Choi, Pui Wah. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 202-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Orientador: Irene Bastos Franceschini Vicentini / Coorientador: Fernanda Antunes Alves da Costa / Banca: Rosicleire Veríssimo Silveira / Banca: Luciene Patrici Papa / Resumo: Sabe-se que em camarões peneídeos, a vitelogenina é sintetizada no ovário e hepatopâncreas, mas a contribuição relativa desses dois tecidos na síntese de vitelogenina ainda é incerta. Este processo pode diferir ainda de acordo com a espécie e estágio vitelogênico ou muda. Assim o presente estudo analisou a localização da produção de vitelogenina através de técnicas imunohistoquímicas, bem como, a quantificação do seu mRNA através do Real Time PCR em fêmeas de Artemesia longinaris em diferentes estágios de maturação ovariana (rudimentar - RU, em maturação - EM e maduro - MA). A espécie de camarão estudada apresenta altíssimo valor econômico. Assim, o estudo dos mecanismos da reprodução podem gerar dados para futuros projetos de produção em escala comercial, contribuindo com a preservação dos estoques naturais. A partir de análises moleculares confirmou-se a síntese de vitelogenina no ovário e hepatopâncreas de fêmeas de A. longinaris durante a maturação ovariana. Os níveis de expressão relativa de transcritos de vitelogenina no ovário e no hepatopâncreas durante a maturação ovariana demonstram que a dinâmica da síntese de vitelogenina é inversamente proporcional. A imunohistoquímica localizou a síntese endógena de vitelogenina nas células foliculares, nos oócitos pré-vitelogênicos e oócitos em vitelogênese inicial. E a síntese exógena de vitelogenina foi localizada nas células R dos túbulos hepatopancreáticos. Ainda, visto a grande contribuição do hepatopâncreas ao ciclo reprodutivo e às demais funções fisiológicas do animal, analisou-se o perfil celular do túbulo hepatopancreático nos estágios de maturação ovariana. Este órgão apresenta comportamento celular que se adapta ao ciclo reprodutivo em fêmeas, ou seja, o ciclo celular do hepatopâncreas além de obedecer a um ciclo digestivo é adaptado ao ciclo reprodutivo / Abstract: It is known that in penaeid shrimp the vitellogenin is synthesized into the ovary and hepatopancreas, but the relative contribution of these two tissues in the synthesis of vitellogenin remains uncertain. This process differs according to the species and vitellogenesis stage or molt. Thus the present study examined the location of vitellogenin production by immunohistochemical techniques, as well as the quantification of its mRNA by Real Time PCR in female Artemesia longinaris at different stages of ovarian maturation (immature - RU, maturing EM and mature - MA). The shrimp species investigated presented a very high economic value. The study of the mechanisms of reproduction can generate data for future projects of commercial scale production contributing with the preservation of wild stocks. The molecular analyzes have confirmed the vitellogenin synthesis in the ovary and hepatopancreas of A. longinaris females during ovarian maturation. the relative expression level of vitellogenin transcripts in the ovary and the hepatopancreas during ovarian maturation show that the dynamics of vitellogenin synthesis is inversely proportional. By immunohistochemistry, it was located the endogenous synthesis of vitellogenin in follicular cells, in pre-vitellogenic oocytes and oocytes in early vitellogenesis. And the synthesis exogenous of vitellogenin was localized in cells R of hepatopancreaticos tubules. Also, given the great contribution of hepatopancreas reproductive cycle and other physiological functions of the animal, we analyzed the cellular profile of the hepatopancreatic tubule at stages of ovarian maturation. This organ has a cellular behavior that adapts to the reproductive cycle in females, in other words, the cellular cycle of hepatopancreas beyond to follow a digestive cycle is adapted to the reproductive cycle / Mestre

Orientador: Gilson Hélio Toniollo / Coorientador: Maricy Apparicio Ferreira / Banca: Elzylene Léga / Banca: Geórgia Modé Magalhães / Resumo: A criopreservação de ovário ou tecido ovariano permite a preservação do material genético de qualquer espécie animal que seja submetido à gonadectomia por indicação preventiva, terapêutica ou, até mesmo, por morte inesperada. O objetivo do presente trabalho foi avaliar se o tamanho do fragmento ovariano influencia ou não a resistência aos crioprotetores. Para tanto, os ovários foram colhidos de 34 gatas domésticas (várias raças, 1-5 anos de idade) por ovariectomia de rotina, transportados ao laboratório e depois seccionados em fragmentos de diferentes tamanhos (3 x 3 x 3mm, 5 x 3 x 3mm e 7 x 3 x 3mm) e destinados aleatoriamente aos grupos de controle (GC3, GC5 e GC7, respectivamente) ou vitrificados (GV3, GV5 e GV7, respectivamente). Os fragmentos vitrificados-aquecidos foram avaliados por histomorfologia e imunohistoquímica (para taxas de apoptose utilizando a caspase-3 clivada). A avaliação histológica demonstrou que 72,97% dos folículos presentes em GV3 e 72,58% nos fragmentos do grupo GV5 eram normais, enquanto que nos fragmentos do GV7 essa taxa foi de apenas 42,86%. A principal alteração morfológica foi o desprendimento das células epiteliais da membrana basal presentes em todos os grupos. Da mesma forma, a avaliação imunohistoquímica, utilizando a caspase 3, revelou uma pequena proporção de células apoptóticas nos fragmentos do grupo GV3 (53%), enquanto que no grupo GV7, 43,58% das células expressaram a caspase 3 clivada. Esses achados indicam que fragmentos seccionado... