Before the consolidation of the germ theory of human diseases at the end of the nineteenth century, medical explanations about disease causation were dominated by the environmental notions of medical geography. This dissertation explores how nineteenth-century Colombian physicians transformed the medical geographical approach using the early concepts and technologies of the emerging Pasteurian germ theory. I follow this transformation in the cases of periodic fevers (yellow fever and malaria), continuous fevers (typhoid fever and typhus) and leprosy. The analysis reveals that by mid century physicians had incorporated neo-Hippocratic versions of disease causation and French medical geographical ideas in order to make sense of disease of the warm, temperate and cold lands. Their conceptual network revolved around the specific, predisposing and occasional causes in which climate and geography played a determinant role. Evidence indicates that this was the case of periodic fevers of the warm lands (yellow fever and malaria). I argue that the “parasitic” hypothesis of yellow fever was accepted during the controversy around the prophylactic inoculations inspired by Pasteurism that were applied in Colombia in 1887. However, doctors struggled to reconcile the medical geographical and the bacteriological perspective of both yellow fever and malaria. Continuous fevers, on the other hand, were also framed within the medical geography scheme of disease causation. I show how during the debates about typhoid fever and typhus happening in the Colombian highlands during the 70s, 80s and 90s, doctors used medical geographical notions and developed anti-pasteurian arguments, while the international scientific community had identified the specific bacilli for typhoid fever. Finally, I argue that the strong interest of Colombian doctors on leprosy –also understood in neo-Hippocratic terms- that foster the search for local treatments based on Pasteurism (antiseptics in the 1880s and serotherapy in the 1890s) also prompted the extension of the bacteriological model and techniques to other diseases in those decades.

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.

SPAG16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the “9 + 2” axoneme. The “9 + 2” axoneme provides the cytoskeletal core of all eukaryotic motile cilia and flagella. In Chlamydomonas, the Pf20 gene encodes a single protein present in the central pair of the axoneme. Loss of Pf20 prevents central pair assembly and results in flagellar paralysis. The murine Spag16 gene encodes two proteins. While 71 kDa SPAG16L is found in all murine cells with motile cilia or flagella, 35 kDa SPAG16S transcript and protein are detected only in male germ cells, suggesting a unique role distinct from general axoneme formation. Transgenic mouse studies published previously by our lab have shown that abrogation of both SPAG16 isoforms causes arrest of spermatogenesis, and the mutant allele is not transmitted to offspring by chimeric males. Mice homozygous for a knock-out of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. The defects seen in chimeric Spag16 mutant mice, unaccounted for by loss of SPAG16L, indicate a possible role for SPAG16S in the specialized process of male germ cell development. Our results demonstrate that SPAG16S is predominantly found in specific regions within the nucleus of round spermatids. These nuclear sub-domains also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. Putative interaction partners of SPAG16S are also shown to play critical roles in the peri-nuclear region during the round spermatid transition to the condensation and elongation stage of spermiogenesis, the final specialization point in sperm development. The distinct localization of SPAG16S at this critical juncture, its interaction with discretely localized proteins at a critical temporal junction in spermatogenesis, and its ability to modulate SPAG16L expression, suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells, and represents an evolutionary distinction in axoneme gene function.

