A cell’s interaction with its extracellular environment is critical to tissue structure and function. This work investigates the effect of substrate topography on selective cell adhesion and morphology. Alterations in cell response to micro- and nanoscale signals and cues can cause changes in downstream functions of proteins and complexes such as invasive and metastatic motility of malignant tumour cells and the differentiation direction of stem cells. Biomaterial surfaces can be modified to provide different chemical and topographical cues and encourage controlled cell-substrate interaction. At the protein level, template substrates have shown and increased affinity for selective binding of the imprinted antigen or antibody. Topography of a cell’s microenvironment may be replicated as a permanent polymer mould by bioimprinting technology, which was developed at University of Canterbury. The resulting high resolution methacrylate polymer samples have been used for imaging and analysis, but have not previously been investigated as cell culture substrates. This work investigates the effect of bioimprint and photolithographic substrate patterning on cell behaviour in culture. Optimisation of a methacrylate co-polymer resulted in a 6:3:1 ethylene glycol dimethacrylate: methacrylic acid: photoinitiator polymer mixture cured by 240 seconds of UV exposure. The polymer was used to replicate cell membrane features into a permanent polymer mould [a bioimprint]. The resulting high resolution methacrylate bioimprints were cleaned and sterilised for use as a secondary cell culture substrate. Ishikawa endometrial cancer cells were cultured on bioimprinted methacrylate polymer substrates. Preliminary results showed preferential cell adhesion to bioimprinted areas over flat areas and also showed three dimensional spheroid growth instead of lateral two dimensional monolayer spreading. At higher seeding densities, preferential adhesion was similarly noted as well as peeling artefacts of shear stresses and cell size variation on flat methacrylate substrate regions. Fluorescent imaging and cell culture stencilling highlighted the association of secondary cells with bioimprint substrate features. To determine whether preferential cell adhesion effects were due to bioimprint features or general topography modification, secondary cancer cells were cultured on comparable photolithographically-defined, geometrically-patterned substrates. Methods for transferring regular pattern arrays into methacrylate polymer substrates were developed. No organisation or preferential adhesion effects were observed in association with pillar and hole patterns between 5-30 µm. However, artefact incidence in methacrylate polymer replication techniques led to development and adaptation of polystyrene patterning techniques. Experimental analysis of substrate-dependent effects on cell culture adhesion and organisation was extended to a non-cancerous cell line model. C2C12 mouse skeletal muscle cells were chosen for these investigations because of their ability to differentiate further, into myocytes or myofibrils. C2C12 myoblasts seeded on common cell culture substrates showed a notable morphology variation and extent of differentiation between cells grown on tissue culture polystyrene [TCPS] and polydimethylsiloxane [PDMS]. Myoblasts were plated on geometrically-patterned polystyrene and PDMS substrates. Significant alignment to grated pattern features was observed on both substrate types, before and after driven differentiation. Peeling artefacts of confluent tissue-like culture from PDMS surfaces which were observed were unreported previously in literature. The results reported in this thesis provide a foundation for potential research and commercial application for surface modification methods. The biomimetic topography provided by bioimprinted substrates can be used to identify and investigate cell activities, including for example the mechanisms of cell adhesion and separation in metastatic and invasive cancer research. Altering the material of the bioimprinted substrates may attune substrate topographies as scaffolds to direct specific stem cell differentiation for regenerative tissue engineering applications.

Age-related degeneration of the equine endometrium is an established and important cause of fertility problems in thoroughbred mares, causing great loss to the industry. As a part of the age-related endometrial degeneration complex, an excessive deposition of collagen leading to endometrial fibrosis is particularly important due to the limitations it causes to uterine function. The consequences include reduced efficacy of uterine defence mechanisms and a decrease in the uterine capacity for foetal nutrition. Extensive research into the process of fibrosis in other organs has shown that this condition results from the malfunction of physiological tissue repair mechanisms. These mechanisms revolve around tissue fibroblasts that due to continuous stimulation secrete excessive amounts of collagen and inhibit the activation of factors essential to the normal collagen degradation occurring in scar resolution. Among these factors are the MMPs, an enzyme family with the ability to degrade extracellular matrix components such as collagen during the normal repair mechanisms following tissue injury. The malfunction in the regulation of these enzymes is important in the development of fibrosis in the liver and other organs. In this study it was demonstrated that MMPs are involved in the acute uterine inflammatory response and that they were secreted by infiltrating inflammatory cells. The cellular mechanisms observed during endometritis in normal mares were comparable to the normal repair mechanisms known to be altered in the fibrosis of other organs. These enzymes were present in equine foetal fluids, and their regulation may be important in the process of abortion and stillbirth. It was demonstrated that inbreeding may be correlated with increased deposition of endometrial collagen in a study population of the Icelandic horse breed even though this breed appears to exhibit less severe endometrial degeneration than what is known in lighter breeds. It is likely that genetic predisposition leads to the disruption of normally self-limiting inflammatory and repair mechanisms in the endometrium, resulting in constant activation of collagen synthesis by local and infiltrating cells. This thesis has shown that tissue repair mechanisms involving MMPs are likely to be involved in endometrial fibrosis in the mare. An inherent alteration in these mechanisms may play a role in the pathogenesis of this condition, and might arise due to genetic predisposition. Further understanding of the pathways leading to excess collagen amounts in the endometrium may produce preventative measures, and even therapeutic targets.

