Dissertations / Theses on the topic 'Endometrial pathology'

The aim of this thesis was to determine the diagnostic accuracy of outpatient endometrial evaluation using endometrial biopsy (EB), ultrasound scan (USS) and hysteroscopy (OPH) by conducting systematic quantitative reviews of the published literature. The optimum diagnostic strategy in terms of cost-effectiveness (cost per life year gained), was then established for the investigation of women with post-menopausal bleeding (PMB) for endometrial cancer, using the review data in a decision analysis designed to reflect current service provision. Meta-analyses showed that a positive test result following EB or OPH was more useful for predicting endometrial disease than USS, whereas a negative test result following USS was more useful for excluding endometrial disease than EB or OPH. The economic model included 12 diagnostic strategies and indicated that a strategy based on initial diagnosis with USS, using a 5mm double layer endometrial thickness cut-off, was the most cost-effective. Sensitivity analyses showed that initial investigation with EB or USS using a 4mm cut-off were also potentially cost-effective (incremental cost-effectiveness ratios under £30,000 per life year gained) at their most favorable estimates of diagnostic performance, in women under 65 years and at disease prevalence of 10% or more. The choice between initial testing with EB or USS will therefore depend upon patient age and preference, disease prevalence and the availability of high quality USS. In most circumstances women presenting for the first time with PMB should undergo initial evaluation with pelvic ultrasound using a threshold of 4mm or 5mm to define abnormal results.

The ADAMTS (A Disintegrin and Metalloproteinase with TromboSpondin Repeats) are a novel family of secreted metalloproteinases. There is increasing evidence that distinct ADAMTS subtypes play key roles in embryonic development, reproduction and cancer. Nineteen ADAMTS subtypes have been identified in humans but most of them have been characterized only at the structural level. ADAMTS-1, -4, -5, -8, -9 and -15 have been subclassified into a subfamily, known as aggrecanases, owing to their ability to cleave the important extracellular matrix components, versican and aggrecan. Previous studies have determined that ADAMTS-1 and -5 are expressed in first trimester decidual cells and are regulated by IL-1ß and TGF- ß1. I have now found that gonadal steroids have complex regulatory effects upon ADAMTS-1 mRNA and ADAMTS-1 levels in endometrial stromal cells during the human menstrual cycle. I further demonstrate that progesterone (P4) and 5α-dihydrotestosterone (DHT), differentially regulated ADAMTS-5, -8, and -9 mRNA and protein levels in human endometrial stromal cells, suggesting that aggrecanases contribute to steroid-mediated ECM remodeling in the endometrium in preparation for pregnancy. My loss- and gain- of function studies have confirmed a function for ADAMTS-1 in endometrial cancer invasion. Overexpression of ADAMTS-1 in well-differentiated ECC-1 endometrial carcinoma cells promoted cell invasion. In contrast, siRNA-mediated silencing of endogenous ADAMTS-1 in poorly differentiated KLE cells decreased their invasive capacity. I have also found that 17ß-estradiol (E2) can up-regulate ADAMTS-1 mRNA and protein levels in ECC-1 cells. This suggests that ADAMTS-1 plays an important role in endometrial cancer progression, and that E2 promotes well-differentiated endometrial cancer cell invasion, at least in part by specific up-regulating ADAMTS-1 expression. Overall, my research provides useful insight into the molecular mechanisms that regulate endometrial physiology and pathology, and additional support for the concept that ADAMTS represent potentially useful prognostic biomarkers of recurrent pregnancy loss and endometrial cancer.

STUDY OBJECTIVE: To evaluate the diagnostic accuracy of transvaginal sonography compared to hysteroscopy in diagnosing benign endometrial pathology. DESIGN: Retrospective cross-sectional study. Canadian Task force classification II – 2 SETTING: Department of Gynaecology, Groote Schuur Hospital, Cape Town, South Africa. PATIENTS: Patients having an office hysteroscopy procedure between January 2014 and December 2016, with a record of a recent transvaginal ultrasound and endometrial histology were included in this study. All malignant cases were excluded. INTERVENTIONS: Transvaginal ultrasound, endometrial biopsy and office hysteroscopy. MEASUREMENTS AND MAIN RESULTS: A total of one hundred and forty two patients, pre- and postmenopausal, were included in this study. The most common indications for hysteroscopy were abnormal uterine bleeding and postmenopausal bleeding. Sensitivity, specificity, positive and negative predictive values were calculated for ultrasonography and hysteroscopy in diagnosing benign endometrial pathology by comparing them to histological diagnosis as gold standard. The most common pathologies identified at histology were polyps and fibroids. For those patients who had a normal endometrium at ultrasound (n=59), hysteroscopy revealed 33.9% polyps, 5.1% submucosal fibroids and 49.2% normal/atrophic endometrium. The remainder of these patients demonstrated proliferative or hyperplastic endometrium, suspicious endometrium and adhesions. For those patients who had a normal hysteroscopy (n=26), ultrasound demonstrated 7.7% polyps, 7.7% submucosal fibroids, 11.5% cystic areas, 3.9% no comment on endometrium and 69.2% normal endometrium. In diagnosing polyps, hysteroscopy had a higher sensitivity (78%) than ultrasound (37.3%). However, ultrasound had a higher specificity (85.5%), compared to that of hysteroscopy which was 71.1%. The negative predictive value of hysteroscopy for polyps was 81.9% and ultrasound, 65.7%. In the diagnosis of submucosal fibroids, ultrasound had a higher sensitivity than hysteroscopy but they both had similar specificity. Ultrasound and hysteroscopy had high negative predictive values and low positive predictive values. The combination of ultrasound and hysteroscopy did not improve sensitivity, PPV or NPV with a small decline in specificity. CONCLUSION: This study demonstrated that hysteroscopy was more accurate in the diagnosis of endometrial polyps than ultrasound with a higher sensitivity and negative predictive value. However hysteroscopy had a lower sensitivity when diagnosing submucosal fibroids.

