does it come automatic for people to appreciate and care for support structures?  I have studied human behaviours towards supportive elements in society; objects, places and services (people) that help us in our everyday life. I asked myself whether there is a collective need to relate differently to these, by carrying notions of support structures, politics of care, housekeeping and hospitality throughout different design iterations.

Quantifizierung des postmortalen RNA-Status im Gehirn mittels Real-time-PCR: Ein Beitrag zur Bestimmung der Leichenliegezeit Der postmortale Nukleinsäureabbau verläuft unterschiedlich: während DNA im Allgemeinen als stabil angesehen wird und erst mit Einsetzen von Fäulniserscheinungen stärkerer Degradation unterliegt, wird RNA mit dem Sistieren der Kreislauftätigkeit relativ rasch abgebaut. Eine Reihe von Studien hat aber gezeigt, dass RNA in bestimmten Geweben eine höhere Stabilität besitzt als ursprünglich angenommen. Dies könnte Bedeutung für die molekulare Medizinforschung besitzen, die auf Genexpressionsstudien in postmortalem Gewebe angewiesen ist. Außerdem könnte eine Quantifizierung der RNA-Degradation z.B. durch Real-time-PCR zur Eingrenzung der Leichenliegezeit genutzt werden. In dieser Studie wurde ein quantitativer Vergleich verschiedener sog. Haushaltsgene (u.a. GAPDH, ß-Actin, FASN) in Gehirngewebe mit einer Leichenliegezeit zwischen 0 und 96 Stunden und unter alternativen Ansätzen zur reversen Transkription (oligo-(dT)-Primer mit und ohne sog. Anker, Random Hexamer Primer) durchgeführt. Zunächst erfolgten systematische Untersuchungen zur Effektivität der RNA-Isolierung, reversen Transkription und der PCR im Hinblick auf eine möglichst präzise Quantifizierung. Es zeigte sich, dass die Resultate der Real-time-PCR ein Maß für die ursprünglich in der Probe vorhandene mRNA-Menge darstellen. Weiterhin stellte sich heraus, dass eine deutliche und evtl. auch zur Liegezeitbestimmung nutzbare RNA-Degradation erst nach 24h einsetzt. Ein wesentlicher Unterschied zwischen Random- und oligo-(dT)-priming der reversen Transkription war dabei nicht festzustellen. Diese Ergebnisse belegen zum einen, dass RNA im frühen postmortalen Intervall relativ stabil ist und als Substrat für quantitative Untersuchungen dienen kann, zum anderen, dass ein zeitabhängiger Abbau besteht, der eine Eingrenzung der Leichenliegezeit z.B. mittels Grenzwerten in ein frühes und mittleres Postmortalintervall zulässt. / Quantification of the postmortem RNA-status in human brain by means of real-time-PCR: A contribution to the determination of the postmortem interval The postmortem degradation of nucleic acid proceeds differently: whereas DNA is generally considered as stable and is only subject to stronger degradation with the beginning of putrefaction, RNA degrades very fast when the circulation is suspended. A series of studies, however, has shown that RNA has a greater stability in certain tissues than originally expected. This could be important for molecular medical research which is dependent on gene expression studies using postmortem tissue. Furthermore, the quantification of RNA-degradation by means of real-time-PCR could be used for the limitation of the postmortem interval. In this study, a quantitative comparison has been made between different so-called housekeeping-genes (e.g. GAPDH, ß-Actin, FASN) in human brain and a postmortem interval between 0 and 96 hours. Different alternative approaches have been used for the reverse transcription (oligo-(dT)-Primer with and without Anker, Random Hexamer Primer). At first, systematic examinations concerning the effectiveness of RNA isolation, reverse transcription and PCR have been undertaken with regard to a preferably exact quantification. It turned out that the results of the real-time-PCR represent a measure for the mRNA amount originally present in the specimen. Moreover, it emerged that a clear RNA degradation, which could possibly be used for the determination of the postmortem interval, begins after 24 hours. An important difference between random and oligo-(dT) priming of the reverse transcription could not be stated. These results demonstrate, on the one hand, that RNA is relatively stable during the early postmortem interval so that it can serve as substrate for quantitative examinations. On the other hand, it is shown that a time-dependent degradation exists which allows a limitation of the postmortem interval into an early and middle postmortem interval by means of threshold values for example.