Endometrial cancer (EC) is the fourth most common cancer affecting women in the UK. Those with a family history of EC have an increased risk compared with the general population. Highly penetrant germline mutations in mismatch repair (MMR) genes and DNA polymerases account for only a small proportion of the familial aggregation. The aim of this thesis is to investigate the genetic susceptibility to EC in the general population using cases and controls of European ancestry. A GWAS meta-analysis totalling 7,737 EC cases and 37,144 controls yielded five novel EC risk loci of genome-wide significance (P < 5x10⁻⁸). In decreasing order of significance, these were at chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). A second independent EC signal was found in the 8q24 locus. The association found in a previous EC GWAS at HNF1B on chromosome 17 was replicated at a higher significance, with the most significant SNP being rs11263763. CYP19A1 SNPs have previously been associated with EC and higher circulating levels of oestrogen from candidate studies, but I confirmed this locus to be genome-wide significant for the first time. Functional annotation and in vitro studies for the EC risk loci at the intergenic region of chromosome 13q22 suggested that the functional SNP sits within a transcriptional repressor for KLF5, with the higher-risk allele reducing repressor activity. The propensity for germline MMR and DNA polymerase muations to cause both EC and colorectal cancer (CRC) prompted me to search for common variants associated with both cancer phenotypes. An EC CRC GWAS meta-analysis showed little evidence of shared susceptibility loci. However, this meta-analysis revealed a novel genome-wide significant risk locus: rs3184504, a missense SH2B3 SNP that has not previously been associated with either EC or CRC. This thesis has enhanced the understanding of genetic susceptibility to sporadic EC and increased the number of genome-wide EC-associated variants to seven.

Endometrial carcinoma is the most common gynaecologic cancer in the United States with about 41,200 new cases projected to occur in 2006. It often presents with abnormal uterine bleeding and spreads to the cervix in 10 to 20% of cases. Whilst early diagnosis is essential for optimal disease treatment, the best investigation for abnormal uterine bleeding remains uncertain. Although hysteroscopy has been reported to have high accuracy in predicting normal or abnormal endometrial histopathology, its accuracy varies with the underlying pathology. The highest accuracy occurs in the diagnosis of intrauterine anatomical pathology such as endometrial polyp whereas it is at its lowest in microscopic histopathology such as endometrial hyperplasia. Hysteroscopy is also potentially useful for detecting tumour spread to the uterine cervix that helps in staging and surgical planning. However, the role of hysteroscopy with guided biopsy in detecting endometrial cancer and the choice of distension medium remain to be determined. As the uterine cavity is a collapsed space, hysteroscopy requires its distension with a gaseous or liquid medium to allow complete visualization of the uterine cavity. The use of such media to rinse the uterine cavity raises the concern that when the endometrium harbours endometrial carcinoma cells, there is a potential risk of retrograde dissemination of these cells into the peritoneal cavity. The work in this thesis has addressed four major issues of diagnostic hysteroscopy in the management of patients with endometrial carcinoma. Firstly, the role of diagnostic hysteroscopy and guided biopsy is limited especially in microscopic tumours. Secondly, the role of diagnostic hysteroscopy to detect cervical invasion in preoperative staging of endometrial carcinoma is proven and the usage of normal saline is more accurate than that which uses carbon dioxide. Thirdly, hysteroscopic dissemination occurs more frequent when using normal saline as opposed to carbon dioxide as the distension medium. Lastly, complete occlusion of both fallopian tubes can effectively prevent the dissemination of endometrial carcinoma cells into the peritoneal cavity during diagnostic hysteroscopy. / Lo, Wing Kit Keith. / "May 2006." / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5873. / Thesis (M.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (167-193). / 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. / School code: 1307.

