Aortic valve (AV) disease is a major cause of cardiovascular-linked deaths globally. In addition, AV disease is a strong risk factor for additional cardiovascular events; however, the mechanism by which it initiates and progresses is not well-understood. We hypothesize that low and oscillatory flow is present on the fibrosa side of the AV and stimulates ECs to differentially regulate microRNA (miRNA) and mRNAs and influence AV disease progression. This hypothesis was tested employing both in vitro and in vivo approaches, high throughput microarray and pathway analyses, as well as a variety of functional assays. First, we isolated and characterized side-dependent, human aortic valvular endothelial cells (HAVECs). We found that HAVECs express both endothelial cell markers (VE-Cadherin, vWF, and PECAM) as well as smooth muscle cell markers (SMA and basic calponin). Using microarray analysis on sheared, side-specific HAVECs, we identified side- and shear-induced changes in miRNA and mRNA expression profiles. More specifically, we identified over 1000 shear-responsive mRNAs which showed robust validation (93% of those tested). We then used Ingenuity Pathway Analysis to identify key miRNAs, including those with many relationships to other genes (for example, thrombospondin and I&B) and those that are members of over-represented pathways and processes (for example, sulfur metabolism). Furthermore, we validated five shear-sensitive miRNAs: miR-139-3p, miR-148a, miR-187, miR-192, and miR-486-5p and one side-dependent miRNA, miR-370. To prioritize these miRNAs, we performed in silico analysis to group these key miRNAs by cellular functions related to AV disease (including tissue remodeling, inflammation, and calcification). Next, to compare our in vitro HAVEC results in vivo, we developed a method to isolate endothelial-enriched, side-dependent total RNA and identify and validate side-dependent (fibrosa vs. ventricularis) miRNAs in porcine aortic valvular endothelium. From this analysis, we discovered and validated eight side-dependent miRNAs in porcine endothelial-enriched AV RNA, including one miRNA previously identified in vitro, miR-486-5p. Lastly, we determined the relationship between important miRNAs (specifically miR-187 and miR-486-5p) and AV disease by modulating levels of miRNAs and performing functional assays. Preliminary studies overexpressing miR-187 in HAVECs have shown a reduction in inflammatory state through monocyte adhesion (p<0.05). Further, miR-486-5p overexpression reveals an increase in migration (p<0.05) and a trend for a decrease in early apoptosis, linking miR-486-5p to tissue remodeling in the AV. Better understanding of AV biology and disease in terms of gene-regulation under different hemodynamic conditions will facilitate the design of a tissue-engineered valve and provide alternative treatment options.

Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 100 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 80-96).

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 19, 2009). Includes bibliographical references (p. 71-80).

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "July 1998." Typescript. Vita. Includes bibliographical references (leaves 215-225). Also available on the Internet.

Title from second page of PDF document. Includes bibliographical references (p. Xx-Xx).

Les ARN messagers (ARNm) fonctionnels sont produits au cours d'un mécanisme complexe qui allie la transcription, qui permet la synthèse d'un pré-ARNm, la maturation de ce transcrit et son export. De plus, ces différentes machineries vont devoir faire face à la structure compacte de la chromatine, nécessitant une activité de décondensation/recondensation de la chromatine qui est notamment régulée par les mécanismes épigénétiques. Un très grand nombre de facteurs sont donc requis pour la production des ARNm fonctionnels . Parmi ces facteurs, les protéines Spt6 et Iws1 sont impliquées dans le mécanisme général de la transcription, dans la modulation de la structure de la chromatine et la maturation et l'export des ARNm. Ces travaux de thèse ont permis de caractériser biochimiquement, structuralement et fonctionnellement ces deux protéines, leur complexe et leur interaction avec d'autres effecteurs de la transcription. Ces travaux ont notamment permis de comprendre en termes moléculaires et fonctionnels (i) comment Spt6 est recrutée par l'ARN polyméraseII au cours de la transcription et (ii) comment le complexe Spt6/Iws 1 est formé. Ils ont également permis d'identifier de nouveaux interactants potentiels de Spt6, et notamment le facteur d'élongation de la transcription TFIIS. Ces travaux ont ainsi permis de révéler le rôle essentiel et extrêmement complexe joué par Spt6 et Iws1 lors de la production d'un ARNm, mais également de permettre l'étude future de leur interaction avec d'autres facteurs transcriptionnels. / Production of functional messenger RNA (mRNA) requires a complex mechanism that couples transcription with maturation and export of the mRNA. In addition to this mechanism, chromatin needs to be unwound to allow the transcription machinery access the DNA, this unwinding being also highly regulated. Thus, production of a functional mRNA requires a huge number of factors implicated in these different processes. Among these proteins Spt6 and Iws1 are participating in the mechanism of transcription, chromatin unwinding, and maturation and export of the mRNA. The work carried out during this thesis has enabled the biochemical, structural and functional characterization of these proteins, their complex and their interaction with other effectors of transcription. This work has specifically enabled the molecular and functional characterization (i) of the recruitment of Spt6 by RNA polymerase II and (ii) of the formation of the Spt6/Iws1 complex. Moreover, this work has identified putative new partners of Spt6, not ably the elongation factor TFIIS. Thus, our work has highlighted the essential and complex role of Spt6 and Iws1 during the production of functional mRNA, and has also enabled future studies of the complexes formed by these two proteins with other transcriptional factors.