The intent of this research was to identify if the degenerative changes within arteries in the endometrium (endometrial angiopathies) correlate with degenerative changes in the uterine arteries and can be used as a predictor of increased risk for uterine artery rupture (UAR). With this objective specimens from 20 mares that died from uterine artery rupture and 21 control mares that died from unrelated causes were obtained from cases submitted to the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL) over a two-year period. Postmortem specimens of each mare were collected from the left and right uterine arteries at the origin, bifurcation, and distal to the bifurcation as well as full thickness uterine wall sections at five different sites. An additional sample was taken from the uterine artery at the site of rupture in the affected mares. Tissue samples were immersed in 10% neutral buffered formalin, routinely processed, and stained with hematoxylin and eosin, Masson’s Trichrome, and Verhoeff´s Van Gieson histochemical stains as well as a smooth muscle-actin immunohistochemical marker. Elastosis, fibrosis, and vascular smooth muscle cell degeneration were identified in this study as potential contributors of vascular degeneration and a scoring system was developed to differentiate the degrees of severity of these specific degenerative changes within the intima and media of the vascular wall. Based on the scoring system, sections of uterine arteries and endometrial arterioles were blindly examined and the scored changes recorded for statistical analysis. Although the degenerative changes in endometrial and uterine arteries were similar within each group, the results could not not be used to predict an increased risk for UAR. Furthermore, we determined the major changes in vascular pathology of the affected uterine arteries and show there is a significant difference in degenerative changes between specific layers of the vascular wall.

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Objetivo: Avaliar a presença do polimorfismo genético do receptor da progesterona (PROGINS) bem como as variáveis clínicas e epidemiológicas de risco para câncer de endométrio em mulheres com pólipos endometriais na pós-menopausa. Casuística e Métodos: Comparou-se em estudo caso-controle 154 mulheres menopausadas com pólipos endometriais benignos e 400 controles normais na pós-menopausa, quanto à presença do PROGINS, por meio da Reação em Cadeia da Polimerase (PCR). O grupo de pólipos endometriais foi comparado a 118 pacientes do grupo controle no tocante às variáveis clínicas e epidemiológicas de risco para câncer de endométrio. Estas variáveis foram também comparadas entre os pólipos benignos e malignos. Resultados: A comparação entre o grupo de pólipos benignos e o grupo controle mostrou significância estatística (p<0,05) para as varáveis: idade (média de 61,7 x 57,5 anos), raça não-branca (44,8% x 22,9%), anos da menopausa (média de 12,9 x 9,2 anos), paridade (média de 4,5 x 3,4 filhos), uso de tamoxifeno (5,2% x 0%), hipertensão arterial (54,5% x 29,7%) e antecedente de câncer de mama (10,4% x 0,8%) respectivamente. Após o ajuste para a idade, permaneceram com significância estatística, apenas a paridade (OR=1,13), a hipertensão arterial (OR=2,19) e o antecedente de câncer de mama (OR=14,44). Seis casos (3,75%), foram diagnosticados como pólipos malignos, nestes casos, sangramento na pós-menopausa e o tamanho grande do pólipo estiveram sempre presentes, enquando que nos pólipos benignos esta frequência foi de 23,4% para sangramento e 54,5% para pólipo grande. A hipertensão arterial foi bem mais frequente no grupo de pólipos malignos, 83,3% x 54,5% nos pólipos benignos. Não houve diferença estatisticamente significante entre os grupos quanto à presença do PROGINS, sendo no grupo de pólipos benignos a distribuição entre homozigoto selvagem, heterozigoto e homozigoto mutado de 79,9%, 19,5% e 0,6% respectivamente. No grupo controle (N=400) esta distribuição foi de 78,8%, 20,8% e 0,5% respectivamente. Conclusões: A presença do PROGINS não mostrou associação significativa com pólipos endometriais. As variáveis epidemiológicas significantemente associadas à presença de pólipos endometriais, após o ajuste para idade, foram a paridade, hipertensão arterial e o antecedente de câncer de mama (implícito o uso de tamoxifeno), além da idade mais avançada. Em nosso estudo, pólipos endometriais malignos estiveram sempre associados à presença de sangramento na pós-menopausa e tamanho grande do pólipo, sendo a hipertensão arterial achado bastante frequente.
