No description available.

No description available.

Keimzellen entwickeln sich aus den sogenannten Urkeimzellen, die auch als primordiale Keimzellen (PGC) bezeichnet werden. PGC entwickeln sich während der Embryonalentwicklung und wandern dann in die Gonadenanlagen, wo sie sich vermehren und bei getrenntgeschlechtlichen Arten in Spermien in Männchen und Oozyten bei Weibchen differenzieren. Während der Geschlechtsdifferenzierung sind die Gonaden anfällig für den Einfluss externer Faktoren, wie z. B. der Wassertemperatur. Eine Erhöhung der Wassertemperatur von 28°C auf 36°C während der kritischen Phase der Geschlechtsdifferenzierung, vom 10. bis zum 20. Tag nach der Befruchtung kann zu einer Vermännlichung genetisch-weiblicher Tilapien führen. In der ersten Studie führte eine entsprechende Temperaturbehandlung bei genetisch weiblichen Tilapien zu einem Anteil funktioneller Männchen von 37%. Makromorphologisch konnten die Gonaden 90 Tage alter Weibchen aus Kontroll- und Behandlungsgruppen als unreife Ovarien klassifiziert werden. Erste Reifungsprozesse bis hin zur Oogenese begannen 120 Tage nach der Befruchtung. Die Oogenese konnte mikro-morphologisch weiterhin in folgende Stadien eingeteilt werden: Chromatin Nukleolus, Peri-Nukleolus, kortikale Alveolus, Vitellogenese und reife Eizelle. Oozyten in den Phasen des Chromatin Nukleolus und des Peri-Nukleolus, welche auch die primäre Wachstumsphase darstellen, wurden bei weiblichen Fischen aller Altersgruppen festgestellt. Während weiter fortgeschrittenen Oozytenstadien erst ab einem Alter von 120 Tagen festgestellt werden konnten. Oozyten in der Phase des kortikalen Alveolus wurden demnach frühestens am 120. Lebenstag gefunden. In der Vitellogenese befindliche Oozyten traten erst nach 150 Tagen auf. Reife Eizellen wurden ab einem Alter von 180 dpf festgestellt. Es konnten keine signifikanten Unterschiede in der Eizellentwicklungsstadien zwischen Kontroll- und Behandlungstieren festgestellt werden. Das Ziel der zweiten Studie war die Identifikation von Keimbahn-Stammzellen bei Tilapien, die während der Geschlechtsdifferenzierung verschiedenen Aufzuchttemperaturen ausgesetzt waren. Die Identifizierung der Keimbahn-Stammzellen erfolgte anhand von Immunohistochemie mittels Vasa und PCNA (Proliferierendes Cell Nuclear Antigen) Antikörperfärbung. Es wurden zunächst histologische Schnitte von in Paraffin eingebettetem Ovargewebe hergestellt. Keimbahn-Stammzellen wurden im Keimepithel der Ovarien identifiziert, wobei diese in einzelner, isolierter oder in Clusterform vorlagen. Keimbahn-Stammzellen wurden sowohl bei weiblichen Tieren aus Kontroll- als auch Behandlungsgruppen identifiziert. Zusammenfassend, konnten erstmalig Keimbahn-Stammzellen und ihre Lage in den Gonaden genetisch weiblicher Tilapien, aus Kontroll- und Behandlungsgruppen, mittels Vasa und PCNA Antikörperfärbung charakterisiert werden.