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Este trabalho tem como objetivo avaliar a estabilidade de genes de referÃncia e a expressÃo das proteÃnas morfogenÃticas Ãsseas (BMP-2, 4, 6, 7 e 15), seus receptores (BMPR-IA, IB e II) e seus mensageiros intracelulares (SMADs-1, 5 e 8) em folÃculos caprinos antes e apÃs cultivo por 18 dias. Para avaliar a estabilidade dos genes de referÃncia e o nÃvel de expressÃo das BMPs, receptores e SMADs, folÃculos com aproximadamente 0,2, 0,5 e 1 mm foram isolados mecanicamente de ovÃrios caprinos. AlÃm disso, folÃculos com aproximadamente 0,2 mm foram isolados e cultivados por 18 dias em meio de cultura suplementado com FSH. ApÃs a extraÃÃo do RNA total e sÃntese de cDNA, foi realizada a quantificaÃÃo do RNAm, por PCR em tempo real, utilizando-se primers especÃficos para genes de referÃncia (β-actina, PGK, GAPDH, β-tubulina, UBQ, RPL-19, rRNA18S), e para as BMPs (2, 4, 6, 7 e 15) receptores de BMPs (BMPR-IA, IB e II) e SMADs (1, 5 e 8). Os resultados mostraram que β-tubulina e PGK sÃo os genes de referÃncia mais estÃveis em folÃculos frescos prÃ-antrais e antrais caprinos. Os RNAs mensageiros para as BMPs (2, 4, 6, 7 e 15), seus receptores (BMPR-IA, IB e II) e SMADs (1, 5 e 8) sÃo expressos em diferentes nÃveis em folÃculos prÃ-antrais e antrais caprinos, sendo que a expressÃo do RNAm para BMP-4, BMP-6 e BMP-7 em folÃculos de 1 mm sÃo significativamente maiores do que em folÃculos de 0,2 e 0,5 mm. Entretanto, os nÃveis de RNAm para BMP-2 foi reduzido em folÃculos de 1 mm, jà os nÃveis de BMP-15 nÃo diferiram entre as categorias foliculares analisadas. Os nÃveis de RNAm para BMPR-IB foram maiores em folÃculos de 0,2 mm do que em folÃculos de 0,5 e 1 mm, enquanto que o RNAm para BMPR-II foi significativamente maior em folÃculos de 0,5 mm do que em folÃculos de 0,2 e 1 mm. Por outro lado, nÃveis de RNAm para BMPR-1A nÃo diferiram entre folÃculos analisados. Os nÃveis de RNAm para SMAD-5 foram significativamente maiores em folÃculos de 0,2 mm do que em folÃculos de 0,5 e 1 mm. Contudo, folÃculos de 0,5 mm mostraram nÃveis maiores de RNAm para SMAD-8 do que folÃculos de 0,2 e 1 mm. Os nÃveis de RNAm para SMAD-1 nÃo diferiram entre os folÃculos. ApÃs as comparaÃÃes dentro de cada categoria folÃcular, BMP-15 foi mais expressa do que BMP-7 em folÃculos de 0,2 e 0,5 mm. Em folÃculos de 0,5 mm a expressÃo do BMPR-IB foi maior do que BMPR-II. Em todas as trÃs categorias foliculares estudadas, a expressÃo da SMAD-5 foi superior a SMAD-8. ApÃs o cultivo, os folÃculos apresentaram reduÃÃo dos nÃveis de RNAm para BMP-2, BMP-4, BMP-7, BMPR-IA e SMAD-5. Em conclusÃo, β-tubulina e PGK sÃo os dois genes housekeeping mais estÃveis para folÃculos frescos caprinos com 0,2, 0,5 e 1 mm de diÃmetro. BMPs, seus receptores e SMADs apresentam padrÃes de expressÃo especÃficos em cada categoria folicular estudada. No entanto, em folÃculos cultivados hà uma variaÃÃo na expressÃo dos componentes do sistema BMP, diferindo da expressÃo in vivo de folÃculos com o mesmo tamanho. / The aims this study to evaluate the stability of reference genes and the expression of bone morphogenetic protein (BMP-2, 4, 6, 7 and 15), their receptors (BMPR-IA, IB and II) and intracellular messengers (SMADs- 1, 5 and 8) in goat follicles before and after culture for 18 days. To evaluate the stability of reference genes and the expression of BMPs, receptors and SMADs, follicles of approximately 0.2, 0.5 and 1 mm were mechanically isolated from goats ovaries. In addition, approximately 0.2 mm follicles were isolated and cultured for 18 days in culture medium supplemented with FSH. Both fresh and cultured follicles were subjected to total RNA extraction and synthesis of cDNA, the quantification of mRNA was carried out by real-time PCR using specific primers for genes of reference (GAPDH, β-tubulin, β-actin, PGK, UBQ, RPL - 19, rRNA18S) and BMPs (2, 4, 6, 7 and 15) receptors of BMPs (BMPR-IA, IB and II) and SMADs (1, 5 and 8). Results showed that β-tubulin and PGK are the most stable reference genes in goats preantral and antral follicles. The messengers RNA for BMP (2, 4, 6, 7 and 15), their receptors (BMPR-IA, IB and II) and Smads (1, 5 and 8) are expressed at different levels in preantral and antral goats, and mRNA expression for BMP-4, BMP-6 and BMP-7 in 1-mm follicles are significantly higher than in follicles of 0.2 and 0.5 mm. However, the levels of mRNA for BMP-2 were reduced in follicles 1 mm, as BMP-15 did not differ between follicular categories. The levels of mRNA for BMPR-IB were higher in follicles of 0.2 mm than in follicles of 0.5 and 1 mm, whereas the mRNA for BMPR-II was significantly higher in follicles than 0.5 mm in follicles of 0.2 to 1 mm. Moreover, mRNA levels for BMPR-1A did not differ between follicles examined. The levels of mRNA for SMAD-5 were significantly higher in 0.2 mm follicles than in follicles of 0.5 and 1 mm. However, follicles of 0.5 mm showed higher levels of mRNA for SMAD-8 than follicles 0.2 and 1 mm. The levels of mRNA for SMAD-1 did not differ between follicles. After the comparisons within each category follicle, BMP-15 expression was higher than BMP-7 in follicles between 0.2 and 0.5 mm. Follicles 0.5 mm in the expression of BMPR-IB was greater than BMPR-II. In all three follicular categories studied, the expression of SMAD-5 was superior to SMAD-8. After culture, follicles showed reduced levels of mRNA for BMP-2, BMP-4, BMP-7, BMPR-IA and SMAD-5. In conclusion, β-tubulin and PGK genes are the two most stable housekeeping for fresh goat follicles 0.2, 0.5 to 1 mm in diameter. BMPs, their receptors and SMADs have specific expression patterns in each category follicular studied. However, in cultured follicles showed a variation in the variation in the expression of BMP system components, differing from in vivo expression of follicles with the same size.