INCUBADORAS DE EMPRESAS: “UNA ESTRATEGIA PARA CONTEMPLAR UN CURSO DE PROMOCIÓN DE DESARROLLO ECONÓMICA EN SANTIAGO DEL ESTERO”.

El presente trabajo puede ser sucintamente descrito como un proyecto institucional que pretende crear inicialmente en el marco del estudiantado de las diferentes carreras, un instrumento que permita brindar tanto individualmente como de manera asociativa un mecanismo de desarrollo productivo y tecnológico con reales posibilidades de inserción en el mercado. Se trata de vincular a la Universidad con su entorno, contribuir a la pertinencia de la institución, conformar un ámbito de construcción de conocimientos, de abrir nuevos campos temáticos e hipótesis de trabajo, generar recursos propios y aportar al desarrollo socio-económico-cultural del entorno. Se propone implementar en la Universidad Nacional de Santiago del estero, una estrategia para contemplar un Curso de capacitación tendiente al desarrollo económico y tecnológico mediante una Incubadora de Empresas. Su función es facilitar, desde los primeros pasos, el seguimiento de nuevos emprendimientos productivos convirtiendo buenas ideas en negocios o actividades o en oportunidades de nacimiento y consolidación de empresas mixtas, compartidas, etc. El objetivo general es que la incubadora podrá presentar asistencia técnica coordinar, planificar acciones. En definitiva, todas las gestiones necesarias para promover el desarrollo de emprendimientos innovadores

Construyendo un modelo adaptativo de aprendizaje : Desarrollo de la plataforma virtual “Edujesel”en la Patagonia

En un escenario regional cada vez más complejo por la realidad social y económica, donde el sector productivo demanda nuevas competencias vinculadas a las nuevas tecnologías, donde se requiere creatividad para salir de su crisis, pero a la vez adaptabilidad estratégica, desde la UNPA y su “Laboratorio de experiencias pedagógicas” del Instituto de investigación en Educación y Ciudadanía se está proponiendo un sistema adaptativo de aprendizaje en torno a una plataforma virtual. Las características geográficas de la patagonia y contingencia social de la región contribuyen a buscar un nuevo formato para el desarrollo de las trayectorias escolares. Las nuevas tecnologías pueden ayudar a encontrar un modelo pedagógico a partir de una plataforma virtual adaptándose a las características del docente, del alumno y su condición social-regional. Esto se traduce en un primer sentido a identificar cuáles son las características de los actores y las destrezas o competencias necesarias para establecer una plataforma virtual entre otros aspectos. Desde el Laboratorio de referencia estamos ensayando la aplicación de la plataforma virtual “Edujesel” que desarrolló la Ing. Marcela Pereyra con aportes pedagógico del equipo de investigación. El presente artículo da cuenta de algunos avances teóricos para la práctica y ejecución.Eje 4 - Mesa 7.Dirección de Educación a Distancia, Innovación en el aula y TI

Construyendo un modelo adaptativo de aprendizaje : Desarrollo de la plataforma virtual “Edujesel”en la Patagonia

En un escenario regional cada vez más complejo por la realidad social y económica, donde el sector productivo demanda nuevas competencias vinculadas a las nuevas tecnologías, donde se requiere creatividad para salir de su crisis, pero a la vez adaptabilidad estratégica, desde la UNPA y su “Laboratorio de experiencias pedagógicas” del Instituto de investigación en Educación y Ciudadanía se está proponiendo un sistema adaptativo de aprendizaje en torno a una plataforma virtual. Las características geográficas de la patagonia y contingencia social de la región contribuyen a buscar un nuevo formato para el desarrollo de las trayectorias escolares. Las nuevas tecnologías pueden ayudar a encontrar un modelo pedagógico a partir de una plataforma virtual adaptándose a las características del docente, del alumno y su condición social-regional. Esto se traduce en un primer sentido a identificar cuáles son las características de los actores y las destrezas o competencias necesarias para establecer una plataforma virtual entre otros aspectos. Desde el Laboratorio de referencia estamos ensayando la aplicación de la plataforma virtual “Edujesel” que desarrolló la Ing. Marcela Pereyra con aportes pedagógico del equipo de investigación. El presente artículo da cuenta de algunos avances teóricos para la práctica y ejecución.Eje 4 - Mesa 7.Dirección de Educación a Distancia, Innovación en el aula y TI

