Com as descobertas das inúmeras aplicações potenciais da biomassa de microalgas é necessário o desenvolvimento de ferramentas que visem auxiliar o aumento da produtividade dos cultivos. A espectrometria por infravermelho com transformada de Fourier (FTIR) é uma técnica versátil e rápida utilizada na identificação, caracterização e quantificação de diversos compostos moleculares. Sua aplicação está definida de acordo com a região espectral a ser analisada. O objetivo deste trabalho é desenvolver uma metodologia que torne possível a utilização da espectroscopia FTIR na região do infravermelho médio (MIR) para quantificar os teores de lipídeos, proteínas, carboidratos e células de Scenedesmus sp. Para tanto, a biomassa de Scenedesmus sp. foi analisada diariamente por métodos tradicionais e através do espectro FTIR. Posteriormente os dados foram correlacionados com as bandas de absorção características de cada composto. O modelo gerado para lipídeos na região entre 3000-2800 cm-1 obteve o maior coeficiente de correlação (R2) de 0,8265. Para os carboidratos, a banda de absorção que melhor representou as ligações características foi entre 1200-950cm-1, com R2=0,8023. Já para proteínas, a escolha do método tradicional não mostrou boa relação com os resultados do FTIR. Quando a concentração celular foi correlacionada com a área total foi obtido R2=0,7900. Por fim, realizaram-se experimentos para validar os modelos preditos, obtendo bons resultados para a quantificação de lipídeos e carboidratos. O FTIR mostra ser uma ferramenta eficiente para estimar os conteúdos de lipídeos e carboidratos de Scenedesmus sp. Além disso, o FTIR permite análise simultânea de múltiplos metabolitos que permitirá o monitoramento mais detalhado do cultivo em um tempo de análise muito mais curto e com alta reprodutibilidade dos resultados. / With the discoveries of the numerous potential applications of microalgal biomass, it is necessary to develop tools to help increase the productivity of cultivation. Infrared spectrometry with Fourier transform (FTIR) is a versatile and fast technique used in the identification, characterization and quantification of several molecular compounds. Its application is defined according to the spectral region to be analyzed. The objective of this work is to develop a methodology that makes possible the use of FTIR spectroscopy in the medium infrared region (MIR) to quantify lipid, protein, carbohydrate and cells contents of Scenedesmus sp. For this, the Scenedesmus sp. biomass was analyzed daily by traditional methods and through the FTIR spectrum. Subsequently the data were correlated with the absorption bands characteristic of each compound. The model generated for lipids in the region between 3000-2800 cm-1 obtained the highest coefficient of correlation (R2) of 0.8265. For carbohydrates, the absorption band that best represented the characteristic bonds was between 1200-950cm-1, with R2=0.8023. For proteins, the choice of the traditional method did not show a good relation with the FTIR results. When the cell concentration was correlated whit the total area an R2=0.7900. Finally, experiments were carried out to validate the predicted models, obtaining good results for the quantification of lipids and carbohydrates. The FTIR shows to be an efficient tool to estimate lipid and carbohydrate contents of Scenedesmus sp. In addition, the FTIR allows simultaneous analysis of multiple metabolites that will enable more detailed monitoring of the cultivation in a much shorter analysis time and with high reproducibility of the results.

