Au Yeung Chi Lam. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 204-221). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Tin-hung Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 137-151. / ACKNOWLEDGEMENT --- p.1 / CONTENT --- p.3 / ABBREVIATIONS --- p.7 / ABSTRACT --- p.8 / Chapter CHAPTER 1 --- INTRODUCTION --- p.10 / Chapter CHAPTER 2 --- LITERATURE REVIEW / Chapter 2.1 --- The cervix and cervical cancer --- p.12 / Chapter 2.2 --- Human papillomaviruses --- p.23 / Chapter 2.3 --- Culture of cancer cells --- p.36 / Chapter 2.4 --- Methods for the detection of HPV infection --- p.40 / Chapter CHAPTER 3 --- MATERIALS AND METHODS / Chapter 3.1 --- Characterization of cervical carcinoma cell lines / Chapter 3.1.1 --- Materials and solutions --- p.47 / Chapter 3.1.2 --- Establishment of cervical carcinoma cell lines --- p.50 / Chapter 3.1.3 --- Morphological studies of cervical carcinoma cells --- p.52 / Chapter 3.1.4 --- Examination of cervical carcinoma cells cultured on collagen gel --- p.54 / Chapter 3.1.5 --- Growth kinetics study --- p.55 / Chapter 3.1.6 --- Plating efficiency test --- p.56 / Chapter 3.1.7 --- Spheroid formation assay --- p.56 / Chapter 3.1.8 --- Chromosome number study --- p.57 / Chapter 3.2 --- Immunocytochemical studies / Chapter 3.2.1 --- Materials and solutions --- p.58 / Chapter 3.2.2 --- Immunocytochemical test for keratin --- p.59 / Chapter 3.2.3 --- Test for HPV capsid antigens --- p.60 / Chapter 3.3 --- Molecular studies of HPV in cervical carcinoma cells / Chapter 3.3.1 --- Materials and solutions --- p.62 / Chapter 3.3.2 --- Preparation of HPV DNA probes --- p.66 / Chapter 3.3.3 --- DNA extraction from the cervical carcinoma cells --- p.74 / Chapter 3.3.4 --- Detection of HPV DNA sequences by the use of hybridization techniques --- p.76 / Chapter 3.3.5 --- Copy number and physical state studies of HPV --- p.81 / Chapter 3.3.6 --- Study of the transcriptional activity of HPV DNA in cultured cervical carcinoma cells --- p.83 / Chapter CHAPTER 4 --- RESULTS / Chapter 4.1 --- Characterization of cervical carcinoma cell lines / Chapter 4.1.1 --- Morphological studies --- p.89 / Chapter 4.1.2 --- Examination of cervical carcinoma cells cultured on collagen gel --- p.90 / Chapter 4.1.3 --- Growth kinetics study --- p.93 / Chapter 4.1.4 --- Plating efficiency test --- p.94 / Chapter 4.1.5 --- Spheroid formation assay --- p.95 / Chapter 4.1.6 --- Chromosome number study --- p.98 / Chapter 4.2 --- Immunocytochemical studies / Chapter 4.2.1 --- Immunocytochemical test for keratin --- p.99 / Chapter 4.2.2 --- Test for HPV capsid antigen --- p.99 / Chapter 4.3 --- Molecular studies of HPV in cervical carcinoma cell lines / Chapter 4.3.1 --- Preparation of HPV DNA probes --- p.101 / Chapter 4.3.2 --- Detection of HPV DNA by the use of hybridization techniques --- p.102 / Chapter 4.3.3 --- Copy number and physical state studies --- p.105 / Chapter 4.3.4 --- Analysis of the transcriptional activity --- p.108 / Chapter CHAPTER 5 --- DISCUSSIONS / Chapter 5.1 --- Characterization of cervical carcinoma cell lines / Chapter 5.1.1 --- Morphological features of two cervical carcinoma cell lines --- p.110 / Chapter 5.1.2 --- Other characteristics of the cell lines --- p.112 / Chapter 5.2 --- Immunocytochemical studies / Chapter 5.2.1 --- Test for keratin antigens --- p.117 / Chapter 5.2.2 --- Test for HPV capsid antigens --- p.117 / Chapter 5.3 --- Molecular studies of HPV in cervical carcinoma cell lines / Chapter 5.3.1 --- Establishment of methods --- p.121 / Chapter 5.3.2 --- Detection of HPV DNA sequences by nucleic acid hybridizations --- p.123 / Chapter 5.3.3 --- Copy number and physical state studies --- p.128 / Chapter 5.3.4 --- Transcriptional analysis of HPV DNA in cervical carcinoma cell lines --- p.132 / CONCLUSION --- p.134 / REFERENCES --- p.137 / ILLUSTRATIONS --- p.152

