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Essa tese teve como objetivo avaliar todos os casos de candidose sistêmica por Candida albicans identificadas através de kit comercial ID 32C® (bioMérieux), diagnosticados no Laboratório de Micologia da Santa Casa de Misericórdia de Porto Alegre/RS, durante o período de 1999 a 2009, buscando identificar a prevalência de C. dubliniensis, bem como avaliar os fatores de virulência e diferença de perfil de suscetibilidade antifúngica entre os isolados clínicos. Foi realizado um levantamento clínico-epidemiológico dos casos incluídos no estudo, avaliando sexo, idade, manifestações clínicas, evolução, região proveniente do paciente, doença de base, condições predisponentes, utilização de corticóides e antibióticos e resposta ao tratamento recebido. Para a diferenciação das duas espécies utilizou-se testes fenotípicos (arranjo dos clamidosporos, teste de termotolerância, formação do tubo germinativo, crescimento em meio hipertônico e niger), molecular (espectrometria de massa) e genotípico (reação em cadeia da polimerase - PCR). Em adição, foi avaliada a eficácia do método de conservação das leveduras estocadas a -20ºC e comparamos quatro substratos (soro fresco, soro congelado, ágar e caldo Mueller-Hinton) para a prova do tubo germinativo. Determinou-se a produção da fosfolipase e proteinase em isolados incluídos no estudo. A atividade in vitro dos antifúngicos fluconazol, anfotericina B e anidulafungina frente aos isolados estudados foi determinada através da concentração inibitória mínima (CIM), a concentração fungicida mínima (CFM) e ponto de corte epidemiológico (ECV). Os casos de candidemia por C. albicans diagnosticados durante 10 anos ocorreram com maior frequência em pacientes adultos com presença de cateteres. Observamos que houve maior chance de ocorrência desta em pacientes oncológicos. O percentual de alta nos pacientes foi baixo. O método utilizado para a conservação de leveduras nesse estudo apresentou taxa de 70% de viabilidade. O ágar e o caldo Mueller-Hinton demonstraram sensibilidade de 90% e especificidade de 100%. Os isolados de C. albicans provenientes de hemocultivos apresentaram produção de fosfolipase em 78% e proteinase em 97% dos isolados. A espécie C. dubliniensis não foi identificada em isolados de hemocultivos, sendo todos os casos de candidemia por C. albicans. Os testes microcultivo em ágar fubá, espectrometria de massa, caldo niger e caldo hipertônico concordaram com o teste genotípico. Os isolados de C. albicans apresentaram maior suscetibilidade a anidulafungina, entretanto, os menores valores obtidos em 90% dos isolados (CIM90) foi pela anfotericina B. E através do ECV, os isolados poderiam ser resistentes ao fluconazol, demonstrando a importância da associação desses dois parâmetros. / The aim this tesis was to evaluate systemic candidiasis cases by Candida albicans through ID 32C® (bioMérieux), at Mycology Laboratory of the Santa Casa de Porto Alegre/RS, during 1999 to 2009, seeking to identify the C. dubliniensis prevalence, as well as evaluating the virulence factors and antifungal susceptibility profile difference of among isolates. The clinical and epidemiological survey was made through gender, age, clinical manifestations, evolution, patient's region, underlying disease, predisposing conditions, steroids and antibiotics use, and response to treatment. The phenotypic tests (tthermotolerance, germ tube, hypertonic and Niger medium), molecular (mass spectrometry) and genotypic (polymerase chain reaction – PCR) was used for two species identification. We also assessed if the mantainance of C. albicans stored at - 20ºC in a freezer with sterile distilled water was usefull.The four substrate (fresh and frozen serum, agar and broth Mueller-Hinton®) were used for germ tube formation and the phospholipase and proteinase activity were evaluated. The in vitro activity of fluconazole, amphotericin B and anidulafungin were compared through the minimum inhibitory concentration (MIC), the minimum fungicidal concentration (MFC) and epidemiological cutoff value (ECV). The candidemia cases by C. albicans for ten years occurred more frequently in adult and catheters use. We observed the more chance this occurrence in cancer patients. The survival percentage was low. The used method in the study for yeast stored had 70% of viability. The agar and broth Mueller-Hinton were 90% sensitivity and 100% specificity. The boodstream isolates of C. albicans produce virulence factors, such the germ tube production and hydrolytic enzymes (78% of phospholipase and 97% of protease) production. The C. dubliniensis was not identified in bloodstream isolates, thus all candidemia cases were by C. albicans. The mass spectrometry, cornmeal agar, Niger and hypertonic broth agreed with genotypic test. The isolates exhibited more susceptibility to anidulafungin, and 90% of them (MIC90) exhibited the lowest values against amphotericin B. Based on ECV and Pfaller classification, isolates could be resistant to fluconazole, demonstrating the importance of the combination of these parameters.