Na fase ativa da leishmaniose visceral (LV) ocorrem alterações no metabolismo de lipoproteínas com redução dos níveis de HDL e aumento de triglicérides. A partir desses dados, focamos neste projeto essas alterações na progressão da infecção e apontamos alguns elementos como seus possíveis desencadeantes. Como essas alterações poderiam resultar de redução de atividade e expressão da lipoproteína lipase (LPL), do receptor alfa do proliferador ativado de peroxissoma (PPAR?) e da proteína transferidora de ésteres de colesteril (CETP), a sua expressão foi avaliada durante a progressão da LV em hamster. Em hamsteres infectados com 2 x 107 amastigotas de L. (L.) infantum observamos aumento de triglicérides nos hamsteres com 55 dias (mediana = 294,0 mg/dL) e 90 dias (303,0 mg/dL ) de infecção comparados aos controles de 55 dias (119,0 mg/dL) e de 90 dias (117,0 mg/dL) (p <= 0,05). Os níveis de colesterol total e de HDL não apresentaram diferença significante entre controles e infectados com 30, 55 e 90 dias de infecção. A expressão de mRNA de PPAR? no fígado com 55 e 90 dias de infecção apresentou tendência de redução nos infectados. Já de CETP no fígado dos hamsteres com 55 dias de infecção, a expressão relativa (CT) estava reduzida nos infectados (0,08) comparados aos controles (1,69) (p <= 0,05) e de LPL no coração dos hamsteres com 90 dias de infecção também estava reduzida (1,43) com relação aos controles (2,61) (p <= 0,05). Há dados na literatura sugerindo a importância de lipídios para o desenvolvimento de amastigotas no hospedeiro vertebrado e é possível que as alterações dos níveis de lipoproteínas contribuam na progressão da infecção. Assim, avaliamos neste estudo o efeito da droga hipolipemiante ciprofibrato no controle do parasitismo na LV em hamster, sabendo-se que ciprofibratos atuam aumentando a expressão de PPAR? e a produção e atividade de LPL. O tratamento com ciprofibrato nos hamsteres com 55 dias de infecção gerou redução de triglicérides (123,0 mg/dL) em relação aos infectados não tratados (294,0 g/dL) (p <= 0,05), além dos níveis de triglicérides nos animais infectados não tratados terem aumentado quando comparados aos controles não tratados (119,0 mg/dL) (p <= 0,05). Houve também, redução de triglicérides nos animais não infectados tratados com ciprofibrato (89,0 mg/dL) comparando-se aos infectados não tratados (p <= 0,05). Os níveis de colesterol nos hamsteres não infectados tratados com ciprofibrato reduziram (53,5 mg/dL) em comparação aos infectados não tratados (93,0 mg/dL) (p <= 0,05). Já naqueles que foram infectados e tratados com ciprofibrato, constatamos redução de colesterol (53,5 mg/dL) quando comparados aos infectados não tratados (p <= 0,05). Os níveis de HDL não aumentaram com ciprofibrato e foram similares entre os hamsteres infectados não tratados e os controles não tratados. A carga parasitária no baço e no fígado não foi reduzida com ciprofibrato. Na leishmaniose visceral em hamster ocorrem alterações do metabolismo lipídico com aumento de triglicérides e redução da expressão da mRNA de LPL e CETP. O tratamento com ciprofibrato foi eficaz no controle das alterações de níveis de lipoproteínas. / In the active phase of visceral leishmaniasis (VL) changes occur