Master of Science / Department of Food Science / Fadi Aramouni / Despite the expansion of the gluten-free (GF) food market, some GF food items are still characterized by an overall mediocre quality. The effects of different types of egg ingredients (fresh, whites, dried) and carob germ flour (CGF) as well as par-baking technology on the quality of dough-based gluten-free sorghum dinner rolls were evaluated. Gluten-free rolls containing 30% of fresh shell eggs or equivalent of egg products and 10% of CGF on a flour basis were evaluated against a control (no egg, no CGF). The feasibility of partial baking of rolls was studied on control as well as fresh eggs and carob germ flour formulas during 5 baking times (0, 8, 10, 12 and 18 minutes). Breads were evaluated for crumb and crust color, specific volume, cell profile, Texture Profile Analysis (TPA) and consumer acceptability. Results showed that rolls containing egg ingredients had higher specific volumes than control (p<0.05) with an increase from 1.45 cm[superscript]3/g to 1.96 cm[superscript]3/g. Carob germ flour did not have a significant effect on specific volume. Eggs also improved cell elongation and produced significantly darker crust (p<0.05). CGF did not appear to have an effect on cell elongation but increased average cell number when combined with egg ingredients, and greatly impacted rolls texture. The combination of fresh eggs or egg whites with CGF reduced significantly (p<0.05) crumb hardness from 2,074 to 1,404g and 1,468g of force respectively. Par-baked dinner rolls displayed similar color, volume, cell profile and texture trends to conventionally baked rolls. Sensory study revealed that acceptability, organoleptic characteristics and willingness to buy of par-baked dinner rolls could be similar to that of conventional wheat products. This research proved that the addition of eggs and CGF to a GF rolls formulation resulted in better overall quality of the product. Moreover, par-baking of the rolls showed great potential to provide safe, convenient and acceptable GF foods to celiac individuals.

The main goal in reversing the allergy epidemic is the development of effective prophylactic strategies. Early life events, such as exposures to microbes, have a major influence on the development of balanced immune responses. Due to their ability to interact with host immune system and to modulate host immune responses probiotics, mainly bifidobacteria and lactobacilli have been used with some success in prevention of allergic disease. In order to be referred to as probiotic, bacterial strain has to undergo rigorous testing. We have selected three new Lactobacillus (L.) strains out of 24 human isolates according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Safety of these strains was proven upon intragastric administration to mice; moreover, we have shown their ability to shift cytokine Th1 - Th2 balance towards non-allergic Th1 response in isolated splenic cells. Allergen specific prophylaxis using probiotics as vehicles for mucosal delivery of recombinant allergen is an attractive concept for development of well-tolerated and effective allergy vaccines. We have shown that neonatal mono-colonization of germ-free mice with the L. plantarum NCIMB8826 strain producing the major birch pollen allergen Bet v 1 attenuates the development of...

Os transplantes de células germinativas e enxertos de testículo podem ser aplicados na reprodução de espécies comerciais e na conservação de espécies ameaçadas de extinção. No entanto, a maior limitação dos transplantes está na sua limitada eficiência, devido à baixa porcentagem de hospedeiros capazes de gerar gametas derivados do doador e da rejeição de enxertos de testículo alogênicos após poucas semanas de sua implantação. O sistema imunológico dos hospedeiros podem estar envolvidos na baixa de eficiência dos transplantes e na rejeição dos enxertos. Desta forma, este trabalho buscou verificar se a administração de drogas imunossupressoras como o tacrolimus e ciclosporina em emulsão poderiam ser utilizados para prolongar a viabilidade de enxertos de testículo em trutas arco-íris. Na primeira parte, experimentos in vitro com leucócitos do sangue periférico demonstraram que ambos o tacrolimus e a ciclosporina impedem a proliferação celular mesmo sob estímulo proliferativo da concanavalina. Nos