INTRODUÇÃO: A propensão do CaP em gerar metástases decorre de mecanismos moleculares específicos em um processo composto por múltiplas etapas, sendo que o remodelamento do meio extracelular através de ações de enzimas proteolíticas denominadas metaloproteinases da matriz (MMP) é uma etapa fundamental. As MMP degradam vários componentes da matriz extracelular, sendo que seu controle pode ser exercido por outras proteínas denominadas TIMPs. Em nível gênico, outro controle pode ser exercido por moléculas chamadas microRNAs. OBJETIVO: O objetivo deste estudo é avaliar a regulação da MMP-9 por miRNAs. A partir de dados da literatura identificamos que a MMP-9 pode sofrer influência do miR-21 e 338-3p. MÉTODOS: Para os experimentos in vitro, linhagens celulares de CaP (DU145, PC3 e LNCaP) foram transfectadas com os miRNAs de interesse e a expressão de MMP-9 foi avaliada por reação em cadeia de polimerase quantitativa com transcriptase reversa (qRT-PCR). O sobrenadante da transfecção foi usado para ensaios de invasão com matrigel, e ELISA. Nos experimentos in vivo, células da linhagem PC-3-luc foram implantadas no subcutâneo de camundongos Balb-c nude e tratadas com injeções de anti-miR-21, miR-338-3p ou a combinação de ambos. RESULTADOS: O miR-21 aumentou expressão de MMP-9 em 72% na PC3. Houve maior invasão celular tanto na PC3 como DU145. In vivo, o bloqueio do miR-21 reduziu em 10% a expressão de MMP-9 nos tumores implantados (p=0,04). O miR-338-3p reduziu a expressão de MMP-9 em 53% na PC3 (p=0,001), 31% na LnCaP (p=0,23) e 24% na DU145 (p=0,16). No ensaio de invasão, menor número de células e colônias foram capazes de invadir a membrana de matrigel. In vivo, houve redução de 27% na expressão de MMP-9 nos camundongos tratados com o miR-338-3p (p=0,07). A combinação anti-miR-21+miR-338-3p reduz a expressão de MMP-9 em maior intensidade tanto in vitro como in vivo. CONCLUSÕES: A expressão de MMP-9 pode ser regulada pelo miR-21 e miR-338-3p. O primeiro se comporta como um oncomiR ao passo que o segundo como um supressor tumoral. A combinação de miRNAs é uma estratégia plausível para ampliar o efeito sobre expressão de genes de interesse / INTRODUCTION: The propensity of CaP to generate metastases results from specific molecular mechanisms in a multiphase process and the remodeling of the extracellular medium through the actions of proteolytic enzymes called matrix metalloproteinases (MMP) is a fundamental step. MMPs degrade several components of the extracellular matrix, and their control can be exerted by other proteins called TIMPs. At the gene level, another control can be exerted by molecules called microRNAs. OBJECTIVE: The objective of this study is to evaluate the regulation of MMP-9 by miRNAs. From literature data we have identified that MMP-9 may be influenced by miR-21 and 338-3p. METHODS: For in vitro experiments, CaP cell lines (DU145, PC3 and LNCaP) were transfected with the miRNAs of interest and the expression of MMP-9 was assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The transfection supernatant was used for matrigel and ELISA invasion assays. For the in vivo experiments, PC3-luc cells were implanted into the subcutaneous Balb-c nude mice and treated with anti-miR-21, miR-338-3p injections or the combination of both. RESULTS: The miR-21 increased MMP-9 expression by 72% in PC3. There was greater cell invasion in both PC3 and DU145. In vivo, miR-21 blockade reduced MMP-9 expression by 10% in implanted tumors (p = 0.04). MiR-338-3p reduced MMP-9 expression by 53% in PC3 (p = 0.001), 31% in LNCaP (p = 0.23), and 24% in DU145 (p = 0.16). In the invasion assay, fewer cells and colonies were able to invade the matrigel membrane. In vivo, there was a 27% reduction in MMP-9 expression in mice treated with miR-338-3p (p = 0.07). The combination of anti-miR-21 + miR-338-3p reduces MMP-9 expression in greater intensity both in vitro and in vivo. CONCLUSIONS: MMP-9 expression can be regulated by miR-21 and miR-338-3p. The former behaves as an oncomyR while the second as a tumor suppressor. The combination of miRNAs is a plausible strategy to extend the effect on gene expression of interest

