Made available in DSpace on 2014-06-12T23:15:04Z (GMT). No. of bitstreams: 2 arquivo9183_1.pdf: 3734643 bytes, checksum: 84c2b156e03dc9e26311a297b5b3d774 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / Este trabalho tem duas partes, ambas envolvendo espectroscopia no infravermelho e quimiometria. Na primeira parte, foram obtidos modelos de calibração multivariada baseados em dados espectrais nas regiões NIR e MIR para prever as principais propriedades de gasolinas comercializadas na região Nordeste. Foram coletadas 160 amostras de gasolinas e os modelos de calibração foram construídos considerando-se dados espectrais da região NIR (em dois caminhos ópticos diferentes - 1 e 10 mm) e MIR, dois algoritmos de calibração (mínimos quadráticos parciais - PLS e regressão linear múltipla - MLR), e diferentes pré-processamentos (derivada, alisamento e seleção de variáveis com o Algoritmo Genético, AG, ou o Algoritmo de Projeções Sucessivas, APS). Analisando-se os erros médios quadráticos relativos de previsão (RMSEPR) para os vários modelos, observou-se que todas as propriedades envolvidas podem ser preditas de forma satisfatória a partir do espectro NIR na faixa 1600-2500 nm (caminho óptico de 1 mm), com calibração por MLR e seleção de variáveis pelo algoritmo genético, com qualquer dos pré-processamentos utilizados.A outra parte trata do problema de transferência de calibração. Propôs-se uma nova estratégia para a construção de modelos de calibração robustos em relação a diferenças entre dois equipamentos. O APS foi utilizado para selecionar variáveis de forma a minimizar o erro de previsão para o conjunto de teste do equipamento primário, mas também para um pequeno conjunto de amostras medidas no equipamento secundário (amostras de transferência). Dois conjuntos de dados foram empregados: espectros MIR de gasolinas C, para previsão da propriedade T90% (temperatura para 90% de amostra destilada); e espectros NIR de amostras de milho para previsão do teor de umidade. Os modelos MLR robustos assim obtidos foram comparados a modelos PLS, utilizando-se padronização direta em etapas (PDS) para corrigir os espectros do equipamento secundário. Os erros de predição no equipamento secundário para os modelos MLR robustos foram comparáveis aos dos modelos PLS-PDS e levemente inferiores aos erros do modelo APSV-MLR