Piezas dentarias retenidas: nuestra experiencia

Un diente retenido es aquel que llegada su época cronológica de erupción, queda incluido en el hueso maxilar, no pudiendo hacer su aparición en la arcada dentaria normalmente. Objetivos: Este trabajo estadístico tiene como finalidad poder clasificar, según diferentes variables, la frecuencia de las piezas dentarias retenidas; lo que a su vez nos permitirá comparar nuestro trabajo con la bibliografía mundial. Materiales y métodos: Se analizaron los casos que fueron intervenidos en el Hospital Dr. Alejandro Korn entre el periodo comprendido de Marzo de 2012 a Marzo de 2013, fijando variables de inclusión y exclusión. Resultados: El estudio retrospectivo arrojó un total de 107 casos en un año de trabajo, pudiendo observarse las diferentes piezas incluidas, posiciones y frecuencia. Conclusiones: Al analizar las intervenciones quirúrgicas de piezas dentarias retenidas que fueron operadas en el servicio pudimos evaluar el nivel académico en el cual nos encontramos con respecto a publicaciones mundiales.A retained tooth is one who arrived the chronological time of eruption, is included in the maxillary bone, failing to make its appearance in the dental arch normally. Objectives: This statistical work is intended to be classified according to different variables, the frequency of impacted teeth; which in turn will enable us to compare our work with the world literature. Materials and methods: analysed cases that were operated on at the Hospital Dr. Alejandro Korn between the period of March 2012 to March 2013, setting variables for inclusion and exclusion. Results: Retrospective study threw a total of 107 cases in a year’s work, and can observe including parts, positions and frequency. Conclusions: analyzing the surgery of impacted teeth that were operated in the service we could evaluate the academic level in which we find ourselves regarding global publications.Facultad de Odontologí

Piezas dentarias retenidas: nuestra experiencia

Un diente retenido es aquel que llegada su época cronológica de erupción, queda incluido en el hueso maxilar, no pudiendo hacer su aparición en la arcada dentaria normalmente. Objetivos: Este trabajo estadístico tiene como finalidad poder clasificar, según diferentes variables, la frecuencia de las piezas dentarias retenidas; lo que a su vez nos permitirá comparar nuestro trabajo con la bibliografía mundial. Materiales y métodos: Se analizaron los casos que fueron intervenidos en el Hospital Dr. Alejandro Korn entre el periodo comprendido de Marzo de 2012 a Marzo de 2013, fijando variables de inclusión y exclusión. Resultados: El estudio retrospectivo arrojó un total de 107 casos en un año de trabajo, pudiendo observarse las diferentes piezas incluidas, posiciones y frecuencia. Conclusiones: Al analizar las intervenciones quirúrgicas de piezas dentarias retenidas que fueron operadas en el servicio pudimos evaluar el nivel académico en el cual nos encontramos con respecto a publicaciones mundiales.A retained tooth is one who arrived the chronological time of eruption, is included in the maxillary bone, failing to make its appearance in the dental arch normally. Objectives: This statistical work is intended to be classified according to different variables, the frequency of impacted teeth; which in turn will enable us to compare our work with the world literature. Materials and methods: analysed cases that were operated on at the Hospital Dr. Alejandro Korn between the period of March 2012 to March 2013, setting variables for inclusion and exclusion. Results: Retrospective study threw a total of 107 cases in a year’s work, and can observe including parts, positions and frequency. Conclusions: analyzing the surgery of impacted teeth that were operated in the service we could evaluate the academic level in which we find ourselves regarding global publications.Facultad de Odontologí

Carrier recombination and transport dynamics in superstrate solar cells analyzed by modeling the intensity modulated photoresponses

The dynamics of carrier recombination and transport of two CuInS2 superstrate solar cells was studied by intensity modulated photovoltage and photocurrent spectroscopy (IMVS and IMPS respectively). For the analysis of the resulting data two different approaches were implemented. In the first approach, the typically used analysis in Dye Sensitized Solar Cells (DSSC) was adapted to obtain the characteristic times of the processes involved. The second approach was based on the fittings of both the IMVS and IMPS data to the solution of the continuity equation. These fittings allow the calculation of different dynamic parameters of the cells. Moreover, consistency between the obtained parameters was observed, in good agreement with the typical analysis for DSSC. The resulting dynamics was associated with the presence and distribution of defect states among the samples. Moreover, from the performed analysis, a relation between the results and the post-treatment applied to the solar cells could be established. The difference in the dynamics of the cells is mainly observed in the difference between the electron lifetimes of both solar cells.Fil: Pereyra, Carlos J.. Universidad de la Republica. Facultad de Ingeniería. Departamento de Física; UruguayFil: Di Iorio, Yesica Dolores. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Berruet, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Vazquez, Marcela Vivian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Marotti Priero, Ricardo Enrique. Universidad de la Republica. Facultad de Ingeniería. Departamento de Física; Urugua