in lipoprotein me-tabolism with reduction in HDL and increase in triglyceride (TG) levels. From these data, in this project we focused these changes during the progression of the infection and we approached some elements as their underlying factors. Since these changes may result from the reduction of the activity and the expression of the lipoprotein lipase (LPL), of the peroxisome proliferator-activated receptor alpha (PPAR?) and of the cholesteryl ester transfer protein (CETP), their expression were evaluated during VL progression in hamster. In 2 x 107 L. (L.) infantum amastigote-infected hamsters we observed an increase in the triglycerides in hamsters with 55 days (median = 294.0 mg/dL) and 90 days (303.0 mg/dL) of infection compared with controls of 55 days (119.0 mg/dL) and of 90 days (117.0 mg/dL) (p <= 0.05). The total cholesterol and the HDL levels did not present significant differences between control and in-fected groups at 30, 55 and 90 days of infection. The expression of mRNA of the PPAR in the liver with 55 and 90 days of infection tended to be reduced in infected animals. However the relative expression (CT) of CETP in the liver of hamsters with 55 days of infection was signicantly reduced in infected (0.08) compared with control animals (1.69) (p <= 0.05). The relative expression (CT) of LPL in the heart of hamsters with 90 days of infection was also reduced (1.43) in relation to controls (2.61) (p <= 0.05). There are data in the literature suggesting the importance of lipids for the development of amastigotes in vertebrate host and it is possible that the changes in the lipoprotein levels contribute for the infection progression. Therefore, we evaluated in this study the effect of the lipid-lowering drug ciprofibrate in the control of parasitism in VL in the hamster, knowing that ciprofibrate acts increasing the expression of the PPAR? and of the LPL production and activity. The treatment with ciprofibrate in infected hamsters at 55 days lead to the reduction of triglyceride level (123.0 mg/dL) in relation to non-treated infected animals (294.0 g/dL) (p <= 0.05). Further the triglyceride levels in the non-treated infected animals were in-creased when compared with untreated controls (119.0 mg/dL) (p <= 0.05). There was also reduction of triglyceride in ciprofibrate treated-non infected animals (89.0 mg/dL) compared with non-treated infected animals (p <= 0.05). The cholesterol lev-els were reduced in the ciprofibrate-treated non-infected hamsters (53.5 mg/dL) in comparison to the non-treated infected ones (93.0 mg/dL) (p <= 0.05). In the ciprofibrate-treated infected ones we found a reduction of cholesterol level (53.5 mg/dL) when compared with non treated infected animals (p <= 0.05). The HDL lev-els did not increase with ciprofibrate and they were similar between the non-treated infected hamsters and non-treated controls. The parasite load in the spleen and liver were not reduced with ciprofibrate. In the visceral leishmaniasis in hamster changes occur in the lipid metabolism with increase in the triglyceride level and the reduction of expression of mRNA of LPL and CETP. The treatment with ciprofibrate was ef-fective in the control of changes in the lipoprotein levels.

