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.

O transplante de células germinativas tem sido uma importante abordagem experimental para o estudo da preservação genética de espécies ameaçadas de extinção ou economicamente importantes. A técnica consiste na remoção das células germinativas indiferenciadas das gônadas do animal doador e na transferência das mesmas para a gônada de um indivíduo receptor. A fim de aumentar a eficiência da técnica, a identificação prévia das células germinativas a serem transplantadas torna-se preferível, visto que a cavidade que as receberá apresenta tamanho limitado. Sendo assim, é importante o desenvolvimento de marcadores moleculares que identifiquem precisamente as células a serem transplantadas na cavidade do individuo receptor, e os genes mais utilizados para esta finalidade são o dead end e o gene vasa, os quais são expressos apenas nas células da linhagem germinativa. Devido à importância do Colossoma macropomum (tambaqui) para a economia brasileira, esta espécie foi escolhida como uma espécie modelo de preservação para este estudo. Através do isolamento e sequenciamento dos genes dead end e vasa, desenvolvemos sondas de hibridização capazes de reconhecer as células onde estes genes são expressos e estudar o seu padrão de expressão nas gônadas. Ambos os genes apresentaram intensa expressão nos oócitos pré-vitelogênicos e fraca expressão em algumas espermatogônias. Pela primeira vez na literatura, diferentes isoformas causadas por splicing alternativo foram identificadas no gene dead end. A quantificação da expressão temporal dos diferentes transcritos mostrou que o padrão de expressão da sequência completa do gene teve uma tendência distintiva comparada ao padrão dos transcritos curtos, sugerindo que as diferentes isoformas desempenham papéis específicos e importantes para o desenvolvimento da linha germinativa nesta espécie. / Germ cell transplantation has been an important experimental approach to the study of the genetic preservation of endangered or economically important species. The technique consists in removing undifferentiated germ cells from the donor animal's gonads and transferring them to the gonad of a recipient individual. In order to increase the efficiency of the technique, the prior identification of the germ cells to be transplanted becomes preferable, since the receiving cavity presents limited size. Therefore, it is important to develop molecular markers to precisely identify the cells to be implanted in the recipient cavity, and the genes most used for this purpose are the dead end and the vasa genes, which are expressed only in germline cells. Due to the importance of Colossoma macropomum (tambaqui) for the Brazilian economy, this species was chosen as a model species for preservation in this study. By isolating and sequencing the dead end and vasa genes, we developed hybridization probes capable of recognizing the cells where these genes are expressed and better studying their expression pattern in the gonads. Both genes presented intense expression in pre-vitellogenic oocytes and poor expression in some spermatogonia. For the first time in the literature, different isoforms caused by alternative splicing were identified in the dead end gene. Quantification of the temporal expression of the different transcripts showed that the expression pattern of the full-length sequence had a distinctive tendency compared to the short transcripts pattern, suggesting that the different isoforms play specific roles for the germline development in this species.