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cryopreservation of ovary or ovarian tissue allows preservation of genetic material of any animal species that is submitted to gonadectomy for preventive, therapeutic or even by unexpected death. The aim of the present study was to investigate whether or not the size of the ovarian fragment influence its resistance to cryostorage. For that purpose, ovaries were collected from 34 queens (various breeds, age 1-5 y) by routine ovariectomy, transported to the laboratory and then sectioned in different sizes (3 x 3 x 3 mm, 5 x 3 x 3 mm and 7 x 3 x 3 mm) and randomly assigned to a control (GC3, GC5 and GC7, respectively) or vitrified (GV3, GV5 and GV7, respectively) groups. Vitrified-warmed fragments were evaluated by histomorphology and immunohistochemistry (for apoptotic rates by using cleaved caspase-3). Histological examination reveal that 72.97% of the follicles in GV3 and 72.58% in GV5 were normal while only 42.86% of the follicles in GV7. The main morphological alteration presented in all groups was a detachment of the epithelial cells by basement membrane. Similarly, immunohistochemistry evaluation using caspase 3 revealed a small proportion of apoptotic cells in GV3 (53%) while in GV7 43.58% of the cells expressed cleaved caspase-3. These findings indicate that fragments sectioned in 3 x 3 x 3 mm (27mm3) seems more adequate for perfusion of the cryoprotectant, causing less damage to the cell after vitrification-warming. / Mestre

Orientador: Marion Burkhardt de Koivisto / Banca: Wilter Ricardo Russiano Vicente / Banca: Cássia Maria Barroso Orlandi / Resumo: O objetivo desse estudo foi avaliar a histologia e morfometria de ovários de 17 gatas classificadas de acordo com as idades G1 (4-12 meses); G2 (1-6 anos) e G3 (> 6 anos). Após realização da ovariosalpingo-histerectomia os ovários foram fixados, processados rotineiramente para a inclusão em parafina e corados com Hematoxilina-eosina. Os parâmetros morfométricos analisados foram: diâmetro (μm), área (μm2) e perímetro (μm) dos folículos e respectivos oócitos de diferentes tipos de folículos. A relação entre o crescimento do folículo e do oócito foi analisada. Os procedimentos estatísticos utilizados foram ANOVA, sendo as médias comparadas pelo teste de Tukey e as medianas pelo teste de Kruskal-Wallis, seguido do teste de comparações múltiplas de Dunn (P< 0,05). Ao todo foram 1039 folículos analisados histologicamente e houve diferença significativa nos diâmetros, áreas e perímetros foliculares e oocitários de folículos primordiais e primários unilaminares. Observou-se padrão bifásico para o crescimento do folículo e do oócito. Conclui-se que as gatas jovens obtiveram maiores valores em folículos primários unilaminares em relação às idosas. Sugere-se que o "pool" de folículos pequenos remanescentes e seus respectivos oócitos diminuem com a idade e que na fase senil esses folículos são de baixa qualidade, quando comparados ao início da vida reprodutiva. Estudos com morfometria do ovário poderão auxiliar pesquisas de tecnologias reprodutivas assistidas, como por exemplo, a maturação in vitro (MIV), já que o tamanho e a qualidade dos folículos e oócitos podem afetar a MIV em felinos domésticos. Adicionalmente, esses estudos poderão auxiliar na preservação de felinos selvagens em extinção / Abstract: The aim of this study was to evaluate the histology and morphometry of ovaries of 17 cats classified according to ages G1 (4-12 months), G2 (1-6 years) and G3 (>6 years). After ovariosalpingohysterectomy the ovaries were fixed in paraformaldehyde 5% prior to embedding in paraffin and staining with haematoxylin-eosin. The morphometric parameters analyzed were: diameter (μm), area (μm2) and perimeter (μm) of follicles and oocytes from different follicles types. The relationship between the growth of the follicle and oocyte was analyzed. The statistical analysis were ANOVA and the means were compared by Tukey test and medians using the Kruskal-Wallis test followed by Dunn's multiple comparisons (P<0.05). A total of 1039 follicles were analyzed histologically and there were significant difference in follicular and oocyte diameter, area and perimeter of primordial and primary unilaminar follicles. It was observed a biphasic pattern for the growth of the follicle and oocyte. In conclusion, young queens had higher values in primary unilaminar follicles than older queens. It is suggested that the remaining pool of small follicles and their oocytes decreases with age and that follicles of older queens has low quality compared to the beginning of reproductive life. Ovarian morphology studies may assist in assisted reproductive technologies such as in vitro maturation (MIV), since it is known that the size and quality of follicles and oocytes may affect the MIV in domestic queens. Additionally, it may assist in the preservation of endangered wild felines / Mestre