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Sejam f:(C^2,0) to (C^3,0) um germe de aplicação holomorfa de coposto 1 e f_t uma perturbação estável de f. Os pontos singulares de f_t são cross-caps, pontos duplos ou pontos triplos. O número de cross-caps e pontos triplos de f_t e o número de Milnor da curva de pontos duplos de f_t são invariantes do germe f. Neste trabalho estudamos fórmulas para obter estes invariantes e no caso dos germes quasi-homogêneos relacionamos estes invariantes com a A_e-codimensão de f. / Let f:(C^2,0) to (C^3,0) be a holomorphic map-germ with corank 1 and f_t a stable perturbation of f. The singular points of f_t are either cross-caps, double points or triple points. The number of cross-caps and the number of triple points of f_t and the Milnor number of the double points curve of f_t are invariants of the germs f. In this work we study formulas to get these invariants and in the case of quasi-homogeneous germs we relate these invariants with the A_e-codimension of f.

L’image du germe, du développement organique, est très importante dans l’œuvre poétique et dramatique de Claudel. Dans ses écrits en prose qui constituent la seconde partie de son œuvre, et sont principalement consacrés à l’étude de la Bible et de ses mystères, cette métaphore devient centrale. Elle est le vecteur privilégié de l’interrogation du poète sur la vocation de l’homme, le sens de sa vie, les réalités spirituelles qui le déterminent souterrainement. La semence devient plus largement le symbole d’un monde dynamique, divinement orienté vers un achèvement parfait tout en restant acteur de son développement et ainsi véritablement créateur. À travers le travail sur cette métaphore, le poète atteint ainsi un équilibre entre la valorisation de la vitalité qu’il admire et célèbre, et la recherche d’une forme parfaite, liée à la reconnaissance d’un Dieu créateur. La mise au point de ce nouvel « art poétique » passe par un travail poétique sur les images et les motifs rencontrés dans la Bible, autour notamment de l’annonce de l’Incarnation. Pour les interpréter, Claudel puise dans la Tradition chrétienne, reçue à travers la liturgie, mais aussi par la lecture des Pères de l’Église ou de théologiens comme saint Augustin et saint Thomas. Mais il exploite également les découvertes scientifiques les plus récentes, et dialogue avec des philosophies plus modernes. L’insistance sur le mouvement et la prise en compte de la spontanéité du vivant et des obscurités de l’homme qu’exprime la métaphore séminale permettent ainsi de rapprocher Claudel, malgré son apparent isolement, de ses contemporains. / The symbol of the germ, and the model of the organic development, play a very important role in Paul Claudel’s poetical and dramatical works. When the poet devoted himself to the study and the poetic commentary of the Bible in the last part of his life, this metaphor becomes central. Claudel uses it particularly in questions such as the meaning of human life and its links with spiritual realities. More broadly speaking, the image expresses the energy and the power of development contained in a world that aims at its complete achievement. Through this image, Claudel celebrates the vigor he admires in nature, and, at the same time, the perfection of a divine realisation. The reading of the Bible leads him to renew his approach of these themes. We also have to consider the influence on him of other sources, especially the Fathers of the Church, great theologists as Thomas Aquinas or Saint Augustine and other spiritual books which he frequently refers to. Nevertheless, we should not forget that he exploits as well the more recent scientific discoveries and discusses contemporary issues. The image of the germ allows Claudel to stress the dynamism of the world, the spontaneity of living things and to illustrate the mystery of man; thus, it is at the heart of his poetical world.