Purpose: To evaluate the genetic polymorphism of the progesterone receptor (PROGINS), as well as clinical and epidemiological risk factors for endometrial cancer in postmenopausal women with endometrial polyps. Methods: A case control study was designed with 154 postmenopausal women with endometrial polyps, compared to a normal control group of 400 postmenopausal women. The genotyping of PROGINS polymorphism was determined by polymerase chain reaction. The group of polyps was compared to 118 normal postmenopausal controls regarding clinical and epidemiological variables. These variables were also compared between benign and malignant endometrial polyps. Results: The epidemiological variables among the group of endometrial polyps and normal control, showed statistical significance (p<0,05) for age: media of 61,7 and 57,5 years, ethnicity non-white 44,8% and 22,9%, time since menopause media of 12,9 and 9,2 years, parity media of 4,5 and 3,4 sons, tamoxifen use 5,2% and 0%, hypertension 54,5% and 29,7% and history of breast cancer 10,4% and 0,8% respectively. After age adjust, statistical significance, remained only for parity (OR=1,13), hypertension (OR=2,19) and history of breast cancer (OR=14,44). Postmenopausal bleeding and large polyps were present in all cases of malignancy. Hypertension was also very frequent in malignant polyps (83,3% and 54,5% respectively). The presence of PROGINS had no statistical significance between the group of polyps and the normal control (N=400). The presence of wild homozygosis genotype, heterozygosis and mutant homozygosis was 79,9%, 19,5% and 0,6% respectively for the polyp group, and 78,8%, 20,8% and 0,5% for the control group (p=0,208). Conclusions: There was no significant association between the presence of PROGINS and endometrial polyps. After age adjust, epidemiological variables significantly associated to endometrial polyps were elderly age, parity, hypertension, and history of breast cancer (implicit tamoxifen use). Malignant polyps in this study were always associated to postmenopausal bleeding, large polyps and frequently associated to hypertension.
TEDE
BV UNIFESP: Teses e dissertações

Estrogen and progesterone receptors were localized in fresh frozen sections of human endometrial tissues, in both health and disease, using the ER-ICA kit and a mouse monoclonal antiprogesterone receptor antibody ($ alpha$PR6), respectively. Estrogen and progesterone receptors were detected exclusively in the nuclei of epithelial and stromal cells of the endometrium. Their highest levels in both components were found during the late proliferative phase of the normal menstrual cycle. Estrogen receptors decreased faster in the stroma than in the epithelium throughout the post ovulatory phase, whereas progesterone receptors decreased more rapidly in the epithelium during the mid and late secretory phases. Estrogen and progesterone receptor levels were high in the epithelium of hyperplasia without cytologic atypia. They were low in the epithelium of endometrial intraepithelial neoplasia (hyperplasia with cytologic atypia) and the majority of invasive carcinomas. The stroma contained relatively high estrogen and progesterone receptors levels, irrespective of whether the epithelium was hyperplastic or neoplastic.

Cheung Ka Wai.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 83-93).
Abstracts in English and Chinese.