Introdução: Prurigo é uma terminologia de conotação sindrômica que tem o prurido como sintoma incondicional. O sinal é dermatológico, caracteristicamente manifestado por pápulas que se distribuem com relativa simetria pelo tegumento. Do ponto de vista clínico é uma condição de fácil suspeita, mas de difícil conclusão causal. Na Fundação Centro de Controle de Oncologia do Amazonas (FCECON), quadro de prurigo em pacientes oncológicos eram ocasionalmente identificados após início da radioterapia para o tratamento do tumor de base, contexto epidemiológico que não é classicamente relacionado ao prurigo. Objetivos: Descrever os aspectos clínicos, histopatológicos e imunoistoquímicos dos casos pósradioterapia. Métodos: estudo transversal, descritivo, de janeiro de 2006 a dezembro de 2012, com seleção de pacientes com CID de prurigo na FCECON. Serão descritas as características clínicas, histopatológicas e imunoistoquimicas dos casos com pesquisa de DNA viral do EBV, CMV e parvovírus B19. Resultados: Foram 35 mulheres e um homem, com idade média de 47 anos, sempre com queixa de prurido. A pelve sempre foi o campo irradiado com mediana de 28 dias para o surgimento dos sintomas. Foram 34 tumores ginecológicos com 31 casos de colo de útero. Ao todo ocorreram 13.339 lesões (média de 371), com 535 do grupo 1 (vesículas e urticas), 4.934 do grupo 2 (pápulas), 4.218 do grupo 3 (escoriações) e 3.652 do grupo 4 (residuais). Os membros inferiores foram acometidos em todos os casos e em 27 casos houve acometimento dos membros superiores. A histologia das 27 lâminas disponíveis identificou epiderme normal em cinco pacientes, atrofia em dois, espongiose em oito e alteração secundária ao prurido nas demais. Espongiose folicular e/ou espongiose do acrossiríngeo foram identificadas por nove ocasiões. Infiltrado perivascular superficial, profundo e intersticial composto por linfócitos e eosinófilos com distribuição perianexial foi o padrão mais encontrado na derme. O padrão do infiltrado foi de linfócitos T CD4 e ausência de linfócitos CD20 e linfócitos CD56. Não foi identificado DNA viral nas amostras examinadas. Discussão: O quadro de prurigo pós-radioterapia possui as mesmas características clínicoepidemiológicas e histológicas da síndrome EPPER. Histologicamente pode ser inclusa dentro das doenças inflamatórias eosinofílicas da pele, com padrão imunoistoquímico do infiltrado sugestivo de reação de hipersensibilidade mediada por linfócitos no padrão Th2. Como reação a picada de mosquitos foi o diagnóstico histológico de todos os casos, postula-se que o prurigo pós radioterapia, ou síndrome EPPER, seja reação imunológica precipitada pela exposição aos antígenos salivares do mosquito em um ambiente imune induzido pelo efeito abscopal secundário a irradiação pélvica. A natureza do estudo não permite confirmar esta conclusão, que deverá ser fruto de pesquisas futuras. Conclusões: O quadro de prurigo pós radioterapia esteve associado a irradiação da pelve, especialmente de tumores ginecológicos, com predomínio do câncer de colo de útero. Clinicamente semelhante a erupção pruriginosa, polimórfica, eosinofílica associada a radioditerapia (EPPER), pode ser classificado como prurigo agudo. As manifestações clínicas, histológicas e imunoistoquímicas são semelhantes ao quadro de hipersensibilidade a picada de mosquito do padrão celular, mediado por linfócitos Th2, sem evidencia de co-participação viral nos casos examinados / Introduction: Prurigo, a terminology used with a syndromic connotation, is characterized by pruritus, as an unconditional symptom, and papules usually distributed with relative symmetry by the integument. From the clinical point of view, it is a condition of easy suspicion, but of difficult causal conclusion. At the Fundação Centro de Controle de Oncologia do Amazonas (FCECON), prurigo was occasionally identified in cancer patients after radiation therapy for the treatment of the underlying tumor, an epidemiological context that is not classically related to prurigo. Objectives: To describe the clinical, histopathological and immunohistochemical aspects of post-radiotherapy prurigo. Methods: a cross-sectional, descriptive study, from January 2006 to December 2012, with the selection of patients with prurigo as classified by international classification of diseases in FCECON. The clinical, histopathological and immunohistochemical characteristics of the cases with viral DNA screening by RT-PCR EBV, CMV and parvovirus B19 is described. Results: There were 35 women and one man, with an average age of 47 years. Pruritus was always present. The pelvis was invariably the irradiated field with a median of 28 days for the onset of symptoms. There were 34 cases with gynecological tumors with 31 of the uterine cervix. A total of 13,339 lesions (mean of 371), with 535 of group 1 (vesicles and urticas), 4,934 of group 2 (papules), 4,218 of group 3 (excoriations) and 3,652 of group 4 (residual). The lower limbs were affected in all cases, and in 27 cases there was involvement of the upper limbs. The histology of the 27 available slides identified normal epidermis in five patients, atrophy in