"Mémoire présenté à la faculté des études supérieures en vue de l'obtention du grade de maîtrise en droit (LL.M.) option droit, biotechnologies et société" / Le transfert d'ooplasme est une nouvelle technique de reproduction (NTR) qUI bouscule les fondements utilisés pour encadrer les modifications génétiques chez l'humain. Par l'intervention dans le matériel génétique contenu dans les mitochondries des cellules, ce nouveau procédé implique la création d'enfant issus du matériel génétique de trois parents. L'exemple est intéressant en ce qu'il permet à la fois d'analyser une situation spécifique aux enjeux éthiques et sociaux considérables, mais également de poser une réflexion plus générale sur les modes d'encadrement des NTR et leur impact sur la liberté de la recherche scientifique. Les théories sociologiques issues de l'analyse de la technoscience permettent de démontrer d'une part un enthousiasme pour la recherche et d'autre part les craintes de sa dérive. L'hypothèse du pluralisme normatif, issue de ces craintes et de l'incapacité du droit à parvenir à les calmer, permet de mettre en lumière la multiplication des normes destinées à encadrer la recherche scientifique. Cette pléthore de normes est responsable d'une confusion dans l'interprétation des différents principes qui les justifient (dignité humaine, innocuité, bienfait thérapeutique), d'autant plus qu'elles doivent être conciliées entre les niveaux international, régional et national. Cette réflexion éthique sur la limitation de la liberté de la recherche par l'encadrement des NTR permet la démonstration des véritables enjeux qu'impliquent la génétique de la reproduction et propose un regard neuf sur la façon de l'envisager. / Ooplasm transfer is a new reproductive technique that jostles the basis of human gene modification. This new fertility treatment involved the transplantation of genetic material included in mitochondrion, and results in new-born with DNA from three different persons. This technique brings important sociological and ethical dilemmas. It also raises a critical discussion on how new reproductive techniques are regulated and how that regulation limits the freedom of research. Sociological theories about technosciences have shown that there is a great enthusiasm for research in society, but also great concerns on its excess. Those concerns have generated a multiplication of norms in order to control possible abuses of researchers. The multiplication of norms limits not only the freedom of research, but is also responsible for the confusion in interpreting the principles that justify them (human dignity, innocuity, health benefits), especial1y when these principles have to be reconciled at the national, regional and international level. This study is an ethical reflection on limits imposed on the freedom of research in the new reproductive genetics area. By using ooplasmic transfer as an example, this work addresses main issues of reproductive genetic and proposes a new way of understanding and considering genetics in the socio-economical context of technoscientific societies.

Em mamíferos, a espermatogênese é mantida pela autorrenovação e diferenciação das células-tronco espermatogoniais (SSC). Apesar da grande importância do SSC para a fertilidade masculina, em Bos taurus pouco se sabe sobre a sua identificação e biologia celular. Para roedores, mais de 30 marcadores para células germinativas indiferenciadas já foram descritos. No entanto, ainda não é conhecido um marcador específico apenas para SSC. Quase todos são também expressos por gonócitos, espermatogônias mais diferenciadas ou mesmo células somáticas. Yin Yang 2 (YY2) é um factor de transcrição expresso nas células com a morfologia de gonócitos e SSC, sendo um candidato a marcador de SSC. Assim, a identificação de novos marcadores para SSC e factores que afectam a sua expressão, tais como a idade, são fundamentais para o desenvolvimento da biotecnologia como transgenia e tratamento de infertilidade, nos quais as SSC poderiam ser ferramentas biológicos importantes. Assim, nesta tese temos duas hipóteses principais: 1) a idade do dador afeta a expressão de marcadores moleculares específicos de SSC bovinas assim como potencial de células-tronco dessas células e que as sequências de DNA em que se associa YY2 regulam a expressão génica de SSC em camundongos. Os objetivos específicos, organizados em 4 artigos científicos, foram: identificar a melhor plaqueamento diferencial para enriquecer SSC bovina (artigo 1), verificar se a expressão de marcadores moleculares de SSC bovina difere entre adultos pré-púberes (artigo 2 e 3), identificar novos marcadores específicos para SSC em Bos taurus (artigo 3), verificar que a idade afeta o potencial de célula-tronco de SSC bovinas (artigo 3), descrever YY2 como um marcador específico para SSC em camundongos e verificar se as sequências DNA associadas YY2 são loci de importância para SSC. Assim, definimos o melhor plaqueamento diferencial para o enriquecimento de SSC bovinas, que idade afeta a expressão marcadores já estabelecidos assim como genes específicos do transcriptoma de SSC bovinas e que idade também afeta o seu potencial de células-tronco (ensaio de repopulação). Concluímos também que YY2 é um marcador para SSC de camundongo em cultivo, em animais adultos e que as sequências do genoma que se associam YY2 possivelmente tem capacidade de regulação génica em SSC murina. / Mammalian spermatogenesis is sustained by self-renewal and differentiation of spermatogonial stem cells (SSC). Despite the importance of the SSC for male fertility, in Bos taurus líttle is known about their identification identity and cell biology. For rodents, more than thirty markers for undifferentiated germ cells have already been described. However, none of these represents a marker specific for SSC, as most are also expressed in gonocytes, differentiated spermatogonia or even somatic cells. Yin Yang 2 (YY2) is a transcription factor specifically expressed in cells with the morphology of gonocytes and SSC in the mouse, being a candidate marker for SSC. For the use of SSC as an important biological tool in the development of biotechnology such as transgenesis and the treatment of infertility, it is important to identify new markers for SSC and factors that affect their expression, such as the age of donors. Therefore, the experimental work described in this thesis was based on two main hypothesis: (1) the donor age affects the expression of specific molecular markers in SSC as well their potential as stem cells and 2) YY2 exerts important functions in SSC and genomic targets correspond to loci relevant for gene regulation or genome management in SSC. The specific goals have been organized in 4 manuscripts as follows: optimization of differential plating to enrich for bovine SSC (Article 1), check if the expression of molecular markers of bovine SSCs differs between prepubertal and adult donors (Article 2 and 3), identify new markers specific for SSC in Bos taurus (Article 3), continuation of the initial description of YY2 as a specific marker for SSC in mice and check if sequences bound by YY2 in vivo harbor the capacity to influence gene expression in SSC. As conclusions, we present an optimized differential plating protocol for the enrichment of bovine SSC, we conclude that age effects the expression of SSC markers, the expression of specific genes of bovine SSC and that age also affects their potential as stem cells (measured in repopulation assays). We also contribute to the description of the restricted expression of YY2 in prepuberal and adult SSC in mice and we show that YY2 binding sites represent genomic sequences relevant for control of gene expression.