Acknowledgments --- p.i
Publications --- p.ii
Awards --- p.iii
List of abbreviations --- p.iv
List of figures --- p.vi
List of tables --- p.vii
Abstract in English --- p.viii
Abstract in Chinese --- p.ix
Table of Contents
Chapter Chapter 1 --- Introduction --- p.1
Chapter Chapter 2 --- literature Review
Chapter 2.1 --- Anatomy and Physiology of Endometrium --- p.2
Chapter 2.2 --- Endometrial cancer --- p.5
Chapter 2.2.1 --- Epidemiology --- p.6
Chapter 2.2.2 --- Etiologies and Risk Factors --- p.7
Chapter 2.3 --- Pathology --- p.11
Chapter 2.3.1 --- Grading of endometrial cancer --- p.14
Chapter 2.3.2 --- Staging of endometrial cancer --- p.14
Chapter 2.4 --- Prevention and Treatment --- p.16
Chapter 2.5 --- Molecular alterations in endometrial cancer --- p.17
Chapter 2.5.1 --- Genetic alterations in endometrial cancer --- p.18
Chapter 2.5.1.1 --- Oncogene activation --- p.19
Chapter 2.5.1.2 --- Tumor suppressor gene inactivation --- p.20
Chapter 2.5.1.2.1 --- Mutation and loss of heterozygosity of tumor suppressor genes in endometrial cancer --- p.22
Chapter 2.5.2 --- Epigenetic alterations --- p.27
Chapter 2.5.2.1 --- CpG islands methylation --- p.29
Chapter 2.5.2.2 --- de novo methylation --- p.29
Chapter 2.5.2.3 --- Detection of gene promoter hypermethylation --- p.31
Chapter 2.5.2.4 --- Epigenetic alteration in endometrial cancer --- p.31
Chapter 2.5.2.5 --- Promoter hypermethylation of tumor suppressor genes in other cancers --- p.39
Chapter 2.5.3 --- Microsatellite instability --- p.42
Chapter Chapter 3 --- The objectives of study --- p.45
Chapter Chapter 4 --- Materials and Methods --- p.46
Chapter 4.1 --- Samples --- p.46
Chapter 4.1.1 --- Formalin fixed paraffin embedded tissues --- p.46
Chapter 4.1.2 --- Cell lines --- p.46
Chapter 4.2 --- Histological grading and staging of samples --- p.47
Chapter 4.3 --- Microdissection on tissue sections --- p.47
Chapter 4.4 --- Extraction of nucleic acid
Chapter 4.4.1 --- Extraction of DNA from paraffin-embedded tissues --- p.48
Chapter 4.4.2 --- Extraction of DNA from cell lines --- p.49
Chapter 4.5 --- DNA methylation analysis --- p.49
Chapter 4.5.1 --- Overview of Methylation-Specific PCR (MSP) --- p.49
Chapter 4.5.2 --- Bisulfite modification of DNA --- p.50
Chapter 4.5.3 --- Methylation specific PCR (MSP) --- p.51
Chapter 4.6 --- Microsatellite Analysis --- p.53
Chapter 4.7 --- Statistical analysis --- p.56
Chapter Chapter 5 --- Results
Chapter 5.1 --- Clinical-pathological features of endometrioid adenocarcinoma --- p.57
Chapter 5.2 --- Promoter hypermethylation in endometrial cancer --- p.57
Chapter 5.3 --- Microsatellite status (MSI) analysis --- p.65
Chapter Chapter 6 --- Discussion
Chapter 6.1 --- Promoter hypermethylation in endometrial cancer --- p.71
Chapter 6.1.1 --- Concurrent hypermethylation of multiple genesin endometrioid adenocarcinoma and its precursor lesions --- p.72
Chapter 6.1.1.1 --- Promoter hypermethylation of E-cad --- p.73
Chapter 6.1.1.2 --- Promoter hypermethylation of APC --- p.73
Chapter 6.1.1.3 --- Promoter hypermethylation of MGMT --- p.74
Chapter 6.1.1.4 --- Promoter hypermethylation of RASSF1A --- p.75
Chapter 6.1.1.5 --- Promoter hypermethylation of hMLH-1 --- p.76
Chapter 6.1.1.6 --- Promoter hypermethylation in ECA coexisting with hyperplasia and not coexisting with hyperplasia --- p.77
Chapter 6.1.2 --- Promoter hypermethylation in SCA --- p.77
Chapter 6.2 --- Microsatellite status analysis --- p.78
Chapter 6.2.1 --- MSI in endometrial cancer --- p.78
Chapter 6.2.2 --- MSI and concurrent promoter hypermethylation --- p.79
Chapter 6.2.3 --- MSI and promoter hypermethylation of hMLH-1 --- p.80
Chapter Chapter 7 --- Conclusion --- p.81
Further studies --- p.82
References --- p.83

子宮内膜癌是最常见的妇科恶性肿瘤，其中有80%属于子宮内膜腺样癌，这一癌症发病的分子机制尚未清楚。研究表明，癌基因表达异常或者功能异常在肿瘤的发生发展过程中具有重要的作用。另一方面，抑癌基因在肿瘤细胞中特异性甲基化失活通常导致细胞恶性转变和肿瘤的发生。本实验将阐明癌基因阴阳1和抑癌基因PCDH10在人类子宮内膜腺样癌发病中的作用。