Mast cells are tissue-resident immune cells known as effector cells for the innate and adaptive immune systems. Mast cells contribute to host defenses against parasites such as large roundworm parasites, bacterial pathogens, and toxins, and participate in wound healing, but they are mostly known for their role in allergic diseases. It has been well established that during allergic diseases, mast cells are stimulated by IgE cross-linkage to release proinflammatory mediators. However, a newly discovered cytokine, IL-33 has also been implicated in allergic disease. Recently, IL-33 has been implicated as a driver of several Type I sensitivities and previous studies have shown that IL-33 can stimulate mast cells in atopic inflammation. Although the importance of IL-33 has been established, there are still several things unknown about IL-33 signaling regulation or treatment. This dissertation will present two separate studies involving the modulation of IL-33-mediated mast cells function In the first study, the effects of fluvastatin are explored. In a previous study, fluvastatin was shown to inhibit proinflammatory functions of IgE crosslinked mast cells. Contrasting to IgE stimulation, fluvastatin augments IL-6 and TNF production in IL-33 stimulated mast cells, but suppressed MCP-1. This phenomenon was seen in mouse and human mast cells in vitro and replicated in a mast cell-dependent murine model of IL-33-induced inflammation in vivo. In the second study, IL-33 was found to induce miR-146a expression in mouse mast cells and mast cell-derived exosomes in vitro, and in plasma exosomes in vivo. IL-33 induced miR-146a was of interest because miR-146a is a known negative regulator of TLR signaling, which shares the MyD88 signaling pathway with IL-33. We found that miR-146a KO mast cells are hyperresponsive to IL-33 stimulation, data that were replicated by suppressing miR-146a-5p in WT mast cells. In an acute mast cell repopulation model, kitW-sh/W-sh mice containing miR-146a KO BMMC had increased IL-33 induced neutrophilia in comparison to their controls. Collectively, these data reveal new IL-33 signaling pathways and means of altering its inflammatory effects on mast cells. Because IL-33 has important roles in allergy and other Th2-mediated diseases, these results advance clinically relevant areas of immunology.

MiR-17-92 is an oncogenic miRNA cluster implicated in the development of several human cancers; however, it remains unknown whether miR-17-92 cluster is able to regulate hepatobiliary carcinogenesis. This study was designed to investigate the biological functions and molecular mechanisms of miR-17-92 cluster in primary liver cancer.<br>In-situ hybridization and qRT-PCR analysis showed that miR-17-92 cluster is highly expressed in human cholangiocarcinoma cells compared to the non-neoplastic biliary epithelial cells. Forced overexpression of the miR-17-92 cluster or its members, miR-92a and miR-19a, in cultured human cholangiocarcinoma cells enhanced tumor cell proliferation, colony formation and invasiveness, in vitro. Overexpression of miR-17-92 cluster or miR-92a also enhanced cholangiocarcinoma growth in vivo in SCID hairless outbred mice. The tumor suppressor PTEN was identified as a bona fide target of both miR-92a and miR-19a in cholangiocarcinoma cells. Accordingly, overexpression of PTEN open reading frame protein (devoid of 3’UTR) prevented miR-92a- or miR-19a-induced cholangiocarcinoma cell growth. Microarray analysis revealed additional targets of miR-17-92 cluster in human cholangiocarcinoma cells, including APAF-1 and PRDM2. Moreover, we observed that the expression of miR-17-92 cluster is regulated by IL-6/Stat3, a key oncogenic signaling pathway pivotal in cholangiocarcinogenesis. Taken together, our findings in this study disclose a novel IL-6/Stat3 miR-17-92 cluster PTEN signaling axis that is crucial for cholangiocarcinogenesis and tumor progression.<br>We also found the miR-17-92 is highly expressed in tumor tissue compared to non-tumor adjacent tissue in hepatocellular carcinoma patient tissue. Forced overexpression of the miR-17-92 cluster in cultured human hepatocellular carcinoma cells enhanced tumor growth in vitro; on contrast, inhibition of miR-17-92 cluster inhibited cell growth. MiR-17-92 cluster promote diethylnitrosamine-induced hepatocarcinogenesis in liver-specific miR-17-92 cluster transgenic mice. Binding sequence and mice whole genome microarray analysis revealed about 300 possible targets. RNA-sequencing data analysis showed both individual miRNAs and the host gene of miR-17-92 cluster was highly expressed in hepatocellular carcinoma patients and had negative correlation with several genes (CREBL2, PRRG1, and NTN4), among which, CREBL2 may play an important role in the hepatocarcinogenesis. / acase@tulane.edu

