The microtubule-associated protein tau forms pathological neuronal filaments in Alzheimer's disease (AD) and other neurodegenerative disorders, known collectively as tauopathies. Previous studies in transgenic mouse models of AD suggest that reducing tau expression may be safe and beneficial for the prevention or treatment of AD and possibly other tauopathies. As a first step toward identifying novel therapeutic strategies to reduce tau levels, the studies presented in this dissertation aim to investigate the role of the human tau 3'-untranslated region (3'-UTR) in regulating tau expression. Tau expresses two 3'-UTR isoforms, long and short, as a result of alternative polyadenylation. The exact sequence of these two 3'-UTR isoforms was determined by rapid amplification of cDNA 3'-ends (3'-RACE), and the two 3'-UTR isoforms were cloned into a luciferase reporter vector. Using these reporter constructs, the expression of these isoforms was found to be differentially controlled in human neuroblastoma cell lines M17D and SH-SY5Y by luciferase assays and quantitative PCR (qPCR). Through an unbiased screen of tau 3'-UTR deletions and fragments using luciferase reporter constructs, several regions in the long tau 3'-UTR isoform that contain regulatory cis-elements were identified. Additionally, several microRNAs were computationally identified as candidates that might bind the long tau 3'-UTR and thereby differentially control the expression of long versus short tau 3'-UTR isoforms. Screening these candidate microRNAs via luciferase reporter assay identified miR-34a, which was subsequently shown to repress the expression of endogenous tau protein and mRNA in M17D cells using Western blot and qPCR, respectively. Conversely, inhibition of endogenously expressed miR-34 family members leads to increased endogenous tau expression. Taken together, these studies suggest that the expression of the two tau 3'-UTR isoforms is differentially regulated and that this differential regulation is due to the presence of regulatory cis-elements found only in the long tau 3'-UTR isoform, including a binding site for miR-34 family members. Improved understanding of the regulation of tau expression by its 3'-UTR may ultimately lead to the development of novel therapeutic strategies for the treatment of Alzheimer's disease and other tauopathies.

In Latin America, a social revolution is statistically far more likely to fail than to succeed. Yet there is little understanding as to the contributory factors of revolutionary failure or success. Many researchers look for commonalities by examining multiple revolutions across the region or even around the globe and throughout large periods of time, but their analysis frequently lacks commonality in the underlying conditions of the insurgencies. The case of Peru, however, provides a unique opportunity to examine multiple revolutions in the fairly homogenous environment of one state during a short and constrained timeframe of thirty years. In the history of the Republic of Peru, there have been only four social revolutions. These insurgencies were contained within two discreet periods of time: the MIR and ELN in the 1960’s, and Shining Path and MRTA in the 1980’s to 1990’s. While each of these revolutions experienced varying levels of success, each ultimately failed due, in no small part, to a particular set of structural and socioeconomic variables. / text