ensaios in vivo, ambas as doses de tacrolimus (0,5 e 1,5 mg/kg) e a mais baixa de ciclosporina (20 mg/kg) inibiram significativamente a expressão de il2 no rim cefálico três dias após a injeção. Já a dose de 40 mg/kg de ciclosporina inibiu a expressão de il2 por até sete dias após a injeção. Na segunda parte, enxertos alogênicos de testículo foram realizados em animais tratados semanalmente com emulsões de tacrolimus. As análises histológicas (coloração H.E.) e as de RT-PCR (vasa e txdnc6) demonstraram a presença de espermatogônias na primeira semana e indicaram a presença de espermátides/espermatócitos na quinta semana, respectivamente, em alguns animais do grupo tratado com a dose de 0,5 mg/kg de tracrolimus. No grupo tratado com a dose mais alta de tacrolimus (1,5 mg/kg) e no grupo controle (sem imunossupressor), células germinativas ou marcadores destas não foram detectados. Estes resultados, portanto, fazem do tacrolimus um imunossupressor promissor para utilização em enxertos alogênicos e também em transplantes de células germinativas de truta arco-íris. A administração concomitante de um outro imunossupressor conjugado ao tacrolimus (na baixa dosagem) para inibir duas ou mais vias de ativação do sistema imunológico, conforme utilizado em transplantes de órgãos em humanos, pode ser uma alternativa para otimizar os efeitos imunossupressivos nos animais receptores. / Germ cell transplantation and testis graft can be applied for the reproduction of commercial or endangered species. However, mechanisms of rejection from the host immune system might limit the production of surrogate gametes and progeny after transplantation and induce rejection of testis allografts within few weeks. In this work, we aimed to administer immunosuppressants-containing emulsions in order to verify whether they are capable to prevent immune rejection and promote testis allograft survival in rainbow trout. In the first part of this study, it was demonstrated in vitro that tacrolimus and cyclosporine were able to inhibit leucocyte proliferation even under concanavalin-induced mitotic stimulation. In in vivo experiments, both dosage of tacrolimus (0,5 and 1,5 mg/kg) and a lower of cyclosporine (20 mg/kg) inhibited significantly the expression of il2 in head kidney at three days post-injection. A higher dosage of cyclosporine (40 mg/kg) was able to inhibit il2 expression for up to seven days post-injection. In the second part, testis allografts were conducted in fish treated weekly with tacrolimus-containing emulsions. Histological (H.E. staining) and RT-PCR (vasa and txdnc6) analysis demonstrated the presence of spermatogonias in the first week and indicated the presence of spermatids/spermatocytes in the fifth week, respectively, in some animals treated with 0,5 mg/kg of tacrolimus. In the group treated with the highest tacrolimus dose (1,5 mg/kg) and in the control group (without immunosupressant), no germ cells or their respective markers were detected. These results suggest that tacrolimus comprise a promising immunosuppressant, with applications to testis allografts or germ cell transplantation in rainbow trout. Co-administration combining tacrolimus (at lower dose) with other immunosuppressive drugs for inhibiting more than two activation pathways of the immune system, as done in human organ transplantation, can be an alternative to optimize the immunossupressive effects in host organisms.

Células germinativas embrionárias são células pluripotentes derivadas das células germinativas primordiais que surgem no período do desenvolvimento embrionário. Sendo precursoras na maturação dos gametas sendo para a proliferação e formação de novas gerações de células germinativas. Estudos em modelos animais com células troncos embrionárias pluripotentes têm sido realizados com sucesso no tratamento de muitas doenças genéticas, principalmente em modelos caninos devido a homologia com humanos. Portanto, objetivamos estabelecer um estudo da biologia das células erminativas caninas para futuras pesquisas, visando sua viabilidade terapêutica. Foram utilizadas fêmeas de cães sem raça definida (SRD), foram submetidas à ovario-salpinge-histerectomia, oriundas de campanhas de castração realizadas por associações e/ou organizações não governamentais na cidade de Pirassununga. Os embriões coletados foram analisados através de um cronograma