This study covers the extraction of antioxidants from wheat germ / which is the byproduct of the flour-milling industry and a rich source of antioxidants / with Ultrasound Assisted (UAE) and Supercritical Carbon Dioxide (SC-CO2) extractions. Extraction conditions were ultrasonication time (1&ndash / 11 min), temperature (20&ndash / 60&deg / C) and ethanol level (5&ndash / 95%) for UAE, and pressure (148&ndash / 602 bar), temperature (40&ndash / 60&deg / C) and time (10&ndash / 60 min) for SC-CO2 extraction. The extraction conditions were optimized based on yield (%), total phenolic contents (TPC, mg GAE/g extract) and antioxidant activities (AA, mg scavenged DPPH&amp / #729 / /g extract) of the extracts, using Central Composite Rotatable Design. Total tocopherol contents (TTC) of the extracts were determined, as well. UAE (at 60&deg / C) with low ethanol level (~5-30%) and short times (1-3 min) provided protein rich extracts with high yield, medium TPC and AA. On the other hand, with high ethanol level (~90%) and long times (6-11 min), waxy structured extracts with low yield but high TPC and AA were obtained. SC-CO2 extraction at 442 bar, 40&ordm / C and 48 min. enabled almost 100% recovery of wheat germ oil (9% yield) but TPC and AA of the extracts were low. On the contrary, the extracts obtained at lower pressures (~150bar) and shorter times (~10 min) at 50-60&ordm / C had high TPC and AA since the oil yield was low. However, TPC and AA of these extracts were only half of those extracted by UAE. Maximum tocopherol (7.142 mg tocopherol/g extract) extraction was achieved at 240 bar, 56&ordm / C for 20 min. Both of the methods extracted high amounts of tocopherols from roasted wheat germ (SC-CO2 extraction / 0.31 mg tocopherol/g germ, UAE / 0.33 mg tocopherol/g germ) but TTC of the extracts obtained by SC-CO2 extraction was superior compared to 1.170 mg tocopherol/g extract obtained by UAE at 9 min, 58&ordm / C and 95% ethanol level. All these extracts with different characteristics have potential uses in cosmetic and food industry depending on the targeted specific application.

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. / "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"

There is a need for improved reproductive toxicology assays that do not require large numbers of animals but are sensitive and informative. Therefore, Staput velocity-sedimentation separation followed by culture of specific mouse testicular cells was used as such a system. The specificity of separation was assessed using immunocytochemistry to identify spermatids, spermatocytes and spermatogonia. The efficacy of the system to detect toxicity was then evaluated by analysing the effects of hydrogen peroxide (H2O2) by the terminal uridine-deoxynucleotide end-labelling (TUNEL) assay to show the rate of apoptosis induced among the different types of germ cells. We found that 2 h of treatment at both 1¿M and 10¿M induced increases of over ~10-fold in the percentage of apoptotic cells (p¿0.001), confirming that testicular germ cells are prone to apoptosis at very low concentrations of H2O2. It was also demonstrated for the first time for this compound that spermatogonia are significantly more susceptible than spermatocytes, which are more affected than spermatids. This reflects the proportion of actively dividing cells in these cell types, suggesting a mechanism for the differential sensitivity. The approach should thus form the basis of a useful test system for reproductive and genetic toxicology in the future.

Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 82-86). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