Advancements in medicine and technology have led to an increasing number of cancer survivors. The development of a second primary cancer is one of the most severe sequelae of a cancer diagnosis, particularly for cancers that lack an effective screening tool as with ovarian cancer. Breast and ovarian cancer are major causes of morbidity and mortality in women; in the U.S., breast cancer has the highest incidence in women and ovarian cancer is the most fatal of gynecological cancers. Further, these two cancers have been found to co-occur. Along with possible treatment effects of the first cancer, shared risk factors, shared genetics, and interactions between these two have been hypothesized to contribute to their co-occurrence. Research on shared risk factors for second cancers is lacking and being able to identify potentially modifiable factors associated with second primary cancer could improve clinical recommendations for cancer survivors. Therefore, this dissertation examined risk factors for the development of double primary breast and ovarian cancer (DPBOC) in three parts 1) a comprehensive review of the literature to identify studies assessing risk factors for DPBOC, 2) a case-control study assessing the association between three potentially-modifiable risk factors (oral contraceptive (OC) use, parity, and breastfeeding), and risk of second primary ovarian cancer following breast cancer (BR-OV), second primary breast cancer following ovarian cancer (OV-BR), single primary ovarian cancer (OV), and single primary breast cancer (BR), and 3) a cohort study assessing OC use, parity, and breastfeeding and risk of BR-OV, OV, and BR. The comprehensive review identified few studies assessing epidemiologic risk factors for the development of DPBOC and most of the findings were not statistically significant. The majority of studies focused on treatment of breast cancer and risk of second primary ovarian cancer. While most of the findings on chemotherapy, radiotherapy, and Tamoxifen were heterogeneous and lacked statistical significance, hormone therapy for breast cancer may be associated with an increased risk of second primary ovarian cancer. The majority of studies on genetic risk factors for DPBOC looked at BRCA1/2 mutations or a crude measure of family history. Both BRCA1/2 and family history were consistently associated with risk of DPBOC, but studies varied on the extent of this risk due to differences in study design, exposure and outcome definition, and statistical power. No studies were identified examining DNA methylation and risk of DPBOC. The case-control study used data from the three clinic-based sites of the Breast Cancer Family Registry (BCFR) which consisted of women from breast and ovarian cancer families. We observed an inverse association with both OC use (OR=0.38, 95% CI: 0.22, 0.60) and breastfeeding (OR=0.52, 95% CI: 0.31, 0.87) and risk of DPBOC, but a positive association with parity (≥2 full-term pregnancies: OR=5.78, 95% CI: 2.82, 14.58), regardless of diagnosis order (BR-OV or OV-BR). We found similar associations for our OV and BR outcomes as well. When we examined differences between high and average risk women (using BRCA1/2 mutation status and predicted lifetime risk of breast or ovarian cancer), the inverse association with OC use only remained in women at average risk while the inverse association with breastfeeding only remained in women at high risk. As the positive association with parity and all of our outcomes disagreed with our hypothesis we conducted several sensitivity analyses to explore this finding. Survivor bias may have influenced our results as we observed differences in our findings between cases diagnosed ≤2 or ≤5 years before the baseline interview (pseudo-incident) and cases diagnosed >2 or >5 years before the baseline interview (prevalent). Specifically, the inverse association with OC use and all of our outcomes, and the positive association with parity and all of our outcomes were attenuated in the pseudo-incident group. To address concerns of selection and information bias in our case-control study, we conducted a cohort study using data from The Breast Cancer Prospective Family Study Cohort (ProF-SC). In contrast to our case-control findings, we observed a suggestive positive association between OC use and risk of BR-OV (HR=1.62, 95% CI: 0.91, 2.90) which became stronger in women at high risk, and an inverse association between having two or more full-term pregnancies compared to nulliparous and risk of BR-OV (HR=0.47, 95% CI: 0.22, 0.97) which did not vary by underlying risk of breast and ovarian cancer. However, our BR-OV results may have similarly been influenced by survivor bias as we observed differences in our results between our pseudo-incident and prevalent BR-OV cases; the association between OC use and BR-OV only remained in the prevalent cases. In summary, the results of this dissertation highlight the methodological challenges in the study of second primary cancers and the importance of considering survivor bias in a cohort of cancer survivors being followed for second cancers. Further, our results are suggestive of a discordant effect of OC use on first primary versus second primary ovarian cancer which should be explored in future studies.