histológico e também mensurados através da medida Crown-Rump(CR), em seguida em condições ésteres micro-dissecados na região paramesonéfrica próximo a região dos somitos. Os fragmentos foram isolados e colocados em cultivo com meio apropriado para o desenvolvimento e propagação das células germinativas primordiais (PGCs). Obteve-se sucesso nos cultivos com embriões em idades gestacionais próximas a 24 á 32 dias de gestação, nos quais demonstraram uma morfologia arredondada, pequena. Na microscopia eletrônica de varredura e transmissão observamos tamanhos variados entre as células aderidas umas as outras, mostrando um núcleo bem evidente e em seu citoplasma observou-se diversos tipos de organelas celulares, principalmente mitocôndrias. A análise imunohistoquímica revelou a presença de marcadores Oct4, sugerindo a presença de células pluripotentes e germinativas. Imunofenotípicamente as células através da citometria de fluxo revelou baixa expressão para o marcadore Oct4 enquanto que para os marcadores CD 34, C-Kit houve expressão.RT-PCR mostrou expressão do marcador para pluripotencialidade Oct4 e Sox2 e pela técnica de Western Blotting identificou a expressão da proteína específica para Oct4. Portanto estes achados sugerem com sucesso o estabelecimento de cultivo e proliferação e desenvolvimento das células germinativas primordiais caninas. / Embryonic germ cells are pluripotent cells derived from primordial germ cells which arise during embryonic development. These cells are precursors in the maturation of gametes and for proliferation and formation of new generations of germ cells. Studies using animal models for pluripotent embryonic stem cells have been conducted successfully in the treatment of many genetic diseases, especially using canine models, due the homology between canine and humans. Therefore, we aimed to establish a study of canine biology of germ cells for future research, aiming viability therapy. For this study, we used female mongrel dogs (SRD), which underwent ovary- salpingo- hysterectomy, arising from castration campaigns run by associations and/or NGOs in Pirassununga city. The embryos collected were analyzed using histology methods following the timeline and measure by measuring Crown-Rump (CR) then in an environment without contaminants (sterile) the embryos were micro-dissected focusing in the paramesonephric region near the region of somite where the germ cells are. The fragments were isolated and placed in culture with a specific media for the development and spread of primordial germ cells (PGCs). Success was achieved in cultures with embryos at a gestational age close to 22 to 30th days of gestation, in which the cells showed a rounded morphology and small. In electron microscopy and transmission analyses, sizes were observed between the cells attached to each other, showing a conspicuous nucleus and in the cytoplasm was observed several types of cell organelles, especially mitochondria. Immunohistochemical analysis revealed the positive immunolabeling for Oct4, suggesting the presence of pluripotent cells and germ cells. The analyses using immunophenotyping by flow cytometry showed low expression for Oct4 while for CD34 and C-kit markers had positive expression. RT-PCR showed expression of the pluripotency markers Oct4 and Sox2. By the technique of Western Blotting identified protein expression specific for Oct4. Thus these findings suggest the successful establishment of culture, proliferation and development of primordial germ cells canine.

O estudo visou elucidar o desenvolvimento embrionário e fetal de pacas (Agouti paca) e demarcação dos sítios germinativos nos embriões em diferentes estágios. Foram utilizados sete espécimes de Agouti paca, sendo dois embriões e três fetos doados do pacário mantido pela UNESP - Jaboticabal e, dois doados do acervo da FMVZ-USP. Os fetos 1 e 2 apresentaram imaturidade facial acentuada, olhos recobertos por uma lente proeminente e lóbulos das orelhas; os fetos 3 e 4, mostravam-se com orelhas bem desenvolvidas, membros torácicos e pélvicos em grau equalitário de desenvolvimento, como vibrissas ao redor das bordas nasais e olhos também protegidos; no feto 5, haviam pêlos distribuídos por todo o corpo, membros torácicos e pélvicos com garras, vibrissas na face, olhos proeminentes e orelhas bem desenvolvidas. O embrião 1, apresentou