La stérilisation des espèces aquacoles est recherchée en aquaculture pour remédier aux problèmes de reproduction intempestive et risques de pollution génétique de la reproduction des poissons d'élevage échappés avec des espèces endémiques. Nous avons cherché à caractériser certains des mécanismes mis en jeu lors d'une stérilisation induite par de fortes températures chez une espèce de poissons thermosensible d'intérêt majeur en aquaculture, le tilapia du Nil, Oreochromis niloticus. L'effet d'une température élevée de 37°C sur le développement gonadique a été étudié pendant la différenciation sexuelle chez des alevins et pendant la maturation sexuelle chez des juvéniles, provenant de descendances mixtes (XXXY) et monosexes femelles (XX) /mâles (XY). L'analyse immunohistochimique de la protéine vasa montre que des traitements à 37°C provoquent une diminution du nombre de cellules germinales (CG) qui résulte d'une augmentation du taux d'apoptose et/ou d'une réduction du taux de prolifération de ces cellules, aboutissant à une stérilité partielle et transitoire ou complète et permanente. L'expression du gène vasa, marqueur des CG, est inhibée dans les gonades des poissons traités à 37°C pendant un minimum de 60 jours ; ceci est corrélé avec la réduction du nombre de CG dans ces gonades. La baisse du niveau d'expression des gènes cyp19a1a et amh, respectivement marqueurs de cellules somatiques femelles et mâles, suggère que la température de 37°C affecte également le nombre ou la fonctionnalité des cellules de la granulosa chez les femelles et de Sertoli chez les mâles. Un traitement de 60 jours est nécessaire pour induire de tels effets et semble impacter préférentiellement les gonades femelles. Ce travail confirme que les fortes températures induisent une réduction du nombre de CGs, en modifiant la balance entre les taux d'apoptose et de prolifération cellulaire, conduisant à des stérilités partielles ou totales. / The sterilization of farmed fishes is searched in aquaculture to remedy in the problems of inconvenient reproduction and risk of genetic pollution of reproduction of escaped farmed fishes into the natural environment with endemic species. We characterized some of the mechanisms involved during induced-sterilization by high temperatures in the Nile tilapia, Oreochromis niloticus, a thermosensitive species of major interest in fish farming. The effect of 37°C-elevated temperature on gonadal development was studied during sexual differentiation in fry and during sexual maturation in juvenile, from mix-sexed (XX/XY), all-genetic female (XX) and male (XY) progenies. The immunochemistry analysis of vasa protein shows that 37°C-treatment causes germ cell (CGs) decrease, resulting from an increase of apoptosis rate and/or reduction of cell proliferation, leading to partial and transitory or complete and permanent sterilization.This work confirms that high temperatures induce a decrease of germ cell number, modifying the balance between rates of cell apoptosis and cell proliferation, leading to partial or complete sterilization.The expression profile of vasa gene, marker of germ cells, is inhibited in gonads of fish treated at least during 60 days at 37°C ; that is correlated with the reduction of germ cell number. The reduction of expression levels of cyp19a1a and amh genes, respectively markers of female and male somatic cells, suggests that the 37°C-temperature also affects the number or function of granulosa cells in females and Sertoli cells in males. A treatment of 60 days is necessary to induce such effects and seems to impact preferentially female gonads.

La metformine est utilisée chez les patients et patientes atteints du diabète de type II. Elle est également administrée lors d’une infertilité liée à une insulino-résistance ou à un désordre métabolique. Mon projet de recherche a été d’analyser les effets de cet antidiabétique sur la fonction gonadique administré au stade foetal ou à la puberté. Nous avons montré par une approche in vivo sur deux modèles animaux: 1) qu'une administration orale de metformine à des poulets pré-pubères entraine un retard de puberté. Il se caractérise par une réduction du poids testiculaire, du diamètre des tubes séminifères et par une baisse de la testostérone sérique. 2) qu’une exposition des foetus mâles à de la metformine, chez la souris, conduit une fois adulte a une diminution de la taille des portées de 30%. Ce dysfonctionnement est associé à des marques épigénétiques sur l’ADN et des anomalies morphologiques de la tête des spermatozoïdes. Des techniques à haut débit (protéomique et métabolomique) réalisées sur des cultures de cellules de Sertoli immatures traitées ou non à la metformine ont permis de démontrer : 1) que le traitement mène à un métabolisme cellulaire de type Warburglike. 2) que des protéines présentent des différences d’abondance entre les deux conditions et qu’elles sont impliquées dans l’organisation du cytosquelette, de l’adhésion cellulaire, de la compaction de l’ADN, de la régulation de la réponse cellulaire à l’hypoxie et de l’immunité. Les résultats obtenus suggèrent que les grandes fonctions de sécrétion, de support et de protection des cellules de Sertoli sont modifiées par cet antidiabétique et qu’une exposition in utero à la metformine pourrait agir sur la fertilité et sur le métabolisme. / Metformin is used to treat patients with type II diabetes, it could be also administered to improve infertility associated to insulin-resistance or metabolic disorder. My research project was to analyse the consequence of a metformin exposure on gonad function from fetal to adult period. We showed by an in vivo approach on two animal models: 1) that oral administration of metformin to young chickens delay the puberty. It is characterized by a reduction in the testis weight, diameter of seminiferous tubule and decrease in testosterone level. 2) that mice, exposed in utero with metformin leads at adulte age to a lower fertility. This dysfunction is associated with morphological abnormalities of the sperm head and epigenetic marks on DNA. The second approach used proteomic and metabolomic strategy on cultured Sertoli cells treated or not with metformin. We demonstrated : 1) that the treatment leads to Warburg-like cellular metabolism. 2) that proteins present differences of abundances are involved in cytoskeleton organization, cell adhesion, DNA compaction, reponsiveness to hypoxia and immunity. The results suggest that the main function of secretion, support and protection of germ cell by Sertoli cells are modified by this antidiabetic, and that in utero exposure to metformin could act on the fertility and metabolism.

