In this study, for the first time we used CRISPR/Cas9 gene editing technology in sturgeons i.e., sterlets (Acipenser ruthenus). The sequences of sgRNA and primers were designed based on published dnd1 sterlet sequence. Each pair of sgRNA oligos after ligation ready duplex DNA fragment was cloned into vector pX330-U6-Chimeric_BB-CBh-hSpCas9 backbone and thereafter the transformation to competent cells Escherichia coli DH5 was done. The plasmid carried sgRNA was extracted for downstream applications. We diluted extracted plasmid with 10% of 2 M KCl and injection into animal pole of fertilized eggs of sterlets at one to four-cell-stage, 4 hours post fertilization (hpf). At the same time, second microinjection with 2.5% FITC-biotin-dextrans was injected into vegetal pole for labelling PGCs. In the control groups, the eggs were only injected by 2.5% FITC into vegetal pole. PGCs of sterlet were visualized and photographed using a uorescent stereo microscope Leica M165 FC. To confirm the presence or deletion/insertion occurring in the target gene, we used MCE-202 MultiNA microchip electrophoresis system for DNA analysis, in which the targeted gene after amplifying by PCR was analyzed. Mutations in both treated and control embryos of sterlet were further assessed by Sanger sequencing of the PCR product. In present study, we successfully established basic protocols such as preparation of competent cells, construction of vector carrying sgRNA and its transformation into competent cells to carry out the CRISPR/Cas9 technology in sturgeons. Less number of PGCs was observed in embryos that were treated with CRISPR/Cas9; however, sequencing did not provide us a reliable evidence for mutation of the targeted gene probably due to an unspecific PCR. Therefore, more authentication of dnd1 knockout should be done in future by more specific PCR and repeated sequencing.

Introdução: a alta prevalência do câncer e o aumento significativo da sobrevivência em longo prazo geraram interesse quanto à preservação da fertilidade em mulheres jovens expostas a quimioterapia e radioterapia. Neste sentido estudos de congelamento de tecido ovariano para posterior transplante, abriram uma nova perspectiva de aplicação no tratamento e prevenção da infertilidade feminina. Objetivos: comparar dois protocolos de congelamento de tecido ovariano, um lento não automatizado e um por vitrificação, com o intuito de avaliar a viabilidade dos tecidos para posterior transplante autólogo. Método: Foram utilizadas 30 camundongos fêmea CF1 com aproximadamente 8 semanas e pesando 29,29g±2,9. •Os ovários extraídos foram vitrificados ou congelados, mantidos em nitrogênio líquido por 30 dias e descongelados. Após o descongelamento, o ovário esquerdo foi destinado às análises histológicas e caracterização por imuno histoquímica para o marcador mouse vasa homologue (MVH) e o ovário direito foi utilizado para os testes de viabilidade celular com exclusão por azul de trypan. Resultados: Nas análises de Hematoxilina e Eosina (HE) foram contados folículos primordiais, primários, pré-antrais e antrais. Não houve diferença significativa na proporção de folículos primordiais, primários e pré-antrais após descongelamento entre os grupos testados. A contagem de folículos antrais foi significativamente maior no grupo de vitrificação (p = 0,004). No ensaio de imunohistoquímica para o marcador MVH, folículos MVH + e MVH- foram contados e comparados com o número total de folículos. O grupo congelamento lento apresentou maior número de células não marcadas (p = 0,012). Conclusão: Embora ambos os protocolos tenham apresentado resultados semelhantes na análise histológica das contagens foliculares, o protocolo de vitrificação foi significativamente melhor para preservar a população de células tronco ovarianas. / Introduction: The high prevalence of cancer and the significant increase in long-term survival have generated interest as the preservation of fertility in young women exposed to chemotherapy and radiotherapy. Experimental techniques have been tried in an attempt to reverse the ovarian failure induced by these treatments. In this regard studies of ovarian tissue freezing for subsequent transplantation disclose a new application perspective in the treatment and prevention of female infertility. Objective: two ovarian tissue freezing protocols were tested, a non-automated slow-freezing and by vitrification, in order to assess the viability of the tissues for subsequent autologous transplantation. Methods: as ovaries donors, were used 30 female CF1 mice approximately 8 weeks and weighing 29,29g±2,9. • The ovaries were vitrified or frozen, stored in liquid nitrogen for 30 days and thawed. After thawing, the left ovary was intended for histological and immunohistochemical characterization by histochemical marker for MVH and right ovary was used for the tests with cell viability by trypan blue exclusion. Results: In HE slides was counting primordial, primary, pre antral and antral follicles. No significant difference was found in the proportion of high-quality primordial, primary and pre antral follicles after thawing/warming in the slow-freezing and vitrification group, respectively. The antral follicle counting was significant higher in vitrification group (p=0,004). In immunohistochemistry assay for MVH Antibody , MVH+ and MVH- follicles were counted and compared with the total number of follicles and slow freeze group had a higher number of not marked cells (p=0,012). Conclusion: Although both protocols showed similar results in the histological analysis for follicular counts, the vitrification protocol was significantly better for preserve the ovarian stem cell population.

