Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer and urgently requires new therapies. Oncolytic viruses (OV) are a strong contender. OVs interact with immune components of the TME, which can be altered due to specific genetic mutations. The present study evaluates the impact of specific tumour mutations on the response to carboplatin, the current standard of care, and VSV∆M51, a promising OV candidate. After a study of genetically diverse models, constitutive KRas activation enhanced VSV∆M51 replication in-vitro and sensitivity in syngeneic in-vivo models. VSV∆M51 prolonged survival in syngeneic tumour- bearing mice with KRas, Trp53 and Pten mutations, including one tumour model that did not respond to carboplatin. Response to VSV∆M51 in-vivo was associated with activation of CD4+ and CD8+ T lymphocytes in the peritoneal TME. In summary, VSV∆M51-based immunotherapy has shown promise in diverse murine models of EOC bearing clinically relevant mutations.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25158号 / 医博第5044号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 小川 誠司, 教授 後藤 慎平 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

INTRODUÇÃO: Os gliomas são tumores neuroepiteliais e correspondem a aproximadamente 24,6% de todos os tumores primários cerebrais. Na última década, grande esforço tem sido feito em meio acadêmico para caracterização molecular dos gliomas na tentativa de descrever comportamento clínico, definir prognóstico e predizer resposta terapêutica. Ferramentas de bioinformática estimam que os microRNAs possam regular cerca de 60% dos genes humanos, incluindo um número significativo de oncogenes, genes supressores tumorais e genes relacionados a quimio e radioressistência. Uma problemática atual na prática clínica é a ausência de ferramentas moleculares para predizer a evolução dos gliomas classificados como baixo grau, uma vez que, durante o seguimento clínico-radiológico pós-cirúrgico, esses pacientes podem ter recidiva tumoral e confirmação histopatológica de glioma de alto grau. A transformação tumoral em glioma de alto grau implica em pior prognóstico e redução das possibilidades terapêuticas. OBJETIVOS: O objetivo deste trabalho é analisar a expressão dos microRNAs miR-124a, miR-138, miR-155, miR-1275 em amostras de tumor primário humano e sangue periférico de pacientes com diagnóstico de gliomas de baixo e alto graus (astrocitoma grau I,astrocitoma grau II, astrocitoma grau III, glioblastoma, oligodendroglioma grau II e oligodendroglioma grau III). PACIENTES E MÉTODOS: As análises da expressão dos microRNAs foram realizadas utilizando-se a técnica de PCR em tempo real. Foram analisados 65 pacientes adultos (entre 18 e 65 anos de idade) com diagnóstico histopatológico confirmado de glioma com material biológico tumoral criopreservado armazenado junto ao Banco de Tumores do Sistema Nervoso Central do HCFMRP. Foram coletadas informações contidas no prontuário médico referentes às características clínicas, epidemiológicas, de evolução clínica e radiológica e tempo para recidiva e óbito. Considerando-se um nível de significância de 5%, a associação das variáveis qualitativas categóricas e expressão de microRNAs foi realizada pelo teste de Mann-Whitney. A análise de sobrevida foi realizada pelo método não-paramétrico de Kaplan-Meier. RESULTADOS: Os microRNAs em estudo não apresentaram hiperexpressão em tecido tumoral de pacientes diagnosticados com Glioblastoma. Apenas o miR-1275 apresentou expressão aumentada em pacientes com diagnóstico de astrocitoma pilocítico e oligodendroglioma grau II. Além disso, tumores de linhagem oligodendroglial, de baixo e alto graus, demonstraram hiperexpressão do miR-1275. Em sangue periférico, observou-se significativa hipoexpressão dos miR-1275, miR-124 e miR-138 em amostra de glioblastoma. Observou-se, ainda, acentuada hipoexpressão do miR-138 em amostras de oligodendroglioma grau III. CONCLUSÃO: Os microRNAs miR-124a, miR-138, miR-155 e miR-1275 não apresentaram hiperexpressão em tecido tumoral de pacientes diagnosticados com GBM. O miR-1275 apresentou expressão aumentada em pacientes com diagnóstico de astrocitoma pilocítico e o miR-155 foi hiperexpresso apenas em amostras de oligodendroglioma grau II. Além disso, tumores de linhagem oligodendroglial, de baixo e alto graus, demonstraram hiperexpressão do miR1275 e miR-124a. Em sangue periférico, observou-se significativa hipoexpressão dos miR-1275, miR-124 e miR-138 em amostra de glioblastoma assim como acentuada hipoexpressão do miR-138 em amostras de oligodendroglioma grau III / INTRODUCTION: Gliomas are neuroepithelial tumors and correspond to approximately 24.6% of