Murine cytomegalovirus (MCMV) encodes a non-coding RNA, m169, that inhibits the cellular miRNA, miR-27. Previous studies have shown that the overexpression of miR-27 in vitro suppresses replication of MCMV and degradation of miR-27 by m169 is important for the viral replication during the lytic stage of infection in vivo. To understand why the virus specifically targets this cellular miRNA for degradation, this thesis focuses on identification of cellular target genes of miR-27 that are involved in viral growth in the lytic infection. Microarray analysis was conducted to globally examine cellular genes differentially expressed following miR-27 overexpression or repression during MCMV infection. Data obtained from the microarray analysis were analysed in order to select potential targets of miR-27 for functional screening. Functional screening involved siRNA knockdown of individual genes followed by infection with a GFP reporter virus (GFP-MCMV) to assess the effects on viral growth. Knockdown of 5 out of 55 genes (Rpl18a, Lyar, Itga5, Mapkapk3 and Pik3r1) led to a significant reduction in GFP expression. Based on luciferase reporter assays, Mapkapk3 was validated as a direct target of miR-27 with a seed site interaction in its 3’UTR. Mutation of this site in the mRNA was shown to eliminate miR-27-mediated repression. Analysis of MAPKAPK3 protein levels upon infection demonstrates that the protein levels are higher in cells infected with wild type MCMV versus the m169 deletion virus (MCMV Δm169). This is in line with the difference in miR-27 levels in the two infections showing a decrease of miR-27 in wild type MCMV and unaltered levels in MCMV Δm169 infection. Mapkapk3 is a direct downstream target of p38 mitogen-activated protein (MAP) kinase within the p38 MAP kinase pathway, which has previously been shown to be an essential pathway for CMV replication. Expression levels of substrates of MAPKAPK3 including HSP27 and ATF1 were examined during infection to evaluate whether they are regulated by miR-27. The level of phosphorylation of HSP27 was shown to correlate with the levels of MAPKAPK3 during infection and was higher in cells infected with wild type MCMV versus MCMV Δm169. This suggests that MAPKAPK3 and its substrate, HSP27, are regulated by miR-27 during MCMV infection. This work provides an important foundation for further functional studies on the role of Mapkapk3 and its substrates in MCMV infection and its capacity to be dynamically regulated by miR-27. Based on the microarray analysis upon miR-27 overexpression, it was shown that miR-27 has an impact on the cell cycle, consistent with previous studies. Functional analysis of miR-27 in the cell cycle using miR-27 mimics and inhibitors demonstrated that the mimics cause an increase of cells in S phase at early time points (12 and 14 h), whereas the inhibition of miR-27 results in a significant reduction in the S phase population and accumulation of cells in G1 phase. Luciferase reporter assays confirmed that two genes known to be associated with the cell cycle are direct targets of miR-27: polycomb ring finger oncogene 1 (Bmi1) and caveolin 1 (Cav1). Knockdown of Bmi1 and Cav1 leads to a significant decrease in the number of cells in S phase and accumulation of cells in the G1 phase; however, this is the opposite result to that observed with the miR-27 mimics. These results suggest that the increase in cells in the S phase induced by miR-27 mimics is unlikely to be associated with targeting of Bmi1 and Cav1. Furthermore, knockdown of Bmi1 and Cav1 does not affect viral replication in vitro. Since miR-27 induces the transition of cells from the G1 to S phase, further studies are required to identify the miR-27 targets involved in this function. To identify direct targets of miR-27 through biochemical methods, one chapter of this thesis was devoted to developing CLASH datasets (cross-linking, ligation and sequencing of hybrid). This technique can directly map miRNA-mRNA interactions within the Argonaute protein (AGO). Initially, a NIH 3T3 stable cell line expressing AGO2 with a double affinity tag at the N terminus was generated. Analysis of the stable cell line revealed no significant alteration of miR-27 regulation or change in permissiveness to MCMV compared to wild type cells, making this amenable to further studies. Using the stable cell line, the CLASH protocol was carried out and preliminary data collected. In summary, this thesis identifies a direct target of miR-27, Mapkapk3, that is an important gene in MCMV replication that requires further investigation. Mapkapk3 is a substrate of p38 in the p38 MAP kinase pathway which is a signal transduction mediating numerous biological processes in response to cellular stresses including CMV infection. Furthermore, miR-27 overexpression was found to stimulate the G1/S transition of the cell cycle, and miR-27 inhibition had the opposite effect. Previous evidence has shown that MCMV and HCMV arrest the cell cycle in the G1 phase and inhibit host DNA synthesis to create an optimal condition for viral gene expression and DNA replication. Given that MCMV arrests host cells in the G1 phase, it is possible that degradation of miR-27 by MCMV contributes to this effect. Since miR-27 regulates both Mapkapk3 and the cell cycle, it seems likely that a number of targets and pathways underlie the antiviral properties of this miRNA.

The incidence of melanoma and non‐melanoma skin cancer is continuing to increase worldwide. Melanoma is the sixth most common cancer in the United States, making skin cancer a significant public health issue. Photo chemoprevention with natural products is an effective strategy for the control of cutaneous neoplastic. Polyphenols from fruits have been shown to protect the skin from the adverse effects of solar UVR, cancer, and the growth of cancer stem cells. In particular, blueberries are known for their high concentration of phenolic compounds that have the high antioxidant capacity, and their effectiveness in reducing UV damage and, therefore, skin cancer. In Matar's lab, we have shown that Polyphenol-Enriched Blueberry Preparation (PEBP), derived from biotransformation of blueberry juice through fermentation, is effective for targeting skin cancer stem cell proliferation in different skin cancer cell lines. We predicted that PEBP affects melanoma skin cancer stem cells (MCSCs) epigenetically by targeting miRNA pathways. We observed the effects of PEBP on sphere growth and cell motility in vitro. We performed RT2-qPCR analyses to determine PEBP influence on miRNA in B16F10 spheres. We transfected B16F10 cells with miR-200b and performed western blotting analyses. Our results demonstrated that PEBP reduced sphere growth and cell migration, and up regulated miR-200b expression in different biological settings. Inhibition of miR-200b increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression. Consequently, PEBP may influence MCSCs through miRNA pathways. Elucidating the mechanisms by which PEBP modulates CSCs biological behavior by controlling miRNAs will enhance our understanding of the molecular mechanisms in skin cancer chemoprevention and might result in their use as natural photo-protectants in skin cancer.