a vesícula óptica com pigmentação da retina, o quarto ventrículo encefálico e curvatura cervical acentuada e broto dos membros em desenvolvimento; o embrião 2, possuiu divisão dos arcos branquiais e neuróporo cranial aberto; presença da área cardíaca e fígado; vesícula óptica sem pigmentação da retina, abertura do tubo neural na região do quarto ventrículo encefálico, rombencéfalo e mesencéfalo em desenvolvimento. Na microscopia de luz, visualizamos a medula espinhal, abertura do 4º ventrículo encefálico, presença das vesículas encefálicas (prosencéfalo, mesencéfalo e rombencéfalo), coluna vertebral, hipófise, cavidades oral e nasal, olho, átrio e ventrículo cardíacos, pulmão e diafragma, além das cristas metanéfrica e mesonéfrica, fígado, intestino e pedículo umbilical. Nas reações de imunohistoquímicas para OCT-4 não houve marcação expressiva em órgãos tais como pulmão, intestino e somitos, o coração apresentou uma leve positividade à reação, enquanto que nas cristas meso e metanéfrica e fígado obtiveram uma marcação expressiva, sendo mais acentuada no último. Nos testes com vimentina todos os órgãos mostraram-se imunopositivos em diferentes áreas; e em se tratando da reação a testes com actina apenas a região de somitos não obteve marcação positiva. Concluímos que o período embrionário/fetal da paca não pode ser comparado com o modelo clássico de roedor; sua embriogênese pode ser comparada à de ratos, Guinea pig, coelhos e humanos. A imunolocalização positiva de OCT-4 apresenta diferenças de acordo com a idade gestacional, devido às mudanças embriológicas dos tecidos. A imunolocalização positiva de OCT-4 apresenta diferenças de acordo com a idade gestacional, devido às mudanças embriológicas dos tecidos. A vimentina como marcador de mesênquima se expressou positivamente em todos os órgãos do embrião de paca. A actina como imunomarcador de músculo liso foi expressiva nas áreas contendo musculatura. / The study aimed elucidates the development of embryonic and fetal of pacas (Agouti paca) and demarcation of the germ sites in embryos at different stages. Seven specimens were used; two embryos and three fetuses from UNESP- Jaboticabal and other two fetuses were from FMVZ-USP collection. The fetuses 1 and 2 showed immaturity facial sharp, eyes covered by a lens and prominent lobes of the ears, the fetuses 3 and 4, showed up with well-developed ears, members thoracic and pelvic in equal level of development, vibrisses around the nasal edges and eye also protected;. The fetus 5 had hairs distributed throughout the body, members thoracic and pelvic with claws, vibrisses on the face, prominent eyes and ears well developed. The embryo 1, presented the optic vesicle with pigmentation of the retina, the fourth encephalic ventricle and cervical curvature and button members in development. The embryo 2, had split the branchial arches and open neuropore cranial; heart and liver were identified, optical vesicle without pigmentation of the retina, the neural tube was opening in the region of the fourth encephalic ventricle, rombencephalon and mesencephalon were in development. Light microscopy, shows the spinal cord, 4 th encephalic ventricle, resence of encephalic vesicles (prosencephalon, mesencephalon and rombencephalon), vertebral column, pituitary gland, oral and nasal cavities, eye, atrium and ventricle heart, lung and diaphragm, as well the metanephron and mesonephron, liver, intestine and mbilical pedicle. The immunohistochemical reactions for OCT-4 were non expressive in organs such as lung, intestine and somites; heart presented a discrete positive reaction, while kidneys and liver obtained an expressive expression, more pronounced in the last one. Vimentina\'s tests showed that all organs stained in different areas, and the reaction whit the actin was negative just in the region of somites. We conclude that the period embryonic/fetal of paca can not be compared with the classical model of rodents; its embryogenesis can be compared with the rats, Guinea pig, rabbits and humans ones. The positive immunolocalization of OCT-4 presents differences according to the gestational age, due to changes embryological tissue. The vimentine as a mesenchyme marker is expressed positively in all organs of the embryo of paca. The actin as immunomarker of smooth muscle was expressive in areas containing muscles.

