Made available in DSpace on 2014-06-12T23:16:08Z (GMT). No. of bitstreams: 2 arquivo943_1.pdf: 1281013 bytes, checksum: ddc6b54734ee04c82c863ce15355d1d6 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O biodiesel, combustível originado de óleos vegetais ou gorduras animais, vem se consolidando como fonte de energia alternativa em substituição ao diesel mineral. Este trabalho reporta o desenvolvimento de métodos analíticos para monitorar a qualidade do biodiesel e de suas misturas com diesel mineral. As propriedades estudadas para o biodiesel puro (B100) foram estabilidade oxidativa, índice de acidez e teor de água. Para as misturas, as propriedades determinadas foram massa específica, temperaturas de destilação (T50% e T85%) e teor de enxofre. Em ambos os casos, foram desenvolvidos modelos de calibração multivariada, usando dados obtidos por espectroscopia na região do infravermelho (IR). As técnicas de regressão por mínimos quadrados parciais (PLS) e a regressão linear múltipla (MLR) foram usadas para a modelagem. No desenvolvimento dos modelos para o controle de qualidade do B100, três técnicas de seleção de variáveis foram avaliadas: o algoritmo das projeções sucessivas (APS) associada à MLR, a regressão por mínimos quadrados parciais por intervalos (iPLS) e o Jack Knifing (PLS). Os resultados obtidos para misturas mostraram que os modelos globais foram adequados para previsão das propriedades tanto das misturas diesel-biodiesel quanto para o diesel puro. Os modelos apresentaram boas correlações que variaram de 0,74 a 0,98, com erros médios quadráticos de previsão (RMSEP) menores que a reprodutibilidade dos métodos de referência, exceto para a massa específica no infravermelho médio (MIR). Em relação ao B100, correlações que variaram entre 0,83 a 0,99 foram obtidas para os modelos desenvolvidos. Os RMSEPs apresentaram-se menores que a reprodutibilidade dos métodos oficiais. De uma forma geral, a espectroscopia na região do infravermelho em ambas as regiões NIR e MIR, combinada com a calibração multivariada mostrou-se eficiente na previsão das propriedades, tanto da mistura diesel-biodiesel, quanto do B100. Foi proposto ainda, empregar um sistema de análise por injeção em fluxo (FIA), usando detecção espectrofotométrica, para determinar fósforo em B100 com amostras digeridas. Uma avaliação sobre os procedimentos de digestão de amostras, em bloco digestor e forno de micro-ondas com cavidade, foi realizada, sendo o procedimento usando bloco digestor escolhido para digerir as amostras que foram usadas no método do FIA-SD. O procedimento com bloco digestor foi escolhido por apresentar menor acidez (2,2 mol L-1) e carbono residual (0,16%) comparando-se ao procedimento usando micro-ondas com cavidade. Para Validar o método, testes de recuperação foram realizados e os resultados mostraram valores entre 98 a 114,8%. O Limite de detecção (LD) foi de 1,4 mg Kg-1 e o Limite de Quantificação (LQ) de 4,6 mg Kg-1. As concentrações de fósforo em biodiesel apresentaram-se semelhantes às encontradas empregando a Espectrometria de Emissão Óptica com Plasma Indutivamente Acoplado (ICP OES) usando amostras digeridas e também amostras dissolvidas em querosene. A espectroscopia IR e o sistema de análise em fluxo (FIA) acoplado a técnica UV-VIS apresentam-se vantajosos, quando comparados aos métodos oficiais, pois possuem custos reduzidos e trabalham com uma quantidade mínima de amostras. Além disso, a espectroscopia IR e o FIA são rápidos e geram pouco resíduo

This thesis explores the design and development of digitally extended, electro- acoustic (EA) pitched percussion instruments, and their use in novel, multi-media performance contexts. The proposed techniques address the lack of expressivity in existing EA pitched percussion systems. The research is interdisciplinary in na- ture, combining Computer Science and Music to form a type of musical human- computer interaction (HCI) in which novel playing techniques are integrated in perfor- mances. Supporting areas include Electrical Engineering- design of custom hardware circuits/DSP; and Mechanical Engineering- design/fabrication of new instruments. The contributions can be grouped into three major themes: 1) non-invasive gesture recognition using sensors and machine learning, 2) acoustically-excited physical mod- els, 3) timbre-recognition software used to trigger idiomatic acoustic actuation. In addition to pitched percussion, which is the main focus of the thesis, application of these ideas to other music contexts is also discussed. / Graduate