Abstract Approximately 5–10% of all breast cancer cases are estimated to result from a hereditary predisposition to the disease. Currently no more than 25–30% of these familial cases can be explained by mutations in the known susceptibility genes, BRCA1 and BRCA2 being the major ones. Additional predisposing genes are therefore likely to be discovered. This study evaluates whether germline alterations in three BRCA1-associated genes, BACH1 (i.e. BRIP1/FANCJ), BARD1 and TOPBP1, contribute to familial breast cancer. Altogether 214 Finnish patients having breast and/or ovarian cancer were analysed for germline mutations in the BACH1 gene. Nine alterations were observed, four of which located in the protein-encoding region. The previously unidentified Pro1034Leu was considered a possible cancer-associated alteration as it appeared with two-fold higher frequency among cancer cases compared to controls. All the other observed alterations were classified as harmless polymorphisms. Mutation analysis of the BARD1 gene among 126 Finnish patients having family history of breast and/or ovarian cancer revealed seven alterations in the protein-encoding region. The Cys557Ser alteration was seen at an elevated frequency among familial cancer cases compared to controls (p = 0.005, odds ratio [OR] 4.2, 95% confidence interval [CI] 1.7–10.7). The other alterations appeared to be harmless polymorphisms. To evaluate further the possible effect of Cys557Ser on cancer risk, a large case-control study was performed, consisting of 3,956 cancer patients from the Nordic countries. The highest prevalence of Cys557Ser was found among breast and ovarian cancer patients from BRCA1/BRCA2 mutation-negative families (p < 0.001, OR 2.6, 95% CI 1.7–4.0). In contrast, no significant association with male breast cancer, ovarian, colorectal or prostate cancer was observed. The current study is the first evaluating the role of TOPBP1 mutations in familial cancer predisposition. The analysis of 125 Finnish patients having breast and/or ovarian cancer revealed one putative pathogenic alteration. The commonly occurring Arg309Cys allele was observed at a significantly higher frequency among familial cancer cases compared to controls (p = 0.002, OR 2.4, 95% CI 1.3–4.2). The other 18 alterations observed were classified as harmless polymorphisms.

Abstract In Western countries, breast and ovarian cancer are among the most frequent malignancies affecting women. Approximately 5–10% of the cases in the general population have been suggested to be attributed to inherited disease susceptibility. BRCA1 and BRCA2 are the main genes associated with predisposition to breast and ovarian cancer. Mutations in these two genes explain a major part of the families displaying a large number of early-onset breast and/or ovarian cancers, but at least one third of the cases appear to be influenced by other, as yet unidentified genes. Therefore, it is likely that defects in other cancer predisposing genes, perhaps associated with lower disease penetrance and action in a polygenic context, will also be discovered. In the present study, the contribution of germline mutations in putative breast and/or ovarian cancer susceptibility genes, based on their biological function, has been investigated in Finnish breast cancer families. The role of large genomic deletions or other rearrangements in the BRCA1 and BRCA2 genes was evaluated by Southern blot analysis, and mutation analysis of TP53, RAD51, the BRC repeats of BRCA2, and 53BP1 was performed by conformation sensitive gel electrophoresis and DNA sequencing. Germline TP53 mutations were searched for in 108 Finnish breast cancer families without BRCA1 or BRCA2 alterations. In this study, the pathogenic TP53 germline mutation, Arg248Gln, was identified in only one family. This family showed a strong family history of breast cancer and other cancers also fulfilling the criteria for Li-Fraumeni-like syndrome. Germline TP53 mutations are expected to be found in cancer families with clinical features seen in Li-Fraumeni or Li-Fraumeni-like syndromes. In this study, large deletions in BRCA1 and BRCA2 were not observed in 82 breast and/or ovarian cancer families. Likewise, no disease-related aberrations were detected in RAD51, the BRC repeats of BRCA2 or 53BP1 in the 126 breast and/or ovarian cancer families studied. The obtained results were validated by comparing to the occurrence in 288–300 female cancer-free control individuals. These results do not support the hypothesis that alterations in these particular genomic regions play a significant role in breast cancer predisposition in Finland. Thus, there are still genes to be discovered to explain the molecular background of breast cancer.

