1,720,978 research outputs found
Identificación y selección de un gen pro-mitótico como potencial blanco de terapia génica para inducir la regeneración cardiaca empleando un vector baculoviral
No full textEl infarto agudo de miocardio (IAM) es una patología causada por el desbalance entre la oferta y la demanda de oxígeno al miocardio, el cual conduce a la isquemia y muerte celular. Como resultado se produce pérdida del tejido contráctil y su reemplazo por tejido fibrótico produciendo un remodelamiento ventricular que conduce a la insuficiencia cardiaca. Ante esta problemática, resulta de cardinal importancia desarrollar una terapéutica que permita inducir la proliferación de los cardiomiocitos para promover la cardiorregeneración. Dicho enfoque terapéutico involucra un cambio de paradigma en el campo de la cardiología regenerativa ya que se basa en la inducción de la autorregeneración miocárdica como una manera de garantizar la integridad funcional del sincicio miocárdico. En este aspecto, la transcriptómica surge como herramienta para la búsqueda y selección de genes involucrados en la regulación del ciclo celular del cardiomiocito, a partir de los cuales se puedan desarrollar terapias génicas orientadas a inducir la regeneración cardiaca. Para ello, se propone como estrategia la utilización de material genético cardiaco de un modelo animal con características biológicas similares a las humanas, tal como lo es el ovino. Asimismo, se presenta como método de delivery génico a vectores basados en baculovirus, los cuales son virus de insectos no infectivos en humanos, que se caracterizan por una gran eficiencia transduccional y un buen perfil de bioseguridad en mamíferos, de modo tal que facilitaran en un futuro la aprobación de protocolos de ensayos clínicos por las agencias regulatorias y, por ende, la traslacionalidad de la terapia desarrollada a la medicina regenerativa humana.
En particular, el presente trabajo final de grado, se enfocó en la identificación de genes involucrados en el ciclo celular del cardiomiocito ventricular a partir del estudio del transcriptoma del corazón ovino y de la búsqueda bibliográfica, en la validación de sus niveles de expresión, y en la posterior selección de uno de ellos a partir de análisis in silico para el diseño y síntesis de una construcción genética que será posteriormente evaluada en modelos in vitro.Fil: Halek, Julia María. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Escuela de Ciencias Agrarias, Naturales y Ambientales; Argentina.Licenciatura en Genétic
Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells
Get PDFBACKGROUND AIMS: Given the close similarity between ovine and human cardiomyocytes, sheep models of myocardial infarction and heart failure are increasingly used in studies of stem cell-mediated heart regeneration. In these studies, mesenchymal stromal cells (MSCs) are frequently employed. To enhance the paracrine effects of these MSCs, ex vivo transfection with genes encoding growth factors has been proposed. Although viral vectors exhibit higher transfection efficiency than plasmids, they entail the risks of uncontrolled transgene expression and immune reactions that preclude repeated administration. Our aim was to optimize the efficiency of plasmid-mediated transfection of ovine MSCs, while preserving cell viability. METHODS: Varying amounts of diverse cationic lipids were used to obtain the reagent-to-DNA mass ratio showing highest luciferase activity. Transfection efficiency (flow cytometry) was tested on plasmid-green fluorescent protein-transfected MSCs at increasing DNA mass. RESULTS: Lipofectamine LTX 5 μL and Plus reagent 4 μL with 2 μg of DNA yielded 42.3 ± 4.7% transfection efficiency, while preserving cell viability. Using these transfection conditions, we transfected MSCs with a plasmid encoding human vascular endothelial growth factor (VEGF) and found high VEGF protein concentrations in the culture supernatant from day 2 (1968 ± 324 pg/mL per μg DNA) through at least day 12 (888 ± 386 pg/mL per μg DNA) after transfection. CONCLUSIONS: Plasmid-mediated transfection of ovine MSCs to over-express paracrine heart-regenerative growth factors is feasible and efficient and overcomes the risks and limitations associated with the use of viral vectors.Fil: Locatelli, Paola. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olea, Fernanda Daniela. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hnatiuk, Anna. Universidad Favaloro; ArgentinaFil: Sepúlveda, Diana Elizabeth. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pérez Sáez, Juan Manuel. Bio Sidus S.A; ArgentinaFil: Argüello, Rafael. Universidad Favaloro; ArgentinaFil: Crottogini, Alberto Jose. Universidad Favaloro; Argentin