Mutations in FGFR2 are common in a subset of endometrial carcinomas. Given the emergence of small molecule inhibitors specific to this receptor tyrosine kinase, FGFR2 is an attractive therapeutic target. However, compensatory and adaptation mechanisms limit the clinical utility of compounds that target nodes in the receptor tyrosine kinase network. Here, we analysed the impact of FGFR inhibition in endometrial cancer cells and observed the emergence of a resistant population in an FGFR2-mutant cell line. To understand the mechanisms underlying this adaptation response, we used a phosphoproteomics approach to measure the kinase network in an unbiased manner. These experiments led to the identification of an AKT-related compensatory mechanism underpinning this resistance. Further dissection of this resistance mechanism utilising gene expression analysis showed PHLDA1, a negative regulator of AKT, was significantly down-regulated in resistant cells. This was further confirmed at the protein level. siRNA knockdown of PHLDA1 conferred immediate drug resistance in the FGFR2-mutant endometrial cancer cell line. Therefore, we identified PHLDA1 down-regulation as a mediator of drug resistance in FGFR2 mutant cancer cells, the first demonstration of the role of PHLDA1 in the acquisition and maintenance of drug resistance. Using a 3D physiomimetic model, we demonstrated that AKT inhibition alone also led to generation of a drug-resistant population. Most importantly, dual-drug therapy inhibited proliferation and induced cell death. Our data highlight how mass spectrometry and microarray gene expression analysis can complement each other in the identification of novel resistance mechanisms in cancer cells. These data suggest that combination treatment of FGFR2-mutant endometrial cancers, targeting both FGFR2 and AKT, represents a promising therapeutic approach.

Orientador: Ricardo da Fonseca / Resumo: Em fêmeas bovinas a liberação de prostaglandina F2α (PGF2α) endometrial pode ser induzida in vivo pelo estradiol (E2), entretanto, seu papel ainda não foi esclarecido. Em estudos prévios realizados pelo grupo, o tratamento com E2 in vivo duas horas antes das vacas serem abatidas no 17º dia do ciclo estral, associado à adição de cálcio ionóforo (CI) in vitro em explantes endometriais, exacerbou a síntese de PGF2α quando comparado ao grupo de vacas não tratadas com E2 e CI. O mesmo foi observado em células endometriais bovinas (BEND) expostas ao E2 e CI simultaneamente. Considerando que as concentrações plasmáticas de PGF2α aumentam a partir de 3,5 horas após da aplicação do E2 in vivo, possivelmente tais ações envolvam a síntese de proteínas. Hipotetizou-se que o E2 estimule a expressão e/ou a atividade das enzimas dependentes de cálcio, especificamente a proteína quinase C (PKC) e fosfolipase A2 citosólica (cPLA2). Assim, objetivou-se analisar o efeito dos inibidores de Proteína G, PKC e PLA2 na síntese de PGF2α induzida pelo 17β-estradiol e CI em células endometriais bovinas BEND (Experimento 1), e avaliar as proteínas PKC e PLA2 e ERK 1/2, pela técnica de Western Blotting, em explantes endometriais obtidos de fêmeas bovinas abatidas 2 horas após a administração de 17β-estradiol no 17o dia do ciclo estral (Experimento 2). No Experimento 1, células endometriais bovinas (BEND) foram submetidas a ausência ou presença de um inibidor de proteína G - 10µM de Guanosine 5’- {B-thio}... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Background Obesity, insulin resistance and type 2 diabetes mellitus (T2DM) have been associated with endometrial cancer (EC). In this systematic review and meta-analysis we evaluated the effect of metformin on clinical outcomes in patients with EC and insulin resistance or T2DM. Methods Four research databases were searched for original articles published in all languages up to 30 October 2016. Outcomes of interest were overall mortality (OM), cancer-specific mortality, disease progression, and metastases. We performed a random effect meta-analysis of adjusted effects expressed as hazard ratios (HR); heterogeneity among studies was described with the I2 statistic. Results Of the 290 retrieved citations, 6 retrospective cohort studies in women with EC (n = 4723) met the inclusion criteria, and 8.9% to 23.8% were treated with metformin; OM data was available from 5 studies. In 4 studies of EC patients (n = 4132), metformin use was associated with a significant reduction in OM in comparison with not using metformin (adjusted HR [aHR] 0.64, 95% CI 0.45–0.89, p = 0.009). In three studies evaluating patients with EC and T2DM (n = 2637), metformin use was associated with a significant reduction in OM (aHR 0.50, 95%CI 0.34–0.74, p = 0.0006). There was low to moderate heterogeneity of adjusted effects across studies. There was no information about the effect of metformin on cancer-specific mortality, disease progression, or metastases. Conclusions Metformin treatment is associated with a significant reduction in OM irrespective of diabetes status in patients with EC. The survival benefit suggests that diabetes screening and maintenance of good glycemic control may improve outcomes in EC. / Revisión por pares