DNA methylation entails the addition of a methyl group to the 5-carbon of the cytosine base of the DNA. This modification is important during many biological processes such as imprinting, X-chromosome inactivation, cell differentiation as well as silencing of transposable elements (TEs). DNA methylation is dynamic during early mammalian development, despite being a more static mark in somatic cells. Global hypomethylation is a hallmark of epigenetic reprogramming in mammalian primordial germ cells (PGCs), the early embryo and in naïve embryonic stem cells (ESCs). Genome integrity is crucial during early development, as the germline DNA needs to be protected for future generations. Therefore, epigenetic reprogramming presents a critical phase for TE defence since presumably alternative silencing pathways need to be employed to limit their activity. In this thesis, I investigate the role of small RNAs to control TEs during global waves of DNA demethylation in cellular reprogramming, naïve pluripotency as well as early mammalian development. Following an introduction to the research questions, in chapter 3 I investigate the mechanism of TE regulation in an in vitro model of Dnmt1 deletion in mouse ES cells to recapitulate in vivo epigenetic reprogramming. I find that certain classes of TEs become transcriptionally upregulated and subsequently resilenced by a mechanism independent of DNA methylation. I identify ARGONAUTE 2 (AGO2) bound siRNAs as the prominent mechanism to control certain classes of TEs, while others appear to be regulated by redistribution of repressive histone modifications. In chapter 4, I construct Dicer constitutive and conditional KO ESCs in the background of the Dnmt1f l/f l ESCs using CRISPR-Cas9. I dissect the role of DNA methylation and of DICER dependent small RNAs on transcriptional changes of ESCs. Additionally, I find that DICER dependent small interfering RNAs (siRNAs) re-silence transcriptionally active TE classes. Finally, in chapter 5, I examine the role of small RNAs in TE silencing in different models of global hypomethylation in vivo and in vitro PGCs, during iPSC reprogramming and in a transition from serum to 2i culturing of mouse ESCs.

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.