L’huître creuse Crassostrea gigas est un mollusque hermaphrodite alternatif dont le cycle dereproduction est annuel. Sa gamétogenèse est soutenue par des réserves énergétiques, stockées dansun tissu conjonctif de réserve entourant la gonade. Des travaux antérieurs ont montré l’implication dusystème insuline dans ce processus liant étroitement alimentation, énergie stockée et gamétogenèse.Le fonctionnement des étapes précoces de gamétogenèse reste encore méconnu chez l’huître. Cetravail a permis d’identifier les cellules germinales souches (GSC) et les progéniteurs potentiels en sebasant sur une approche d’histologie quantitative couplée au marquage des cellules germinales par unanticorps homologie anti-Oyvlg (Oyster vasa-like gene). Certains éléments constitutionnels de la nichegerminale ont également été identifiés, notamment la présence d’une cellule somatique associée à lacellule germinale souche potentielle qui présente un marquage par un anticorps hétérologue anti-BMP2/4. En amont de l’étude concernant la régulation des étapes précoces de gamétogenèse par lesvoies des insulines, le criblage in silico des bases de données a permis d’identifier des ligands, unrécepteur et de nombreux effecteurs du signal insuline conservés chez C. gigas. Six IRPs (InsulinRelated Peptides) ont été caractérisés ce qui a permis de retracer l’histoire évolutive des IRPs chez lesmollusques. D’après leur profil d’expression en qPCR et HIS, Cg-mip123 et Cg-ilp sont susceptiblesd’être impliqués dans le contrôle de la reproduction. Ces IRPs peuvent se lier à un récepteur CIR (C.gigas Insulin Receptor) dont la séquence complète a été décrite. Les acteurs des voies de signalisationde l’insuline sont également conservés chez l’huître et exprimés dans la gonade. Pour aller plus loin surle rôle de ces IRPs dans les étapes précoces de gamétogenèse, un conditionnement alimentaire (à jeunvs alimentés en Isochrysis galbana) a été réalisé sur des huîtres en première gamétogenèse. Lesrésultats de ce conditionnement montrent que l’apport nutritif augmente la différenciation des GSCainsi que les proliférations goniales. L’implication du signal insuline dans ce contrôle devra êtreprécisée. / The Pacific oyster Crassostrea gigas is an alternative hermaphrodite mollusc with an annualreproduction cycle. Its gametogenesis is supported by energy reserves, store in a conjunctive storagetissue surrounding the gonad. Previous studies have shown the insulin system involvement in thisprocess closely connecting diet, energy reserves and gametogenesis. The functioning of earlygametogenetic stages stays unknown in the oyster. This work allows the identification of putative germstem cells (GSC) et progenitors on the basis of histological quantitative approach combined with agerm cells labelling by homologous antibody against Oyvlg (Oyster vasa-like gene). The maincomponents of the germinal niche have also been identified including a somatic cell, associated to theputative germ stem cell, with a heterologous antibody against BMP2/4 labelling. Above the study ofthe early gametogenetic stages regulation by insulin signalling, the genomic and transcriptomic-widescreening allows the identification of ligands, receptor et several effectors conserved in C. gigas. SixIRPs (Insulin Related Peptides) have been characterized which inform about the evolutionary history ofmolluscan IRPs. According to the expression profiles, in qPCR and ISH, Cg-mip123 and Cg-ilp may beinvolved in the reproduction process. These IRPs are able to bind the CIR (C. gigas Insulin Receptor)receptor whose sequence has been described. The insulin signalling effectors are also conserved in C.gigas and expressed in the gonad. To better understand the involvement of IRPs in the early stagesfunctioning, a food conditioning (unfed vs fed with Isochrysis galbana) has been done with oysters intheir first gametogenesis. The results showed that nutrient intake increases GSC and gonial mitosis.The involvement of insulin signalling has to be clarified.