MicroRNAs (miRNAs) are a group of small, noncoding RNAs that modulate the translation of genes into proteins by binding to specific target sites in messenger RNAs. This study investigated the biological function and molecular mechanism of microRNA-21 (miR-21) in human cholangiocarcinoma. In situ hybridization analysis of human cholangiocarcinoma tissues showed increased miR-21 in cholangiocarcinoma cells compared to the noncancerous biliary epithelial cells. Forced overexpression of miR-21 by lentivirus transduction enhanced human cholangiocarcinoma cell growth and clonogenic efficiency in vitro, whereas inhibition of miR-21 decreased these parameters. MiR-21 overexpression also promoted cholangiocarcinoma growth in a tumor xenograft model. The NAD+-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme that converts the pro-tumorigenic prostaglandin E2 (PGE2) to biologically inactive metabolite, was identified as a direct target of miR-21 in cholangiocarcinoma cells. In parallel, cyclooxygenase-2 (COX-2) overexpression and PGE2 treatment increased miR-21 expression and enhanced miR-21 promoter reporter activity in human cholangiocarcinoma cells. Our results disclose a novel cross-talk between COX-2/PGE2 and miR-21 signaling pathways that converges at 15-PGDH which is crucial in cholangiocarcinogenesis and tumor progression. The enhancer of zeste homolog 2 (EZH2) is the catalytic subunit in the PRC2 complex catalyzing the trimethylation of histone3 lysine27 (H3K27) and mediates gene silencing of the target genes. The biological function of EZH2 in cholangiocarcinoma was investigated in this study. Immunohistochemistry staining of EZH2 on human cholangiocarcinoma tissues showed increased EZH2 expression in cholangiocarcinoma cells. Pharmacologically inhibition of EZH2 by EZH2 inhibitors decreased cholangiocarcinoma growth and induced G1 arrest. The CD133, one of the putative cancer stem cell markers, was found to express in the cholangiocarcinoma cell lines we used. Inhibition of EZH2 decreased CD133+ population and the sphere forming ability of cancer cells. Our results indicate that EZH2 may represent a promising target for targeting the tumor-initiating cell population and future cholangiocarcinoma therapy. / acase@tulane.edu

xviii, 419 leaves / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1972)--University of Adelaide, Dept. of French Language and Literature, 1972

Notre étude a porté, d'une part sur l'effet du bicalutamide, un inhibiteur du récepteur aux androgènes, sur la réponse de trois lignées de cancer de prostate en association avec les rayonnements ionisants, d'autre part sur la recherche d'une corrélation entre l'expression des microARN miR-210 et miR-373 sur la tolérance à l'hypoxie et la réponse au rayonnement.Nous montrons que le bicalutamide induit un effet cytostatique et cytotoxique dans la lignée LNCaP, qui exprime le récepteur aux androgènes. Les lignées DU145 et PC3, qui n'expriment pas ou peu le récepteur, sont sensiblement plus résistantes mais sont cependant affectées par les concentrations élevées de bicalutamide. Cette sensibilité résiduelle suggère l'existence d'un mécanisme secondaire, indépendant de la voie de signalisation du récepteur aux androgènes. L'inhibition de la prolifération produite par le bicalutamide s'accompagne d'un blocage du cycle cellulaire en phase G1 avec une augmentation de l'expression de la protéine p27 et une diminution de l'expression de la protéine HER2. L'association concomitante au bicalutamide se traduit par un effet radioprotecteur dans la lignée LNCaP. Cette observation nous conduit à déconseiller l'association concomitante du bicalutamide avec la radiothérapie, notamment en cas d'irradiation hypofractionnée.Facteur bien connu de radiorésistance dans les tumeurs solides, l'hypoxie est associée à un mauvais pronostic dans les cancers de la prostate. Nos données montrent qu'en sus de l'induction de marqueurs classiques comme HIF-1α, CA9 et VEGF, l'hypoxie promeut l'expression du microARN miR-210, (mais non de miR-373) indépendamment de l'expression du récepteur aux androgènes. Les données suggèrent que miR-210, dont l'expression apparaît corrélée à la résistance à l'hypoxie, pourrait constituer un bon biomarqueur pronostique dans le cancer de la prostate. En revanche, l'inhibition de l'expression de miR-210 n'a aucun effet sur la radiosensibilité des cellules en condition d'hypoxie.