two, spongiosis in eight and alteration secondary to pruritus in the others. Follicular spongiosis or spongiosis of the acrosyringium were identified on nine occasions. A superficial, deep and interstitial perivascular infiltrate composed of lymphocytes and eosinophils with perianexial distribution was the most common pattern found in the dermis. The composition of the infiltrate was of CD3 CD4 T lymphocytes and absence of CD20 lymphocytes and CD56 lymphocytes. There were no viral DNA identified in the samples examined. Discussion: Postradiotherapy prurigo has the same clinical-epidemiological and histological characteristics of eosinophilic, polymorphic, pruritic associated with radiotherapy (EPPER) syndrome. Histologically it can be included within the inflammatory eosinophilic diseases of the skin, and the immunohistochemestry pattern of the infiltrate suggests a hypersensitivity reaction mediated by Th2 lymphocytes. Since a reaction to mosquito bites was the clinicopathological diagnosis of all cases, the authors postulate that post-radiation prurigo, or EPPER syndrome, is an immunological reaction precipitated by exposure to the mosquito\'s salivary antigens in an immune environment induced by the abscopal effect secondary to irradiation of the pelvis. The nature of the study does not allow this conclusion, which should be the result of future research. Conclusions: Post-radiation prurigo was associated with irradiation of the pelvis especially to treat gynecological tumors, with cancer of the cervix predominance. Clinically similar to EPPER syndrome, it can be classified as acute prurigo. Clinical, histological and immunohistochemical manifestations are similar to the hypersensitivity of Th2 lymphocytemediated mosquito bites, with no evidence of viral co-participation in the cases examined

Os métodos minimamente invasivos têm sido cada vez mais utilizados para o tratamento do leiomioma e, a embolização da artéria uterina, tem se destacado como método seguro e efetivo. O objetivo deste estudo foi avaliar, pela ressonância magnética da pelve, os fatores preditores da diminuição dos leiomiomas de pacientes submetidos a embolização da artéria uterina. Métodos: Estudaram-se 50 mulheres sintomáticas com leiomioma uterino, na menacme, que foram submetidas a embolização da artéria uterina. Acompanhou-se, por meio da ressonância magnética o volume do útero e dos leiomiomas. Foram examinados 179 leiomiomas nestas pacientes, um mês antes e seis meses depois do procedimento. Resultados: Seis meses após o tratamento, a redução média do volume uterino foi de 38,91%, enquanto os leiomiomas tiveram redução de 55,23%. Nos leiomiomas submucosos e/ou com a relação nódulo/músculo em T2 mais elevada, a redução do volume foi ainda maior (maior que 50,00%). Conclusões: As pacientes portadoras de leiomiomas e submetidas à embolização da artéria uterina apresentaram redução de volume dos nódulos superior a 50,00%, à ressonância magnética, quando eram submucosos e/ou tinham uma relação nódulo/músculo em T2 mais elevada / Objective : Minimally invasive methods are being an alternative to treat leiomyomas, including the uterine artery embolization that has emerged as a safe and effective method. The aim of this study was to evaluate the magnetic resonance imaging predictors of decrease in leiomyomas of patients who underwent uterine artery embolization. Methods: This study followed 50 symptomatic premenopausal women with uterine leiomyoma who underwent uterine artery embolization. Treatment was accompanied by magnetic resonance imaging of both the volume of the uterus and the leiomyomas. We examined 179 leiomyomas in that 50 patients, one month before and six months after of the procedure. Results: Six months after treatment, the mean reduction in uterine was 38.91%, while leiomyomas decreased by 55.23%. In submucosal leiomyomas and/or with a higher node/muscle ratio in T2, the volume reduction was even higher (greater than 50.00%). Conclusions: The patients with leiomyomas and underwent uterine artery embolization, showed reductions in the volume of nodes greater than 50,00%, on the magnetic resonance imaging, when they were submucosal and / or had a higher node-to-muscle ratio in T2