Rezidivierte und refraktäre Hodentumoren waren bis zu Beginn der 80er Jahre nur selten kurativ behandelbar. Mit Einführung der Hochdosischemotherapie in Verbindung mit autologer Stammzellreinfusion, konnte eine kurative Behandlungsoption auch in dieser prognostisch ungünstigen Situation in der Klinik etabliert werden. Die vorliegende Arbeit beschreibt die Ergebnisse der ersten Phase I/II Studie zur klinischen Etablierung dieses Therapieverfahrens ebenso wie verschiedene nachfolgende Untersuchungen zur Optimierung der Hochdosischemotherapie. Eine "matched-pair" Analyse konnte zumindest im retrospektiven Vergleich, den Nutzen dieses neuen Therapieverfahrens im Vergleich zu einer konventionell-dosierten Behandlung belegen. / Until the beginning of the 1980ies relapsed and refractory germ-cell tumors were rarely cured. With the introduction of high-dose chemotherapy in combination with autologous stem cell reinfusion, a curative treatment option could be established in this prognostically unfavorable situation. The present work describes the results from the initial phase I/II studies that established this new treatment as well as the results of several subsequent trials to optimize this new procedure. Finally, the results of a "matched-pair" analysis is presented that demonstrates the superiority of this new treatment as compared to conventional-dose chemotherapy.

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.

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.

Genomic instability underlies diseases of unregulated cell growth that result in cancers and developmental abnormalities in humans. Similar genome destabilizing mechanisms are used to create genetic variety in crops for use in breeding and trait development. Errors that occur during DNA replication may cause mutations if they are not corrected before further cell divisions. DNA mismatch repair (MMR) corrects misinsertions and insertion/deletion DNA loop-outs that arise during DNA replication in plants, animals, prokaryotes, and some archaea, all of which incur mutations at rates 100 to 1,000-fold greater when subjected to inherited or somatic-mismatch repair deficiencies. An understanding of the effects of mismatch repair on somatic and germ-line cells in Arabidopsis thaliana is critical to the development of this plant as a model system for the study of genomic instability. Insertions and deletions of multiples of two base pairs in dinucleotide repeat sequences (microsatellites) occur more frequently in the absence of mismatch repair, and the mismatch-repair status of an individual, tissue, or cell may be inferred on the basis of microsatellite mutation frequency. Single-template PCR analysis measured microsatellite mutation frequencies in leaves and shoot-apical-meristem stem cells, and allowed me to address for the first time an important question: Do plants relax mismatch repair in vegetative tissues relative to meristematic germ-line and floral tissue? Analyses of four microsatellite loci in mismatch repair-deficient and wild type plants surprisingly suggest that there is little difference in mismatch repair activity between leaves and seeds. Mismatch-repair-deficient leaves displayed only two-fold higher microsatellite mutation frequency compared to wild type, and wild-type leaves also displayed a two-fold higher microsatellite mutation frequency compared to shoot-apical- meristems. The high frequency of microsatellite mutation in these wildtype tissues is unexpected, and it suggests that plants relax mismatch repair in differentiated tissues while maintaining genetic fidelity in a small set of stem cells in the shoot apical meristem (SAM). Genome sequencing of msh2⁻/⁻ mutation accumulation A. thaliana lines provides an estimated germ-line mutation rate of 3.9 × 10⁻⁷ in the absence of mismatch repair. Comparison of the rates of base substitution mutation per chromosome in mismatch repair-deficient plants with rates reported for wild-type plants suggests mismatch repair is more efficient on chromosome 5 than on chromosomes 1-4. Bias towards G:C → A:T mutations among transitions is maintained but increased nearly 100-fold in the absence of mismatch repair. / Graduation date: 2012