No description available.

In recent decades there has been an increased interest in the carbon content of Earth’s geochemical reservoirs due to the impact of atmospheric carbon on the habitability of our planet. Earth’s interior likely hosts a greater mass of carbon than that of the oceans, atmosphere and crust combined, which has buffered the carbon content of the atmosphere over geological time. Yet only a few direct measurements of carbon from the upper mantle, and none from the lower mantle, have been made. Undegassed basalts erupted at mid-ocean ridges have previously been used to estimate the carbon content of the upper mantle. However, due to the low solubility of carbon within silicate melt, these undegassed basalt suites are rare. The majority of basalts have lost their mantle carbon information en route to eruption through the crust. Various crustal processes act to modify the geochemistry of melts before eruption, therefore it is important to be able to characterise the effect of these processes to better interpret the volatile signals preserved in erupted products. Pressure, and therefore depth, is a key parameter controlling volatile solubility and can be estimated using a variety of igneous barometers. This thesis presents results from crys- tallisation experiments conducted on basaltic glass from the Miðfell eruption, Iceland. The experiments provide new data that has been used to test a variety of barometers and crystalli- sation models used by igneous petrologists, and could aid future barometer recalibration. A key part of this work was the development of an experimental method for stabilising 5 kbar conditions in a piston cylinder apparatus. The experiments have shown that clinopyroxene- liquid barometry is more reliable than multi-reaction barometry. However, knowledge of equilibrium clinopyroxene compositions is crucial for accurately determining pressure using the clinopyroxene-liquid barometer. More experiments conducted at mid-crustal pressures are required for a full recalibration of these barometers. The results of testing igneous barometers and crystallisation models have been applied to two suites of olivine-hosted melt inclusions from the Kistufell and Miðfell eruptions to help determine the melt evolution history of these basalts. These eruptions were targeted due to previously measured noble gas isotopic ratios that suggest a primordial mantle component present in their melting regions, and therefore evoking the possibility that they could hold information about deep mantle carbon. Barometry suggests that Miðfell phases equilibrated, and therefore crystallised, at mid-crustal pressures (5–7 kbar), which could allow for the entrapment of undegassed melt inclusions within olivine. The two melt inclusion suites were found to differ in trace element variability, with the observation that the more trace element enriched eruption, Kistufell, had lower relative trace element variability than the more depleted eruption, Miðfell. Several processes, both in the crust and the mantle, are likely responsible for the level of trace element enrichment and variability, including extent of mantle melting, source heterogeneity, and melt transport. The depleted nature of the Miðfell melt inclusions has allowed them to preserve some of the highest CO$_2$/Ba and CO$_2$/Nb ratios ever recorded in basaltic glass, with ratios over five times greater than undegassed mid-ocean ridge basalt values. This carbon enrichment is not due to any crustal melt modification process, but rather pertaining to lower mantle carbon-rich lithologies that have been tapped by the Icelandic mantle plume. The carbon reservoir beneath Miðfell is estimated to contain 744 $\pm$ 188 ppm carbon, 15 times greater than the depleted upper mantle. This value matches estimates of bulk mantle carbon from planetary mass balance calculations and provides evidence for carbon-rich domains within the Earth.