A resposta imune desencadeada em pacientes com leishmaniose tegumentar Americana (LTA) é predominantemente celular. Assim, o estudo dos mecanismos imunológicos responsáveis pela formação, progressão e cura desta infecção é de grande importância, considerando a deformação que ela pode causar em indivíduos infectados. Este estudo investigou a expressão de mRNA de IFN-?, IL-10, IL-27, TNF-?, TGF-? e IL-6 em 40 biopsias de parafina (FFPE) fixadas em formalina de pacientes com diagnóstico clínico e laboratorial para LTA. Um grupo de 10 biópsias FFPE coletadas de pacientes com diagnóstico clínico de leishmaniose mucosa (LM) foi utilizado para o controle da evolução da infecção. Inicialmente, foi padronizado a extração de RNA, a síntese de cDNA e a escolha do gene endógeno em 9 biopsias frescas e 8 FFPE. Após essas padronizações, as moléculas de RNA de fragmentos de biopsias parafinadas, foram extraídas com kit específico, tratadas com DNase, quantificadas por fluorimetria e sintetizadas para cDNA. A expressão relativa de cada citocina foi determinada (em duplicata) por PCR em tempo real. As reações foram normalizadas frente ao padrão de expressão do gene endógeno GAPDH, que foi previamente padronizado. O padrão de expressão de cada gene alvo foram determinados segundo a fórmula do \"CT comparativo\" (2-??CT), na qual calcula-se quantas vezes mais ocorre a expressão do gene alvo em relação a indivíduos normais. Também incluído nesse estudo 5 amostras de biopsia de pele de indivíduos normais (grupo IV). Os resultados foram expressos em média de quantificação relativa, a análise estatística foi realizada utilizando foi determinada por Teste t não pareado e teste F para comparação de variâncias, onde três grupos de pacientes foram formados. Grupo I, 35 pacientes com LTA, cuja expressão relativa de IFN-? foi inferior a 100. Grupo II, 5 pacientes com LTA cuja expressão relativa de IFN-? foi superior a 100. Grupo III, 10 pacientes com leishmaniose mucosa. Os níveis de expressão para IFN-? foram 33,34; 214,1 e 129,6 vezes maiores para os Grupos I, II e III, respectivamente do que as amostras do controle normal. Para o TNF-? foram 11,22; 2,37 e 4,59 vezes superiores ao normal nos Grupos I, II e III, respectivamente. Para IL-10 foram 5,14; 11,54 e 2,51 vezes superiores aos valores normais nos Grupos I, II e III, respectivamente. Para IL-27 foram 15,81; 85,29 e 17,29 vezes superiores ao normal nos Grupos I, II e III, respectivamente. Para o TGF-? foram 5,38; 4,33 e 4,72 vezes superiores ao normal nos Grupos I, II e III, respectivamente. Para a IL-6 foram 5,92; 5,69 e 2,09 vezes superiores aos valores normais nos Grupos I, II e III, respectivamente. Estes resultados foram semelhantes a resultados de estudos que analisam a expressão de citocinas já sintetizadas. No geral, os resultados de expressão gênica das citocinas estudadas sugerem que a exacerbação da resposta imune do perfil Th1 (IFN-?) poderia levar ao surgimento de lesões mais graves nos pacientes dos Grupos II e III. Os níveis elevados de mRNA de IL-10, observado nos pacientes do Grupo II podem promover baixa ativação macrofágica, colaborando com a replicação do parasita e assim culminar com a progressão da doença. A expressão de mRNA de IL-27 não interferiu na expressão de mRNA de IFN-? nas células dos pacientes com leishmaiose tegumentar ou leishmaniose mucocutânea. Em conclusão, estes resultados podem sugerir que os pacientes do Grupo II podem desenvolver formas mais graves de LTA quando comparados aos do Grupo I. / The immune response triggered in patients with American cutaneous leishmaniasis (ACL) is predominantly cellular. Thus, the study of the immunological mechanisms responsible for the formation, progression and cure of this infection is of great importance, considering the deformation that it can cause in infected individuals. This study investigated the expression of IFN-?, IL-10, IL-27, TNF-?, TGF-? and IL-6 mRNA in 40 formalin-fixed paraffin-embedded paraffin biopsies (FFPE) from patients with clinical and laboratory diagnosis for LTA. A group of 10 FFPE biopsies collected from patients with clinical diagnosis of mucosal leishmaniasis (MCL) was used to control the evolution of the infection. Initially, RNA extraction, cDNA synthesis and the choice of the endogenous gene were standardized in 9 fresh biopsies and 8 FFPE. After these standardizations, the RNA molecules of fragments of paraffin-shaped biopsies were extracted with a specific kit, treated with DNase, quantified by fluorimetry and synthesized for cDNA. The relative expression of each cytokine was determined (in duplicate) by real-time PCR. Reactions were normalized against the expression pattern of the endogenous GAPDH gene, which was previously standardized. The expression pattern results for each target gene were determined by the \"comparative CT\" (2-??CT) formula, in which it is calculated how many times more the expression of the target gene occurs in relation to normal individuals. Also included in this study 5 skin