An ovine model of postinfarction dilated cardiomyopathy in animals with highly variable coronary anatomy
Get PDFStudies on cardiac regeneration require large mammalian models of dilated cardiomyopathy (DCM) after acute myocardial infarction (AMI), and pig and sheep models are increasingly used in this field of preclinical research. Given the large interindividual variability in ovine left anterior descending artery (LAD) anatomy, protocols based on the coronary arteries to be ligated often lead to significant variation in infarct sizes and hence to heterogeneous results, ranging from no ventricular remodeling to acute, lethal left ventricular (LV) failure. We designed an ovine model of postinfarction DCM based on estimated infarct size rather than on a predetermined menu of coronary artery ligatures. In seven adult sheep we induced an anterolateral AMI of approximately 25% of the LV mass by ligating the branches of the LAD that, by visual inspection, would lead to such an infarct size. In 10 to 12 weeks, LV end-diastolic volume more than doubled and LV end-systolic volume almost tripled. LV ejection fraction decreased dramatically, as did LV percent fractional shortening and LV percent wall thickening. Infarct size (planimetry) was approximately 25% of the LV endocardial surface. We conclude that in sheep, an anterolateral AMI of approximately 25% of the LV mass--regardless of the coronary branches ligated to attain that infarct size--results in a model of postinfarction DCM that may prove useful in preclinical research on myocardial regeneration.Fil: Locatelli, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Favaloro; ArgentinaFil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Favaloro; ArgentinaFil: Mendiz, Oscar. Fundación Favaloro; ArgentinaFil: Salmo, Fabián. Fundación Favaloro; ArgentinaFil: Fazzi, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Favaloro; ArgentinaFil: Hnatiuk, Anna. Universidad Favaloro; ArgentinaFil: Laguens, Rubén. Fundación Favaloro; ArgentinaFil: Crottogini, Alberto Jose. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Favaloro; Argentin
Early to late VSV-G expression in AcMNPV BV enhances transduction in mammalian cells but does not affect virion yield in insect cells
No full textBackground/Objectives: Baculoviruses represent promising gene delivery vectors for mammalian systems, combining high safety profiles with substantial cargo capacity. While pseudotyping with vesicular stomatitis virus G-protein (VSV-G) enhances transduction efficiency, optimal expression strategies during the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection cycle remain unexplored. This study investigates how VSV-G expression timing affects pseudotype incorporation into budded virions (BVs) and subsequent transduction efficacy. Methods: Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (ie1, gp64, and p10) and GFP via a CMV promoter. VSV-G incorporation was verified by Western blot, while transduction efficiency was quantified in mammalian cell lines (fluorescence microscopy/flow cytometry) and rat hind limbs. Viral productivity was assessed through production kinetics and plaque assays. Results: All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The p10 promoter drove the highest VSV-G expression and transduction efficiency. Crucially, BV production yields and infectivity remained unaffected by VSV-G expression timing. The in vivo results mirrored the cell culture findings, with p10-driven constructs showing greater GFP expression at low doses (104 virions). Conclusions: Strategic VSV-G expression via very late promoters (particularly p10) maximizes baculoviral transduction without compromising production yields. This study establishes a framework for optimizing pseudotyped BV systems, demonstrating that late-phase glycoprotein expression balances high mammalian transduction with preserved insect-cell productivity—a critical advancement for vaccine vector development.Instituto de BiotecnologíaFil: Simonin, Jorge Alejandro. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); ArgentinaFil: Simonin, Jorge Alejandro. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); ArgentinaFil: Cuccovia Warlet, Franco Uriel. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); ArgentinaFil: Cuccovia Warlet, Franco Uriel. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); ArgentinaFil: Bauzá, María del Rosario. Universidad Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB). Laboratorio de Medicina Regenerativa Cardiovascular; ArgentinaFil: Bauzá, María del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Plastine, María Del Pilar. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Plastine, María Del Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Alfonso, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olea, Fernanda Daniela. Universidad Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB). Laboratorio de Medicina Regenerativa Cardiovascular; ArgentinaFil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cerrudo, Carolina Susana. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); ArgentinaFil: Cerrudo, Carolina Susana. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); ArgentinaFil: Belaich, Mariano Nicolás. Universidad Nacional de Quilmes. Instituto de Microbiología Básica y Aplicada. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM); ArgentinaFil: Belaich, Mariano Nicolás. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC); Argentin
Long-term effects of growth hormone on infarct size and left ventricular function in sheep with coronary artery occlusion
Get PDFThe effects of growth hormone (GH) on infarct size and left ventricular (LV) function in experimental acute myocardial infarction (AMI) have been controversial. Moreover, little, if any, information exists regarding long-term evaluation of therapeutic doses of GH in large mammalian models of AMI. We therefore aimed to assess the effect of therapeutic doses of GH over 3.5 months on infarct size and heart function in sheep with AMI. After coronary artery ligation, sheep received subcutaneous human GH 8 IU/d (n = 8) or vehicle (n = 8) over 100 days. Infarct area was similar in GH (16.9% 6 3% of LV area) and placebo (16.5% 6 3.7%, P = not significant) sheep. At 3 days of treatment onset, but not at later times, GH sheep had higher LV shortening fraction (30.7% 6 3.5% vs. 24.8% 6 6.1%, P , 0.04), systolic anterior wall thickness (10.1 6 0.8 vs. 8.6 6 1.2 mm, P , 0.02), and cardiac index (3.8 6 0.6 vs. 2.8 6 0.7 L min21 m22 , P , 0.01). This evolution of function parameters paralleled that of serum insulin-like growth factor 1 levels, which differed significantly only during the first week, suggesting a direct effect of GH on LV contractility. These results may suggest the usefulness of therapeutic doses of GH at the early phases of AMI but do not support maintaining the treatment for longer time.Fil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Favaloro; ArgentinaFil: De Lorenzi, Andrea. Universidad Favaloro; ArgentinaFil: Cortés, Claudia. Universidad Favaloro; ArgentinaFil: Cabeza Meckert, Patricia. Universidad Favaloro; ArgentinaFil: Cendoya, Oscar. Universidad Favaloro; ArgentinaFil: Barra, Juan G.. Universidad Favaloro; ArgentinaFil: Bercovich, Andrés. Biosidus S. A.; ArgentinaFil: González, Eliseo. Biosidus S. A.; ArgentinaFil: Laguens, Rubén. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Crottogini, Alberto José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Favaloro; Argentin
High-dose Erythropoietin Has No Long-term Protective Effects in Sheep with Reperfused Myocardial Infarction
Get PDFHigh-dose erythropoietin has been claimed to be cardioprotective in experimental acute myocardial infarction. In large mammals, however, results are controversial and long-term follow-up data are lacking. We thus assessed the long-term effects of high-dose erythropoietin on left ventricular infarct size and function in an ovine model of reperfused myocardial infarction. After 90 minutes of coronary occlusion followed by reperfusion, sheep received recombinant human erythropoietin (rhEPO) 3000 units/kg on 3 consecutive days (rhEPO group, n=7) or vehicle (placebo group, n=6). Ten weeks later, ventricular function was assessed by echocardiography and catheterization. Infarct size, evaluated as percent fibrotic myocardium (morphometry) and by hydroxyproline