Submitted by RENATO DA COSTA SANTOS null (renato.costa87@gmail.com) on 2016-03-02T19:44:58Z No. of bitstreams: 1 Tese.pdf: 1888991 bytes, checksum: c77d5b52416f56a3c90be7e3f7ef385f (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-04T12:43:11Z (GMT) No. of bitstreams: 1 santos_rc_dr_ift.pdf: 1888991 bytes, checksum: c77d5b52416f56a3c90be7e3f7ef385f (MD5) / Made available in DSpace on 2016-03-04T12:43:11Z (GMT). No. of bitstreams: 1 santos_rc_dr_ift.pdf: 1888991 bytes, checksum: c77d5b52416f56a3c90be7e3f7ef385f (MD5) Previous issue date: 2016-02-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nesta tese a Teoria das Perturbações Cosmológicas é revisada e três tópicos originais, incluídos neste grande ramo da cosmologia teórica, são apresentados. Começamos introduzindo e deduzindo as fórmulas necessárias partindo de primeiros princípios no capítulo 2. Em seguida, no capítulo 3, estudamos em detalhe Teorias Quânticas de Campos em de Sitter que contêm correntes de spin alto. Mostramos que a existência de correntes de spin alto - mesmo em teorias com interação - pode colocar mais vínculos na função de n-pontos, tornando a teoria assintoticamente gaussiana no futuro longínquo. Este resultado pode ser interpretado como o análogo do teorema de ColemanMandula para o espaço-tempo de de Sitter. O capítulo 4 é dedicado a modelos inflacionários conformes com o campo de Higgs fazendo o papel de Inflaton. Modelos com simetria de Weyl e com simetria SO(1, 1) para valores altos da energia são construídos. Verificamos quais as condições necessárias para que se obtenha um valor arbitrário para a razão escalar tensorial, parâmetro que mede a intensidade de ondas gravitacionais primordiais em um dado modelo. Introduzimos também um acoplamento diferente do valor conforme para a interação do campo escalar com o tensor de curvatura. Isto quebra a simetria de Weyl, mas verificamos que existe um forte atrator na direção da linha de Starobinsky. No último capítulo, aplicamos o efeito do ‘back reaction’ dos modos com comprimentos de onda longo (maiores que o raio de Hubble) em alguns modelos inflacionários e no cenário Ekpyrótico. Checamos se este efeito pode prevenir a inflação eterna nas regiões onde efeitos estocásticos são importantes nestes modelos. Alguns apêndices, com cálculos detalhados, são incluídos no final. / In this thesis the Theory of Cosmological Perturbations is reviewed and three original topics, that are part of this huge branch of theoretical cosmology, are presented. We start by reviewing and deducing the needed formulas from first principles in chapter 2. After it, in chapter 3, we study in detail Quantum Field Theories in de Sitter spacetime that contain Higher Spin currents. We show that the existence of Higher Spin currents - even in the interacting case - can put further constraints on the n-point function, making it asymptotically gaussian in the far future. This result can be interpreted as the analog of Coleman-Mandula theorem for de Sitter spacetime. Chapter 4 is devoted to conformal inflationary models with the Higgs field playing the role of the Inflaton field. We construct models with a Weyl symmetry and a SO(1, 1) symmetry at high energies. It is verified what are the conditions to get an arbitrary value for the tensor to scalar ratio, which measures the amplitude of primordial gravitational waves in a given model. Also, we introduce a coupling, different from the conformal one, for the scalar field and the curvature tensor. This breaks the Weyl symmetry but we verify that there is a strong attractor towards the Starobinsky line. In the last chapter, we apply the back-reaction effect of long wavelength modes (modes with wavelength bigger than the Hubble radius) in some inflationary models and in the Ekpyrotic scenario. We check if this effect could prevent eternal inflation in the region where stochastic effects are important for these models. Some appendices, with detailed calculations, are also included in the end.

