京都大学 / 0048 / 新制・論文博士 / 博士(社会健康医学) / 乙第13292号 / 論社医博第13号 / 新制||社医||10(附属図書館) / (主査)教授 川上 浩司, 教授 平井 豊博, 教授 伊達 洋至 / 学位規則第4条第2項該当 / Doctor of Public Health / Kyoto University / DFAM

This diploma thesis dealt with the comparison of different methods of amination of polycaprolcatone in terms of their effectiveness for tissue engineering. A polycaprolactone membrane was prepared by an electrospinning method, which was subsequently modified by three different amination methods. Selected types of amination were plasma polymerization with cyclopropylamine monomer, hybrid modification using plasma and N-allylmethylamine monomer, and chemical amination using aminolysis with diaminohexane. Surface amines were subsequently characterized by electron scanning microscopy (SEM), X-ray photoelectron spectroscopy (XPS), attenuated total reflection infrared spectroscopy (ATR-FTIR) and contact angle measurement. A cell culture designated A375 (Human malignant melanoma cell lines A375® CRL-1619®) was cultured on the thus modified membranes, which was analyzed by optical microscopy, and a proliferation assay was performed by determining the relative amount of ATP. Based on the experimental results, we can confirm the success for all types of amination. In terms of efficiency for tissue engineering, the amination method by plasma polymerization with the monomer cyclopropylamine has the most satisfactory results.

Trends and patterns of cancer mortality in Kazakhstan in comparison with some selected European countries from 1986 to 2008 Abstract This thesis primarily addresses mortality patterns and trends by main causes of death and by major neoplasms in Kazakhstan in comparison with the selected European countries: the Czech Republic, France and Sweden during 1986-2008. Within the whole group of main causes of death, the changes of mortality levels in different groups of neoplasms are analyzed. The analysis is accompanied with the comparison of mortality levels from major neoplasms within the selected countries. Afterwards the thesis focuses on cancer causes and risk factors in the countries under observation. The analysis concludes that the current mortality situation in Kazakhstan follows up long term adverse mortality trends of the past two decades and neoplasms remain an important public health problem in Kazakhstan. Key Words: mortality, cancer, malignant neoplasms, cause of death, standardized mortality rates, comparison, Kazakhstan, Czech Republic, France, Sweden

Le mésothéliome pleural malin (MPM) est une tumeur primitive de la plèvre, rare, très agressive, avec un pronostic sombre. Nous avons souhaité au cours de ce travail de thèse, identifier de nouveaux biomarqueurs du MPM en testant l’influence de l’inactivation des membres de la famille RASSF/Hippo sur la survie des 448 patients inclus dans l’essai clinique MAPS (IFCT-GFPC-0701). Nous souhaitions également comprendre quelles fonctions et signalisations essentielles à l’homéostasie cellulaire, auxquelles participe la voie de signalisation RASSF/Hippo, sont perturbées lors du processus de transformation des cellules mésothéliales. L’inactivation des membres de la voie a été étudiée par PCR spécifique de méthylation (MS-PCR) et leur influence sur la survie des 448 patients inclus dans l’essai clinique MAPS testée en analyse uni- et multivariée avant d’être validée par boostrap. D’autre part, nous avons mimé in cell, l’inactivation par ARN interférence de plusieurs membres de la voie Hippo dans des cellules de lignées mésothéliales humaines (MSTO-211H, H2452, H28 et H2052). Nous avons pu identifier plusieurs biomarqueurs du MPM : i) la kinase MST1 dont l’inactivation est un facteur de mauvais pronostic, ii) l’amphiréguline dont l’expression cytoplasmique est au contraire un facteur de bon pronostic et enfin iii) le CD44 dont l’expression élevée constitue un outil diagnostique du MPM. Les approches in cell, nous ont permis de démontrer que les altérations de la voie RASSF/Hippo induisent une activité inappropriée de l’effecteur terminal YAP : le moins bon pronostic des patients présentant une inactivation de MST1 s’explique ainsi par le fait qu’en régulant l’activité de YAP, MST1 contrôle la balance apoptose/prolifération et prévient l’invasion et la croissance sans adhésion. En son absence, ces processus cellulaires sont dérégulés. Ce travail démontre l’importance de l’axe CD44/RASSF1A/MST1 dans le contrôle d’une activité appropriée de YAP et de l’homéostasie des cellules mésothéliales. La compréhension des désordres cellulaires induits par la dérégulation de le voie RASSF/Hippo, désigne YAP comme cible thérapeutique potentielle chez les patients atteints de MPM et présentant des altérations de cette voie de signalisation. / Malignant pleural mesothelioma (MPM) is a rare, very aggressive, primary tumor with a poor prognosis. During this thesis, we wanted to identify new biomarkers of MPM by testing the influence of the RASSF/Hippo pathway inactivation on the survival of the 448 patients included in the clinical trial MAPS (IFCT- GFPC-0701). We also wanted to understand which functions and signals essential to cellular homeostasis, linked to RASSF/Hippo signaling pathway, are disturbed during the mesothelial cell transformation process. Inactivation of RASSF/Hippo members was studied by methylation-specific PCR (MS-PCR) and their influence on the survival of the 448 patients included in the MAPS clinical trial tested in uni- and multivariate analysis before being validated by bootstrap. We also mimed in cell, by RNA interference, several members of the Hippo pathway inactivation in human mesothelial cells lines (MSTO-211H, H2452, H28 and H2052). We have identified several biomarkers of MPM: i) MST1 kinase whose inactivation is a factor of poor prognosis, ii) amphiregulin whose cytoplasmic expression is on the contrary a factor of good prognosis and finally iii) CD44 whose high expression is a diagnostic tool for MPM. In cell we demonstrate that RASSF/Hippo pathway alterations induce an inappropriate activity of YAP, one Hippo end effector: the poorer prognosis of patients with inactivation of MST1 is thus explained by the fact that, by regulating YAP activity, MST1 controls the apoptosis/proliferation balance and prevents invasion and growth without adhesion from mesothelial cells. In its absence, these cellular processes are deregulated. This work finally demonstrates the importance of the CD44/RASSF1A/MST1 axis in controlling appropriate YAP activity and mesothelial cell homeostasis. The understanding of the cellular disorders induced by the of the RASSF/Hippo pathway deregulation designates YAP as a potential therapeutic target in patients with MPM and presenting alterations of this signaling pathway.

Most cardiac tumors are metastatic tumors, which are 20–40 times more common than primary tumors of the heart. Most primary tumors of the heart are benign, with atrial myxomas being the most common. Primary cardiac tumors are extremely rare with an incidence of less than 0.1 percent. Virtually all types of sarcomas have been reported in the heart as isolated case reports. We present a rare case of biatrial high-grade osteosarcoma. A fifty-four-year-old Hispanic female presented with shortness of breath and was cyanotic on the exam while visiting Mexico. Due to abnormal chest x-ray, echocardiogram concerning for bilateral atrial myoma she was referred to a cardiothoracic surgeon. She underwent bi atrial intracardiac tumor resection in Mexico. Several months prior to her resection she noted numbness on the side of the face that was evaluated by her physicians in the United States with a Brain MRI and carotid Ultrasound/Doppler that was unrevealing. She also remembered an episode of uncontrolled hypertension two years prior to surgery requiring admission to a local hospital in East Tennessee with cardiology evaluation that did not include an echocardiogram. Surgical pathology showed extensive undifferentiated spindle cell proliferation with multifocal osteoid production and foci of osseocartilaginous differentiation. There was prominent necrosis and moderately high mitotic rate (10-19/HPF). Tumor cells were positive for SatB2 and negative for vascular, muscular, or neural markers. This is consistent with a primary cardiac high-grade osteosarcoma. These occur very rarely, usually in the atria, and behave aggressively. Post resection staging PET-CT showed hypermetabolic mixed lytic and sclerotic lesion of T10 concerning for metastasis disease. She received approximately 6 cycles of adriamycin and ifosfamide chemotherapy. Adriamycin was discontinued due to left ventricular dysfunction with an ejection fraction of 30-35%, multiple segmental abnormalities, diffuse left ventricular hypokinesis, and moderate to severe mitral valve regurgitation. Despite intracardiac tumor, resection, concern for metastatic disease, chemotherapy, and systolic dysfunction patient is asymptomatic and does have robust performance status. A follow-up PET-CT five months after cessation of treatment reveals no significant evidence of uptake other than abnormalities in the T10 vertebra. A repeat echocardiogram continues to reveal a depressed ejection fraction of 35%. The patient is completely asymptomatic, seemingly fit with an ECOG performance status of 0-1. Osteosarcomas are aggressive with a high incidence of recurrence and metastasis. Fewer than 50 cases of primary cardiac osteosarcomas have been reported in the literature. Currently, it is postulated that they arise from undifferentiated mesenchymal stem cells in the endocardium that transform into active osteoblasts. Even though complete resection can be achieved in some cases, long-term results are usually poor. No standard therapy has been established due to the tumor's low incidence rate and lack of clinical trial data. Our case highlights the importance of evaluating common symptoms thoroughly since it may be a harbinger of rare and serious disorders. This case reflects the heterogeneous nature of sarcoma histology, the consequent tumor biology and hence varied clinical course and prognosis.

Major capside protein of Polyomaviridae family viruses is able to selfassemble into virus-like particle (VLP) even without the presence of minor proteins, bind exogenous DNA non-specifically and recognise the receptor on the cellular surface. These characteristics determine its use as vector in gene therapy or immunotherapy. It was discovered before that MPyV VLPs significantly stimulate immune system and have strong adjuvant effect. Chimeric VLP derived from mouse polyomavirus carrying exogenous antigene or epitop is supposed to elicit specifically targeted immune response after immunisation. The main obstacle is choice of immunogene that is strong enough to cause adequate immune response. The goal of this thesis was to construct chimeric particles carrying epitop of malignant melanoma, one of the most immunogenic tumours, on their surface, using methods of genetic engineering. For future research of particle's immunogenic properties three types of particles were developed - particles with human and mouse melanoma epitopes, respectively and control particles with ovalbumine epitop. For the purpose of production of chimeric protein was used baculovirus expression system. It was verified then, with the use of electron microscopy, that introduction of tumour antigen into one of surface loops of VP1...

Background: Carotid body tumor (CBT) is a rare lesion of the neuroendocrine system. Chronic hypoxia has long been recognized as an etiology of CBT and other paragangliomas. Recent biogenetic discoveries reveal that mutations in oxygen-sensing genes are another etiology, accounting for approximately 35% of cases, and that these 2 etiologies are probably additive. Data Sources: (1) A retrospective analysis of fifteen cases of CBT in a 6-year period occurring in the mountains of Southern Appalachia; (2) an extensive review of the literature on the surgery of CBT and on the expansive biogenetic understanding of the disease. Conclusions: Improved imaging, vascular surgical techniques, and understanding of the disease have vastly improved outcomes for patients. The necessities for long-term follow-up and appropriate genetic testing and counseling of patients and their families are documented. Surgeon and institutional competence are critical in achieving maximal outcomes.

Malignant catarrhal fever (MCF), a disease that is usually fatal in cattle, is caused by two distinct but related bovine herpesviruses which are members of the genus Macavirus. The wildebeest-associated alcelaphine herpesvirus-1 (AlHV-1) occurs mainly in East and southern Africa, whereas the sheep-associated ovine herpesvirus-1 (OvHV-2) has an almost worldwide distribution. The natural hosts or carriers of these two viruses are subclinically infected. The 130 kilobase pair (kbp) AlHV-1 double stranded DNA genome consists of 18 open reading frames (ORFs) coding for structural proteins and approximately 50 ORFs coding for non-structural proteins. The 18 structural ORFs encode for 4 capsid proteins, 5 tegument proteins, 8 glycoproteins and a minor capsid scaffold protein. ORF8 encoding for glycoprotein B, is the most conserved of the proteins amongst gammaherpesviruses, whereas the minor capsid protein encoded by ORF65, is amongst the most variable. Thus, the minor capsid protein is one of the antigens of choice for the development of an ELISA for detection of AlHV-1 reactive antibodies and glycoprotein B could be of importance in developing a cross-protective vaccine for gammaherpesviruses. The naming and annotation of most of the AlHV-1 ORFs is based on comparison with related gammaherpesviruses and bioinformatics. Most of these ORFs are putative as there is no direct experimental evidence confirming that they code for any particular protein. In order to investigate whether the ORFs code for any proteins, two ORFs were targeted for in vitro heterologous expression. AlHV-1, isolate C500, was grown in fetal bovine turbinate (BT) cell culture and viral genomic DNA extracted. ORF8, the putative glycoprotein B, was amplified with a PCR assay and inserted into a mammalian expression vector, pCI. VERO cells were transfected with the recombinant vector. Expression of ORF8 was confirmed by an indirect immunofluorescence assay (IFA) with AlHV-1 polyclonal sera and rabbit anti-bovine IgG (whole molecule) FITC conjugate. Truncated forms of ORF8 were further expressed as baculovirus recombinants using the Bac-to-Bac baculovirus expression system. Expression of the truncated ORF8 was confirmed by SDS-PAGE and Western blot. AlHV-1 ORF65, the minor capsid protein gene, was amplified with a PCR assay from the viral genomic DNA and cloned in frame with a histidine tag in a bacterial expression vector, pCOLD I. Expression of the minor capsid protein was confirmed by SDS-PAGE and Western blot with the histidine tag monoclonal as well as AlHV-1 polyclonal sera. Orf65 was expressed in large quantities and column purified using the histidine tag. Orf65 was also expressed as a baculovirus recombinant using the Bac-to-Bac baculovirus expression system. Expression of the protein was confirmed by SDS-PAGE and Western blot with the histidine tag and AlHV-1 polyclonal sera. ORF65 expression in the baculovirus Bac-to-Bac expression system was up-scaled and the expressed protein column purified. Antibodies raised in chicken against the purified antigen were used successfully in an indirect immunoassay to detect AlHV-1 infected cells. An indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against AlHV-1 was developed. It is based on the use of the AlHV-1 minor capsid protein as the capture antigen for antibodies. The primary antibodies are detected by the addition of enzymelabelled (horseradish peroxidase) protein G which detects bovid, ovid and wildebeest antibodies. Addition of a substrate of the enzyme, in this case, 3,3’,5,5’- tetramethylbenzidine (TMB), results in a colour reaction which is measured using spectrophotometric procedures. At a selected cut-off point of 18, the ELISA test has a sensitivity of 100% and a specificity of 100% and has been shown to detect AlHV-1 antibodies in cattle and wildebeest. The ELISA showed no cross-reactivity with sera raised in cattle against related viruses such as ovine herpesvirus 2, bovine herpesvirus 1, 2 and 4. The two expressed proteins used in this study were found to be amongst the antigens expressed in cattle suffering from malignant catarrhal fever. The experimental AlHV-1 indirect ELISA needs further validation and this research may be extended to determine the performance of these antigens as candidate subunit vaccines. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted

京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第23385号 / 人健博第92号 / 新制||人健||6(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 田村 恵子, 教授 稲富 宏之, 教授 溝脇 尚志 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

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