Mitad Argentina, mitad la republica de Bangkok, de esa mezcla radial entre realidad y la ficción Lalo Mir y compañía gestaron uno de los programas radiales humorísticos más innovadores de la historia de la radio. Corrían los tiempos de la primavera democrática en la Argentina, Daniel Grinbank dirigía la FM Rock & Pop y confió en Lalo para la conducción de un simple programa de discos. Era el furor de pasar música las 24hs del día, un concepto original en los tiempos de la primavera democrática Argentina. Pero la palabra que representaba “la voz de la calle” le ganaría la pulseada a la música, a la seriedad vacía de contenido, a un modo de hacer que estaba contra las cuerdas. Un Renault 12 convertido de manera provisoria en hogar para un Lalo Mir separado, fue el ambiente móvil donde junto a Quique Prossen y Bobby Flores y luego Douglas Vinci, sus compañeros de ruta. Ellos le dieron forma a unas palabras que se asomaron por el eter radial el jueves 23 de Abril de 1987: “Aquí Radio Bangkok”. En el programa “Radio Bangkok” trabaja un ya genial conductor y editor de radio, Lalo Mir, dos musicalizadotes que eran Boby Flores y Enrique (Quique) Prosen, y un creativo, un artista plástico, Douglas Vinci que se integra en tercer término. Tanto Boby como Quique pasaron al aire con Lalo luego de unas semanas con un solo conductor. El programa pasaba a tener tres voces y luego se sumaría Douglas. Era un programa donde las voces dejaban de ser meticulosas y cuidadas como lo venían siendo hasta entonces. El ciclo se identificaba con la trasgresión. Era una época de cambios vertiginosos. Los cuatro eran jóvenes que venían de sufrir una etapa oscura y tenían algo que decir, a su modo, con un lenguaje urbano, como en la casa, adaptado a la radio, con un mensaje cifrado en el código juvenil de trato informal, en confianza, tratando los temas que tocaban os grupos de amigos. Rock & Pop puso en evidencia la fuerte presencia de los jóvenes en la radio, con un humor surrealista destinado a un tipo de joven de clase media ilustrada, porque había un código cómplice que entender y era complejo. Después fue bajando el nivel social y el mensaje se hizo más abierto. El otro tipo de público se sentía identificado dentro de diferentes radios que apostaban a pocas palabras y a una música un tanto más comercial. Bangkok era trasgresor en tanto producía un discurso propio volcado al humor y la ironía, explotando los recursos de la radio con efectos, jingles, producciones artísticas creativas con la tecnología del momento. El lenguaje era trasgresor para lo concebido como forma de hablar al aire. Se expresaban como la gente, hasta incluso si era necesario se escuchaban algunas “puteadas”. La acidez de Lalo Mir se activaba frente al micrófono cada vez que su voz se detiene en observaciones irónicas y absurdas, de lo que pasaba en los noticieros. La provocación que hizo de Bangkok el primer quiebre en el estilo de conducción por frecuencia modulada e inspiró a una nueva generación de conductores, cuyo referente más visible es hoy Mario Pergolini. Lalo Mir, "Ese programa partía siempre del diario, pero visto desde Bangkok, y tomando esa realidad con el absurdo y la ridiculez más grandes." Así resume Mir un estilo que consistía en parodiar las noticias del día, vistas desde la capital de Tailandia. Radio Bangkok lo hacíamos cuatro pibes que éramos de la calle. No estábamos bien organizados para sostener algo “serio”, pero con Lalo, Quique y Douglas teníamos años de radio encima y dentro de ese caos sabíamos perfectamente lo que hacíamos. A favor no teníamos nada: ni plata, ni influencias, ni buena imagen, pero compartíamos el gusto con la gente. Nadie se bancaba más la radio que había en los 80s, era como un boxeador cuando lo ves que tuvo su momento de gloria y ya no gana hace 2 años, la estructura radial era esa y justo aparecimos nosotros. En esa época en la AM habían pesos pesados como Héctor Larrea y Antonio Carrizo, pero cuando arrancamos en FM a la mañana no había nada, era como hoy tener un programa en Internet. No había un plan pensado sobre como lograr un gran éxito, todos teníamos un laburo más importante y nos divertíamos haciendo Radio Bangkok. “Bangkok era genial que estuviera, no éramos genios. Escuchabas temas que la AM no permitía, pasábamos música como The Cult, The Doors y las cosas se decían como la hablábamos nosotros, con el “lenguaje” de la calle. Nosotros habíamos sufrido una dictadura militar donde tenías que comportarte de determinada manera para que no te jodieran, porque si no directamente te mataban, eso generó gente como nosotros: una generación golpeada. Los realizadores de Radio Bangkok ven a la distancia que era una época muy difícil para preproducir, Douglas Vinci recuerda que: “era otro mundo, otro acceso a la música, no había información, no conocíamos la cara de Eric Clapton, no sabíamos si Bob Marley era negro o blanco, hoy con Internet te parece increíble pero era así”. No sé cómo se dio, pero hubo un momento en el qué explotó todo y nosotros ya estábamos adentro. Nunca supimos por donde empezó esa explosión pero sí vivimos los efectos, venía la gente de la tele a hacernos notas, las modelos a sacarse fotos, una verdadera locura. Hay que tener en cuenta que fuimos la primer radio generada en democracia, en ese momento Rock & Pop era lo que la gente quería, alguien tenía que rehacer la radio. Éramos uno de los emergentes de una sociedad callada que catapultó a personajes como Jorge Lanata, Ricardo Piglia, Rodrigo Fresán, Los Redondos y muchos más que hoy son referentes. / Tesis de producción sonora acompañada de una carpeta que complementa el trabajo con una indagación sobre los consumos culturales de los años 80.