本实验第一部分研究多功能转录因子阴阳1（YY1）在子宮内膜腺样癌发病过程中的作用。首先本实验证实YY1在子宮内膜腺样癌临床标本和癌细胞系中均明显表达上调，并且上调的程度与肿瘤的FIGO分期相关。接着体外细胞培养和裸鼠荷瘤模型的实验均提示抑制YY1 表达可抑制癌细胞增殖和体外迁移，而过表达YY1则促进癌细胞增殖。这些结果表明YY1在子宮内膜腺样癌发病中具有促进作用。进一步全细胞基因组转录谱分析提示YY1 介入子宮内膜腺样发病的各个方面，并通过抑制抑癌基因APC的表达发挥发挥重要作用。深入的分子机制研究发现一个新的表观抑制作用模型：YY1可募集EZH2等多梳蛋白到APC启动子区并导致后者组蛋白3赖氨酸27上三甲基化，从而抑制APC基因转录。此外，本实验还发现YY1在子宮内膜腺样癌的表达增高是由于微小RNA，miR-193a-5p，在此癌中表达下降所导致的。所以，本实验第一部分的结果揭示了miR-193a-5p-YY1-APC这条全新的信号通路在子宮内膜腺样癌发病中发挥重要作用，并可作为潜在的治疗靶点。
本实验第二部分鉴定出PCDH10作为子宮内膜腺样癌一个新的抑癌基因。通过5-氮杂-2'-氧胞嘧啶处理和亚硫酸氢钠测序的方法，我们证实抑癌基因PCDH10在子宮内膜腺样癌中失活是由于其启动子区DNA甲基化所致，并且这种DNA甲基化介导的PCDH10表达沉默在子宮内膜腺样癌临床标本和癌细胞系中很常见，但不存在于正常子宮内膜组织。另外，在子宮内膜腺样癌细胞系体外实验中恢复PCDH10的表达可抑制细胞增殖、单细胞克隆形成，促进细胞凋亡。 同时在体实验荷瘤模型中恢复PCDH10的表达也可抑制肿瘤细胞增殖，这些结果与其肿瘤抑制功能相符。
总之，本实验结果阐明了YY1和PCDH10在子宮内膜腺样癌发病过程中新的作用，拓展了子宮内膜腺样癌发病分子机制的研究并为其药物治疗提供了潜在的靶点。
Endometrial cancer is the most common gynecologic malignancy and about 80% of these cancers are endometrial Endometrioid carcinoma (EEC). The molecular mechanisms underlying EEC tumorigenesis are under-explored. Aberrant expression and function of oncogenes promote tumor development by modulating many aspects of tumor cell growth. On the other hand, tumor specific promoter methylation on tumor suppressor genes (TSG), which are generally unmethylated in normal cells, usually initiate and promote malignant transformation and cancer initiation. Our study aims to characterize the functions of an oncogenic transcription factor Yin Yang 1 (YY1) and a novel tumor suppressor gene PCDH10 in Human Endometrioid Endometrial Adenocarcinoma.
In the first part of our study, we investigated the function of a multifunctional TF, YY1 in EEC tumorigenesis. We demonstrated YY1 is up-regulated in EEC cell lines and primary tumors and its expression is associated with FIGO stages. Depletion of YY1 inhibits EEC cell proliferation and migration both in vitro and in vivo whereas over-expression of YY1 promotes EEC cell growth. These results suggest that YY1 functions as an onocogenic factor in EEC. Transcriptome analysis revealed a significant effect of YY1 on critical aspects of EEC tumorigenesis and its down-regulation of APC transcripts. Further mechanistic investigation uncovered a new epigenetic silencing mode of Adenomatosis Polyposis Coli (APC) by YY1 through recruitment of EZH2 and trimethylation of histone 3 lysine 27 in its promoter region. Additionally, YY1 over-expression was found to be a consequence of miR-193a-5p down-regulation through direct miR-193a-5p-YY1 interplay. Our results therefore established a novel miR-193a-5p-YY1-APC regulatory axis contributing to EEC development, which may serve as future intervention target.
In the second part of our study, we identified PCDH10 as a novel tumor suppressor gene in EEC. By using bisulfate genomic sequencing combined with pharmacologic demethylation drug treatment, we elucidated that PCDH10 inactivation in EEC is a consequence of DNA hypermethylation on its promoter region. Further study suggested that hypermethylation-mediated PCDH10 silencing was a common event in EEC cell lines and clinical samples, but not in normal endometrial tissues. Restoration of PCDH10 expression in EEC cells suppressed cell proliferation, inhibited single cell colony formation and induced cell apoptosis; moreover, overexpression of PCDH10 inhibited EEC xenograft tumor growth in vivo.These results suggest PCDH10 acts as a tumor suppressor.
Together, our results reveal the novel functions of YY1 and PCDH10 in EEC. These findings add novel insights into the molecular mechanisms of EEC development and progression, which may serve as potential therapeutic targets for this disease.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Yang, Yihua.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 170-186).
Abstracts also in Chinese.