In order to effectively treat disorders whose pathology is marked by neovascularization, a better understanding of the pathways that mediate the processes involved in angiogenesis is needed. To this end we have identified two important pathways that regulate endothelial cell capillary morphogenesis, a key process in angiogenesis. We have identified the small GTPase RhoB as being induced by vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs). Depletion of RhoB inhibited endothelial cell VEGF - mediated migration, sprouting, and cord formation. Cells depleted of RhoB showed a marked increase in RhoA activation in response to VEGF. Defects in cord formation in RhoB - depleted cells could be partially restored through treatment with the Rho inhibitor C3 transferase or ROCK I/II inhibitors, indicating increased RhoA activity and enhanced downstream signaling from RhoA contribute to the phenotype of decreased cord formation observed in cells depleted of RhoB. Interestingly, we did not observe a significant change in RhoC activity in RhoB - depleted cells suggesting differential regulation of RhoA and RhoC by RhoB in HUVECs. We have also identified microRNA - 30b (miR - 30b) as being negatively regulated by VEGF and as being a negative regulator of HUVEC capillary morphogenesis. Overexpression of miR - 30b significantly reduced HUVEC cord formation in vitro, while inhibition of miR - 30b enhanced cord formation. Neither overexpression nor inhibition of miR - 30b affected migration or viability of endothelial cells. Interestingly, miR - 30b regulated the expression of TGFβ2 but not TGFβ1, with overexpression of miR - 30b inducing expression of TGFβ2 mRNA and protein, and inducing phosphorylaton of Smad2 , suggesting TGFβ2 produced in response to miR - 30b overexpression functions in an iii autocrine manner to stimulate HUVECs . MiR - 30b effects on TGFβ2 expression were found to be regulated to an extent by ATF2, as miR - 30b overexpressing cells exhibited increased levels of phosphorylated ATF2 , with depletion of ATF2 via siRNA resulting in inhibition of miR - 30b - induced TGFβ2 expression. Treatment of HUVECs with TGFβ2 inhibited cord formation, while TGFβ1 had no effect, indicating a major difference in how endothelial cells respond to these two related growth factors. Inhibition of TGFβ2 with a neutralizing antibody restored cord formation in miR - 30b overexpressing cells to levels similar to control cells, thus identifying TGFβ2 expression as contributing to the inhibitory effects of miR - 30b overexpression on capillary morphogenesis. Thus, we have identified two signaling pathways regulated by VEGF in HUVECs that further our understanding of the process of angiogenesis and may provide novel targets for therapeutic intervention into diseases involving angiogenesis.