all primary brain tumors. In the last decade, great effort has been made in academic circles to characterize gliomas in an attempt to describe clinical behavior, define prognosis and predict therapeutic response. Bioinformatics tools estimate that microRNAs can regulate about 60% of human genes, including a significant number of oncogenes, tumor suppressor genes, and chemo and radioresistance genes. A current problem in clinical practice is the absence of molecular tools to predict the evolution of gliomas classified as low grade, since during postoperative radiological follow-up these patients may have tumor recurrence with histopathological confirmation of high glioma degree. Tumor transformation in high-grade glioma has a worsening of prognosis and reduction of therapeutic possibilities OBJECTIVES: The objective of this project is to analyze the expression of miR124a, miR-138, miR-155, miR-1275 in human primary and peripheral blood samples from patients diagnosed with low and high grade gliomas (astrocytoma grade I, astrocytoma grade II, astrocytoma grade III, glioblastoma, oligodendroglioma grade II and oligodendroglioma grade III). PATIENTS AND METHODS: MicroRNA expression analyzes were performed using the real-time PCR technique. Sixty-five adult patients (18-65 years of age) with confirmed histopathological diagnosis of glioma with cryopreserved tumor biological material stored at the Bank of Tumors of the Central Nervous System of HCFMRP were analyzed. Information collected in the medical records regarding clinical, epidemiological, clinical and radiological characteristics and time for recurrence and death were collected. Considering a level of significance of 5%, the association of categorical qualitative variables and microRNA expression were performed by the Mann-Whitney test. Survival analysis was performed using the Kaplan-Meier non-parametric method. RESULTS: The microRNAs under study did not present hyperexpression in tumor tissue of patients diagnosed with glioblastoma. Only miR-1275 showed increased expression in patients diagnosed with pilocytic astrocytoma and grade II oligodendroglioma. In addition, tumors of low and high grade oligodendroglial lineage demonstrated overexpression of miR-1275. In peripheral blood, significant hypoexpression of miR-1275, miR-124 and miR-138 were observed in a glioblastoma sample. There was also a marked hypoexpression of miR-138 in samples of grade III oligodendroglioma. CONCLUSION: The microRNAs miR-124a, miR-138, miR155 and miR-1275 did not show hyperexpression in tumor tissue of patients diagnosed with GBM. MiR-1275 showed increased expression in patients diagnosed with pilocytic astrocytoma and miR-155 was hyperexpressed only in samples of grade II oligodendroglioma. In addition, tumors of oligodendroglial lineage, of low and high grades, demonstrated hyperexpression of miR-1275 and miR-124a. In peripheral blood, significant hypoexpression of miR-1275, miR-124 and miR-138 were observed in the GBM sample as well as marked hypoexpression of miR-138 in samples of grade III oligodendroglioma

The survival for high-grade glioma patients is poor and the treatment may cause severe side effects. A common obstacle in the treatment is chemoresistance. To improve the quality of life and prolong survival for these patients prognostic biomarkers and new approaches for chemotherapy are needed. To this end, a strategy to evade chemoresistance was evaluated by combining chemotherapeutic drugs with agents inhibiting resistance mechanisms identified by a bioinformatic analysis (paper I). The prognostic value of 13 different proteins was analyzed in this thesis (papers II-IV). Two of them, p38 mitogen-activated protein kinase (MAPK) and protein tyrosine phosphatase non-receptor type 6 (PTPN6, also known as SHP1) were analyzed for their potential as targets in combination chemotherapy (in paper III and IV, respectively).   We found that: PTPN6 expression and methylation status may be important for survival of anaplastic glioma patients, p38 MAPK phosphorylation may be a potential negative prognostic biomarker for high-grade glioma patients and FGF2 expression may be a potential negative prognostic biomarker for proneural glioma patients. PTPN6 may be a useful target for combination chemotherapy with cisplatin, melphalan or bortezomib in high-grade gliomas. The following drug combinations; camptothecin combined with an EGFR or RAC1 inhibitor, imatinib combined with a Notch or RAC1 inhibitor, temozolomide combined with an EGFR or FAK inhibitor and vandetanib combined with a p38 MAPK inhibitor may be useful combination chemotherapy for high-grade gliomas.