Introdução: Leiomiomas uterinos ou miomas são tumores benignos que se desenvolvem no miométrio e acometem cerca de 50% da população feminina. Têm como principais sintomas, sangramento excessivo e dor pélvica inespecífica. Convencionalmente o estrogênio é considerado o responsável pelo início da proliferação tumoral, mas recentes evidências clínicas e bioquímicas sugerem que a progesterona apresenta um papel importante no desenvolvimento desses tumores. Além disso, mecanismos epigenéticos parecem estar envolvidos na etiologia dos leiomiomas uterinos, como a metilação de DNA e acetilação de histonas. Somente nos EUA são realizadas 240 mil histerectomias/ano para tratar essa doença sendo considerado um problema de saúde pública. Frente a esses dados é imprescindível o entendimento dos mecanismos moleculares envolvidos no desenvolvimento desses tumores e a busca de tratamentos menos invasivos. A mifepristona (RU 486), um modulador seletivo dos receptores de progesterona e glicocorticoides, é capaz de diminuir o tamanho dos tumores e amenizar os sintomas associados. Objetivos: verificar se (1) nos tecidos de leiomiomas uterinos e miométrio, os mecanismos epigenéticos como a metilação global do DNA e a acetilação de histonas estão alterados (2) se em cultura primária de leiomioma uterino e miométrio o tratamento com estradiol e progesterona é capaz de modular a expressão gênica dos receptores de progesterona total e B, assim como a atividade das enzimas histona acetiltransferase e desacetilase, e (3) se a mifepristona (RU 486) é capaz de modular diretamente a expressão gênica dos receptores de progesterona total e B após tratamento com os hormônios estradiol e progesterona. Métodos: para análise de metilação global do DNA e acetilação de histonas foram utilizadas 25 amostras teciduais para cada grupo de leiomioma uterino e miométrio oriundos de pacientes submetidas à histerectomia no Hospital de Clínicas de Porto Alegre. A metilação global do DNA e a atividade da enzima histona acetiltransferase foram dosadas pelo método de ELISA e a enzima histona desacetilase por detecção fluorimétrica. Para verificar o efeito dos hormônios sexuais ovarianos e o efeito da mifepristona (RU 486) sobre a expressão gênica dos receptores de progesterona total e B foram realizadas 7 culturas primárias de leiomiomas uterinos e miométrio. Resultados: (1) Foram observadas hipermetilação (P = 0,022) e hipoacetilação (P = 0,04) em leiomiomas uterinos quando comparado ao miométrio. Não houve diferença estatística entre estes tecidos em relação à atividade da histona acetiltransferase. (2) Houve aumento da expressão gênica do receptor de progesterona total em cultura primária de leiomioma uterino quando tratado com estradiol (P = 0,028) e o receptor de progesterona B teve sua expressão aumentada quando tratado com estradiol, progesterona e estradiol + progesterona (P = 0,001). (3) O tratamento com mifepristona (RU 486) na dose de 10-6M não foi capaz de diminuir a expressão gênica dos receptores de progesterona total e B em células de leiomiomas uterinos e miométrio. A atividade da enzima histona desacetilase foi maior nas células de leiomioma uterino quando tratadas com estradiol (P = 0,034) e estradiol + progesterona + RU 486 (P = 0,001) quando comparado às células de miométrio, já a atividade da enzima histona acetiltransferase não foi detectada, devido a sua baixa quantidade. Conclusão: Nesse estudo foi observado uma hipermetilação e hipoacetilação nos tecidos de leiomiomas uterinos. Esses resultados sugerem que mecanismos epigenéticos podem estar contribuindo para a diminuição transcricional de genes relacionados ao funcionamento normal do miométrio e, com isso, colaborando para o crescimento desses tumores. Além disso, nossos resultados sugerem que estradiol e progesterona são capazes de modular a expressão gênica dos receptores de progesterona total e B e o medicamento RU 486 na dose de 10-6M não foi capaz de diminuir diretamente a expressão desses receptores. / Introduction: Uterine leiomyoma, also known as fibroids, is a smooth muscle cell tumor, and is clinically apparent in up to 50% of reproductive-age women. Clinical symptoms include abnormal uterine bleeding and pelvic pain. Estrogen has been considered a primary growth promoter of uterine leiomyoma, however clinical and biochemical evidence has suggested that progesterone plays a critical role in the development of these tumors. Furthermore, epigenetic modifications may be involved with etiology of theses tumors, through DNA methylation and histone acetylation. In the United States, 240.000 hysterectomies/year are performed to treat uterine leiomyomas which is being considered to be a public public health problem. The understanding of molecular mechanisms involved in the development of uterine