L’hématopoïèse est un processus actif, ordonné, et hautement régulé faisant intervenir des étapes de prolifération, de différenciation et d’apoptose et permettant la production de toutes les cellules sanguines matures à partir d’un nombre restreint de cellules souches hématopoïétiques. La dérégulation des mécanismes intervenant dans l’hématopoïèse induit le développement d’hémopathies, notamment de leucémies. De nombreux facteurs de transcription et microARN (miARN) ont été identifiés en tant que des régulateurs essentiels à l’établissement des différents lignages hématopoïétiques. Mon travail de thèse a porté sur l’étude du rôle des miARN dans la régulation de la différenciation macrophagique humaine. Nous avons reproduit le processus de différenciation macrophagique in vitro à partir de monocytes issus du sang périphérique traités avec du CSF-1 (colony stimulating factor-1). Suite à l’analyse du profil d’expression des miARN au cours du processus de différenciation, notre projet s’est orienté sur l’étude de miR-142-3p dont le taux d’expression diminue le plus fortement au cours de cette différenciation. Nous avons montré que miR-142-3p forme une boucle d’auto-régulation négative avec EGR2 (early growth response 2), un facteur de transcription connu pour réguler positivement la différenciation macrophagique. Cette boucle est essentielle au bon déroulement de la différenciation. Par ailleurs, nous avons observé une altération de cette boucle de régulation dans les monocytes de patients atteints d’une LMMC (leucémie myélomonocytaire chronique) suggérant que ce mécanisme puisse être impliqué dans la leucémogenèse. Au cours de ce projet, nous avons également initié une étude in vivo via l’utilisation du modèle que représente le Poisson-Zèbre. L’hématopoïèse du Poisson-Zèbre est très similaire à celle des mammifères que ce soit au niveau des populations hématopoïétiques ou des mécanismes de régulation impliqués. L’inhibition de l’expression du miR-142a-3p, homologue du miR-142-3p humain, se traduit par une absence de monocytes et de macrophages au niveau de l’ICM (intermediate cell mass), organe primaire de l’hématopoïèse, ainsi que par une diminution de l’expression de la myéloperoxydase, marqueur des granulocytes chez le Poisson-Zèbre. Ainsi, miR-142-3p semble être un inducteur de la formation des granulocytes et monocytes. / Hematopoiesis is an active process, orderly and highly regulated, involving proliferation, differentiation and apoptosis steps, and allowing the production of mature blood cells from a restricted number of hematopoietic stem cells. Deregulation of mechanisms involved in hematopoiesis leads to the development of leukemias. Many transcription factors and microRNAs (miRNAs) have been identified as essential regulators in the establishment of different hematopoietic lineages. My thesis investigated the role of miRNAs in the regulation of human macrophage differentiation. We examined macrophage differentiation in vitro, from peripheral blood monocytes treated with CSF-1 (colony stimulating factor-1). After the analysis of miRNAs expression profile, our project has focused on the study of miR-142-3p whose expression levels decreased most strongly during macrophage differentiation. We showed that miR-142-3p involved in a negative feedback loop with EGR2 (early growth response 2), a transcription factor known to favor macrophage differentiation. This molecular circuitry is necessary for the normal processus of differentiation. Furthermore, we observed an alteration of this regulation circuitry in monocytes of CMML (chronic myelomonocytic leukemia) patients, suggesting that this mechanism may be involved in leukemogenesis. During this project, we also initiated a study in vivo through the use of the zebrafish model. Zebrafish hematopoiesis is very similar to that in mammals both at the level of hematopoietic populations or regulatory mechanisms involved. The inhibition of miR-142a-3p expression, homolog of the human miR-142-3p, gave rise to an absence of monocytes and macrophages in ICM (intermediate cell mass), the primary organ of hematopoiesis and a decreased expression of myeloperoxidase, a marker of granulocytes in the zebrafish. Thus, miR-142-3p appears to be an inducer of granulocytes and monocytes formation.

Endometrial cancer is the fourth most common cancer in women and the most common gynecological malignancy. While patient outcome has improved for the majority of cancers, the outlook for endometrial cancer has steadily decreased. In order to address this problem, we must better understand the different mechanisms involved in endometrial cancer development and progression. To this end, we quantified expression of 667 miRNAs in four endometrioid adenocarcinoma and four serous adenocarcinoma using Taqman Low Density Arrays (TLDAs). miR-888 was one of the most highly overexpressed miRNAs in both endometrial cancer subtypes. Analysis of miR-888 expression across multiple cancer types using the The Cancer Genome Atlas database revealed that miR-888 was selectively expressed in endometrial cancer, with a significant association to invasive and high grade tumors. In addition, miR-888 was most predominantly expressed in endometrial carcinosarcoma, a rare but deadly form of endometrial cancer. Therefore, we conclude that miR-888 expression marks an aggressive endometrial tumor phenotype. One of the top predicted targets of miR-888 by TargetScan is the progesterone receptor (PR). PR is a potent tumor suppressor of the endometrium whose expression is often lost in advanced endometrial cancers. We quantified PR mRNA expression in a panel of endometrial tumors and found a statistically significant, negative correlation between miR-888 and PR mRNA expression. Furthermore, overexpression of miR-888 in endometrial cancer cell lines was capable of decreasing PR at the protein level. To determine if miR-888 directly targets PR, we cloned each of the four miR-888 binding sites downstream of Renilla luciferase into the psiCHECK2 reporter vector. miR-888 overexpression was capable of decreasing luciferase activity for all four binding sites, with the second and third binding sites producing the most prominent results. Here we describe a novel mechanism by which miR-888 inhibits PR mRNA translation to negatively regulate PR expression in endometrial tumors. To determine the endogenous function of miR-888 in human cells, we quantified miR-888 in a panel of 21 normal human tissues. Interestingly, miR-888 was highly expressed in testes, with minimal or absence of expression in all other tissues investigated. The restricted expression pattern of miR-888 in testes and cancer suggested that miR-888 may qualify as a novel cancer-testis (CT) antigen. CT-antigens are a large class of genes that demonstrate selective expression normally in testes germ cells and abnormally in various types of cancer. Furthermore, CT-antigen genes are predominantly located on the X chromosome and are part of evolutionarily novel multicopy gene families. Indeed, miR-888 is part of a multicopy, primate-specific miRNA gene family located on the X-chromosome. Furthermore, miRNA in situ hybridization localized miR-888 expression to the early stages of spermatogenesis, as is often observed for CT antigens. Together, these data identify miR-888 as the first miRNA CT antigen and expand the CT antigen field to noncoding RNAs.