Orientador: Wilter Ricardo Russiano Vicente / Banca: Elzylene Léga / Banca: Marcos Lania de Araujo / Resumo: Na prática da clínica de pequenos animais, os médicos veterinários são freqüentemente confrontados com o quadro ou com o diagnóstico diferencial da piometra em cadelas, e devem decidir rapidamente sobre a melhor forma de tratamento, pois se trata de uma situação de risco para a vida da paciente. Esta revisão tem como objetivo a descrição dos conceitos práticos e atuais da etiopatogenia da hiperplasia endometrial cística em cadelas, a qual precede o desenvolvimento do quadro de piometra. Abordam-se tanto a classificação quanto os sintomas, os achados clínicos, bem como aspectos diagnósticos dessa síndrome. Com relação ao tratamento, são consideradas tanto a abordagem cirúrgica quanto a medicamentosa, bem como as vantagens e desvantagens de cada opção. / Abstract: In clinics, pyometra in bitches is one of the most common disorders. Veterinarians must decide about the therapeutical approach and the best way to conduct treatment. So, it is important to know the complex physiopathogeny of this disorder, as well as the concomitant diseases, laboratorial alterations and types of treatment. The aim of this dissertation is to describe the new concepts of the etiopathogenia of the cystic endometrial hyperplasia-pyometra complex in bitches, evaluate the clinical and laboratorial alterations and also the options of the treatments found in animals consulted at the obstetrics department at the Veterinary Hospital "Governador Laudo Natel", FCAV-UNESP-Jaboticabal and confront them with the results published in literature and also with cases from other national and international universities. Thus, it was described the classification of clinical signs, diagnosis and therapeutics of this syndrome. / Mestre

Physiological inflammation occurs in the female reproductive tract, but pathological inflammation is implicated in reproductive pathologies such as preterm labour and endometrial cancer. Preterm labour (PTL, before 37 weeks of gestation) is the leading cause of preterm birth, neonatal mortality and perinatal morbidities. Endometrial cancer is the commonest gynaecological cancer, and its pathogenesis is characterised by chronic inflammation. The overall aims of this thesis were (i) to develop an in vitro model of myometrial-monocyte interactions to replicate the events occurring in the myometrium in preterm labour (ii) to determine the effects of potential therapeutics such as lipoxins, IL-10 and progesterone, on inflammation, and (iii) to characterise the lipoxin pathway in endometrial adenocarcinoma. Macrophages infiltrate the pregnant myometrium during labour; however the role of these cells is unclear. A myometrial-monocyte coculture model was developed either using non-pregnant primary myometrial smooth muscle cells (UtSMCs), or immortalised pregnant human myometrial cells (PHM1-41), with primary monocytes from term (38-41 weeks of gestation), non-labouring pregnant women. Cultures were stimulated with the toll-like receptor 4 agonist lipopolysaccharide (LPS), in the presence or absence of each of lipoxins, IL-10 and progesterone. A significant and synergistic increase in IL-6 and IL-8 secretion was found in the UtSMC/monocyte coculture after stimulation with LPS for 24 hours, compared to LPS-treated UtSMCs, or monocytes alone, but the increase in IL-6 and IL-8 secretion was not inhibited by lipoxin, epi-lipoxin or benzo-lipoxin. The PHM1-41/monocyte coculture both alone and in response to LPS treatment generated significantly increased IL-6 and IL-8 secretion, compared to vehicle treatment in the coculture and compared to the culture of either cell type alone. IL-1β and TNFα secretion were only detected from the PHM1/monocyte coculture, and monocytes alone. Use of a TNFα blocking antibody partially suppressed LPS-induced IL-6 and IL-8 secretion in the coculture. Coculture of PHM1/monocytes resulted in increased secretion of multiple mediators including pro-inflammatory cytokines, chemokines and growth factors compared to culture of either PHM1 cells or primary monocytes separately, both with vehicle and with LPS. IL-10 inhibited LPS-induced IL-6 and IL-8 secretion from the coculture, as