A top-down analysis approach used for over 17 thousand of euro-denominated corporate bond issuances executed in the European primary bond market between 1999 and 2013. The main findings reveal not only the most active corporate issuers in Europe, but they also show the decomposition of issuance by coupon type, rated versus unrated issuance, debut and perpetual issuances, high grade as well as high yield new bond issues split by sector, rating and maturity. I used the opensource statistical package Gretl and applied the ordinary least square technique in order to quantify the association between euro-denominated corporate issuance and credit spreads.

The tumour microenvironment comprises the non-cancerous cells present in the tumour mass (fibroblasts, endothelial, and immune cells), as well as signalling molecules and extracellular matrix. Tumour growth, invasion, metastasis, and response to therapy are influenced by the tumour microenvironment. Therefore, characterising the cellular and molecular components of the tumour microenvironment, and understanding how they influence tumour progression, represent a crucial aim for the success of cancer therapies. High-grade serous ovarian cancer provides an excellent opportunity to systematically study the tumour microenvironment due to its clinical presentation of advanced disseminated disease and debulking surgery being standard of care. This thesis first presents a case report of a long-term survivor (>10 years) of metastatic high-grade serous ovarian cancer who exhibited concomitant regression/progression of the metastatic lesions (5 samples). We found that progressing metastases were characterized by immune cell exclusion, whereas regressing metastases were infiltrated by CD8+ and CD4+ T cells. Through a T cell - neoepitope challenge assay we demonstrated that pre- dicted neoepitopes were recognised by the CD8+ T cells obtained from blood drawn from the patient, suggesting that regressing tumours were subjected to immune attack. Immune excluded tumours presented a higher expression of immunosuppressive Wnt signalling, while infiltrated tumours showed a higher expression of the T cell chemoattractant CXCL9 and evidence of immunoediting. These findings suggest that multiple distinct tumour immune microenvironments can co-exist within a single individual and may explain in part the hetero- geneous fates of metastatic lesions often observed in the clinic post-therapy. Second, this thesis explores the prevalence of intra-patient tumour microenvironment het- erogeneity in high-grade serous ovarian cancer at diagnosis (38 samples from 8 patients), as well as the effect of chemotherapy on the tumour microenvironment (80 paired samples from 40 patients). Whole transcriptome analysis and image-based quantification of T cells from treatment-naive tumours revealed highly prevalent variability in immune signalling and distinct immune microenvironments co-existing within the same individuals at diagnosis. ConsensusTME, a method that generates consensus immune and stromal cell gene signatures by intersecting state-of-the-art deconvolution methods that predict immune cell populations using bulk RNA data was developed. ConsensusTME improved accuracy and sensitivity of T cell and leukocyte deconvolutions in ovarian cancer samples. As previously observed in the case report, Wnt signalling expression positively correlated with immune cell exclusion. To evaluate the effect of chemotherapy on the tumour microenvironment, we compared site-matched and site-unmatched tumours before and after neoadjuvant chemotherapy. Site- matched samples showed increased cytotoxic immune activation and oligoclonal expansion of T cells after chemotherapy, unlike site-unmatched samples where heterogeneity could not be accounted for. In addition, low levels of immune activation pre-chemotherapy were found to be correlated with immune activation upon chemotherapy treatment. These results cor- roborate that the tumour-immune interface in advanced high-grade serous ovarian cancer is intrinsically heterogeneous, and that chemotherapy induces an immunogenic effect mediated by cytotoxic cells. Finally, the different deconvolution methods were benchmarked along with ConsensusTME in a pan-cancer setting by comparing deconvolution scores to DNA-based purity scores, leukocyte methylation data, and tumour infiltrating lymphocyte counts from image analysis. In so far as it has been benchmarked, unlike the other methods, ConsensusTME performs consistently among the top three methods across cancer-related benchmarks. Additionally, ConsensusTME provides a dynamic and evolvable framework that can integrate newer de- convolution tools and benchmark their performance against itself, thus generating an ever updated version. Overall, this thesis presents a systematic characterisation of the tumour microenvironment of high grade serous ovarian cancer in treatment-naive and chemotherapy treated samples, and puts forward the development of an integrative computational method for the systematic analysis of the tumour microenvironment of different tumour types using bulk RNA data.

ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

<p>Sexuality has an important role in human life and there are many opinions about how to handle questions about sexuality. The attitudes have change over time and differ between different cultures, gender and age. This study deals with the question about youth attitudes to sex and personal relationships and what impact the education in this filed have on the youth. We have also studied how the education is organized. To get answers to these questions we have performed a questionnaire study among 116 students in grade seven to nine in an elementary school in Western Sweden. We have also interviewed two teachers in nature science in high grade and one school nurse. The analyses about the youth attitudes to sex and personal relationship are based on theories about sexuality, gender, socialization, culture differences, globalization, and consumption society. The study shows that the youth in general are equal in their attitudes to sexuality. Nearness, respect, love, self-knowledge are important, regardless gender. The attitudes about intercourse were different between boys and girls. They didn´t value the factors about sexual intercourse in the same way. Internet and sms plays an important role in how come in touch with each other. Boys thought that internet are important in sexual education.</p>

Sexuality has an important role in human life and there are many opinions about how to handle questions about sexuality. The attitudes have change over time and differ between different cultures, gender and age. This study deals with the question about youth attitudes to sex and personal relationships and what impact the education in this filed have on the youth. We have also studied how the education is organized. To get answers to these questions we have performed a questionnaire study among 116 students in grade seven to nine in an elementary school in Western Sweden. We have also interviewed two teachers in nature science in high grade and one school nurse. The analyses about the youth attitudes to sex and personal relationship are based on theories about sexuality, gender, socialization, culture differences, globalization, and consumption society. The study shows that the youth in general are equal in their attitudes to sexuality. Nearness, respect, love, self-knowledge are important, regardless gender. The attitudes about intercourse were different between boys and girls. They didn´t value the factors about sexual intercourse in the same way. Internet and sms plays an important role in how come in touch with each other. Boys thought that internet are important in sexual education.

The recent substantial increase in world demand for energy and raw material resources has accelerated oil and gas exploration and production. At the same time, the depletion of onshore and shallow water oil resources presents a challenge to engineers to develop new means of harvesting and transporting oil and gas from harsh and remote areas. Sandwich Pipe (SP) is a relatively new design concept developed to address the transportation of oil in deep and ultra-deep waters as well as in cold environments. The main focus of this thesis is on the characterization of the structural performance of these novel systems. Deep and ultra-deep water offshore pipelines are subjected to excessive hydrostatic external pressure during installation and operation. In this research, an innovative analytical solution was developed to evaluate the external pressure capacity of SPs by calculating the linear eigenvalues of the characteristic equations of the system. In the proposed solution, the interface condition between the layers of the system is accounted for in the governing equations. As well, a set of comprehensive parametric studies using the Finite Element (FE) method was developed to investigate both the elastic and plastic buckling response of SPs. The influence of various structural parameters such as the material, geometrical and intra-layer interaction properties on the characteristic behavior and the buckling pressure of SPs was examined. In addition to the proposed analytical solution, two sets of semi-empirical equations based on the FE analysis results were recommended in calculating the elastic and plastic buckling pressure of SPs. As bending represents an important loading state in the installation and service life of SPs, it should be considered a governing loading scenario. In this thesis, the behavior of SPs under bending was investigated using a comprehensive set of parametric studies. SP systems with a wide practical range of physical parameters were analyzed using the FE method, and the influence of various structural parameters on the characteristic response and bending capacity of the system was explored, including pipe geometry, core layer properties, material yield anisotropy of high-grade steel pipes, and various intra-layer adhesion configurations.