Répression des genes par le dépôt de la marque H3K27me3. Divers cofacteurs contrôlent sa fonction dans des cellules de différentes origines, comme les gametes. Au cours de ma thèse, j'ai utilisé des modèles murins ou un tag a été introduit dans les gènes Ezh2 et Ezh1, j'ai isolé des extraits nucléaires de testicules adultes entiers et identifié un nouveau polypeptide interagissant avec PRC2. Ce dernier est spécifiquement exprimé dans les gonades et sa fonction est inconnue. J'ai confirmé son interaction avec PRC2 et montré qu'il pourrait recruter PRC2 à la chromatine. Grâce à un modèle de souris knock-out, j'ai démontré que la protéine est nécessaire pour la fertilité féminine, alors que son ablation apporte une augmentation globale de la marque associée à PRC2, dans les cellules germinales masculines avec peu de conséquences sur la fertilité. J'ai également contribué à la caractérisation de l'interaction entre le long ARN non-codant HOTAIR et PRC2. Nombreux ARNnc ont été proposés pour moduler l'action des complexes modifiant la chromatine. Avec l'aide d'un nouveau système de recrutement artificiel d'ARN, l'expression induite par HOTAIR provoque une répression transgénique indépendamment de PRC2. La surexpression forcée de HOTAIR a également peu d'impact sur le transcriptome dans des cellules cancéreuses. En conclusion, la liaison PRC2 à l'ARN n'est pas requise pour le ciblage de la chromatine. / The Polycomb Repressive Complex 2 (PRC2) plays an essential role in development by maintaining gene repression through the deposition of H3K27me3. A variety of cofactors have been shown to control its function in cells of various origins however little is known about PRC2 regulation during gametogenesis. During my PhD, I took advantage of murine models where Ezh2 and Ezh1 were knocked-in, I isolated nuclear extracts from whole adult testis and, identified a new polypeptide interacting with PRC2. This protein is specifically expressed in gonads, is of unknown function and does not contain any conserved domain. I have confirmed its interaction with PRC2, identified the domain of interaction with PRC2 and shown that it could tether PRC2 to chromatin. Thanks to a knockout mouse model, I demonstrated that the protein is required for female fertility, whereas its ablation brings to a global increase of H3K27me3 PRC2-associated mark in male germ cells with little consequences on male fertility. I also contributed to the characterization of the interplay between the long non-coding RNA (lncRNA) HOTAIR and PRC2 complex. Many lncRNAs have been proposed to modulate chromatin-modifying complexes action on chromatin. With the help of novel RNA-tethering system, HOTAIR inducible expression causes transgene repression independently from PRC2. Forced overexpression of HOTAIR also has little impact on transcriptome in breast cancer cells. Generally, PRC2 binding to RNA is not required for chromatin targeting. Taken together these results shed light to the mechanism of a new-identified cofactor regulating PRC2 in the gonads and contribute to dissect PRC2-RNA relationship at molecular level.

Cette thèse est divisée en trois parties. Les groupes de première partie des contributions à l'étude des équilibres corrélés. Nous nous concentrons sur les propriétés et les applications de la réduction de la double (Myerson, 1997) et la géométrie de Nash équilibres et des équilibres corrélés. La deuxième partie traite de l'évolution dynamique. Nous étudions le lien entre les stratégies appartenant à l'appui de Nash ou équilibres corrélés et les stratégies de survie dans le long terme. Nous constatons que la dynamique de beaucoup, y compris le réplicateur et les plus dynamiques de réponse peut éliminer toutes les stratégies à l'appui des équilibres corrélés. Élimination de toutes les stratégies à l'appui des équilibres de Nash est jugée encore plus universelle, et peuvent se produire à partir de presque toutes les conditions initiales. Le troisième partie se compose d'un seul article co-écrit, qui appartient au domaine de la biologie théorique. Nous aspects de l'étude de la transition de l'unicellulaire à organismes multi-cellulaires, en particulier les facteurs de conduite germe-soma spécialisation chez les algues vertes volvocine. introductions longues sont donnés au début de chaque partie. Le Bibliographie de la partie I et partie II est disjointe de la bibliographie de la partie III, et est offert à la fin de la partie II. Bien que reliées entre elles, les chapitres sont essentiellement autonome. En particulier, les notations et quelques définitions sont rappelés à chaque fois. Cela explique certaines répétitions.