Introdução: a alta prevalência do câncer e o aumento significativo da sobrevivência em longo prazo geraram interesse quanto à preservação da fertilidade em mulheres jovens expostas a quimioterapia e radioterapia. Neste sentido estudos de congelamento de tecido ovariano para posterior transplante, abriram uma nova perspectiva de aplicação no tratamento e prevenção da infertilidade feminina. Objetivos: comparar dois protocolos de congelamento de tecido ovariano, um lento não automatizado e um por vitrificação, com o intuito de avaliar a viabilidade dos tecidos para posterior transplante autólogo. Método: Foram utilizadas 30 camundongos fêmea CF1 com aproximadamente 8 semanas e pesando 29,29g±2,9. •Os ovários extraídos foram vitrificados ou congelados, mantidos em nitrogênio líquido por 30 dias e descongelados. Após o descongelamento, o ovário esquerdo foi destinado às análises histológicas e caracterização por imuno histoquímica para o marcador mouse vasa homologue (MVH) e o ovário direito foi utilizado para os testes de viabilidade celular com exclusão por azul de trypan. Resultados: Nas análises de Hematoxilina e Eosina (HE) foram contados folículos primordiais, primários, pré-antrais e antrais. Não houve diferença significativa na proporção de folículos primordiais, primários e pré-antrais após descongelamento entre os grupos testados. A contagem de folículos antrais foi significativamente maior no grupo de vitrificação (p = 0,004). No ensaio de imunohistoquímica para o marcador MVH, folículos MVH + e MVH- foram contados e comparados com o número total de folículos. O grupo congelamento lento apresentou maior número de células não marcadas (p = 0,012). Conclusão: Embora ambos os protocolos tenham apresentado resultados semelhantes na análise histológica das contagens foliculares, o protocolo de vitrificação foi significativamente melhor para preservar a população de células tronco ovarianas. / Introduction: The high prevalence of cancer and the significant increase in long-term survival have generated interest as the preservation of fertility in young women exposed to chemotherapy and radiotherapy. Experimental techniques have been tried in an attempt to reverse the ovarian failure induced by these treatments. In this regard studies of ovarian tissue freezing for subsequent transplantation disclose a new application perspective in the treatment and prevention of female infertility. Objective: two ovarian tissue freezing protocols were tested, a non-automated slow-freezing and by vitrification, in order to assess the viability of the tissues for subsequent autologous transplantation. Methods: as ovaries donors, were used 30 female CF1 mice approximately 8 weeks and weighing 29,29g±2,9. • The ovaries were vitrified or frozen, stored in liquid nitrogen for 30 days and thawed. After thawing, the left ovary was intended for histological and immunohistochemical characterization by histochemical marker for MVH and right ovary was used for the tests with cell viability by trypan blue exclusion. Results: In HE slides was counting primordial, primary, pre antral and antral follicles. No significant difference was found in the proportion of high-quality primordial, primary and pre antral follicles after thawing/warming in the slow-freezing and vitrification group, respectively. The antral follicle counting was significant higher in vitrification group (p=0,004). In immunohistochemistry assay for MVH Antibody , MVH+ and MVH- follicles were counted and compared with the total number of follicles and slow freeze group had a higher number of not marked cells (p=0,012). Conclusion: Although both protocols showed similar results in the histological analysis for follicular counts, the vitrification protocol was significantly better for preserve the ovarian stem cell population.