quantification, was similar in both groups (morphometry: rhEPO: 22.1 +/- 5.5%, placebo: 18.1 +/- 3.3%, P not significant; hydroxyproline: rhEPO: 6.6 +/- 1.3 microg/mg wet weight, placebo: 7.1 +/- 0.9 microg/mg, P not significant). Ventricular function was diminished in the rhEPO group, as indicated by lower septal wall thickening at the infarct border zone (rhEPO: -1.9 +/- 16.4%, placebo: 20.5 +/- 17%, P<0.04), higher end systolic volume (rhEPO: 47 +/- 14.3 mL, placebo: 32.6 +/- 7.3 mL, P<0.05), and higher end diastolic pressure (rhEPO: 17 +/- 6.5 mm Hg, placebo: 10.1 +/- 2.8 mm Hg, P<0.03). In the rhEPO group, left ventricular endocardial area was larger, suggesting dilatation. High-dose erythropoietin has no cardioprotective effects in sheep with reperfused myocardial infarction.Fil: Olea, Fernanda Daniela. Fundación Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vera Janavel, Gustavo. No especifíca;Fil: De Lorenzi, Andrea. Fundación Favaloro; ArgentinaFil: Cuniberti, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Favaloro; ArgentinaFil: Yannarelli, Gustavo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Favaloro; ArgentinaFil: Cabeza Meckert, Patricia. Fundación Favaloro; ArgentinaFil: Cearras, Martín. No especifíca;Fil: Laguens, Rubén. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Crottogini, Alberto Jose. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Reference values for echocardiographic parameters and indexes of left ventricular function in healthy, young adult sheep used in translational research: comparison with standardized values in humans
Get PDFOvine models of ischemic heart disease and cardiac failure are increasingly used in translational research. However, reliable extrapolation of the results to the clinical setting requires knowing if ovine normal left ventricular (LV) function is comparable to that of humans. We thus assessed for echocardiographic LV dimensions and indexes in a large normal adult sheep population and compared them with standardized values in normal human adults. Bidimensional and tissue Doppler echocardiograms were performed in 69 young adult Corriedale sheep under light sedation. LV dimensions and indexes of systolic and diastolic function were measured. Absolute and body surface areanormalized values were compared to those for normal adult humans and their statistical distribution was assessed. Normalized dimensions (except for end diastolic diameter) as well as ejection fraction and fractional shortening fell within the ranges established by the American Society of Echocardiography and European Association of Echocardiography for normal adult humans. Normalized end diastolic diameter exceeded the upper normal limit but got close to it when correcting for the higher heart mass/body surface area ratio of sheep with respect to humans. Diastolic parameters also fell within normal human ranges except for a slightly lower mitral deceleration time. All values exhibited a Gaussian distribution. We conclude that echocardiographic parameters of systolic and diastolic LV performance in young adult sheep can be reliably extrapolated to the adult human, thus supporting the use of ovine models of human heart disease in translational research.Fil: Locatelli, Paola. Universidad Favaloro. Instituto de Invest.en Cs.basicas. Departamento D/cs.fisiológicas,farmacológicas y Bioquímicas. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olea, Fernanda Daniela. Universidad Favaloro. Instituto de Invest.en Cs.basicas. Departamento D/cs.fisiológicas,farmacológicas y Bioquímicas. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: De Lorenzi, Andrea. Fundación Favaloro; ArgentinaFil: Salmo, Fabián. Fundación Favaloro; ArgentinaFil: Vera Janavel, Gustavo. Universidad Favaloro. Instituto de Invest.en Cs.basicas. Departamento D/cs.fisiológicas,farmacológicas y Bioquímicas. Cátedra de Fisiología; ArgentinaFil: Hnatiuk, Anna. Universidad Favaloro. Instituto de Invest.en Cs.basicas. Departamento D/cs.fisiológicas,farmacológicas y Bioquímicas. Cátedra de Fisiología; ArgentinaFil: Guevara, Eduardo. Fundación Favaloro; ArgentinaFil: Crottogini, Alberto Jose. Universidad Favaloro. Instituto de Invest.en Cs.basicas. Departamento D/cs.fisiológicas,farmacológicas y Bioquímicas. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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