A key problem in multiple sclerosis (MS) is the diminished capacity for myelin repair. Although oligodendrocyte (OL) precursors can be seen at the lesion site, their ability to differentiate appears inhibited. MicroRNAs are key regulators of OL differentiation, and have been observed to be misregulated in MS lesions compared to healthy white matter. Thus, aberrant microRNA expression in MS lesions may disrupt the ability of incoming oligodendrocyte progenitor cells (OPC s) to differentiate. Specifically, a microRNA known as miR - 145 is downregulated as OPCs progress to OLs, but is found at unusually high levels in MS lesions. In this study, we investigated how misregulation of miR - 145 affects OL differentiation in vitro. Bioinformatic analysis revealed that putative targets of miR - 145 are significantly enriched for factors which promote actin cytoskeleton organization and myelination. An immortalized OL cell line was transduced with an inducible lentivirus to create stable lines that overexpress miR - 145. These stable lines were characterized while proliferating, early in differentiation and late in differentiation. Immunofluorescence was used to quantify changes in proliferation rate, apoptosis, branching ability and myelin gene expression. qPCR arrays were used to quantify changes in microRNA target expression levels between induced and uninduced cells. Two stable lines were created: ON - 145 - 1 and ON - 145 - 2, which upon induction, over - express miR - 145 ~33 - fold and ~11 - fold, respectively. When proliferating, no significant morphological differences nor target expression differences could be detected between induced and uninduced cells. Proliferation was significantly decreased in ON - 145 - 1 induced cells, but not in ON - 145 - 2. No changes in apoptosis frequency were detected. In contrast, during early and late differentiation, both induced cell lines showed significant morphological defects characterized by a reduction in both iii primary and secondary branching. Further, significant differences in branching ability were observed between induced cells of ON - 145 - 1 and ON - 145 - 2, suggesting a dose - dependent response to miR - 145 overexpression. Expression of MAG, a myelin marker, was also significantly lowered in induced cells of both cell lines. Finally, we found that multiple miR - 145 targets involved in promoting cytoskeletal organization and myelination were significantly decreased both early and late in differentiation. These results suggest that overexpression of miR - 145 during OL differentiation may disrupt actin organization and myelin gene expression required for successful process extension and subsequent myelinating ability. Thus, the increase in miR - 145 in MS lesions may be a significant contributing factor to the loss of myelin repair in MS lesions.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18856号 / 医博第3967号 / 新制||医||1007(附属図書館) / 31807 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 小川 誠司, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19271号 / 医博第4035号 / 新制||医||1011(附属図書館) / 32273 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 松田 秀一, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21695号 / 医科博第99号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 Shohab YOUSSEFIAN, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

miRNAs are important post-transcriptional regulators of gene expression which play vital roles in the arrays of physiological processes, including skin and hair follicle (HF) development. In this study, the role for miR-214 in the skin and HF development and their postnatal physiological regeneration was investigated. miR-214 exhibits discrete expression patterns in the epidermis and HF in developing and postnatal skin, and is highly expressed in the epithelial stem cells and their lineage-committed progenies. The effects of miR-214 on HF morphogenesis and cycle progression were evaluated by using doxycyclineinducible miR-214 transgenic mice (K14-rtTA/TRE-miR-214). Keratinocyte specific miR-214 overexpression during skin embryogenesis resulted in the partial inhibition of HF induction and formation of the HF reduced in size producing thinner hair. Overexpression of miR-214 in telogen skin caused retardation of the anagen progression and HF growth. Inhibitory effects of miR- 214 on HF development and cycling were associated with supressed activity of stem cells, reduced proliferation in the hair matrix, and altered differentiation. miR-214 induced complex changes in gene expression programs in keratinocytes, including inhibition of cyclins and cyclin-dependent kinases and several essential components of Wnt, Edar, Shh and Bmp signalling pathways, whereas 􀁅-catenin acts as a novel conserved miR-214 target. Indeed, the inhibitory effects of miR-214 on HF development were rescued by intracutaneous delivery of pharmacological Wnt activator. Thus, this study demonstrated that by targeting 􀁅-catenin and, therefore, interfering with Wnt signalling activity miR-214 may act as one of the upstream effectors of the signalling cascades which govern HF morphogenesis and cycling.

No description available.