leiomyoma may provide not only opportunities for a diagnostic, but also generation of novel therapeutic approaches. The mifepristone (RU 486), a synthetic steroid that has affinity for progesterone and glucocorticoid receptors has been reported to induce regression of uterine leiomyoma and reduce the symptoms. Objective: verify if (1) DNA global methylation and histone acetylation patterns are altered in uterine leiomioma tissue compared with myometrium; (2) the treatment with estradiol and progesterone are able to modulated de gene expression of total and B progesterone receptors and histone acetylation patterns in primary culture of uterine leiomyoma cells and myometrium as well as (3) the mifepristone (RU 486) are able to modulate the gene expression of total and B progesterone receptors after the treatment with ovarian steroids hormones. Methods: DNA global methylation and histone acetylation patterns were analyzed in 25 tissue samples from uterine leiomioma and myometrium. The DNA global methylation and the activity of histone acetyltransferase were performed using ELISA method. In order to evaluate histone deacetylase activity fluorimetric detection was used. To verify the effect of estradiol and progesterone on total and B progesterone receptors gene expression, as well as the influence of RU486 on this parameters, seven cultured cells from uterine leiomyoma and myometrium cells were performed. Results: (1) Hypermethylation (p = 0.022) and hypoacetylation (p = 0.04) in uterine leiomioma tissues compared with myometrium were observed. There was no statistic difference between these tissues in histone acetyltransferase activity. (2) We observed increased gene expression of total progesterone receptor in culture cells of uterine leiomyoma when treated with estradiol (p = 0.028). There was an increase of B progesterone receptor mRNA when treated with hormones, estradiol and progesterone (p = 0.001). The treatment with RU 486 was not able to modulate progesterone receptors. The histone desacetilase activity was elevated in uterine leiomyoma cells when treated with estradiol (p = 0.034) and estradiol + progesterone + RU 486 (p = 0.001). The histone acetyltransferase activity was barely detectable. Conclusion: In our study we found hypermethylation and hypoacetylation in uterine leiomyoma tissues suggesting that this process may lead to a decreased transcriptional activity of important genes associated with normal myometrium function contributing to the development of these tumors. Furthermore, our results suggest that ovarian steroids hormones increase progesterone receptors expression, being mifepristone (RU 486) at dose of 10-6M unable to decrease total and B progesterone receptors in uterine leiomyoma cells.

by Yam Hin Fai. / "June 1998." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 118-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstract in Chinese.

O objetivo do presente trabalho foi comparar os efeitos dos distintos ambientes endócrinos peri-ovulatórios na proliferação celular, no metabolismo das poliaminas e no ambiente redox do útero bovino durante o diestro inicial. Para isso, controlou-se farmacologicamente o crescimento do folículo objetivando induzir a ovulação de folículos de maior diâmetro (grupo folículo grande-CL grande, FG-CLG) ou de menor diâmetro (grupo folículo pequeno-CL pequeno, FP-CLP). Trinta vacas multíparas Nelore, foram pré-sincronizadas, metade destes animais foram destinados para o grupo FG-CLG e receberam uma dose de prostaglandina F2&#945; (PGF) e um dispositivo de progesterona, juntamente com benzoato de estradiol no D10. No momento da retirada dos dispositivos de progesterona (entre D1,75 e D2,5) todos os animas receberam uma dose de PGF. A ovulação foi induzida com acetato de buserelina (D0). O que diferiu entre os tratamentos foi que os animais do grupo FP-CLP não receberam uma dose de PGF no D10 e o momento da retirada dos dispositivos foi entre D1,25 e o D1,5. No D7 um subgrupo dos animais foi abatido e amostras de endométrio e lavado uterino foram coletadas para as análises laboratoriais. Os animais do grupo FG-CLG apresentaram no D0 um folículo 1,2 vezes maior e estradiol 2,3 vezes maior em relação ao FP-CLP. Consequentemente, as vacas do FG-CLG desenvolveram CL maiores (1,6 vezes) e mais pesados (1,4 vezes) o que proporcionou no D7 concentrações de P4 1,5 vezes maior em relação ao FP-CLP. Primeiramente, uma análise do transcriptoma endometrial foi realizada e indicou que processos