Background: Recent evidence suggests that the prostate cancer (PCa)-specific up-regulation of certain genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 could be associated with an aberrant expression of non-coding microRNAs (miRNA). Methods: In silico analyses were used to search for miRNAs being putative regulators of PCa-associated genes. The expression of nine selected miRNAs (hsa-miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660) as well as of the aforementioned PCa-associated genes was analyzed by quantitative PCR using 50 malignant (Tu) and matched non-malignant (Tf) tissue samples from prostatectomy specimens as well as 30 samples from patients with benign prostatic hyperplasia (BPH). Then, correlations between paired miRNA and target gene expression levels were analyzed. Furthermore, the effect of exogenously administered miR-26a on selected target genes was determined by quantitative PCR and Western Blot in various PCa cell lines. A luciferase reporter assay was used for target validation. Results: The expression of all selected miRNAs was decreased in PCa tissue samples compared to either control group (Tu vs Tf: -1.35 to -5.61-fold; Tu vs BPH: -1.17 to -5.49-fold). The down-regulation of most miRNAs inversely correlated with an up-regulation of their putative target genes with Spearman correlation coefficients ranging from -0.107 to -0.551. MiR-186 showed a significantly diminished expression in patients with non-organ confined PCa and initial metastases. Furthermore, over-expression of miR-26a reduced the mRNA and protein expression of its potential target gene AMACR in vitro. Using the luciferase reporter assay AMACR was validated as new target for miR-26a. Conclusions: The findings of this study indicate that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction.

Biological systems, their physical structure and their functions, are built, maintained, and controlled by the activity of enzymes. Understanding how enzymes contribute to the regulation of various pathways and processes allows us to gain a deeper understanding of the entirety of the biological system. As changes in enzyme activity are often essential for the pathogenesis of multiple and varied diseases, identifying these changes represents a crucial step to both understanding the disease and preventing its progression within the individual. Enzymes’ functional output can be controlled by numerous different mechanisms, including control of transcription and translation, subcellular localisation, co-factor interactions, or chemical modification to specific amino acids. Activity-based protein profiling allows the potential for activity of target enzymes to be measured, thereby gaining a more accurate representation of the functional state of the biological system. In this work, profiling differential enzyme activity allows the discovery of previously unknown links between metabolic regulatory enzymes and infection by the hepatitis C virus (HCV). The novel probe wortmannin-yne is described and is shown to be able to report on the activity multiple kinases, including MAPK1, whose activity is dysregulated during HCV replication. Novel probes designed to target a smaller selection of kinases, phosphatidylinositol kinases, are reported and are shown to be capable of measuring HCV-induced changes to not only kinase activity but also regulatory protein-protein interactions with the phosphoinositide kinases. Lastly, the role of microRNA-27b in the HCV-induced dysregulation of lipid metabolic enzymes is examined. Three novel targets of microRNA-27b are identified, and their dysregulation is shown to have an effect on the life cycle of HCV. Altogether, this work has developed new tools for the study of metabolic enzymes and identified new avenues of investigation into the dysregulation of lipid metabolism.

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Neutrophils, known primarily as key players in defense against invading pathogens, represent an essential component of both the innate and adaptive immunity. Continuous production of large quantities of neutrophils is ensured by a complex process termed granulopoiesis. In order to maintain a stable neutrophilic population, granulopoiesis requires to be tightly regulated. Moreover, impaired granulopoiesis may lead to aberrant bone marrow function and, ultimately, give rise to acute myeloid leukemia (AML). Despite decades of research, the mechanisms regulating granulopoiesis are still unclear. In particular, the CCAAT/enhancer binding protein (C/EBP) family of transcription factors plays a critical role in this process. C/EBPα acts as a master regulator of granulopoiesis mainly by orchestrating expression of its target genes, which will mediate granulocytic differentiation. Thus, characterization of novel C/EBPα target genes is critical for a better understanding of the molecular mechanisms that regulate granulopoiesis. Previously, we showed that another C/EBP member, CEBPG, is a direct target of C/EBPα. In the first part of the present work, we addressed the unknown role of C/EBPγ in granulopoiesis. We observed that Cebpg conditional knockout (KO) mice, which have the Cebpg gene ablated specifically...