TITLE --- p.I
ABSTRACT --- p.III
ACKNOWLEDGEMENTS --- p.VII
PUBLICATION --- p.IX
ABBREVIATIONS --- p.X
LIST OF FIGURES --- p.XIII
LIST OF TABLES --- p.XVI
TABLES OF CONTENT --- p.XVII
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Endometrioid Endometrial Adenocarcinoma (EEC) --- p.1
Chapter 1.1.1 --- Epidemiology --- p.2
Chapter 1.1.2 --- Etiology and risk factors --- p.3
Chapter 1.1.3 --- Treatment and prognosis --- p.8
Chapter 1.1.4 --- Molecular Mechanisms --- p.9
Chapter 1.1.5 --- APC and Wnt/β-catenin signaling pathway --- p.12
Chapter 1.1.6 --- Summary --- p.18
Chapter 1.2 --- Epigenetic modifications and EEC --- p.20
Chapter 1.2.1 --- Epigenetic modifications --- p.20
Chapter 1.2.2 --- Epigenetic and cancer --- p.21
Chapter 1.2.5 --- Summary --- p.30
Chapter Chapter 2 --- Material and Method --- p.31
Chapter 2.1 --- Tissue samples --- p.31
Chapter 2.2 --- Cell culture --- p.32
Chapter 2.3 --- Cell proliferation assays --- p.33
Chapter 2.4 --- Cell migration assay --- p.34
Chapter 2.5 --- 3-deazaneplanocin A (Dznep) or 5-aza-2'-deoxycytidine (5-aza) treatment --- p.34
Chapter 2.6 --- Computational prediction --- p.35
Chapter 2.7 --- Cell cycle assay --- p.35
Chapter 2.8 --- Apoptosis assay --- p.36
Chapter 2.9 --- Total RNAs, Total proteins and Genomic DNA extraction --- p.36
Chapter 2.10 --- Bisulfite Genomic Sequencing --- p.38
Chapter 2.11 --- Oligonucleotides --- p.39
Chapter 2.12 --- RT-PCR, Semi-quantitative PCR and Real-time PCR --- p.41
Chapter 2.13 --- microRNA validation --- p.43
Chapter 2.14 --- Plasmid construction --- p.43
Chapter 2.15 --- Transfection --- p.45
Chapter 2.16 --- Luciferase reporter assay --- p.45
Chapter 2.17 --- Western blotting --- p.46
Chapter 2.18 --- Immunofluorescence ( IF ) --- p.48
Chapter 2.19 --- Immunohistochemistry (IHC) --- p.50
Chapter 2.20 --- ChIP assay --- p.53
Chapter 2.21 --- Sequencing and base calling --- p.55
Chapter 2.22 --- Read mapping to genome with splice-aware aligner sequenced --- p.55
Chapter 2.23 --- Xenograft mouse model --- p.55
Chapter 2.24 --- Statistical analysis --- p.57
Chapter Chapter 3 --- Yin Yang 1 Plays an Oncogenic Role in Human Endometrioid Endometrial Adenocarcinoma --- p.58
Chapter 3.1 --- YIN YANG 1(YY1) --- p.58
Chapter 3.1.1 --- YY1 structure --- p.58
Chapter 3.1.2 --- YY1 function --- p.59
Chapter 3.1.3 --- YY1 and epigenetic --- p.61
Chapter 3.1.4 --- YY1 and cancer --- p.62
Chapter 3.1.5 --- Regulation of YY1 expression and activity --- p.66
Chapter 3.2 --- Results --- p.68
Chapter 3.2.1 --- YY1 is up-regulated in EEC lines and localizes in nuclei of EEC cells --- p.68
Chapter 3.2.2 --- YY1 expression level is associated with EEC clinicopathological features --- p.72
Chapter 3.2.3 --- Knock-down of YY1 by RNAi inhibits EEC cell proliferation and migration --- p.77
Chapter 3.2.4 --- Ectopic expression of YY1 promotes EEC cell proliferation --- p.84
Chapter 3.2.5 --- YY1 does not affect EEC cell cycle and cell apoptosis --- p.91
Chapter 3.2.6 --- Genome-wide characterization of YY1-mediated transcriptome changes --- p.94
Chapter 3.2.7 --- Gene Ontology analysis of YY1 targets on EEC tumorigenesis --- p.98
Chapter 3.2.8 --- YY1 inhibits APC gene expression and functions --- p.101
Chapter 3.2.9 --- YY1 inhibits APC expression through recruiting EZH2 and causing H3K27me3. --- p.105
Chapter 3.2.10 --- Knock-down of YY1 does not change DNA methylation status of CpG island of APC gene --- p.117
Chapter 3.2.11 --- SiYY1 oligo injection inhibits tumor grows in vivo --- p.119
Chapter 3.2.12 --- miR-193a-5p is down-regulated in EEC cell lines and clinical samples --- p.126
Chapter 3.2.13 --- miR-193a-5p targets YY1 3’UTR and inhibits YY1 expression --- p.128
Chapter 3.2.14 --- miR-193a-5p inhibits tumor grow in vivo --- p.133
Chapter 3.3 --- Discussion --- p.136