celulares como proliferação, composição de matrix extracelular, processos metabólicos e processos de oxirredução foram afetados pelo modelo experimental. Para avaliar a condição proliferativa e/ou apoptótica endometrial análises de imunomarcação foram realizadas (Ki-67, marcador de proliferação e caspase 3 ativada, marcador de apoptose). O número de células positivas para Ki-67 no epitélio luminal foi 4,1 vezes maior no FP-CLP, porém não houve diferença na marcação da caspase 3 ativada. No epitélio glandular tanto o ki-67 (1,4 vezes) como a caspase 3 ativada (2,6) estavam aumentados no grupo FG-CLG. Em relação à síntese das poliaminas não houve diferença entre os grupos nas concentrações das poliaminas e abundância dos transcritos e proteína das enzimas de síntese. Por outro lado, foi detectada uma forte associação entre a expressão do gene SAT1 com o gene ODC1 (r2: 0,73; P<0,01) e também entre os genes AZIN1 e AMD1 (r2: 0,61; P<0,01). Quanto ao ambiente redox não houve diferença entre os tratamentos no lavado uterino mas no endométrio, no grupo FP-CLP, houve redução da atividade das enzimas Catalase (0,5 vs 0,79 U/mg proteína; P<0,01), glutationa peroxidase (2,0 vs 2,43 nmol NADPH/min/mg proteína; P<0,01), aumento da atividade da Superóxido dismutase (44,77 vs 37,76 U; P=0,04) e da peroxidação lipídica (28,5 vs 17,43 nmol/MDA/mg proteína; P<0,001), mas sem alterar as quantidades da espécies reativas. De acordo com os resultados obtidos, pode-se concluir que o ambiente endócrino peri-ovulatório regula a proliferação endometrial mas sem alterar a via de síntese das poliaminas e modula também o ambiente redox uterino / This work aimed to compare the effects of the different periovulatory endocrine milieu on the several molecular pathways: as cellular proliferation, polyamine metabolism and redox environment of the bovine uterus during the early diestrus. For this, we controlled pharmacologically the follicular growth to induce the ovulation of larger follicle (Large Follicle-Large Corpus lutem group LF-LCL) or smaller follicle (Small follicle-Small Corpus luteum group SF-SCL). Thirty multiparous Nelore cows were synchronized. Fifteen cows were assigned for each group. On the LF-LCL, the cows received a dose of prostaglandin F2&#945; (PGF), a progesterone device and an estradiol benzoate on D10. On the time of progesterone device was withdraw (between D1.75 e D2.5) all the animals received a dose of PGF. The ovulation was induced with buserelin acetate (D0). The unique difference between the groups was that animals of SF-SCL did not received a dose of PGF on D10 and the progesterone device withdraw was between D1.25 e o D1.5. On D7 a subset of animals was slaughtered and the endometrial and uterine washings samples were collected for laboratorial analyses. On D0 the animals of LF-LCL had a 1.2 times larger follicles and 2.3 times higher levels of estradiol than animals of SF-SCL. Firstly, an endometrial transcriptome analyses was realized and suggested that several cellular processes as proliferation, metabolic processes and oxi-reduction processes were affected by experimental model. To evaluate the proliferative and/or apoptotic condition, endometrial immunohistochemistry analyses were realized (ki-67, proliferation marker and caspase 3 activated, apoptose marker). The number of Ki-67 positive cells in the luminal epithelium was 4.1 times higher on SF-SCL. However, there was no difference in caspase 3 activated analyses. On the glandular epithelium both ki-67 (1.4) and caspase 3 activated (2.6) were increased on LF-LCL. About polyamine synthesis pathway there was no difference in polyamines levels and gene and protein expression of synthesis enzymes between the groups. In other hand, it was detected a strong association between SAT1 gene expression and ODC1 gene expression (r2: 0.73; P<0.01) and also between AZIN1 and AMD1 gene expression (r2: 0.61; P<0.01). On the redox environment analysis there was no difference between the groups on the uterine washings but the SF-SCL group had lower endometrial catalase (0.5 vs. 0.79 U/mg protein, P<0.001) and glutathione peroxidase (GPx; 2.0 vs. 2.43 nmol NADPH/min/mg protein, P=0.04) activity, as well as higher lipid peroxidation (28.5 vs. 17.43 nmol MDA/mg of protein, P < 0.001) and superoxide dismutase (SOD) activity (44.77 vs. 37.76 U; P=0.04). There were no differences in the endometrial reactive species (RS) between the groups. In conclusion, the periovulatory endocrine milieu regulates the endometrial proliferation but without alterations in polyamine metabolism and also modulates the uterine redox environment in beef cattle