Wong Wai Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.iii / Abbreviation --- p.vii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-1 / Chapter 1.1 --- Classification of common epithelial ovarian tumors --- p.1-2 / Chapter 1.1.1 --- Serous tumors --- p.1-4 / Chapter 1.1.2 --- Mucinous tumors --- p.1-5 / Chapter 1.1.3 --- Endometrioid tumors --- p.1-6 / Chapter 1.1.4 --- Clear cell tumors --- p.1-6 / Chapter 1.1.5 --- Cancer staging --- p.1-7 / Chapter 1.1.6 --- Tumor grading --- p.1-8 / Chapter 1.2 --- Etiology --- p.1-10 / Chapter 1.2.1 --- Factors associated with increased risks --- p.1-10 / Chapter 1.2.2 --- Factors associated with decreased risks --- p.1-12 / Chapter 1.2.3 --- Other factors --- p.1-13 / Chapter 1.3 --- Understanding of progression of ovarian carcinoma --- p.1-13 / Chapter 1.4 --- Current screening test for ovarian cancer --- p.1-15 / Chapter 1.4.1 --- Transvaginal utrasound --- p.1-15 / Chapter 1.4.2 --- Serum tumor markers --- p.1-16 / Chapter 1.5 --- Molecular basis of ovarian cancer --- p.1-18 / Chapter 1.5.1 --- Loss of heterozygosity --- p.1-18 / Chapter 1.5.2 --- Microsatellite instability --- p.1-19 / Chapter 1.5.3 --- Oncogenes --- p.1-19 / Chapter 1.5.4 --- Tumor suppressor genes --- p.1-21 / Chapter 1.6 --- Microarray gene expression profiling analysis --- p.1-25 / Chapter 1.6.1 --- Princeple of DNA micorarray --- p.1-26 / Chapter 1.6.2 --- Types of microarray --- p.1-29 / Chapter 1.7 --- Gene expression profiling of ovarian cancer --- p.1-29 / Chapter 1.7.1 --- Up-regulated genes in ovarian cancer --- p.1-30 / Chapter 1.7.2 --- Down-regulated genes in ovarian cancer --- p.1-32 / Chapter 1.8 --- Project aims --- p.1-35 / Chapter CHPATER 2 --- MATERIALS AND METHODS --- p.2-1 / Chapter 2.1 --- Materials --- p.2-1 / Chapter 2.1.1 --- Patients --- p.2-1 / Chapter 2.1.2 --- Ovarian tissue specimen --- p.2-1 / Chapter 2.2 --- Methods --- p.2-2 / Chapter 2.2.1 --- Preparation of OCT-embedded Specimen Sections --- p.2-2 / Chapter 2.2.2 --- Microdissection of Tumor Cells from Specimen Sections --- p.2-3 / Chapter 2.2.3 --- Disruption of normal ovarian frozen tissue --- p.2-3 / Chapter 2.2.4 --- Total RNA Extraction --- p.2-3 / Chapter 2.2.4.1 --- RNA Isolation --- p.2-4 / Chapter 2.2.4.2 --- DNase I Digestion --- p.2-4 / Chapter 2.2.4.3 --- RNA Cleanup and Elution --- p.2-5 / Chapter 2.2.5 --- Oligonucleotide Microarray --- p.2-6 / Chapter 2.2.5.1 --- Two-Cycle cDNA Synthesis --- p.2-6 / Chapter 2.2.5.2 --- Synthesis of Biotin-Labeled cRNA --- p.2-9 / Chapter 2.2.5.3 --- Fragmenting the cRNA for Target Preparation --- p.2-9 / Chapter 2.2.5.4 --- Target Hybridization --- p.2-10 / Chapter 2.2.5.5 --- "Array Washing, Staining, and Scanning" --- p.2-11 / Chapter 2.2.5.6 --- Statistical Analysis of Microarray Data --- p.2-11 / Chapter 2.2.6 --- Quantitative Real-time Polymerase Chain Reaction --- p.2-13 / Chapter 2.2.6.1 --- Primer and Probe --- p.2-13 / Chapter 2.2.6.2 --- Reverse-transcription --- p.2-13 / Chapter 2.2.6.3 --- Plate Setup --- p.2-14 / Chapter 2.2.6.4 --- Fluocogenic PCR --- p.2-14 / Chapter 2.2.6.5 --- Statistical Analysis of Quantitative Real-time PCR Data --- p.2-15 / Chapter CHAPTER 3 --- RESULTS --- p.3-1 / Chapter 3.1 --- Microarray gene expression data analysis --- p.3-1 / Chapter 3.1.1 --- Unsupervised Gene Selection --- p.3-1 / Chapter 3.1.2 --- Supervised Gene Selection --- p.3-3 / Chapter 3.1.2.1 --- Gene expression profiles distinguish Serous Epithelial Ovarian Tumor from Normal Ovary and identifydifferentially expressed genes --- p.3-3 / Chapter 3.1.2.2 --- Gene expression profiles distinguish Advanced Stage Serous Epithelial Ovarian Tumor from Early Stage Serous Epithelial Ovarian Tumor and identify differentially expressed genes --- p.3-22 / Chapter 3.1.2.3 --- Gene expression profiles distinguish Metastatic Serous Epithelial Ovarian Tumor from Primary Serous Epithelial Ovarian Tumor and identify differentially expressed genes --- p.3-24 / Chapter 3.2 --- Validation of microarray data by quantitative Real-time PCR --- p.3-27 / Chapter 3.2.1 --- Fold change of candidate genes --- p.3-27 / Chapter 3.2.2 --- Correlation between microarray and quantitative real-time PCR results --- p.3-29 / Chapter 3.2.3 --- Comparison of the expression of candidates genes among the different histological types of epithelial ovarian tumors --- p.3-32 / Chapter CHAPTER 4 --- DISCUSSION --- p.4-1 / Chapter 4.1 --- Global gene expression profiling using oligonucleotide microarray --- p.4-1 / Chapter 4.1.1 --- "Sensitivity, specificity and reproducibility of the Affymetrix GeneChip® microarray" --- p.4-1 / Chapter 4.1.2 --- Microarray analysis software --- p.4-3 / Chapter 4.1.2.1 --- DNA-Chip Analyzer software --- p.4-3 / Chapter 4.1.2.2 --- Comparison of statistical methods for analysis of Affymetrix GeneChip® microarray data --- p.4-5 / Chapter 4.2 --- Validation of microarray data --- p.4-7 / Chapter 4.2.1 --- Advantages of using real-time PCR for mRNA quantification --- p.4-8 / Chapter 4.2.2 --- Comparison of mRNA gene expression by RT-PCR and DNA microarray --- p.4-9 / Chapter 4.3 --- Gene expression profiling in serous ovarian cancer compared with normal ovarian epithelium --- p.4-10 / Chapter 4.3.1 --- Potential biomarkers or therapeutic targets in ovarian cancer --- p.4-12 / Chapter 4.4 --- Gene expression profiling in advanced serous ovarian cancer compared with early ovarian cancer --- p.4-16 / Chapter 4.4.1 --- Potential prognostic markers or therapeutic targets in advanced ovarian cancer --- p.4-17 / Chapter 4.5 --- Gene expression profiling in metastatic cancer compared with primary ovarian cancer --- p.4-22 / Chapter 4.5.1 --- Potential predictive markers or therapeutic targets in metastatic cancer of ovary origin --- p.4-23 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.5-1 / Chapter CHAPTER 6 --- FUTURE PROSPECT --- p.6-1 / REFERENCES --- p.R-1

Another important gene, heat shock factor (MeHSF) was also cloned using homology based PCR because it was suggested to participate in the transcriptional regulation of many essential components of ovarian maturation including vitellogenin gene and several proteins for hormones metabolism. The complete cDNA sequence of MeHSF was 2211 bp in length, which encoded a 622 amino acid protein. The translated MeHSF protein shared high similarity with those of other species, especially in the N terminal region. RT-PCR showed that MeHSF was universally expressed in most of the female tissues investigated including ovary, central nervous system, heart, gill, gut and muscle. However, its expression was not detectable in eyestalk and hepatopancreas. MeHSF was highly expressed in immature ovaries, and decreased dramatically with the progress of ovarian maturation. Since the synthesis of vitellogenin in ovary showed an opposite trend, the result suggested that MeHSF probably functioned as a transcriptional repressor to vitellogenin. Four HSFs isoforms generated from alternative splicing were obtained in immature ovaries, suggesting a possible universal role of HSF in coordinating transcription of different target genes during shrimp ovarian maturation. / As an important component of enzymatic scavenger systems, glutathione peroxidases (GPx) play important roles in maintaining the balance between reactive oxygen species (ROS) production and cellular scavenging ability. In this research, a full length GPx gene (MeGPx) which had been identified using RAP-PCR previously was cloned and characterized. MeGPx might play a pivotal role in preventing oocytes from oxidative damage and balancing ROS production. The present data on shrimp GPx provides insights on the regulation of ROS in the ovarian maturation process. / Four candidate genes possibly participating in the regulation of ovarian maturation were obtained by random sequencing the libraries, including metallothionein, two zinc finger proteins and member 4 of wingless-type MMTV integration site family (WNT4). The zinc finger protein containing a plant homeodomain, was only expressed in the eyestalk of female with immature ovaries, but not that of female with early mature and mature ovaries. The full length cDNA sequence of shrimp WNT4 gene (MeWNT4) was obtained using RACE technique. RT-PCR showed that the expression of MeWNT4 in eyestalk decreased with the maturation of shrimp ovaries. Interestingly, MeWNT4 was strongly expressed in the central nervous system and gut of both female and male shrimp. It was suggested that WNT4 could antagonize the testis determining factor (SRY), and play an essential role in suppressing the formation of testis, and at the same time, controlling of female development. Thus, the identification WNT4 from crustacean would contribute to our understanding on the sex determination mechanism. / In this thesis, two genes, heat shock protein 90 and heat shock factor, possibly playing important roles in shrimp ovarian maturation were identified and characterized. / Shrimp farming plays an important economic role in Southeast Asian countries. Yet further development of this industry is seriously restricted by the environmental deterioration and the prevalence of fetal diseases. Moreover, the failure of sexual maturation of cultured female shrimp forms a bottleneck to the further development of shrimp aquaculture. With the aim to produce shrimp without totally depending on the wild stocks, many studies have been focused on the endocrine regulation of shrimp ovarian maturation. In order to enhance our understanding on the molecular events occurred during ovarian maturation, in this research, several candidate genes are identified, and their potential roles in ovarian maturation are studied