The objective of this research is double: 1) to study the factors associated with the problems of quality with drinking water on the physicochemical and biological level in the municipality of Abomey-Calavi in Benin ; 2)to study its relationships with public health. The goal is to bring on one hand, the tools of improvement to the quality of water and public health in this commune. In addition, to contribute to the reduction of the rate of morbidity and mortality generated by the diseases related to drinking water contamination in this locality Methods: For the purpase of this study, we made a preliminary investigation in the municipality (households 55898 and almost all the households have the traditional wells). In the same way the information provided by the National Society of Water in Benin gave the indications on the level koverage of drinkable water conveyance of the commune. This study carried out within a general logical framework of the durable development based on an DPSIR analysis in the municipality of Abomey-Calavi of the Atlantic district in Benin made it possible to provide information on the main sources of contamination of the drinking water. We also carried out a random sampling of 110 wells and 110 households by taking account into the geographical distribution of the households in the commune. We selected 60 taps and 60 households by random sampling for those using water drink distributed by the national Society of Water in Benin. In each zone or district, three households consuming either well water or tap water were drawn randomly. A retrospective study on the number of cases of the hydrous diseases and other symptoms of hydrous diseases was carried out by consultation of medical registers of the commune from 2007 to 2009. Cases of diarrhoeas used as basic variable of health were compared with the other cases of hydrous diseases available in the files. Results: The results show that: the most al the well water taken in the households has temperatures beyond the recommendation of the World Health Organization (25 °C) and 98.2 % have a pH below the normal which is 6.5 to 8.5. We note a strong mercury and cadmium, complete iron, aluminum, nitrate nitrite pollution of the sampled wells (n=110) which is respectively 32.73%, 11.82%,10%, 43.64%, 6.36% and 14.55%. The results of the bacteriological analysis of water revealed that the totality of the wells is contaminated. The germs identified during the analysis of the well water are: Escherichia coli, fecal streptococcus species, bacterial salmonella ,Shigella , Clostridium perfringens, staphylococcus species, Klebsiella pneumoniae and Pseudomonas aeruginosa Most frequent among these germs are: Escherichia coli (100%), Klebsiella pneumoniae (97.27%), bacterial salmonella and Shigella (96,36%) with regard to the coliformes and enterobacteries. As for the cocci with Gram (+) they are staphylococcus species (97.27%) and the fecal streptococcus (88.18%). It is useful to stress that all the wells are strongly contaminated by Clostridium perfringens (95.45%). The differences between the respective prevalence of parasitic coprology, uroculture, diarrhoeas, pains abdominal and saddles bloody for exposed (45.4%, 71.8%, 76.4% and 59.1%) : those who consume the water of wells not exposed (8.3%, 1.7%,13.3%, 16.7): those who consume tap water, are statistically significant (p<0.001). For diarrhoeas according to the characteristics of the people reached of hydrous diseases and other symptoms of hydrous diseases we have: • in 2007: - 18.9 %, 20%, 35,1% and 33.9 % of the patients respectively between 0 at 3 year, more 3 at 5 year more 5 at 55 year and more 55 at 85 years had presented diarrhoea with a statistically significant difference (p<0.05); - 45.2% of those which made an ascariasis had presented the diarrhoea with a statistically significant difference; - 87.7 % of those which presented a salmonellose made the diarrhoea with a statistically significant difference; - 52.2% those which presented gastro-enteritis had presented diarrhoea with a statistically significant difference; - and finally other infections found not being correlated with the variable diarrhoea, just as the profession, educational level and sex. • in 2008: - 40.5% of those which make the diarrhoeas present an ascariasis with a statistically significant difference; - 40 % of those which make the diarrhoeas present an amoebiasis with a statistically significant difference; - 43.5% of the subjects which make the diarrhoeas present anaemias with a statistically significant difference. • in 2009 - 74.2%, 67.8%, 46.9%, 71.4% of those which make the diarrhoeas are respectively not provided education for, of primary education, secondary level and of higher level with a statistically significant difference; - 74.7%, 62.5%, 55.6%, 45.8% of those which make the diarrhoeas are respectively without profession, of the workmen, tradesmen and