Cervical cancer is the third leading cause of cancer death in women worldwide. Although cervical cancer is commonly infected with human papillomavirus (HPV), HPV infection alone is insufficient to induce malignant changes. Many characteristic genetic and epigenetic alterations have been identified in invasive cervical carcinomas but relatively little is known about the specific genetic and molecular alterations that allow pre-invasive epithelial cells to acquire the ability to progress to invasive squamous cell carcinomas. Recently, a family of small non-coding RNAs termed microRNAs (miRNAs) with specific inhibitory functions on target gene expression has been suggested to play an important role in the pathogenesis of human cancers including lung and breast cancer but remain undefined in cervical cancer. / Genome wide chromosomal copy number changes in cervical cancer by Agilent high-density array Comparative Genomic Hybridization demonstrated that only a very limited number of genomic imbalances have an impact on the miRNA profile in cervical cancer cells, although a high proportion of genomic loci containing miRNA genes exhibited DNA copy number alterations in other cancers. The impact of the genomic aberration on their mRNA expression was then confirmed by Aligent Whole Human Genome gene expression array. This suggests that the regulation of miRNA and mRNA expression may be different in cervical cancer. / In conclusion, our global miRNA profiling identified the common differentially expressed and genomic aberration independent miRNAs in cervical cancer. We further revealed the inhibition of hsa-miR-182 reduced tumor cell growth in vitro and in vivo through apoptosis and cell cycle mechanism. This provides new evidence that hsa-miR-182 may contribute to the pathogenesis of cervical cancer. / Keywords. MicroRNA, Cervical Cancer, Tumor Growth / To identify microRNA(s) associated with the tumorigenesis of cervical cancer, we firstly used the TaqMan MicroRNA Assays to survey and quantify a panel of 157 known human miRNAs in cervical cancer cell fines and micro-dissected normal cervical epithelium cells. We identified 2 microRNAs that were differentially up-regulated (fold change > 2, p &lt; 0.05) and 9 differentially down-regulated (fold change > 2, p &lt; 0.05) in cervical cancer cell lines comparing with normal cervical epithelium. Further investigation in tumor samples confirmed these two up-regulated miRNAs (hsa-miR-182 and -183 ) and 3 down-regulated miRNA (hsa-miR-145, 150, 195) from 4 investigated downregulated miRNAs (hsa-miR-145, 150, 195 and 328). / To investigate the biological function of those aberrantly expressed microRNAs, we chose one of the most aberrantly up-regulated microRNA ( hsa-miR-182, fold change > 10) for further investigation. Inhibition of hsa-miR-182 by antisense oligonucleotides inhibited HeLa cervical cancer cell growth in vitro and reduced tumor cell volume in vivo. Gene expression array analysis of HeLa cells with hsa-miR-182 knockdown and over-expression showed specific hsa-miR-182 targeting pathway in apoptosis and cell cycle. It indicated the roles of hsa-miR-182 in cervical cancer growth through apoptosis and cell cycle functions. / Tang, Tao. / Adviser: Richard K W Choy. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3446. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 155-169). / 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.

Fung, Man See Joyce. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 111-120). / Abstracts in English and Chinese. / Table of Contents / Acknowledgements --- p.I / Abstract --- p.II / 論文摘要 --- p.VII / Table of Contents --- p.X / List of Figures --- p.XIV / List of Tables --- p.XVI / Abbreviations --- p.XVII / Chapter Chapter 1 - --- Introduction --- p.l / Chapter 1.1 --- Biology of HPV --- p.2 / Chapter 1.1.1 --- History --- p.2 / Chapter 1.1.2 --- Classification --- p.2 / Chapter 1.1.3 --- Genome structure --- p.3 / Chapter 1.2 --- HPV and cervical cancer --- p.8 / Chapter 1.2.1 --- Classification of cervical lesions --- p.8 / Chapter 1.2.2 --- Natural history of development of cervical cancer --- p.9 / Chapter 1.2.3 --- Risk factors --- p.11 / Chapter 1.3 --- Prevention of cervical cancer --- p.12 / Chapter 1.3.1 --- Vaccination --- p.12 / Chapter 1.3.2 --- Screening --- p.12 / Chapter 1.3.2.1 --- Pap test --- p.12 / Chapter 