Le développement de sources lasers monomodes et accordables constitue un enjeu important dans plusieurs domaines en fort développement telle que la métrologie, les senseurs optiques, la spectroscopie, le traitement optique de l'information ou la médecine. Dans cette thèse nous faisons le point sur les limites des technologies actuelles, puis nous montrons comment les surpasser à travers la conception et l'étude physique de lasers à semiconducteur de haute cohérence émettant dans l'infrarouge. Nous nous intéressons particulièrement aux VECSEL dans le but d'obtenir un fonctionnement monofréquence accordables et robustes. Nous traitons de la conception et de la caractérisation de ces lasers, depuis l'optimisation du milieu à gain jusqu'à l'élaboration de prototypes laser fonctionnels. Nous effectuons ensuite une étude approfondie des propriétés physiques de la source. Les différents sujets abordés traitent de nombreux aspects de l'optoélectronique tels que la physique du solide, l'optique traditionnelle et quantique, la technologie des matériaux, la thermique, ect. Les travaux présentés ici ouvrent la voie à de nombreux développements liés aux VECSEL et à leurs applications.

<p>Proteins of the band 4.1 superfamily are characterized by their sequence similarity to the ERM proteins ezrin, radixin and moesin, which are involved in cell motility, adhesion of cells, and signal transduction events. Little is however known of the function of ERM proteins in the nervous system, though an essential role for radixin and moesin in neuronal growth cone motility has been suggested. </p><p> This thesis is focused on the cloning, functional characterization and description of the tissue distribution in rat brain of MIR, a novel member of the band 4.1 superfamily. </p><p> The cDNA of MIR encods a protein of 445 amino acids which is composed of an ERM-homology domain and a RING finger, separated by an interregion. To reveal the cellular function of MIR, PC12 cell lines overexpressing MIR was generated and observed to inhibit NGF stimulated neurite outgrowth. </p><p> To elucidate the signal transduction of MIR by which it exerts its physiological activity, the yeast two-hybrid system was employed to screen for proteins that interact with MIR. A number of interactors known to regulate the cytoskeleton was obtained - among them myosin regulatory light chain-B which controls the actomyosin complex - and a novel type 2 membrane protein denoted NSAP for its similarity to saposin A-D. Overexpressed NSAP induced neurite outgrowth in PC12 cells and enhanced cell adhesion in fibroblasts. </p><p> The tissue distribution of MIR in rat brain, as determined by immunohistochemistry studies, showed that MIR is localized especially to neurons in hippocampus and cerebellum. The chromosomal localization of the MIR gene was assessed to 6p22.3-23, a region lost in the 6p23 deletion syndrome.</p><p> These results suggests that MIR is expressed in neurons in discrete regions of rat brain where it may regulate neurite outgrowth by modulating the cytoskeleton.</p>

Systemic lupus erythematosus (SLE) is a chronic and complex autoimmune disorder characterized by a failure in the mechanism of self-tolerance and production of autoantibodies, potentially affecting any organ in the body. The genetic factors behind the disease have been extensively studied in the past years and to date a list of more than 30 loci have been associated with SLE. However, very little is known about the functional significance of the risk variants. In this thesis, we focused on the analysis of SLE-associated variants in three genes: interferon regulatory factor 5 (IRF5), CD226 and the microRNA 146a. In paper I, we analyzed four polymorphisms in the IRF5 gene in a large set of individuals from different populations. We replicated a strong association of a promoter indel in our meta-analysis, but expression analysis indicated that it is rather another variant, SNP rs10954213 in the poly(A) signal of the gene that is in fact the major contributor to the altered gene expression in leukocytes. In manuscript II, we further characterized the regulation of IRF5 expression, showing that this gene can be up-regulated by estrogen in PBMCs and monocytes, regardless of the genotype, which could to some extent, explain the sex-bias of SLE. In paper III, we investigated the association of CD226 with SLE and the potential functional effect of the associated variants. The genetic analysis showed an association of a three-SNP-haplotype located at the 3’UTR region of the gene. The risk haplotype correlated with lower CD226 protein expression on the surface of cytotoxic and helper T cells, as well as in NK T cells. Reporter assays pointed to rs727088 in the 3’UTR as the main responsible variant for altered gene expression. In paper IV, we described the association of a variant in microRNA miR-146a, involved in the interferon pathway, with SLE in Europeans, which could in addition be correlated with decreased expression of both mature and primary miR-146a in leukocytes. In summary, we have investigated the genetic association of three genes with SLE in a large cohort of individuals and identified variants responsible for functional alterations of these genes, providing further insight into the pathogenesis of SLE.