in the shrimp Metapenaeus ensis. / Since heat shock protein 90 gene is one of the essential components of steroid hormone signal cascades in vertebrates, it was cloned and isolated by homology cloning strategy. The complete cDNA sequence of shrimp Hsp90 ( MeHsp90) was 2524 by in length, which encoded a 720 amino acid polypeptide. The MeHsp90 coding region was interrupted by four introns. MeHsp90 was differentially expressed in eyestalk, ovary and hepatopancreas at different ovarian maturation stages, and consistently expressed in other tissues including heart, gill, gut, muscle and central nervous system. In vitro ovary explant assay revealed that MeHsp90 expression in immature ovary could be induced by the addition of exogenous estradio1-17beta. MeHsp90 was highly expressed in pre-vitellogenic oocytes, and its expression decreased with the progress of maturation, and finally stopped in late-vitellogenic oocytes. The co-regulation of MeHsp90 and vitellogenin by estrogen hormone suggested a possible regulatory role of Hsp90 in vitellogenin synthesis of the shrimp. / Wu, Long Tao. / "May 2008." / Adviser: Chu Ka Hou. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1409. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 92-113). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Ovarian cancer is the 5th leading cause of cancer death in women in the United States and is the most fatal gynecological malignancy. High grade serous ovarian cancer (HGSC) is the most common and deadly type of ovarian cancer largely due to the rapid metastasis throughout the peritoneum (abdominal cavity wall and organ lining). Metastatic spread of ovarian cancer usually occurs before diagnosis and can lead to bowel obstruction, organ failure, ascites, cachexia, infection and sepsis, and pulmonary embolism all causing death. Current methods to detect early stage ovarian cancer do not increase overall survival. A better understanding of the metastatic ability of ovarian cancers and the mechanism of cancer cell dissemination are critical to the development of new treatments for this devastating disease. In particular, investigation of pathways that affect early metastasis may indicate treatments that will lower disease burden and may suggest biomarkers of recurrent and/or chemotherapy resistant disease. Notch3 expression correlates with worse prognosis, chemotherapy resistance, and increased tumorigenic cell behaviors in HGSC. Here, we demonstrate that Notch3 acts to promote early stages of metastasis in a model of HGSC using the murine ID8 IP2 ovarian surface epithelial cell line. ID8 IP2 cells have little to no endogenous Notch3 expression and model metastatic disease when introduced intraperitoneally. We investigated the role of Notch3 by ectopically expressing the intracellular domain of murine Notch3 to induce constitutive Notch3 signaling in ID8 IP2 cells and verified Notch signal activation by target gene assessment. Induction of Notch3 signaling in ID8 IP2 reduced survival and accelerated disease burden, as measured by ascites accumulation, after intraperitoneal introduction of cells into nude mice. We interrogated downstream targets in Notch3 activated cells by RNA-Seq and found that Notch3 induced a significant enrichment of adhesion and extracellular matrix pathways. Notch3 active cells showed increased ITGA1 expression and increased adhesion on collagens I and IV in vitro, suggesting that increased adhesion to collagen-rich peritoneal surfaces drives the observed increase in tumor burden. Notch3 active cells showed reduced migration on surfaces coated with multiple types of extracellular matrix and no detectable increase in invasion through extracellular matrix, indicating that Notch3 effects may be specific to the initial adhesion of tumor cells and not the later stages of metastasis. These results demonstrate that Notch3 upregulates the expression of specific adhesion genes in ovarian cancer cells and this promotes increased attachment to the collagen-rich extracellular matrix. The implications of this study are that oncogenic Notch signal activation, as documented in human disease, may promote dissemination and metastasis of primary and/or recurrent HGSC by increasing attachment to the peritoneal lining.