Chapter 3.3.1 --- YY1 oncogenic functions in EEC --- p.136
Chapter 3.3.2 --- YY1 epigenetically silences APC --- p.138
Chapter 3.3.3 --- miR-193a-5p down-regulates YY1 in EEC --- p.139
Chapter 3.4 --- Conclusion --- p.141
Chapter Chapter 4 --- The tumor suppressive functions of PCDH10 in Human Endometrioid Endometrial Adenocarcinoma --- p.143
Chapter 4.1 --- Introduction --- p.143
Chapter 4.1.1 --- PCDH10 structure and function --- p.143
Chapter 4.1.2 --- PCDH10 and tumor --- p.146
Chapter 4.1 --- Results --- p.149
Chapter 4.2.1 --- PCDH10 is down-regulated in EEC cell lines and clinical samples --- p.149
Chapter 4.2.2 --- PCDH10 is hypermethylated in EEC cell lines and clinical samples --- p.150
Chapter 4.2.3 --- Pharmacologic demethylation restores PCDH10 expression in EEC cell lines --- p.152
Chapter 4.2.4 --- Ectopic over-expression of PCDH10 inhibits EEC cell proliferation --- p.154
Chapter 4.2.5 --- PCDH10 over-expression induces EEC cell apoptosis --- p.161
Chapter 4.2.6 --- PCDH10 over-expression inhibits tumor grows in vivo --- p.166
Chapter 4.3 --- Discussion and future plan --- p.169
REFERENCE --- p.170

Στις μετεμμηνοπαυσιακές γυναίκες με καρκίνο μαστού, θετικό για οιστρογονικούς υποδοχείς, μετά από χειρουργική θεραπεία, η μακροχρόνια χορήγηση της ταμοξιφαίνης (SERM πρώτης γενεάς), έχει αποδειχθεί ευεργετική. Ο σκοπός της παρούσης μελέτης, ήταν να διαπιστώσει, εάν οι πολυμορφισμοί του γονιδίου των ER (PvuII & XbaI του ERα και οι RsaI & AluI του Erβ), οι οποίοι έχουν συσχετισθεί με καρκίνο μαστού, σχετίζονται με το στάδιο του καρκίνου του μαστού ή την ανταπόκριση του ενδομητρίου στην μακροχρόνια αγωγή με ταμοξιφαίνη, στις μετεμμηνοπαυσιακές γυναίκες με καρκίνο του μαστού. Η μελέτη περιέλαβε 87 μετεμμηνοπαυσιακές γυναίκες με καρκίνο μαστού θετικό για οιστρογονικούς υποδοχείς, στις οποίες χορηγήθηκε ταμοξιφαίνη. Η μέση ηλικία των ασθενών ήταν 58,7± 4,7 έτη, και η μέση διάρκεια της αγωγής με ταμοξιφαίνη ήταν 3,9 ± 1,1 έτη. Το γονιδιακό DNA απομονώθηκε από τα λευκά αιμοσφαίρια δειγμάτων περιφερικού αίματος με την κλασσική μέθοδο φαινόλης - χλωροφορμίου. Τα κλάσματα των γονιδίων των ERα και Erβ, τα οποία συμπεριελάμβαναν τις θέσεις των πολυμορφισμών, πολλαπλασιάσθηκαν με την αλυσιδωτή αντίδραση πολυμεράσης (PCR). Ο προσδιορισμός της παρουσίας των πολυμορφισμών στο DNA πραγματοποιήθηκε με την χρήση ενζύμων περιορισμού. Συμπερασματικά, στις Ελληνίδες μετεμμηνοπαυσιακές γυναίκες με καρκίνο του μαστού υπό αγωγή με ταμοξιφαίνη, οι πολυμορφισμοί των οιστρογονικών υποδοχέων, δεν συνδέθηκαν, ούτε με την παρουσία παθολογίας του ενδομητρίου, ούτε με το στάδιο του καρκίνου του μαστού.
In postmenopausal women with estrogen receptor (ER) positive breast cancer, after surgical treatment long term tamoxifen administration has been proved beneficial. The aim of the present study was to identify whether these ER gene polymorphisms are associated with breast cancer stage or endometrial responsiveness to long-term tamoxifen treatment in postmenopausal women with breast cancer. The study included 87 postmenopausal women with estrogen receptor positive breast cancer treated with tamoxifen. The mean age of patients was 58,7 ± 4,7 years and the mean duration of Tamoxifen treatment was 3.9 ± 1,1 years. Genomic DNA was extracted from peripheral blood leukocyte samples by the standard phenol/chloroform procedure. Fragments of the ERα and ERβ genes encompassing the polymorphic sites were amplified by the polymerase chain reaction (PCR). The determination of presence of polymorphisms in the DNA was realised with restriction endonucleases. Ιn conclusion, in Greek postmenopausal women with breast cancer under tamoxifen treatment, Estrogen Receptors polymorphisms were not linked to either the presence of endometrial pathology or the stage of breast cancer.