biopsy samples from normal subjects (group IV). The results were expressed as mean relative quantification, statistical analysis was performed using was determined by unpaired t test and F test for comparison of variances, where three groups of patients were formed. Group I, 35 patients with ACL, whose relative expression of IFN-? was less than 100. Group II, 5 patients with ACL whose relative expression of IFN-? was greater than 100. Group III, 10 patients with mucosal leishmaniasis. The expression levels for IFN-? were 33,34; 214,1 and 129,6 times higher for Groups I, II and III, respectively than the samples from the normal control. For TNF-? were 11,22; 2,37 and 4,59 times higher than normal in Groups I, II and III, respectively. For IL-10 were 5,14; 11,54 and 2,51 times higher than the normal values in Groups I, II and III, respectively. For IL-27, 15,81; 85,29 and 17,29 times higher than normal in Groups I, II and III, respectively. For TGF-? were 5,38; 4,33 and 4,72 times higher than normal in Groups I, II and III, respectively. For IL-6 were 5,92; 5,69 and 2,09 times higher than the normal values in Groups I, II and III, respectively. These results were similar to the results of studies that analyze the expression of cytokines already synthesized. In general, the gene expression results of the cytokines studied suggest that the exacerbation of the Th1 profile (IFN-?) immune response could lead to more serious lesions in patients in Groups II and III. Elevated levels of IL-10 mRNA observed in Group II patients may promote low macrophage activation, assisting in the replication of the parasite and thus culminating in the progression of the disease. Expression of IL-27 mRNA did not interfere in the expression of IFN-? mRNA in the cells of patients with tegumentary leishmaiosis or mucocutaneous leishmaniasis. In conclusion, these results may suggest that patients in Group II may develop more severe forms of ACL when compared to those in Group I.

Le 5-Fluorouracile (5-FU) est un anti-métabolite intensément utilisé dans les traitements chimio-thérapeutiques de nombreux cancers. Cependant, les mécanismes moléculaires de l'action de cet agent anti-cancer, son impact sur la biologie cellulaire et les processus de résistance restent encore largement à déterminer. Nous avons proposé que le 5-FU, en s'intégrant dans l'ARN induit une altération de la traduction. Dans cette étude, nous avons déterminé pour la lignée de cellules de cancer colorectal HCT116 la dose et le temps de traitement qui induit une modification du comportement cellulaire sans conduire à une mort cellulaire massive. Nous avons ensuite analysé le translatome de ces cellules traitées et celui de cellules non traitées. Pour cela, les fractions cytoplasmiques ont été purifiées et séparées sur gradients de saccharose pour séparer les ARNm qui sont associés aux polysomes de ceux associés aux monosomes et des ARN libres. L'analyse des modifications du translatome induites par le 5-FU montre que cette drogue est capable de stimuler la traduction d'un grand nombre d'ARNm qui codent pour des protéines possédant diverses fonctions. Cette activation traductionnelle est probablement médiée, au moins en partie, par une action du 5-FU sur les microARN. C'est ainsi que nous avons démontré que la stimulation de la traduction de l'ARNm du gène HIVEP2 est un mécanisme dépendant du microARN miR-155 / 5-Fluorouracil (5-FU) is an anti-metabolite intensely used in chemotherapeutic treatments in various cancers. The cellular and molecular mechanisms of action of this anti-cancer agent still remain to be determined. Because 5-FU is incorporated within all classes of RNA, knowledge of the different levels of gene expression regulation affected by 5-FU will help to decipher its mode of action. We hypothesized that the translational control is altered by 5-FU treatment as a consequence of disrupted RNA metabolism. In this study, the colorectal cancer cell line HCT116 has been treated or not by different doses of 5-FU for different periods of time to determine the time and dose window that induces modifications of cell behavior without leading to an extensive cell death. Translational reprogramming was then analyzed during this time and dose window. For this, cytoplasmic fractions were purified and separated through sucrose gradients to distinguish the actively translated mRNAs that are associated with polysomes from the inactive mRNAs associated with monosomes or free mRNAs. A microarray analysis was then performed to identify the mRNAs presented in monosome and polysome fractions with and without treatment. This polysome profiling approach reveals that 5-FU treatment did not turn-off the global translation efficiency, but rather modulates translation efficiency of specific mRNAs. Secondly, more than 640 mRNAs were found to be up-translated following 5-FU treatment. Finally, we have demonstrated that 5-FU induced up-regulation of HIVEP2 by a molecular mechanism involving an action of 5-FU on miR-155