Doctor of Philosophy / Department of Biology / Susan J. Brown / All arthropods share a segmented body plan. Detailed studies on segmentation mechanisms in the long-germ insect Drosophila melanogaster identified a segmentation hierarchy composed of maternal, gap, pair-rule, and segment polarity genes. In this hierarchy, pair-rule genes play an important role to translate gradients of regional information from maternal and gap genes into segmental expression of segment polarity genes. However, our understanding of the role of pair-rule genes in other short-germ insects and basally branching arthropods is still limited. To gain insights into the role of pair-rule genes in short-germ segmentation, I analyzed genetic interactions as well as expression patterns and functions of homologs of Drosophila pair-rule genes in the short-germ insect Tribolium castaneum. Interestingly, despite the pair-rule like expression patterns of Tribolium homologs of almost all eight canonical Drosophila pair-rule genes, only five have a segmentation function. Knock-down of primary pair-rule genes caused asegmental and truncated phenotypes while knock-down of secondary pair-rule genes caused typical pair-rule phenotypes. Epistatic analysis between the genes revealed that primary pair-rule genes form a gene circuit to prepattern a two-segmental unit, and secondary pair-rule genes are downstream targets of the gene circuit. The typical pair-rule phenotypes observed in secondary pair-rule gene RNAi embryos led to a detailed comparative analysis of the role of paired (prd) and sloppy-paired (slp) between Drosophila and Tribolium. This study revealed that prd is functionally conserved while the functional parasegmental register for Tribolium slp is opposite that of Drosophila slp. The fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning. Despite this flexibility in pair-rule patterning between Drosophila and Tribolium, segmental expression of engrailed (en) and wingless (wg) at parasegmental boundaries is conserved in both insects. Analysis of double and triple RNAi for pair-rule genes in Tribolium revealed that the primary pair-rule genes even-skipped and runt are redeployed to directly regulate en and wg with prd or slp at parasegmental boundaries. This redeployment of primary pair-rule genes seem to compensate for the apparently fewer number of functional secondary pair-rule genes in Tribolium segmentation.

Fxrα est le récepteur nucléaire des acides biliaires, exprimé majoritairement dans le foie, l'intestin, les reins et les glandes surrénales. L'intérêt pour ce dernier est devenu croissant au cours des dernières années, de part le rôle central qu'il joue dans le contrôle de l'homéostasie du cholestérol, des acides biliaires, des triglycérides ou encore du glucose. Plus récemment, Fxrα ainsi que ses ligands, les acides biliaires, ont été localisés dans le testicule, soulevant la question du rôle potentiel de Fxrα dans cet organe, et plus généralement dans la fonction de reproduction mâle. Mais les études menées à ce sujet restent jusqu'à présent peu nombreuses, et focalisées sur son implication dans le contrôle du métabolisme des stéroïdes : l'activation in vivo de Fxrα par un agoniste synthétique conduit ainsi chez l'adulte à court terme à une répression de la stéroïdogenèse. Outre son rôle dans le contrôle de l'activité endocrine des cellules de Leydig, l'impact de l'activation in vivo de Fxrα sur la physiologie plus globale du testicule n'a jamais été abordé à ce jour. De telles études seraient pourtant pertinentes étant donné que Fxrα est ciblé pour le traitement de pathologies métaboliques telles que la dyslipidémie ou le diabète. Dans ce contexte, l'objectif de ce travail de thèse était d'étudier le rôle de Fxrα dans la physiologie et la pathophysiologie du testicule, en s'appuyant sur l'analyse d'un modèle murin dont le gène codant Fxrα a été invalidé. Nos résultats démontrent que : 1) la perte de Fxrα prédispose le testicule à une sur-mortalité des cellules germinales dans un contexte pathologique de cholestase ; 2) la sur-activation de la signalisation Fxrα au cours de la puberté conduit à un défaut de la différenciation germinale, associée à une altération de la fonction endocrine du testicule ; 3) outre la régulation de la stéroïdogenèse dans les cellules de Leydig, Fxrα participe au contrôle des fonctions sertoliennes et de la prolifération et / ou différenciation des cellules germinales souches. L'ensemble de ces données définissent Fxrα comme un nouvel acteur impliqué dans le contrôle de la physiologie testiculaire et devraient être prises en considération quant-à l'utilisation de molécules agonistes et / ou antagonistes de Fxrα dans le cadre du traitement de pathologies métaboliques. / Fxrα is the bile acid nuclear receptor, predominantly expressed in liver, intestine, kidney and adrenal glands. In recent years, interest in Fxrα has been increasing due to its central role in