Endometriosis is a reproductive disease affecting women in their prime reproductive years characterized by the presence of endometrial glands and stroma in ectopic locations. Animal models have been proven to be indispensable not only for the study of the disease but also to develop better non-invasive diagnostic imaging modalities. A major limitation with the diagnosis of endometriosis is the lack of a specific and sensitive non-invasive diagnostic test. Our objective was to develop a domestic animal model of endometriosis suitable for serial diagnostic imaging procedures and assessment of therapies. Two major studies were conducted to achieve this objective. First study involved in vitro whole tissue-explant culture and surgical induction of endometriosis in dog, pig and sheep to choose the most suitable model. For in vitro co-culture, dog, pig and sheep endometrium was placed on visceral peritoneum for 24 to 72 h to assess the degree of attachment and adhesion characteristics of endometrium (epithelium, glandular and stromal cells). Surgical induction of endometriosis was tested in dog, sheep (n=5 each) and pig (n=4) using autologous endometrial (n=4 grafts per animal) and fat grafts sutured to visceral (urinary bladder surface in dog and pig, uterus in sheep) and parietal (abdominal wall) peritoneum. Sham surgeries were performed in control group animals (dog and sheep n=5, pig n=3) using fat grafts alone. Animals were euthanized between 80-110 days post-surgery. Size, gross characteristics and histopathologic features of endometriotic lesions were recorded. During in vitro explant culture, surface epithelial, stromal and glandular cells of endometrium were capable of attaching to visceral peritoneum within 24 hours with and without an intact layer of mesothelial lining in dog, pig and sheep. The proportion of successful endometrial attachments were greater at 24h compared to 72h (15/18 vs. 7/18, p=0.008; data combined among species) with intermediate attachment at 48h (12/15). Following surgical induction, there was no difference (p>0.05) in proportion of successful tissue grafts placed on serosal surface of visceral vs. parietal peritoneum in dog (10/10 vs. 10/10), pig (7/8 vs. 8/8) or sheep (7/10 vs. 8/10). A variety of outcomes (endometriotic cysts with sero-sangunous fluid, solid lesions, vesicles, absence of lesions) were found. The proportion of cystic lesions was greater (p<0.01) in dog (19/20 grafts) than in pig (8/16) and sheep (5/20). Further, the area of endometriotic lesions at euthanasia was larger (0.89 ± 0.11 cm2) compared to that at the time of surgery (0.50 ± 0.09 cm2) in dog, whereas, the size of lesions decreased by half or more (p<0.05) in pig and sheep. Combined among grafting sites (visceral and parietal peritoneum) and species, a greater proportion (p=0.015) of surgical sites had adhesions in treatment (12/14) versus control group animals (5/13). The wall of majority of endometrial cysts in dogs were characterized by simple cuboidal/columnar epithelium, endometrial glands (normal, dilated and cystic), subepithelial capillary network and presence of stromal and smooth muscle cells. Hemorrhage and/or hemosiderin-laden macrophages were observed in the cystic lesions in dog. Development of a greater proportion of growing lesions in the form of endometriotic cysts in dogs compared to sheep and pig led us to conclude dog as a better suitable domestic animal model for endometriosis than sheep and pig. Second study involved assessing the usefulness and limitations of ultrasonography, magnetic resonance imaging (MRI) and positron emission tomography-computed tomography (PET-CT) in detecting cystic endometriotic lesions in dog and sheep. Surgical induction of endometriois was performed in dogs (n=5) and sheep (n=5) using autologous endometrial grafts (n=4 grafts per animal) and fat grafts sutured to visceral peritoneum (urinary bladder in dogs, uterus in sheep) and parietal peritoneum (ventral abdominal wall). Weekly ultrasonography was performed from Week 1-9 post-surgery and day of euthanasia (Week 14-15). T1 and T2 weighted weighted MRI images (n=2 each for dog and sheep) and PET-CT (n=3 dog, n=1 sheep) using 18F-fluorodeoxyglucose (18F-FDG) as radiolabel was performed between Week 13-15 post-surgery in dog and sheep. Gray-scale B-mode ultrasonography was able to detect endometriotic cysts (0.25-1.75cm) on urinary bladder and abdominal wall in dogs; endometrial grafts Week 1 post-surgery appeared as a homogenous, hypoechoic masses, following which they grew larger with evidence of cyst formation by Week 5. Cysts were undetectable from Week 10-13, whereas they appeared as homogenous masses with a hypoechoic fluid-filled cavity with diffuse hyperechoic echoes and low vascularisation (Color-Doppler imaging) by Week 14-15. In sheep, endometrial grafts were detected as hypoechoic masses Week 1 post-surgery that became smaller until no detectable lesions were visible beyond Week 6-7. Cysts in dogs and sheep appeared hyperintense on T2 and hypointense on T1 weighted images. 18F -FDG PET-CT did not show hypermetabolic activity in endometriotic cysts in dogs and sheep. In conclusion, MRI appeared to provide the most definitive diagnostic images of endometriotic cysts in dogs and sheep, particularly for lesions in sheep which were not evident by ultrasonography. However, ultrasonography was sufficient to characterize most endometriotic cysts in dogs. Further research needs to be carried out to develop specific PET tracers for endometriosis.