Cervical cancer is the third most common cancer among women worldwide, and the fourth leading cause of cancer mortality. Despite significant declines in the incidence and mortality rates of cervical cancer in Canada, it remains the 4th most common cancer in women aged 20-29 years. In order to gain novel insights into cervical cancer tumourigenesis and clinical outcome, we investigated and characterized the alterations in microRNA (miRNA) expression in this disease. Firstly, we performed global miRNA expression profiling of cervical cancer cell lines (n=3), and patient specimens (n=79). From this analysis, we identified miR-196b to be significantly down-regulated in cervical cancer, and characterized its role in regulating the HOXB7~VEGF axis. The global miRNA expression data also led to the development of a candidate 9-miRNA signature that was prognostic for disease-free survival in patients with cervical cancer, although we were unable to validate this signature in an independent cohort. This report describes important considerations concerning the development and validation of microRNA signatures for cervical cancer. Our investigations also led us to a comparison of three methods for measuring miRNA abundance: the TaqMan Low Density Array, the NanoString nCounter assay, and single-well quantitative real-time PCR. Our findings demonstrated limited concordance between the TLDA and NanoString platforms, although each platform correlated well with PCR, which is considered the gold standard for nucleic acid quantification. Furthermore, we examined biases created by amplification protocols for microarray studies. Our analysis demonstrated that performing a correction using the LTR-method (linear transformation of replicates) could help mitigate, but not completely eliminate such biases. Overall, this report presents insights into the role of miRNAs in cervical cancer, as well as an evaluation of technical considerations concerning miRNA and mRNA expression profiling studies.

Implanted biomaterials often induce inflammation that frequently leads to the foreign body response, fibrosis, and the failure of the implant. Thus, it is important to evaluate how cells interact with materials to promote a more regenerative response. It is critical to determine how to modulate the response of tissue resident innate immune cells, as they are among the first cells to interact with implanted materials. Among tissue resident innate immune cells are mast cells, which are inflammatory sentinels that degranulate and orchestrate the fate of other cell populations, such as monocytes/macrophages and lymphocytes. Mast cells have also been reported to play a vital role in the foreign body response of implanted biomaterials as well as angiogenesis. The goal of this study was to determine how to modulate mast cell responses to electrospun scaffolds by altering scaffold architecture and composition to promote anti-inflammatory and regenerative cell-scaffold interactions. Scaffold architecture was manipulated by changing either fiber diameter or pore diameter and mast cell responses were mediated by endogenous and exogenous DAMPs (i.e. IL-33 and LPS, respectively). Particularly in response to IL-33, scaffolds with increased fiber and pore diameter promoted less inflammatory cytokine and chemokine release while increasing angiogenic cytokine release. Additionally, taking scaffolds that promoted increased inflammatory cytokine expression and increasing the pore diameter alone dampened inflammatory cytokine expression. The next question we wanted to answer was how might the degradative byproducts of scaffolds alter mast cell inflammatory responses. Given the widespread use of polylactic acid, we decided to investigate this question using lactic acid as a degradative byproduct. In the presence of physiologically relevant levels of lactic acid, IL-33- and IgE-mediated inflammatory cytokines and chemokines are suppressed, while angiogenic cytokines are enhanced. This response was shown to be pH- and MCT1-dependent and was recapitulated in primary human skin mast cells as well as in vivo. In summary, scaffold architecture and the presence of select polymer degradative byproducts have the potential of selectively suppressing inflammatory cytokines and enhancing angiogenic cytokines.