1.3.2.2 --- HPV DNA test --- p.13 / Chapter 1.3.2.3 --- Methylation pattern as a novel marker --- p.13 / Chapter 1.4 --- Biology of Methylation --- p.14 / Chapter 1.4.1 --- Definition --- p.14 / Chapter 1.4.2 --- Silencing effect --- p.18 / Chapter 1.4.3 --- Roles in normal development --- p.20 / Chapter 1.5 --- Methylation and human diseases --- p.20 / Chapter 1.5.1 --- Genetic diseases --- p.20 / Chapter 1.5.2 --- Cancers --- p.21 / Chapter 1.5.3 --- Methylation and oncogenic viruses --- p.23 / Chapter 1.5.4 --- Potential of methylation pattern as a novel biomarker of cancer --- p.24 / Chapter 1.5.5 --- Epigenetic therapy --- p.25 / Chapter 1.6 --- Methylation and HPV --- p.25 / Chapter 1.6.1 --- History --- p.25 / Chapter 1.6.2 --- Potential roles in transcription regulation of HPV --- p.26 / Chapter 1.6.3 --- Viral gene methylation --- p.27 / Chapter Chapter 2 - --- "Hypotheses, Objectives and Study Design" --- p.28 / Chapter 2.1 --- Hypotheses --- p.29 / Chapter 2.2 --- Objectives --- p.30 / Chapter 2.3 --- Study Design --- p.30 / Chapter Chapter 3 - --- Materials and Methods --- p.34 / Chapter 3.1 --- Work flow --- p.35 / Chapter 3.2 --- Study subjects --- p.37 / Chapter 3.2.1 --- Invasive cervical cancer group --- p.37 / Chapter 3.2.2 --- Low-grade group --- p.37 / Chapter 3.2.3 --- Cell lines --- p.38 / Chapter 3.3 --- DNA extraction --- p.38 / Chapter 3.4 --- HPV genotyping --- p.39 / Chapter 3.5 --- PCR of HPV16 LCR --- p.39 / Chapter 3.6 --- Sequencing of HPV 16 LCR --- p.42 / Chapter 3.6.1 --- Purification of PCR products --- p.42 / Chapter 3.6.2 --- Cycle sequencing reaction --- p.42 / Chapter 3.6.3 --- Purification of cycle sequencing products --- p.43 / Chapter 3.6.4 --- Sequencer and data analysis --- p.43 / Chapter 3.7 --- Bisulfite modification --- p.43 / Chapter 3.8 --- PCR of bisulfite modified LCR --- p.45 / Chapter 3.9 --- Cloning --- p.48 / Chapter 3.9.1 --- Ligation --- p.48 / Chapter 3.9.2 --- Transformation --- p.48 / Chapter 3.9.3 --- Colony PCR --- p.49 / Chapter 3.10 --- Sequencing of clones --- p.51 / Chapter 3.10.1 --- Purification of PCR products --- p.51 / Chapter 3.10.2 --- Cycle sequencing reaction --- p.51 / Chapter 3.10.3 --- Purification of cycle sequencing products --- p.52 / Chapter 3.10.4 --- Sequencer and data analysis --- p.52 / Chapter 3.11 --- Statistical methods --- p.52 / Chapter Chapter 4 - --- Results --- p.54 / Chapter 4.1 --- Sample selection --- p.55 / Chapter 4.2 --- HPV16 LCR PCR and sequencing --- p.57 / Chapter 4.3 --- Methylation patterns --- p.61 / Chapter 4.3.1 --- Cell lines --- p.61 / Chapter 4.3.2 --- Cancer group --- p.63 / Chapter 4.3.2.1 --- Overview --- p.63 / Chapter 4.3.2.2 --- Methylation pattern of the cancer samples --- p.66 / Chapter 4.3.2.3 --- Methylation pattern of the promoter region --- p.74 / Chapter 4.3.3 --- Low-grade group --- p.76 / Chapter 4.3.3.1 --- Overview --- p.76 / Chapter 4.3.3.2 --- Methylation pattern of the low-grade samples --- p.79 / Chapter 4.3.4 --- Comparison of the methylation patterns of the cancer samples and the low-grade samples --- p.84 / Chapter Chapter 5 - --- Discussion --- p.95 / Chapter 5.1 --- Sequence variations of HPV 16 LCR --- p.96 / Chapter 5.2 --- Methylation patterns of CaSki and SiHa cell lines --- p.98 / Chapter 5.3 --- Methylation pattern of the cancer samples --- p.99 / Chapter 5.4 --- Methylation pattern of the low-grade samples --- p.100 / Chapter 5.5 --- Comparison of methylation patterns of the cancer samples and the low-grade samples --- p.101 / Chapter 5.5.1 --- Promoter region in 3' LCR --- p.102 / Chapter 5.5.1.1 --- SP1 binding site --- p.102 / Chapter 5.5.1.2 --- E2BS3 and E2BS4 --- p.103 / Chapter 5.5.2 --- Silencer region --- p.104 / Chapter 5.5.3 --- Enhancer region in central LCR --- p.105 / Chapter 5.5.4 --- CpG sites within 5' LCR --- p.106 / Chapter 5.6 --- Role of methylation in HPV 16 --- p.107 / Chapter 5.7 --- Potential as novel biomarker --- p.108 / Chapter 5.8 --- Conclusions --- p.109 / References --- p.111 / Appendix A

by Yang Lei. / Publication date from spine. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 110-126). / Acknowledgement --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Table of Contents --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.4 / Chapter Chapter 3 --- Materials and Methods --- p.41 / Chapter Chapter 4 --- Results --- p.58 / Chapter Chapter 5 --- Discussion --- p.86 / References --- p.110 / Appendix --- p.120 / Publications --- p.125 / Illustrations --- p.127