A critical step in mammalian ovarian follicle development is the transition of gonadotropin-independent preantral follicles to the gonadotropin-dependent antral follicles. However, the molecular mechanisms underlying the transition or early follicle activation are largely unknown. Using zebrafish as the model, we have recently identified Y-box binding protein 1 (YB-1, Ybx1/ybx1), a transcription factor and mRNA binding protein, in early developing oocytes whose expression level was very high in the gonadotropin-independent primary growth (PG) stage but drastically diminished at the beginning of the gonadotropin-dependent secondary growth (SG) stage, i.e., previtellogenic (PV) stage. This has raised interesting questions on the role of Ybx1 in follicle activation as well as how it is controlled. To provide clues to these issues, we first analyzed the regulation of Ybx1 during PG-to-PV transition under IGF-I treatment and the associated signaling pathways. IGF-I, an endocrine/paracrine factor in the growth axis, stimulats Ybx1 phosphorylation via PI3K/Akt but not MAPK pathway in PG follicles. Interestingly, the phosphorylation correlated well with the decline of Ybx1 protein level and the activation of the follicle from the PG follicle pool. This, together with our finding that zebrafish Ybx1 is exclusively produced in PG oocytes in large amount but suddenly disappears during PG-to-PV transition, has prompted us to wonder what the relationship between Ybx1 phosphorylation and degradation. Further experiments showed that Akt directly binds and phosphorylates Ybx1, leading the regulation of Ybx1, including its phosphorylation, cleavage, translocation and degradation, which in turn regulates gene expression and protein synthesis. / In summary, as a multifunctional protein that may play a critical role in early follicle development, Ybx1 is subject to regulation by external factors such as IGF-I, which stimulated Ybx1 phosphorylation via PI3K/Akt but not MAPK pathway. Once Ybx1 is phosphoylated by Akt in the cytoplasm of PG follicle, on one hand, it will be cleaved and translocated to the nucleus to regulate gene expression. On the other hand, the phosphor-Ybx1 can also be degraded through the Ub-proteasome pathway, leading the release of free mRNA to further translation. All these promote the synthesis of many growth- and differentiation-related proteins, which will facilitate early follicle activation. Our findings suggest that the oocyte may serve as the headquarter to programme follicle activation and that the oocyte Ybx1 protein may play a critical role in this event. The delineation of the signaling pathways involved in IGF-I-induced Ybx1 phosphorylation and the regulation of Ybx1 as well as its function in gene transcription and protein synthesis during PG-to-PV transition will provide insight into the mechanism of early follicle activation and puberty initiation. / 哺乳动物卵巢卵泡发育的一个关键步骤是从促性腺激素非依赖的窦前卵泡向促性腺依赖的窦状卵泡的转变过程。但是这一早期卵泡激活的分子机制却不是非常清楚。利用斑马鱼为模型，我们在早期发育的卵母细胞中发现一种名叫Y-box结合蛋白1 （YB-1, Ybx1/ybx1）的转录因子和mRNA 结合蛋白，它在促性腺激素不依赖的初级生长期卵泡（PG）中大量表达，但是在促性腺激素依赖的第二生长期卵泡（SG）,也叫卵黄发生前期(PV)中表达量大大降低。这引发我们猜想YB-1 可能在早期卵泡激活（PG-to-PV 转变）中发挥着重要作用，并且想知道它的这一表达量的巨变是如何被调控的。为了弄清楚这些问题，我们首先分析了IGF-I 处理下Ybx1 在PG-to-PV 的转变中是怎样被调控的，以及相关的信号通路。我们发现在PG 阶段，IGF-I 这种存在于生长轴中的内分泌／旁分泌因子，通过PI3K/Akt 而不是MAPK 通路促进Ybx1 的磷酸化。有趣的是，这种磷酸化的升高正好伴随着Ybx1 蛋白水平的下降以及PG 卵泡的激活。结合我们之前的发现：斑马鱼Ybx1 只在PG 卵母细胞中大量表达但在PG-to-PV 的转变过程中突然消失，促使我们猜想Ybx1 磷酸化和它的降解之间应该存在一定的关系。进一步的实验表明Akt 激酶直接结合并磷酸化Ybx1,导致一系列对Ybx1 调控，包括它的磷酸化，切割，转位以及降解，所有这些又将促进基因的表达调控及蛋白的合成。 / 总之， 多功能蛋白Ybx1 可能在早期卵泡发育过程中发挥着至关重要的作用。外界刺激因子，如IGF-I，通过PI3K/Akt 而非MAPK 途径促进Ybx1 磷酸化。一旦Ybx1 在PG 卵泡细胞质中被Akt 磷酸化，一方面Ybx1 将会被切割并且转位到细胞核中去调节基因表达，另一方面，磷酸化的Ybx1 还会通过泛素蛋白酶途径被降解，从而释放出mRNA 去进一步的翻译。所有这些将促进许多生长和分化相关的蛋白合成，从而促进早期卵泡的激活。我们的研究结果表明，卵母细胞很可能是程序性卵泡激活的核心部分，存在于卵母细胞中的Ybx1 蛋白在这一过程中起着关键作用。研究IGF-I 参与诱导的Ybx1 磷酸化的信号通路以及在PGto-PV 转变过程中对Ybx1 蛋白的调控和它在基因表达及蛋白合成中的作用，将有力的帮助我们弄清早期卵母细胞激活及青春期的启动机制。 / Zhang, Lingling. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 104-127). / Abstracts also in Chinese. / Title from PDF title page (viewed on 06, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Yu, Man Ying Susana. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-117). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of contents --- p.viii / Symbols and abbreviations --- p.xii / Scientific names --- p.xiv / List of figures --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Structure of ovarian follicles --- p.1 / Chapter 1.2 --- Regulation of ovarian follicle development --- p.3 / Chapter 1.2.1 --- Endocrine regulation --- p.3 / Chapter 1.2.1.1 --- Gonadotropins- FSH and LH --- p.3 / Chapter 1.2.1.2 --- Co-gonadotropin- growth hormone --- p.5 / Chapter 1.2.2. --- Paracrine regulation --- p.6 / Chapter 1.2.2.1 --- Activin --- p.6 / Chapter 1.2.2.2 --- Insulin-like growth factor I (IGF-I) --- p.7 / Chapter 1.3 --- The GH-IGF-I axis --- p.7 / Chapter 1.3.1 --- The somatomedin hypothesis --- p.8 / Chapter 1.3.2 --- "Structure and signaling of GH, GHR" --- p.8 / Chapter 1.3.3 --- Structure and signaling of IGF system --- p.9 / Chapter 1.3.4 --- Role of GH-IGF system in reproduction --- p.11 / Chapter 1.3.5 --- GH action in ovarian functions --- p.12 / Chapter 1.3.6 --- IGF-I action in ovarian functions --- p.13 / Chapter 1.3.7 --- The mini GH-IGF axis within the ovary --- p.14 / Chapter 1.4 --- Objectives of present study --- p.14 / Chapter Chapter 2 --- "Expression Profiles