the control of cholesterol, bile acids, triglycerides or glucose homeostasis. More recently, Fxrα and its ligands, bile acids, have been detected in the testis pointing out its potential involvement in this tissue and more widely in the male reproductive functions. However, the few studies on this topic focused essentially on Fxrα involvement in the control of steroids metabolism. Indeed, activation of Fxrα in vivo with a synthetic agonist leads to short-term steroidogenesis repression in the adult. In vivo the impact of alteration of Fxrα signaling on the global testis physiology has never been explored so far. Such studies would be pertinent considering that Fxrα is a target for the treatment of metabolic diseases such as dyslipidemia or diabetes. In this context, the aim of my work was to study the implication of Fxrα in testis physiology and physiopathology by analyzing a knock out mouse model for Fxrα. Our results show that: 1) the loss of Fxrα increase germ cell mortality in the testis in a disease context of cholestasis ; 2) over-activation of Fxrα signaling during puberty leads to germ cell differentiation defects, associated with an alteration of testis endocrine function ; 3) besides steroidogenesis control in Leydig cell, Fxrα is involved in Sertoli cell functions and spermatogonial stem cell proliferation and/or differentiation. Taken together, these data define Fxrα as a new actor involved in the control of testis physiology, and should be taken into consideration regarding the use of Fxrα agonistic or antagonistic ligands for the treatment of metabolic diseases.

Le but de ce travail était d’accroitre notre connaissance des mécanismes qui gouvernent la formation, la spécification et la différentiation des cellules souches, à l’aide du modèle métazoaire non-bilatérien Clytia hemisphaerica. Clytia possède une population particulière de cellules multipotentes appelées cellules interstitielles (i-cells). Ces cellules, présentes au cours du développement larvaire et chez la méduse adulte, sont capables de donner naissances a des cellules somatiques et aux gamètes. Chez les bilatériens, la signalisation Wnt/β-caténine (Wntβc) régule les processus développementaux fondamentaux ainsi que la prolifération, la spécification et la différenciation des cellules souches. Mon travail s’est porté sur l’implication de la signalisation Wntβc dans les dynamiques des i-cells. Mes résultats suggèrent que la signalisation Wntβc est impliquée dans la dernière étape de la différenciation de certains neurones, mais pas pour la spécification du destin cellulaire. D’autre part, j’ai aussi observé qu’en condition contrôle, la formation des i-cells est Wntβc-indépendante et probablement entraînée par le héritage de plasma germinatif contenant les ARNm localisées au pôle animal. Cependant, suite à des expériences de microdissection, j’ai observe que la reformation des i-cells dans la moitie végétative des embryons requérait l’activation de la voie Wntβc. En conclusion, deux mécanismes distincts peuvent conduire à la formation des i-cell pendant l'embryogenèse. Globalement, les résultats obtenus ont fourni une meilleure idée de la façon dont i- cellules et leurs dérivés se posent lors de l'embryogenèse et le développement larvaire. / The aim of this work was to extend our understanding of the mechanisms regulating stem cell formation, specification and differentiation by studies in the non-bilaterian metazoan model Clytia hemisphaerica. Clytia, like other hydrozoan cnidarians, possess a particular population of multipotent stem cells called interstitial cells (i-cells), present during larval development and in the adult medusa, which are able to give rise both to somatic cell types and to gametes.In bilaterian animals Wnt/β-catenin signalling regulates fundamental developmental processes such primary body axis specification, but also regulates stem cell proliferation, lineage specification and differentiation. I investigated the role of Wnt/β-catenin signalling in i-cell specification and differentiation. The results obtained suggest that Wnt/β-catenin signalling is involved in the last step of differentiation for certain neuronal cell types, but not for somatic cell fate choice. In the second part of my study I investigated the role of Wnt/β-catenin signalling in i-cell formation during embryogenesis. The results indicated that during normal development i-cell formation is Wnt/β-catenin independent and probably driven by inheritance of germ plasm containing localised mRNAs from the egg animal pole. In contrast in embryo re-patterning following embryo bisection, Wnt/β-catenin signalling appears to be necessary for de novo i-cell formation in the absence of germ plasm. Thus two distinct mechanisms can lead to i-cell formation during embryogenesis. Overall the results obtained provided a better picture of how i-cells and their derivatives arise during embryogenesis and larval development.