It was previously demonstrated that miR-199a was down-regulated in testicular germ cell tumor (TGCT) partly caused by hypermethylation of its promoter. More detailed analyses showed that miR-199a-5p, one of its two derivatives, suppressed TGCT invasiveness and proliferation via directing targeting PODXL and MAFB. The biological role of the other derivative, miR-199a-3p in TGCT, remains largely uncharacterized. In this project we identified DNMT3A, the de novo methyltransferase, as a direct target of miR-199a-3p using a 3’-UTR reporter assay. In NT2 (NTera 2) and HT (Hs 1.Tes) cells, miR-199a-3p regulated the expression of endogenous DNMT3A (both DNMT3A1 and DNMT3A2 isoforms), especially DNMT3A2 isoform. In clinical samples, the expression of DNMT3A2 and miR-199a-3p were reciprocally regulated. However, DNMT3A did not regulate miR-199a expression. Further characterization of miR-199a-3p revealed that it negatively regulated DNA methylation partly through targeting DNMT3A. MiR-199a-3p could restore the expression of APC and MGMT via de-methylation in their promoters. Our studies demonstrated the dysregulation of miR-199a-3p in TGCT may provide novel mechanistic insights into TGCT carcinogenesis and suggested a potential therapeutic use of synthetic miR-199a-3p oligonucleotides as effective demethylation agent in the treatment of TGCT. However, since DNMT3A expression did not regulate miR-199a expression, the mechanism of promoter DNA hypermethylation of miR-199a in TGCT needs further investigation. / MiR-199a is encoded by two loci in the human genome, namely, miR-199a-1 on chromosome 19 and miR-199a-2 on chromosome 1. Another microRNA, miR-214, also locates on chromosome 1. Previous study revealed that it is co-transcribed with miR-199a-2, which is directed by miR-199a-2 promoter. However, the biological significance of the co-expression of miR-199a and miR-214 remains largely unknown. In this project, it was determined that miR-199a and miR-214 were concordantly expressed in TGCT. Silencing of DNMT1 increased the expression of miR-199a and miR-214, accompanied by de-methylation in the promoters of miR-199a-1/2. Overexpression of TP53 down-regulated the expression of DNMT1 and increased the expression of mature miR-199-3p/5p and miR-214. In addition, silencing of PSMD10 up-regulated the expression of TP53, while miR-214 over-expression resulted in PSMD10 down-regulation and TP53 up-regulation. Collectively, our findings highlighted a miR-199a/miR-214/PSMD10/TP53/DNMT1 self-regulatory network, which caused the down-regulation of miR-199a, miR-214 and TP53, as well as the up-regulation of DNMT1 and PSMD10 in TGCT. These observations partly explain the mechanism of promoter DNA hypermethylation in miR-199a in TGCT. They also suggest a potential therapeutic approach by targeting the miR-199a/miR-214/PSMD10/TP53/DNMT1 regulatory network in the treatment of TGCT. / 先前的研究證實miR-199a在睾丸生殖細胞腫瘤 (簡稱睾丸癌) 中是低表達的，部分歸因於其啟動子區域過度甲基化。對其功能研究發現miR-199a能抑制睾丸癌細胞的生長，侵襲和轉移，且miR-199a的抑癌屬性應歸功於它的兩個衍生物之一miR-199a-5p。然而，miR-199a的另一個衍生物miR-199a-3p在睾丸癌中的生物學功能仍然在很大程度上是未知的。此研究中，DNMT3A被鑒定為miR-199a-3p的直接靶定目標。在NT2和HT細胞中，miR-199a-3p能調控內源性DNMT3A(DNMT3A1和DNMT3A2)的表達水準，尤其是DNMT3A2。在臨床樣本中，DNMT3A2的表達水準與miR-199a-3p的表達水準呈負相關。但DNMT3A並不能調控miR-199a的表達水準。進一步研究顯示過表達miR-199a-3p能減少APC和MGMT啟動子區域甲基化而恢復其表達水準。