Les microARNs sont des petits ARNs non codants d'environ 22 nucléotides qui régulent négativement la traduction de l'ARN messager cible (ARNm) et ont donc des fonctions cellulaires. Le microARN-16 (miR-16) est connu pour ses effets antiprolifératifs. Nous avons observé que l’expression de miR-16 est diminuée dans les cellules endothéliales humaines sénescentes et quiescentes en comparaison à des cellules prolifératives. Une analyse informatique des sites potentiels de liaison de miR-16 prévoit que GLUT-4, un transporteur du glucose insulinodépendant, pourrait être une cible potentielle du miR-16. Nous avons donc testé l'hypothèse que miR-16 régule négativement le métabolisme du glucose cellulaire. Dans des HUVEC, l'inhibition de miR-16 endogène avec des anti-miRNA oligonucléotides (AMO) augmente les niveaux protéiques de GLUT-4 de 1,7 ± 0,4 fois (p=0,0037 ; n=9). Dans des souris nourries avec un régime alimentaire normal ou riche en graisse et en sucre, l’expression de GLUT-4 dans le muscle squelettique a tendance à corréler négativement avec les niveaux de miR-16 (p=0,0998, r2=0,3866, n=4). Ces résultats suggèrent que miR-16 est un régulateur négatif de GLUT-4 et qu’il pourrait être impliqué dans la régulation du métabolisme cellulaire du glucose. / MicroRNAs are small noncoding RNAs of approximately 22 nucleotides that negatively regulate translation of the target messenger RNA (mRNA) and therefore have cellular functions. MicroRNA-16 (miR-16) is known to display anti-proliferative effects. We observed that miR-16 was down-regulated in non-proliferative human senescent endothelial cells. Computational analysis of the potential binding sites of miR-16 predicted that GLUT-4, an insulin-dependent glucose transporter, is a potential target of miR- 16. We therefore tested the hypothesis that miR-16 down-regulates cellular glucose metabolism. In HUVEC, inhibition of using anti-miRNA oligonucleotides (AMO) endogenous miR-16 up-regulated GLUT-4 protein levels 1,7 ± 0,39 folds (p=0,0037; n=9). In mice fed a regular or high fat diet, skeletal muscle expression of GLUT-4 tended to negatively correlate with miR- 16 levels (p=0,0998, r2=0,3866, n=4). These results suggest that miR-16 is a negative regulator of GLUT-4 and may be involved in the regulation of cellular glucose metabolism.

Prostate cancer is the most common cancer affecting men today. Therefore, there is a strong need for accurate biomarkers and successful therapeutic treatments. A novel approach combining a computationally built protein-protein interaction network of proven microRNA protein targets with high throughput proteomics identified ErbB2 and ErbB3 as key proteins in prostate cancer. These results coupled with microRNA array screening of an androgen-independent prostate cancer progression model, substantiated by single microRNA analysis, suggested miR125b as a key tumor suppressor contributing to prostate cancer progression. miR125b expression was shown to be substantially increased in the non-tumorigenic P69 cell line compared to its highly tumorigenic, metastatic M12 variant. Luciferase reporter gene assays including the entire 3’UTR of either ErbB2 or ErbB3 revealed a 2.8- and 2.4-fold decrease (respectively) compared to control vector. Thus, this combinatorial approach has suggested an additional microRNA and its target involved in prostate tumor progression.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Colon and breast cancers are amongst the leading causes of cancer deaths in the United States, mostly attributed to metastasis and resistance to therapy. Hence, there is a critical need to identify novel biomarkers for effective prognosis and to design targeted therapies to combat the metastatic diseases. Loss of heterozygosity (LOH) at chromosome 18q and inactivation of the target gene, SMAD4, corresponds to resistance to the common chemotherapeutic agent, 5-fluorouracil (5-FU), in colon cancer. Our examination of the therapeutic resistance phenomenon in SMAD4-negative colon cancer cells with the three common agents revealed significant resistance to both 5-FU and irinotecan but not to oxaliplatin. We also followed up with the earlier findings from our group, which suggested that SMAD4 might interact with metastasis-promoting factors to suppress metastatic progression and render sensitivity to chemotherapy. Co-immunoprecipitation and mass spectrometry analysis revealed that SMAD4 interacts with and inhibits RICTOR, a component of mTORC2 that activates oncogenic AKT via phosphorylation at Serine 473. Overexpression of SMAD4, depletion of RICTOR, or inhibition of AKT signaling restores sensitivity to irinotecan in SMAD4-negative colon cancer cells in vitro. Furthermore, as expected pharmacological inhibition of AKT sensitizes these cells to irinotecan in vivo. Interestingly, high RICTOR/AKT expression correlates with worse survival in colon cancer patients, suggesting them as novel prognostic biomarkers and therapeutic targets. On the other hand, triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer due to lack of effective targeted therapies. Using miRNA expression profiling of a model for epithelial-mesenchymal transition in TNBC, we found suppression of miR-4417 during the progression from non-malignant to malignant stage. Furthermore, localization of miR-4417 to chromosome 1p36, a region corresponding to high frequency of LOH in multiple cancers and low-level expression in TNBC patients associated with poor overall survival is consistent with its likely role as a tumor suppressor. Interestingly, we found that overexpression of miR-4417 is sufficient to inhibit migration and tumorigenecity of TNBC cells in vitro. Overall, our findings suggest miR-4417 exerts a tumor-suppressive effect and could serve as a novel prognostic biomarker and therapeutic tool against TNBC. / 2021-02-20T00:00:00Z

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.