The dissertation deals with a progressive method of primordial writing - improvement of teaching writing in the first year at primary school. We suggest an alternative teaching method of writing, which should assure the fastest and the most effective progress in pupils´ scribal skills. The suggested procedure of improvement in teaching method of writing in an elementary class is grounded, in the theoretical and application field, on the knowledge laid out by J.P. Galperin in the ``theory of formation of intellectual operations{\crqq} and further elaborated by docent PhDr. J.Šemberova , CSc. The thesis is divided into two sections. The difference between the classical method of writing and the suggested progressive method is outlined in the theoretical part of the thesis. Its second section is practical. It is possible to find here a draft of the worksheets for teaching of primordial writing.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Os objetivos deste estudo foram investigar o efeito de diferentes concentraÃÃes de jacalina e da interaÃÃo de jacalina e FSH sobre a sobrevivÃncia, ativaÃÃo e expressÃo gÃnica em folÃculos primordiais caprinos cultivados in vitro. Para isto, os fragmentos do cÃrtex ovariano foram cultivados em Meio Essencial MÃnimo (MEM) suplementado com diferentes concentraÃÃes de jacalina (0, 10, 25, 50 e 100 μg/mL - experimento I), por um e seis dias. ApÃs o tÃrmino do perÃodo de cultivo, os fragmentos de cÃrtex ovariano foram fixados para histologia clÃssica. Em seguida, avaliou-se a percentagem de folÃculos primordiais ou em desenvolvimento no controle nÃo cultivado e no tecido ovariano cultivado nos diferentes tratamentos. ApÃs a determinaÃÃo da concentraÃÃo de jacalina mais eficiente (50 μg/mL), realizou-se o cultivo de fragmentos de cÃrtex ovariano em MEM suplementado com a jacalina (50 μg/mL), FSH (50 ng/mL) ou ambos (experimento II). ApÃs 6 dias de cultivo, os fragmentos ovarianos foram fixados para histologia clÃssica. AlÃm disso, para cada tratamento, foram coletadas amostras de tecido para avaliar o perfil de expressÃo de RNAs mensageiros para BMP-15, KL, c-kit, GDF-9 e PCNA em folÃculos ovarianos caprinos cultivado in vitro por 6 dias. Os resultados demonstraram que apÃs seis dias de cultivo, a presenÃa de 50 μg/mL de jacalina no meio de cultivo promoveu um aumento de folÃculos morfologicamente normais, bem como uma reduÃÃo da percentagem de folÃculos primordiais e aumento de folÃculos em desenvolvimento, quando comparado ao meio controle. No experimento II, demonstrou-se que jacalina ou FSH estimulam a ativaÃÃo dos folÃculos e contribuem para a manutenÃÃo da viabilidade, mas nÃo foi observada uma interaÃÃo positiva entres essas duas substÃncias. A expressÃo de RNAm para a BMP-15 e KL apÃs o cultivo in vitro de fragmentos de ovÃrio nos diferentes tratamentos por 6 dias nÃo foi alterada. No entanto, a presenÃa de FSH aumentou os nÃveis de RNAm para o c-kit e para o PCNA, enquanto que o GDF-9 teve sua expressÃo reduzida em meio suplementado com jacalina. Em conclusÃo, jacalina e FSH foram capazes de promover a sobrevivÃncia e ativaÃÃo de folÃculos primordiais caprinos apÃs 6 dias de cultivo. A presenÃa de FSH aumentou a expressÃo do RNAm para PCNA e c-kit, enquanto que a presenÃa da jacalina reduziu a expressÃo do GDF-9. / The aims of this study were to investigate the effect of different concentrations of jacalin and the interaction of jacalin and FSH on survival, activation and gene expression of goat primordial follicles cultured in vitro. For this, fragments of ovarian cortex were cultured in Minimum Essential Medium (MEM) supplemented with different concentrations of jacalin (0, 10, 25, 50 and 100 mg/mL - experiment I) for one and six days. After the end of cultured period, the fragments of ovarian cortex were fixed for histology. Then, the percentage of primordial follicles in uncultured or cultured ovarian tissue in different treatments were evaluated. After determining the most effective concentration of jacalin (50 μg/mL), ovarian cortex fragments were cultured in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (experiment II). After 6 days of culture, the ovarian fragments were fixed for histology. Furthermore, for each treatment, tissue samples were collected to evaluate the expression profile of mRNA for c-kit, KL, GDF-9, BMP-15 and PCNA in goats ovarian follicles cultured in vitro for 6 days. The results showed that after six days of culture, the presence of 50 μg/ml jacalin in culture medium increased the percentage of normal follicles, and promoted a reduction in the percentage of primordial follicles and increase of developing follicles, when compared to control medium. In experiment II, jacalin or FSH stimulated primordial follicle activation and contributed to maintain follicle viability, but there was no positive interaction between these two substances. The levels of mRNA for BMP-15 and KL after in vitro culture of ovarian fragments in different treatments was not altered. However, the presence of FSH increased levels of mRNA for c-kit and PCNA, while the GDF-9 expression was reduced in medium supplemented with jacalin. In conclusion, jacalin and FSH were able to promote the survival and activation of goat primordial follicles after 6 days of culture. The presence of FSH increased expression mRNA of PCNA and c-kit, while the presence of jacalin reduced the expression of GDF-9.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21663号 / 医博第4469号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 万代 昌紀, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22880号 / 医博第4674号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 近藤 玄, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM