MicroRNA-9 (miR-9) modulates gene expression and demonstrates high structural conservation and wide expression in the central nervous system. Bioinformatics analysis predicts almost 100 ion channels, membrane transporters and receptors, including genes linked to primary familial brain calcification (PFBC), as possible miR-9-5p targets. PFBC is a neurodegenerative disorder, characterized by bilateral and symmetrical calcifications in the brain, associated with motor and behavioral disturbances. In this work, we seek to study the influence of miR-9-5p in regulating genes involved in PFBC, in an osteogenic differentiation model with SaOs-2 cells. During the induced calcification process, solute carrier family 20 member 2 (SLC20A2) and platelet-derived growth factor receptor beta (PDGFRB) were downregulated, while platelet-derived growth factor beta (PDGFB) showed no significant changes. Significantly decreased levels of SLC20A2 and PDGFRB were caused by the presence of miR-9-5p, while PDGFB showed no regulation. We confirmed the findings using an miR-9-5p inhibitor and also probed the cells in electrophysiological analysis to assess whether such microRNA might affect a broader range of ion channels, membrane transporters and receptors. Our electrophysiological data show that an increase of the miR-9-5p in SaOs-2 cells decreased the density and amplitude of the output ionic currents, indicating that it may influence the activity, and perhaps the expression, of some ionic channels. Additional investigations should determine whether such an effect is specific to miR-9-5p, and whether it could be used, together with the miR-9-5p inhibitor, as a therapeutic or diagnostic tool.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20985号 / 医博第4331号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 湊谷 謙司, 教授 野田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Myocardial ischemia/reperfusion (I/R) injury is a clinically fatal disease, caused by restoring myocardial blood supply after a period of ischemia or hypoxia. However, the underlying mechanism remains unclear. Recently, increasing evidence reveal that microRNAs (miRs) participate in myocardial I/R injury. This study aimed to investigate whether miR-128-1-5p contributed to cardiomyocyte apoptosis induced by myocardial I/R injury. Here, we showed that the expression of miR-128-1-5p was decreased in mice following myocardial I/R injury. Down-regulation of miR-128-1-5p was also showed in H9c2 cardiomyocytes after hypoxia/reoxygenation (H/R), and in neonatal rat cardiomyocytes (NRCMs) with H2O2 treatment. Importantly, we found that overexpression of miR-128-1-5p ameliorates cardiomyocyte apoptosis both in H9c2 cardiomyocytes and NRCMs. Moreover, we also found that growth arrest DNA damage-inducible gene 45 gamma (Gadd45g) is identified as a direct target of miR-128-1-5p, which negatively regulated Gadd45g expression. Additionally, silencing of Gadd45g inhibits cardiomyocyte apoptosis in H9c2 cardiomyocytes and NRCMs. These results reveal a novel mechanism by which miR-128-1-5p regulates Gadd45g-mediated cardiomyocyte apoptosis in myocardial I/R injury.

Objective: Up-regulated expression of transcription-factor E2F1 in human visceral adipose tissue (VAT) characterizes a dysmetabolic obesity sub-phenotype. An E2F1-miRNA network has been described in multiple cancers. Here we investigated whether elevated VAT-E2F1 in obesity is associated with VAT-miRNA alterations similar to, or distinct from, those described in cancer. Furthermore, we assessed if E2F1-associated miRNA changes may contribute to the link between high- VAT-E2F1 and a dysmetabolic obesity phenotype. Methods: We assembled a cohort of patients with obesity and high-VAT-E2F1, matched by age, sex, ±BMI to patients with low-VAT-E2F1, with and without obesity (8 patients/groupX3 groups). We performed Nanostring -based miRNA profiling of VAT samples from all 24 patients. Candidate E2F1-related miRNAs were validated by qPCR in an independent cohort of patients with extreme obesity, with or without type-2-diabetes (T2DM) (n = 20). Bioinformatic tools and manipulation of E2F1 expression in cells were used to establish the plausibility of the functional VAT-E2F1-miRNA network in obesity. Results: Among n = 798 identified miRNAs, 17 were differentially expressed in relation to E2F1 and not to obesity itself. No evidence for the cancer-related E2F1-miRNA network was identified in human VAT in obesity. In HEK293-cells, overexpression/downregulation of E2F1 correspondingly altered the expression of miRNA-206 and miRNA-210-5p, two miRNAs with reported metabolic functions consistent with those of E2F1. In VAT from both cohorts, the expression of both miRNA-206 and 210-5p intercorrelated, and correlated with the expression of E2F1. In cohort 1 we did not detect significant associations with biochemical parameters. In cohort 2 of patients with extreme obesity, all those with high VAT-E2F1 showed a diabetes-complicated obesity phenotype and higher expression of miRNA-206 and miRNA-210-5p, which also correlated with fasting glucose levels (both miRNAs) and fasting insulin (miRNA-210-5p). Conclusions: Whilst the previously described cancer-related E2F1-miRNA network does not appear to operate in VAT in obesity, miRNAs-206 and 210-5p may link high-E2F1 expression in VAT with diabetes-complicated extreme obesity phenotype.

Introdução: A epilepsia pode ser considerada uma desordem neurológica causada pela anormalidade da transmissão de impulsos nervosos, devido ao aumento da excitação nervosa e/ou diminuição da sua inibição. Dentre as síndromes epilépticas, destaca-se a Epilepsia do Lobo Temporal Mesial (ELTM) devido a sua alta prevalência e refratariedade ao tratamento medicamentoso. Com intuito de implementar novas possibilidades de tratamento torna-se necessário uma maior compreensão das bases moleculares da ELTM. Dentro desta perspectiva, destacam-se os microRNAs, que possuem papel regulatório nas células, inclusive as do Sistema Nervoso Central. Com base em dados da literatura e num experimento de microarray, realizado no laboratório de Biologia Molecular do Departamento de Cirurgia e Anatomia da Faculdade de Medicina de Ribeirão Preto, foram escolhidos 3 dos 10 microRNAs, cujos alvos preditos estão relacionados à epileptogênese e que se apresentam diferencialmente expressos em hipocampos de pacientes com ELTM. Objetivos: avaliar a expressão diferencial de três microRNAs de interesse em hipocampos de pacientes operados por ELTM refratária ao tratamento clínico a fim de correlacionar os resultados em relação aos controles das crises epilépticas após a cirurgia. Metodologia: Foram utilizadas 15 amostras de hipocampo de pacientes com ELTM classificados como Engel I (boa evolução - nenhuma ou poucas crises convulsivas após lobectomia temporal parcial) e 15 amostras de hipocampos de pacientes classificados como Engel III/IV (má evolução - sem melhora evidente após tratamento cirúrgico). Como controles, foram utilizadas 10 amostras de hipocampo de pacientes sem doenças neurológicas, obtidas de necropsias do Serviço de Patologia do Hospital das Clínicas de Ribeirão Preto (HCFMRP). Por meio de técnica de PCR quantitativo em tempo real (RT-qPCR) foram realizadas as avaliações das expressões diferenciais dos seguintes microRNAs: miR-184, miR-190a-5p e miR-493-3p. Resultados: Em relação aos controles, o miR-184 e o miR190a-5p apresentaram-se, respectivamente, com redução significativa (p = 0,001) e com tendência a baixa expressão (p = 0,24) nos hipocampos de pacientes com ELTM. No que se refere ao controle das crises, o miR-184 apresentou-se significativamente mais expresso nos hipocampos de pacientes com pior evolução (Engel III/IV vs Engel I, p = 0,003). A expressão do miR-493-3p não se correlacionou com o controle de crises com significância estatística. Conclusão: Dentro os microRNAs avaliados, a expressão do miR-184, que possui importante papel na regulação de componentes celulares relacionados a apoptose e morte celular, correlacionou-se inversamente com o controle das crises após cirurgia (mais expresso em pacientes com má evolução) tornando-se potencial biomarcador e com valor preditivo / Introduction: Epilepsy is a neurological disorder caused by nerve impulse transmission abnormalities, by increasing nerve excitation and/or decreasing nerve inhibition. Among the epileptic syndromes, one of the most important ones is Mesial Temporal Lobe Epilepsy (MTLE) due to its high prevalence and resistance to drug treatment. A greater understanding of the epilepsy molecular basis is necessary aiming to implement new treatments possibilities. Within this field of study, it is possible to highlight the microRNA molecules, which have a regulatory role in cell regulation including in the Central Nervous System (CNS). Based on literature data and in an microarray experiment, performed in the Anatomy and Surgery Departament Molecular Biology laboratory of the Ribeirao Preto Medical School, we have chosen three out of ten microRNAs, with predicted targets related to epileptogenesis and that presented themselves differencially expressed in patients hippocampus with MTLE. Objective: To evaluate three microRNAs of interest which were differentially expressed in the hippocampus of MTLE drug-resistant operated patients in order to correlate the results with the clinical evolution based on the seizures control after surgery. Methodology: Fifteen hippocampus samples from patients with MTLE classified as Engel I (good evolution - without seizures or few seizures after surgical resection) and 15 samples from patients classified as Engel III/IV (bad evolution - no evident improvement after surgery) were used in this study. In the control group, we used ten samples of hippocampus fragments from patients without neurological diseases, that were obtained from the Ribeirao Preto Clinical Hospital Pathology and Legal Medicine Service. The differential expression validation from microRNAs-184, -190a-5p and -493-3p were performed by quantitative real time PCR (RT-qPCR) technique. Results: Regarding the controls, miR-184 and miR- 190a-5p showed, respectively, a significant expression reduction (p=0,001) and a decreased expression tendency (p=0,24) in MTLE patients. If we consider seizure control, miR-184 presented itself significantly upregulated in bad evolution hippocampus patients (Engel III/IV vs Engel I, p=0,003). miR-493-3p did not show expression differences with statistical significance. Conclusions: After evaluating the expression of these microRNAs, it was possible to conclude that microRNA-184, which regulates cellular death and apoptosis related components, was the best molecule that could differentiate the patients regarding seizure control after surgery, and that fact makes this microRNA a potential biomarker with predictive value

Introdução: A epilepsia pode ser considerada uma desordem neurológica causada pela anormalidade da transmissão de impulsos nervosos, devido ao aumento da excitação nervosa e/ou diminuição da sua inibição. Dentre as síndromes epilépticas, destaca-se a Epilepsia do Lobo Temporal Mesial (ELTM) devido a sua alta prevalência e refratariedade ao tratamento medicamentoso. Com intuito de implementar novas possibilidades de tratamento torna-se necessário uma maior compreensão das bases moleculares da ELTM. Dentro desta perspectiva, destacam-se os microRNAs, que possuem papel regulatório nas células, inclusive as do Sistema Nervoso Central. Com base em dados da literatura e num experimento de microarray, realizado no laboratório de Biologia Molecular do Departamento de Cirurgia e Anatomia da Faculdade de Medicina de Ribeirão Preto, foram escolhidos 3 dos 10 microRNAs, cujos alvos preditos estão relacionados à epileptogênese e que se apresentam diferencialmente expressos em hipocampos de pacientes com ELTM. Objetivos: avaliar a expressão diferencial de três microRNAs de interesse em hipocampos de pacientes operados por ELTM refratária ao tratamento clínico a fim de correlacionar os resultados em relação aos controles das crises epilépticas após a cirurgia. Metodologia: Foram utilizadas 15 amostras de hipocampo de pacientes com ELTM classificados como Engel I (boa evolução - nenhuma ou poucas crises convulsivas após lobectomia temporal parcial) e 15 amostras de hipocampos de pacientes classificados como Engel III/IV (má evolução - sem melhora evidente após tratamento cirúrgico). Como controles, foram utilizadas 10 amostras de hipocampo de pacientes sem doenças neurológicas, obtidas de necropsias do Serviço de Patologia do Hospital das Clínicas de Ribeirão Preto (HCFMRP). Por meio de técnica de PCR quantitativo em tempo real (RT-qPCR) foram realizadas as avaliações das expressões diferenciais dos seguintes microRNAs: miR-184, miR-190a-5p e miR-493-3p. Resultados: Em relação aos controles, o miR-184 e o miR190a-5p apresentaram-se, respectivamente, com redução significativa (p = 0,001) e com tendência a baixa expressão (p = 0,24) nos hipocampos de pacientes com ELTM. No que se refere ao controle das crises, o miR-184 apresentou-se significativamente mais expresso nos hipocampos de pacientes com pior evolução (Engel III/IV vs Engel I, p = 0,003). A expressão do miR-493-3p não se correlacionou com o controle de crises com significância estatística. Conclusão: Dentro os microRNAs avaliados, a expressão do miR-184, que possui importante papel na regulação de componentes celulares relacionados a apoptose e morte celular, correlacionou-se inversamente com o controle das crises após cirurgia (mais expresso em pacientes com má evolução) tornando-se potencial biomarcador e com valor preditivo / Introduction: Epilepsy is a neurological disorder caused by nerve impulse transmission abnormalities, by increasing nerve excitation and/or decreasing nerve inhibition. Among the epileptic syndromes, one of the most important ones is Mesial Temporal Lobe Epilepsy (MTLE) due to its high prevalence and resistance to drug treatment. A greater understanding of the epilepsy molecular basis is necessary aiming to implement new treatments possibilities. Within this field of study, it is possible to highlight the microRNA molecules, which have a regulatory role in cell regulation including in the Central Nervous System (CNS). Based on literature data and in an microarray experiment, performed in the Anatomy and Surgery Departament Molecular Biology laboratory of the Ribeirao Preto Medical School, we have chosen three out of ten microRNAs, with predicted targets related to epileptogenesis and that presented themselves differencially expressed in patients hippocampus with MTLE. Objective: To evaluate three microRNAs of interest which were differentially expressed in the hippocampus of MTLE drug-resistant operated patients in order to correlate the results with the clinical evolution based on the seizures control after surgery. Methodology: Fifteen hippocampus samples from patients with MTLE classified as Engel I (good evolution - without seizures or few seizures after surgical resection) and 15 samples from patients classified as Engel III/IV (bad evolution - no evident improvement after surgery) were used in this study. In the control group, we used ten samples of hippocampus fragments from patients without neurological diseases, that were obtained from the Ribeirao Preto Clinical Hospital Pathology and Legal Medicine Service. The differential expression validation from microRNAs-184, -190a-5p and -493-3p were performed by quantitative real time PCR (RT-qPCR) technique. Results: Regarding the controls, miR-184 and miR- 190a-5p showed, respectively, a significant expression reduction (p=0,001) and a decreased expression tendency (p=0,24) in MTLE patients. If we consider seizure control, miR-184 presented itself significantly upregulated in bad evolution hippocampus patients (Engel III/IV vs Engel I, p=0,003). miR-493-3p did not show expression differences with statistical significance. Conclusions: After evaluating the expression of these microRNAs, it was possible to conclude that microRNA-184, which regulates cellular death and apoptosis related components, was the best molecule that could differentiate the patients regarding seizure control after surgery, and that fact makes this microRNA a potential biomarker with predictive value

This master´s thesis deals with compiling a marketing plan for L 410 NG aircraft, manufactured by Aircraft Industries a.s.. The first part includes general theoretical knowledge, focusing on the process of formulating a plan by analyzing the external environment SLEPTE , Porter's five forces model, SWOT analysis and marketing mix 5P. In the second analytical part, the fundamental theory is applied to the company. On the basis of the results achieved, the individuals steps of a new marketing plan, including costs and time schedule are formulated in the final part of the thesis.

La classification du cancer de l'endomètre, basée sur l'histologie, conditionne la prise en charge thérapeutique alors qu'elle ne montre pas une pertinence suffisante pour prédire l'envahissement ganglionnaire. La détermination de profils d'expression biologiques corrélés au statut ganglionnaire et à d'autres facteurs pronostiques majeurs tels que les emboles apparait donc fondamentale pour mieux adapter la prise en charge. Nous avons montré, par une analyse de puce à partir des ARNs extraits de tissus sous paraffine de cancer de l'endomètre de type endométrioide, de grade 1-2, supposé limité à l'utérus, que les niveaux d'expression de cinq micro-ARNs (miR-34c-5p, -375, -184, -34c-3p, et -34b-5p) étaient plus faibles dans les tissus tumoraux avec envahissement ganglionnaire. Les niveaux d'expression de trois micro-ARNs (miR-34c-5p, -23b-5p, et -23c) étaient plus faibles dans les tissus tumoraux avec emboles lymphovasculaires. Une analyse par RT-qPCR a permis de déterminer des seuils de micro-ARNs corrélés aux facteurs pronostiques. Les patientes avec seuil d'expression du micro-ARN-184 <0,30 avaient un envahissement ganglionnaire dans 60% des cas, contre 11,5% pour les patientes avec un seuil d'expression de micro-ARN-184 >0,30, p=0,006. Les patientes avec seuil d'expression du micro-ARN-34c-5p <0,15 avaient un risque plus élevé d'avoir des emboles (92,3%) que les patientes avec un seuil d'expression de micro-ARN-34c-5p >0,15 (0,0%), p<0,001. Ces profils d'expression de micro-ARNs fournissent les bases pour de nouvelles études sur la fonction des micro-ARNs dans le cancer de l'endomètre et pourraient constituer un nouvel outil au diagnostic du statut ganglionnaire. / Current histological classification of early-stage endometrial cancer (EC) may show insufficient accuracy to precisely predict lymph node metastases leading to potential over or under treatment. Hence, additional highly sensitive and specific molecular prognostic biomarkers correlated with prognostic factors, such as nodal involvement and lymphovascular space involvement (LVSI), are needed to better adapt surgical management and adjuvant therapies. We fstudied by microarray analysis microRNA expression profiles of formalin-fixed paraffin-embedded grade 1–2 supposed early-stage endometrioid adenocarcinomas specimen. The expression levels of 5 microRNAs (miR-34c-5p, -375, -184, -34c-3p, et -34b-5p) were lower in the EC with positive nodal status compared to those with negative nodal status. The expression levels of 3 microRNAs (miR-34c-5p, -23b-5p, et -23c) were lower in the EC with positive LVSI compared to those with negative LVSI. A quantitative reverse transcriptase–PCR assay was used to determine micro-RNAs thresholds correlated with prognostic factors. Women with a microRNA-184-fold change <0.30 were more likely to have positive lymph node (n=6; 60.0%) compared with those with a microRNA-184-fold change >0.30 (n=3; 11.5%), p=0.006. Women with a microRNA-34c-5p fold change <0.15 were more likely to have positive LVSI status (n=12; 92.3%) compared with those with a microRNA-34c-5p fold change >0.15 (n=0; 0.0%), p<0.001. These microRNA expression profiles may provide a basis for further studies of the micro-RNA function in endometrioid adenocarcinoma, and be used as a diagnostic tool.

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民國九十七年二月底，就學貸款的總申貸戶已突破九十萬人、總核貸金額也超過一千五百億台幣，雙雙創下歷史的新高；但在同時，學生逾期未繳的呆帳也成長到四十四億的台幣，逾放比相較一般銀行的2%，更是高達4.59%，中央銀行更認為其逾放比高達16%，這造成政府在財政上的龐大的負擔。 反觀近幾年被熱烈討論的微型貸款，其在貸款上的創新、和就學貸款同樣具有社會服務性質，將貸款提供給予貧困、一般商業銀行不願意接受的民眾，但又可以維持高度獨立的財務支撐性而為人稱奇。1970年代開始發展的微型貸款，其首位推行者Yunas於2006年時受到諾貝爾和平獎的肯定，其在孟加拉創辦的Grameen Bank更是創下97~99%的還款率。 是什麼因素造成原本商業銀行不接受的微型貸款可以成功，而實行良久的就學貸款卻面臨貸款難以回收的問題？本文希望透過商品及消費者的角度、而非傳統社會福利的角度來審視微型貸款和就學貸款；利用過去的文獻及個案，以及訪談在台灣的微型貸款計畫－創業鳳凰微型貸款、嬌生彭婉如貸款，來比較分析貸款成功的關鍵因素，以給予就學貸款改進上的建議。 本文利用現今普遍被銀行採用由Paul H. Hunn提出的信用貸款5P原則：People貸款戶、Purpose貸款用途、Payment還款來源、Protection債權保障、Perspective貸款戶展望；以及由Jerome McCarthy提出、經Philip Kotler推廣的行銷4P：Product 產品規劃、Price定價、Promotion產品促銷、Place通路劃分；透過這4P及5P原則來建立架構、分析比較個案。 本研究經由訪談個案相關人員、以及過去國內外文獻資料，瞭解到成功的貸款必須要注意三個關鍵的要素。一、計畫的設計必須配合當地環境：貸款的回收是透過借款人創造正向的經濟循環產生的收入，所以必須要有一個適合發展的政治社會環境；二、篩選願意接受改變的借款人並賦權：給予貸款者適當的能力及資源以幫助他們成功，這必須透過挑選有意願改變的人、給予需要的資源，包括貸款及訓練、或是諮詢顧問；三、透過社群來建立社會支援系統：透過建立借款人間正式或非正式的社群網絡，可以幫助資訊的交流及人脈的建立，提高借款人成功的機會及貸款計畫影響持續的時間。