did progesterone, which also inhibited GM-CSF, MCP-1 and CXCL5 secretion. Myocyte contraction, measured by PHM1-41 cells embedded in collagen was increased by primary monocyte treatment. This suggests that not only do infiltrating monocytes increase myometrial inflammation but they can induce myometrial smooth muscle contraction. In endometrial adenocarcinoma, the lipoxin synthesis enzymes, ALOX-5 and -15 and FPR2 mRNA expression were upregulated compared to proliferative phase endometrium, with FPR2, a reported lipoxin receptor, immunolocalised in endometrial adenocarcinoma tissue. Additionally, TNFα treatment of Ishikawa endometrial adenocarcinoma cells increased FPR2 mRNA expression, and upregulation of FPR2 mRNA also occurred in xenograft tumours from CD1 nude mice, compared to the Ishikawa cells from which they originated. These findings highlight FPR2 expression in endometrial adenocarcinoma, and suggest this receptor could mediate inflammatory signals, and lipoxins could be produced by ALOX-5 and ALOX-15. Collectively, these data describe the novel effects of monocytes in the regulation of myometrial smooth muscle cell inflammation, and demonstrate a mechanism by which myometrial inflammation during both term and preterm labour is triggered by infiltrating macrophages. This myocyte/monocyte inflammation is regulated in part by TNFα, and can be suppressed by both IL-10 and progesterone co-treatment. Components of the lipoxin pathway are present in endometrial adenocarcinoma, but their role in regulation of inflammation is still to be elucidated. Future research to clarify the processes, by which leukocyte recruitment is regulated at labour and the role of monocyte/macrophages in altering myocyte properties, could help to elucidate the mechanisms coupling inflammation to labour and provide more appropriate targets for the treatment of PTL.

Objetivo do estudo: Estimar a prevalência de lesões malignas e prémalignas nos pólipos endometriais e correlacionar com fatores associados com risco de neoplasia de endométrio. Métodos: Revisados dados sobre características clínicas e resultado anatomopatológico dos pólipos ressecados em histeroscopias cirúrgicas com polipectomia realizados entre janeiro de 2005 e julho de 2013 no Hospital de Clínicas de Porto Alegre (HCPA). Resultados: Incluídas 359 pacientes submetidas a polipectomias histeroscópicas. 87,2% das pacientes apresentaram pólipos benignos e 9,9% apresentaram hiperplasia sem atipias. Pólipos com hiperplasia atýpica corresponderam a 2,6% da amostra, enquanto que adenocarcinoma de endométrio foi encontrado de 0,3% dos casos. Verificou-se correlação de resultado maligno/pré-maligno dos pólipos com idade da paciente, seu status menopausal e a presença de sangramento uterino anormal. Todas as mulheres com resultados malignos/pré-malignos apresentaram sangramento uterino anormal. Observou-se maior frequência de malignidade dos pólipos entre usuárias de tamoxifeno, porém sem significância estatística (p 0,059%). Não houve correlação com hipertensão arterial, diabetes mellitus, obesidade, uso de terapia hormonal, espessura endometrial ou diâmetro do pólipo. Conclusão: A prevalência de lesões malignas/pré-malignas nos pólipos endometriais é baixa, tendo sido nula nas pacientes sem sangramento. Não se recomenda a exérese rotineira dos pólipos em pacientes assintomáticas. / Objective: To estimate the prevalence of malignant and premalignant lesions among endometrial polyps and correlate this prevalence with risk factors for endometrial neoplasms. Methods: Review of clinical and histopathological data on polyps resected during hysteroscopic polypectomies performed from January 2005 through July 2013 at Hospital de Clínicas de Porto Alegre (HCPA), Brazil. Results: The sample comprised 359 patients who underwent hysteroscopic polypectomy. Overall, 87.2% of patients had benign polyps and 9.9% had hyperplasia without atypia. Polyps with atypical hyperplasia were found in 2.6% of patients, and endometrial adenocarcinoma, in 0.3%. Polyp malignancy/premalignancy correlated with patient age, menopausal status, and presence of abnormal uterine bleeding. All women with malignant/premalignant lesions had abnormal uterine bleeding.The rate of polyp malignancy was higher among tamoxifen users, although the difference did not reach statistical significance (p=0.059). There was no correlation with hypertension, diabetes mellitus, obesity, hormone replacement therapy, endometrial thickness, or polyp diameter. Conclusion: The prevalence of malignancy/premalignancy among endometrial polyps is low; no cases were identified in patients without uterine bleeding. Routine excision of asymptomatic polyps cannot be recommended.