Submitted by Hellen Monção de Carvalho Santana (hellenmcarvalho@hotmail.com) on 2016-03-21T13:04:10Z No. of bitstreams: 1 Versão Final Dissertação (com dia).pdf: 33709454 bytes, checksum: b55274e623607677efb4f7d385bf4e3e (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-22T17:36:33Z (GMT) No. of bitstreams: 1 santana_hmc_me_sjrp.pdf: 33709454 bytes, checksum: b55274e623607677efb4f7d385bf4e3e (MD5) / Made available in DSpace on 2016-03-22T17:36:33Z (GMT). No. of bitstreams: 1 santana_hmc_me_sjrp.pdf: 33709454 bytes, checksum: b55274e623607677efb4f7d385bf4e3e (MD5) Previous issue date: 2016-03-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste trabalho é estudar curvas reduzidas. Associado a elas, Buchweitz e Greuel definem um número, chamado número de Milnor de curvas reduzidas, pois no caso de curvas planas este coincide com o número de Milnor definido por Milnor. Este número é obtido através de um importante objeto algébrico: o módulo dual de Grothendieck. Com o intuito de facilitar a obtenção deste número, mostraremos que ele está relacionado com outro número, chamado delta, mais fácil de ser calculado. Por fim, mostraremos que, de maneira análoga, Nuño-Ballesteros e Tomazella definem um número associado a germes de função finita definidos em curvas reduzidas. Este número está relacionado com o grau deste germe e com o número de Milnor da curva reduzida associada. / The aim of this work is to study reduced curves. Associate to them, Buchweitz and Greuel define a number, called Milnor number once that in the case of plane curves, this number coincides to the Milnor number defined by Milnor. This number is obtained through an important algebraic object: dual module of Grothendieck. In order to make it easier to obtain this number, we will prove that it is related to another number, called delta, easier to be computed. At last, we prove that, in the same way, Nuño-Ballesteros and Tomazella define a number associate to finite function germs defined over reduced curves. This number is related to the degree of this germ and to the Milnor number of the reduced curve associated to it.

Made available in DSpace on 2014-06-11T19:24:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2003-05-21Bitstream added on 2014-06-13T20:13:02Z : No. of bitstreams: 1 manente_jcpp_me_sjrp.pdf: 685880 bytes, checksum: cb9d50daf3a657ec5c57bcb533adeba6 (MD5) / Foi monitorada a hidratação dos germes dos grãos de milho macerados a 52ºC e 180 rpm, em 9 soluções formadas pelas combinações de três níveis de SO2 (0,0 ; 0,1 e 0,2%) e três níveis de ácido lático (0,0 ; 0,55 e 1,0%) em intervalos de tempos de 10 minutos até 36 horas. Para determinar os coeficientes efetivos de difusão da água no germe foi utilizada a solução proposta por Crank (1975) para a Segunda Lei de Fick. Os coeficientes de difusão foram da ordem de 10-6 cm2/s, valores até 10 vezes maiores que os reportados na literatura para o grão inteiro. Verificou-se que o ácido lático dificultou a hidratação nas primeiras 2 horas e facilitou em 24 horas de maceração, ao passo que o SO2 dificultou a hidratação em 24 horas de maceração. Numa segunda etapa, macerou-se 1kg de grãos de milho em 4 tratamentos que apresentaram maior hidratação e no tratamento convencional (36 horas, 0,2% SO2 e 0,55% ácido lático), determinando-se o rendimento, a porcentagem de danificação e o teor de óleo nos germes. Foi verificado que a primeira moagem do processo úmido deve ocorrer somente após o germe atingir umidade próxima de 50% b.u. e que o SO2 e o ácido lático são imprescindíveis para se obter germe de boa qualidade. / Moisture content was measured at 15 steep times, from 10 minutes to 36 hours at 9 steep solutions made by combination between three levels of SO2 (0,0; 0,1; 0,2%) and three levels of lactic acid (0,0; 0,55; 1,0%). The solution of Fick's Second Law proposed by Crank (1975) was used to find the water effective diffusivity coefficients. The coefficients were in the order of 10-6cm2/s, and were up to 10 times faster them the values reported on the literature for diffusivity in the whole grain. Lactic acid decreased germ hydration during the first two hours of steeping and increased hydration at steeping time of 24 hours. SO2 decreased hydration for steeping time of 24 hours. On the second part of this work, 1kg of corn grains on the four treatments that showed to be of highest hydration and the conventional steeping treatment (36 hours, 0,2% SO2 and 1,0% lactic acid) were wet milled to recovery germ fraction. Percentage of germ yields, damages and oil content were determined and compared. Results showed that the first milling of the corn wet milling processes must occur only after the germ moisture content achieves approximately 50% w. b., and that SO2 and lactic acid are indispensable to obtain a high quality germ.