of the civils servant with a statistically significant difference; - 81.7 % of those which make the diarrhoeas present a salmonellose with a statistically significant difference; - 90% those which make the diarrhoeas present an amoebiasis with a statistically significant difference. Conclusions: This study had enabled us to demonstrate that well water with domestic use in the municipality of Abomey-Calavi in Benin is of public health concern insofar as most of the wells were contaminated with in particular an incidence of 17,4% diarrhoeas. It arose that the problem of the availability of drinking water continued to be a major concern for the population. It is urgent to implement suitable technical provisions in order to improve the access to drinkable water in this community. As the drinkable water supply is a public prerogative, the authority which they came within province of health, hydraulics, or political world should develop anet work of terminals fountains of drillings equipped with pumps with arm for the water supply of good quality,a regional planning to avoid pathologies of hydrous origin and to improve health and water quality for the inhabitant in this commune. /L’objectif de cette recherche est double : 1) étudier les facteurs associés à la problématique de la qualité de l’eau de boisson sur le plan physico-chimique et biologique dans la commune d’Abomey-Calavi au Bénin ; 2) étudier ses liens avec la santé de la population. Le but est d’apporter d’une part, les outils d’amélioration à la qualité de l’eau et à la santé des populations dans cette commune. D’autre part, de contribuer à la réduction du taux de morbidité et de mortalité engendrée par les maladies liées à la contamination de l'eau de boisson dans cette commune. Méthodes: Pour la réalisation de cette étude, nous avons fait une enquête préliminaire dans la commune qui nous a permis d’avoir une idée sur le nombre de ménages (55898) et de constater que presque tous les ménages disposent des puits traditionnels. De même les renseignements fournis par la Société Nationale des Eaux du Bénin ont donné les indications sur le niveau de couverture d’adduction d’eau potable de la commune. Cette étude réalisée dans un cadre logique général du développement durable basé sur une analyse DPSIR dans la commune d’Abomey-Calavi du département de l’Atlantique au Bénin, a permis de fournir des informations sur les principales sources d’eau de boisson. Nous avons également procédé à un tirage aléatoire de 110 puits et de 110 ménages en tenant compte de la répartition géographique des ménages de la commune. Nous avons sélectionné 60 robinets et 60 ménages par tirage aléatoire pour ceux qui sont alimentés par l’eau de boisson distribuée par la société nationale des eaux du Bénin. Dans chaque zone ou quartier, trois ménages consommant de l’eau de puits ont été tirés au hasard et ensuite un puits et robinet ont été tirés au sort parmi les puits et robinets de ces 3 ménages. L’étude rétrospective sur le nombre de cas des maladies hydriques et autres symptômes de maladies hydriques a été réalisée par consultation de registres sanitaires de la commune de 2007 à 2009. Les cas de diarrhées utilisés comme variables principales de santé ont été comparés aux autres cas de maladies hydriques dont les données étaient disponibles dans les dossiers. Résultats : Les résultats avaient montré que : la quasi-totalité des eaux de puits prélevées dans les ménages avaient des températures au-delà de la recommandation fixée par l’Organisation Mondiale de la Santé qui était de 25 °C et 98,2 % avait un pH en dessous de la normale qui était de 6,5 à 8,5. Sur les 110 puits prélevés, nous avions noté une forte pollution en nitrites en nitrates, en aluminium, en fer total, en cadmium et en mercure qui étaient respectivement de 32,73%, 11,82%,10%, 43,64%, 6,36% et 14,55% . Les résultats de l’analyse bactériologique des eaux avaient révélé que la totalité des puits prélevés étaient contaminés. Les germes indice de pollution bactériologique que nous avions identifiés au cours de l’analyse de l’eau de puits étaient : Escherichia coli, Streptococcus faecalis, Salmonella Spp ,Shigella Spp ,Clostridium perfringens, Staphylococcus aureus, Klebsiella pneumoniae et Pseudomonas aeruginosa . Les plus fréquents parmi ces germes étaient : Escherichia coli (100%), Klebsiella pneumoniae (97,27%) , Salmonella Spp et Shigella Spp (96,36%) en ce qui concernait les coliformes et entérobactéries. Quant aux cocci à Gram(+) il s’agissait des Staphylococcus aureus (97,27%) et des streptococcus faecalis (88,18%). Il était utile de souligner que tous les puits étaient fortement contaminés par le Clostridium perfringens (95,45%). Les différences entre les prévalences respectives de coprologie parasitaire, uroculture, diarrhées, douleurs abdominales et selles sanguinolentes chez les exposés (45,4%, 71,8%, 76,4% et 59,1%) c’est-à-dire ceux qui consommaient l’eau