研究證實異常表達的miR-199-3p可能在睾丸癌的癌變過程中發揮作用，並提出一個潛在的治療方案，即使用miR-199a -3p作為有效的去甲基化藥劑治療睾丸癌。然而睾丸癌中導致miR-199a啟動子區域過度甲基化的機制有待進一步研究。 / 在人類基因組中，miR-199a-1(位於19號染色體)和miR-199a-2(位於1號染色體)都編碼miR-199a。同時miR -214也位於1號染色體，研究表明miR-214與miR-199a-2由miR-199a-2啟動子介導共同轉錄，但miR-199a和miR- 214共同表達的生物學意義仍未知。此研究中，miR-199a和miR-214在睾丸癌中的表達呈現一致性。沉默DNMT1後miR-199a和miR-214的表達水準顯著提高，並伴隨著miR-199a-1/2啟動子區域的DNA去甲基化。在NT2細胞中。過表達TP53能下調DNMT1的表達水準，同時上調miR-199-3p/5p和miR- 214的表達水準。此外，過表達miR -214能導致PSMD10表達水準的下調以及TP53表達水準的上調。綜上所述，我們提出一個miR-199a/miR-214/PSMD10/TP53/DNMT1自我調控網路，此調控通路能引起睾丸癌中miR-199a，miR-214和TP53表達水準的下調，以及DNMT1和PSMD10表達水準的上調，且部分解釋睾丸癌中miR-199a啟動子區域過度甲基化的機制，同時該調控網路可作為治療睾丸癌的一個潛在靶點。 / Chen, Bifeng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 103-127). / Abstracts also in Chinese. / Title from PDF title page (viewed on 20, December, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Fetal germ cell development is a key stage of female reproductive life. The DAZ family proteins (DAZ, DAZL and BOLL) are RNA-binding proteins with critical roles in murine germ cell development but their expression and potential targets in the human are largely unknown. The studies in this Thesis investigated the expression and function of DAZL and BOLL in human fetal ovary. Both DAZL and BOLL mRNA are increased dramatically at the time of entry into meiosis. Immunohistochemical analysis with specific meiotic markers suggested that DAZL and BOLL have distinct spatial-temporal expression patterns, with minimal co-expression – BOLL expression was transient prior to follicle formation. This pattern was shown not to be present in the mouse fetal ovary, where Dazl and Boll are co-expressed, indicating a limitation of the mouse for exploring the function of Boll. Two human cell lines, embryonic kidney derived HEK293 cells and germ cell tumour derived TCam-2 cells were used as models to identify the mRNA targets of DAZL and BOLL after transfection of DAZL or BOLL vectors. In HEK293 cells, TEX19 and TEX14 were confirmed as potential targets of both DAZL and BOLL, and CDC25A as a potential DAZL target. Further experiments indicated that DAZL and BOLL did not increase target mRNA transcription but increased stabilisation. A DAZL/GFP co-transfection-FACS system for TCam-2 cells was established as this cell line has very low transfection efficiency. TEX14 and SYCP3 significantly increased in GFP+ve-DAZL+ve cells when compare to the GFP-ve-DAZL-ve cells, whilst SOX17 and DNMT3L significantly decreased in the GFP+ve-DAZL+ve cells. A 3'-UTR luciferase assay confirmed regulation of TEX14 and SOX17 by DAZL through their 3'-UTR. RNA immunoprecipitation further demonstrated direct binding between human TEX14, TEX19, SYCP3, SOX17 mRNA and DAZL protein, and that TEX14 binding is through its 3'-UTR. Dual fluorescence immunohistochemistry showed that SOX17 and DMNT3L are expressed in early germ cells with DAZL, and are later down-regulated co-incident with that of DAZL, consistent with the novel repressive effect of human DAZL on these two potential targets. These studies indicate that DAZL and BOLL are associated with different key meiotic stages of germ cell development in human fetal ovary. Several potential mRNA targets of DAZL and BOLL, and a novel repression function of human DAZL on its mRNA targets were identified giving further insight into the role of these factors in human ovarian development.