of the GH-IGF System in the Ovary of Zebrafish, Danio rerio" --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Material and Methods --- p.21 / Chapter 2.2.1 --- Animals --- p.21 / Chapter 2.2.2 --- Isolation of tissues and different stages of follicles from the zebrafish --- p.22 / Chapter 2.2.3 --- Separation of somatic follicle layers and oocytes --- p.22 / Chapter 2.2.4 --- Primary follicle cell culture --- p.22 / Chapter 2.2.5 --- Total RNA extraction --- p.23 / Chapter 2.2.6 --- Reverse transcription --- p.23 / Chapter 2.2.7 --- "Validation of semi-quantitative RT-PCR assays for GH (gh), GHR (ghr), IGF-I (igf1), IGF-II (igf2), and IGF-I receptor (igf1r)" --- p.24 / Chapter 2.2.8 --- Data analysis --- p.25 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Validation of semi-quantitative RT-PCR assays --- p.25 / Chapter 2.3.2 --- Spatial expression of GH-IGF in different tissues of zebrafish --- p.26 / Chapter 2.3.3 --- "Localization of gh, ghr, igf1, igf2 and igf1r within the zebrafish follicle" --- p.26 / Chapter 2.3.4 --- Temporal expression profiles of GH-IGF system during folliculogenesis --- p.28 / Chapter 2.4 --- Discussion --- p.28 / Chapter Chapter 3 --- Regulation of the GH-IGF-I System and Its Cross-talk with the Activin System in the Zebrafish Ovary --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Material and methods --- p.45 / Chapter 3.2.1 --- Animals --- p.45 / Chapter 3.2.2 --- Chemicals and hormones --- p.45 / Chapter 3.2.3 --- Primary follicle cell culture --- p.45 / Chapter 3.2.4 --- Preparation of ovarian fragments --- p.45 / Chapter 3.2.5 --- Total RNA extraction --- p.45 / Chapter 3.2.6 --- RT-PCR --- p.47 / Chapter 3.2.7 --- Construction of real-time PCR standards --- p.47 / Chapter 3.2.8 --- Real-time PCR and semi-quantitative RT-PCR --- p.48 / Chapter 3.2.9 --- Data analysis --- p.49 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- "Expression of growth hormone (gh), growth hormone receptors (ghr1 and ghr2\ IGF-I (igf1), IGF-II (igf2), IGF-I receptor (igf1ra and igf1rb), activin subunits (inhba and inhbb) and follistatin (fst) in cultured zebrafish ovarian fragments" --- p.49 / Chapter 3.3.2 --- "Establishment of real-time RT-PCR for zebrafish inhba, inhbb and bactin" --- p.50 / Chapter 3.3.3 --- GH regulation of activin expression in cultured zebrafish follicle cells --- p.50 / Chapter 3.3.4 --- GH regulation of IGF-I in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.5 --- IGF-I regulation of activin expression in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.6 --- Activin regulation of IGF system --- p.52 / Chapter 3.4 --- Discussion --- p.52 / Chapter Chapter 4 --- Production of recombinant zebrafish growth hormone --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Material and Methods --- p.71 / Chapter 4.2.1 --- Animals --- p.71 / Chapter 4.2.2 --- Construction of expression plasmids pPIC9K/zfGH --- p.71 / Chapter 4.2.3 --- Production of recombinant zebrafish GH using Pichia pastoris --- p.73 / Chapter 4.2.4 --- SDS-PAGE and silver staining --- p.74 / Chapter 4.2.5 --- Purification --- p.74 / Chapter 4.2.6 --- Primary follicle cell culture --- p.75 / Chapter 4.2.7 --- Zebrafish hepatic cell culture --- p.76 / Chapter 4.2.8 --- RNA extraction and RT-PCR --- p.76 / Chapter 4.2.9 --- Real-time PCR --- p.77 / Chapter 4.2.10 --- Cell culture and transient transfection --- p.78 / Chapter 4.2.11 --- Luciferase reporter gene assay --- p.78 / Chapter 4.2.12 --- Data analysis --- p.79 / Chapter 4.3 --- Results --- p.79 / Chapter 4.3.1 --- Production of recombinant zebrafish GH --- p.79 / Chapter 4.3.2 --- Effect of recombiant zfGH on the expression of activin β Aand βB in cultured zebrafish follicle cells --- p.80 / Chapter 4.3.3 --- Effect of zfGH on the expression of igf1 in cultured zebrafish hepatic cells --- p.80 / Chapter 4.3.4 --- Luciferase reporter gene assay --- p.81 / Chapter 4.4 --- Discussion --- p.81 / Chapter Chapter 5 --- General Discussion --- p.94 / Chapter 5.1 --- Overview --- p.94 / Chapter 5.2 --- Major achievements of the present study --- p.95 / Chapter 5.2.1 --- Demonstration of a local mini-GH-IGF-I axis within the zebrafish ovary --- p.96 / Chapter 5.2.2 --- Differential expression profiles of the GH-IGF system during folliculogenesis --- p.96 / Chapter 5.2.3 --- The inter-relationship of GH-IGF and activin-follistatin systems --- p.96 / Chapter 5.2.4 --- Production of recombinant zebrafish GH --- p.97 / Chapter 5.3 --- Future prospects --- p.97 / References --- p.102 / Symbols and Abbreviations / Symbols / α Alpha / β Beta / Abbreviations / 20β-HSD 20β-hydroxysteroid dehydrogenase / bp Base pair / cAMP Cyclic adenosine monophosphate / cDNA Complementary cDNA / CHO Chinese hamster ovary / "DHP 17α, 20β-dihydroxy-4-prenane-3 -one" / DNA Deoxyribonucleic acid / EGF Epidermal growth factor