de puits et chez les non exposés (8,3%, 1,7%, 13,3%, 16,7) c’est-à-dire ceux qui consommaient l’eau de robinet étaient statistiquement significatives (p<0,001). Pour le pourcentage de diarrhées en fonction des caractéristiques des personnes atteintes de maladies hydriques et autres symptômes de maladies hydriques nous avions : • en 2007 : - 18,9 % ; 20% ; 35,1% et 33,9 % des patients respectivement entre 0 à 3 ans, plus de 3 ans à 5 ans plus de 5 à 55 ans et plus de 55 à 85 ans avaient présenté de la diarrhée avec une différence statistiquement significative (p<0,05); - 45,2% de ceux qui faisaient une ascaridiose avaient présenté la diarrhée avec une différence statistiquement significative; - 87,7 % de ceux qui présentaient une salmonellose avaient présenté de la diarrhée avec une différence statistiquement significative ; -52,2% de ceux qui présentaient une gastro-entérite avaient présenté de la diarrhée avec une différence statistiquement significative ; - et enfin les autres infections trouvées n’étant pas corrélé avec la variable diarrhée, de même que le niveau d’instruction, la profession et le sexe. • en 2008 : - 40,5% de ceux qui présentaient une ascaridiose avaient présenté les diarrhées avec une différence statistiquement significative; - 40 % de ceux qui présentaient une amibiase avaient présenté des diarrhées avec une différence statistiquement significative ; - 43,5% des sujets qui présentaient des anémies avaient présenté des diarrhées avec une différence statistiquement significative. • en 2009 : - 74,2% ; 67,8% ; 46,9% ; 71,4% de ceux qui étaient respectivement non scolarisé, de niveau primaire, secondaire et de niveau supérieur avaient présenté des diarrhées avec une différence statistiquement significative; - 74,7% ; 62,5% ; 55,6% ; 45,8% de ceux qui étaient respectivement sans profession, des ouvriers, commerçants et des fonctionnaires avaient présenté des diarrhées avec une différence statistiquement significative; - 81,7 % de ceux qui présentaient une salmonellose présentaient les diarrhées avec une différence statistiquement significative ; - 90% ceux qui présentaient une amibiase avient présenté les diarrhées avec une différence statistiquement significative. Conclusion : Cette étude nous a permis de noter à travers les résultats obtenus que la qualité des eaux de puits à usage domestique dans la commune d’Abomey-Calavi au Bénin pose un problème de santé publique dans la mesure où l’ensemble des puits sont contaminés avec une incidence notamment de diarrhées de 17,4%. Il ressort que le problème de la disponibilité de l’eau potable continue d’être une préoccupation majeure pour la population. Il est urgent de mettre en œuvre des dispositions techniques appropriées afin d’améliorer l’accès à l’eau potable dans cette communauté. Comme l’alimentation en eau potable est une prérogative publique, les autorités qu’elles soient du domaine de la santé, de l’hydraulique, ou du monde politique doivent développer l’installation de bornes fontaines, l’implantation de forages équipés de pompes à bras pour l’alimentation en eau de bonne qualité , l’aménagement du territoire dans cette commune pour éviter les pathologies d’origine hydrique et améliorer la santé et la qualité de l’eau pour les habitants.

Phthalates are industrial plasticizers with a wide range of applications. Di-(2-ethylhexyl) phthalate (DEHP) is one of the most highly produced and frequently studied phthalates. Its metabolite, mono-(2-ethylhexyl) phthalate (MEHP) is known as a testicular toxicant. The objective of this study was to examine expression of the genes of interest in testicular germ cells exposed to MEHP in a dose- and time-dependent manner at concentrations of 1µM, 10µM, and 100µM at 24, 48, 72 and 96hr time points. The genes consisted of Testisin, GSPT1, and MGMT genes which are a tumor suppressors, phase II xenobiotic metabolizing enzyme and DNA repair gene respectively. These genes were analyzed by Quantitative Real Time PCR (RT-PCR). The results revealed an overall down-regulation for each gene as the concentration and/or time increased. Testisin was the focus of the gene expression analysis. Testisin is epigenetically silenced in testicular germ cell tumors (TGCT) by DNA methylation at the 5’CpG island of the gene. To investigate if MEHP is capable of DNA hypermethylation, a co-exposure with 5-azacytidine (demethylating agent) was conducted. Compared with the 5-azacytidine treatment alone, there was a significant down-regulation of the Testisin gene in the co-exposure. This suggests that MEHP may down-regulate Testisin gene expression by DNA methylation. These findings provide evidence that MEHP can alter the expression of Testisin, GSTP1 and MGMT, genes that are associated in the risk of developing testicular germ cell tumors. In addition, results indicated that MEHP may cause DNA methylation leading to the down-regulation/silencing of genes such as Testisin.

ABSTRACT ENCAPSULATION OF WHEAT GERM OIL Yazicioglu, Basak M.Sc., Department of Food Engineering Supervisor: Prof. Dr. Serpil Sahin Co- Supervisor: Prof. Dr. G&uuml / l&uuml / m Sumnu February 2013, 82 pages Wheat germ oil is a rich source of omega 3 and omega 6, octacosanol and tocopherol which has vitamin E activity. Due to these properties it is beneficial for health but it is prone to oxidation in free form. The aim of this study was to encapsulate wheat germ oil in micron size and determine the best encapsulation conditions by analysing encapsulation efficiency, particle size distribution and surface morphology of the capsules. The effects of core to coating ratio, coating materials ratio and ultrasonication time on encapsulation of wheat germ oil were investigated. Maltodextrin (MD) and whey protein concentrate (WPC) at different ratios (3:1, 2:2, 1:3) were used as coating materials. Total solid content of all samples was 40% (w/w). Five different core to coating ratios (1:8, 1:4, 2:4, 3:4, 4:4) were experimented. Ultrasound was used at 320 W and 20 kHz frequency for three different times (2, 5, 10 min). Prepared emulsions were frozen and then freeze dried for 48 hours to obtain microcapsules. Encapsulation efficiency analysis, particle size analysis and scanning electron microscopy (SEM) analysis were performed. Increasing WPC content in coating led to an increase in encapsulation efficiency. Microcapsules prepared with MD:WPC ratio of 1:3 were found to have higher encapsulation efficiencies (65.62%-89.62%) than the other ratios. Increase in oil load led to decrease in encapsulation efficiency thus 1:8 core to coating ratio gave better results. The best conditions for microcapsules were determined as ultrasonication time 10 min, core to coating ratio of 1:8 and MD:WPC ratio 1:3.

<p>Germ cells represent the immortal line: they are guardians of a totipotent genome and are essential for the genetic survival of an individual organism and ultimately a species. An error at any stage in development (specification, migration, colonization, differentiation, adult maintenance) can lead to one of two disastrous outcomes: (1) germ cell death or (2) unchecked growth and proliferation leading to tumorigenesis. The work in this dissertation utilizes a classic mouse model (<italic>Ter</italic>) resulting in both of these phenotypes to further explore the molecular mechanisms important for development of germ cells. </p> <p>A homozygous nonsense mutation (<italic>Ter</italic>) in murine <italic>Dnd1</italic> (<italic>Dnd1<super>Ter/Ter</super></italic>) results in a significant (but not complete) early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129/SvJ background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost, develop into teratomas, or what factors directly control the strain-specific phenotype variation. </p> <p>Work here demonstrates that <italic>Dnd1</italic> expression is restricted to germ cells and that the <italic>Ter</italic> mutant defect is cell autonomous. The early loss of germ cells is due in part to BAX&ndash;mediated apoptosis which also affects the incidence of tumorigenesis on a mixed genetic background. Moreover, tumor formation is-specific to the male developmental pathway and not dependent on sex chromosome composition of the germ cell (XX vs. XY). Despite normal initiation of the male somatic pathway, mutant germ cells fail to differentiate as pro&ndash;spermatogonia and instead prematurely enter meiosis.</p> <p>Results here also reveal that, on a 129/SvJ background, many mutant germ cells fail to commit to the male differentiation pathway, instead maintain expression of the pluripotency markers, NANOG, SOX2, and OCT4, and initiate teratoma formation at the stage when male germ cells normally enter mitotic arrest. RNA immunoprecipitation experiments reveal that mouse DND1 directly binds a group of transcripts that encode negative regulators of the cell cycle, including <italic>p27Kip1</italic>, which is not translated in <italic>Dnd1<super>Ter/Ter</super></italic> germ cells. Additionally, overexpression of DND1 in a teratocarcinoma cell line leads to significant alteration of pathways controlling the G1/S checkpoint and the RB tumor suppressor protein. This strongly suggests that DND1 regulates mitotic arrest in male germ cells through regulation of cell cycle genes, serving as a gatekeeper to prevent the activation of a pluripotent program leading to teratoma formation. Furthermore, strain&ndash;specific morphological and expression level differences possibly account for sensitivity to tumor development.</p> / Dissertation

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 49-59). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 49-59). Also available in print.