La Extensión Universitaria como práctica de incidencia en derechos de niñas, niños y jóvenes

Esta ponencia tiene como finalidad la presentación del Programa de Extensión: “Aportes para la intervención con Infancia y Juventudes. Enfoque de Derechos”, cuyo objetivo tiende a contribuir al desarrollo de prácticas comunitarias en relación a la Infancia y Juventud, teniendo como eje la efectivización de derechos, en  zonas urbanas y rurales. Concebimos a la extensión como un espacio de interacción entre la Universidad y los diversos ámbitos de la comunidad, en la cual aquella está inserta, mediante el intercambio de conocimientos, saberes y experiencias, en forma dialéctica; y donde se generan acciones conjuntas de los sujetos involucrados, posibilitando la efectivización de sus derechos. Este Programa está compuesto por dos proyectos de extensión: “Piedra Libre para todos. Promoción y prácticas socioeducativas en derechos de niños, niñas y jóvenes”, (Villa Mercedes), y  “La Infancia Rural. Un abordaje comunitario. Zona “Los Manantiales”, (Paso Grande, Las Vertientes, Las Aguadas, Las Chacras, Villa Praga), de la provincia de San Luis. El primer proyecto refiere a la protección de los derechos de niñas, niños y jóvenes, donde intervienen docentes, no docentes, alumnos y graduados de la FICES, desde dos ejes de trabajo orientados a los niños/as, jóvenes y sus familias, que concurren a la Asociación “Lección de Vida” de nuestra ciudad. El primer eje radica en el abordaje de promoción de derechos,  mediante talleres de reflexión, coordinados por docentes y alumnos; y el segundo, consiste en la orientación y asesoramiento psicopedagógico y social, de los docentes (pedagoga y trabajadoras sociales) y profesionales externos al proyecto (psicóloga, médicos, etc.), con la colaboración de los alumnos de la carrera. El segundo proyecto tiene como finalidad “adentrarnos” en las diversas formas de vida de los habitantes campesinos, a través de un abordaje comunitario, con niñas, niños y adolescentes, mediante entrevistas en profundidad, observaciones y espacios de diálogo, (talleres lúdicos y cortos documentales), dando “lugar a la palabra”, para contar, narrar, advertir, reflexionar y exigir derechos que puedan ser percibidos como vulnerados, poniendo énfasis en el derecho a la identidad y a la participación en la comunidad. Estas intervenciones extensionistas se fundan en el paradigma de Derechos Humanos, entendiendo a niñas, niños y jóvenes, como sujetos ciudadanos, con una identidad construida en un espacio geopolítico y socio-histórico coyuntural, como prácticas de incidencia en derechos sociales, desde la Universidad, en su función de compromiso social.

Efficiency of Sperm-Mediated Gene Transfer in the Ovine by Laparoscopic Insemination, In Vitro Fertilization and ICSI

Abstract. Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/ plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6%) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60% of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors. Key words: Green fluorescent protein, Sheep, Transgenesis (J. Reprod. Dev. 57: [188][189][190][191][192][193][194][195][196] 2011) ransgenesis in mammals constitutes an important tool for recombinant protein production Evidence of heterologous DNA interaction with mammalian spermatozoa and subsequent delivery into the oocyte during fertilization was first reported in 1971 The ovine is a good model in the field of animal transgenesis and biotechnology due to its relatively short pregnancy period compared with the bovine. Moreover, its significant saliva and milk production make the salivary and mammary glands interesting targets for recombinant protein expression SPERM-MEDIATED GENE TRANSFER IN THE OVINE techniques using sperm incubated with DNA for a long (2 h to 17 C) or short (5 min to 0-5 C) period of time. In addition, the interaction between sperm and labeled DNA was analyzed by fluorescent microscopy in order to interpret differences between treatments. Presence of exogenous plasmid DNA in LI, IVF and ICSI embryos was determined by PCR, and localization of exogenous DNA into their nuclei was confirmed by FISH in ICSIderived embryos. Chemical activation following nontransgenic ICSI has been reported to improve bovine embryo development Materials and Methods Animal care and governmental authorization Animal usage and care treatments were approved by the Institutional Committee of Care and Use of Laboratory Animals of the Buenos Aires University (Resolution No. 2007/15). The Secretariat of Agriculture, Livestock, Fisheries and Foods of Argentina, as counseled by the Comisión Nacional de Biotecnología Argentina (CONABIA), approved the animal confinement conditions for LI (Resolution No. 250). Chemicals and media Except where otherwise indicated, all chemicals were obtained from Sigma Chemical (St. Louis, MO, USA). DNA construction The plasmid used was pCX-EGFP (kindly provided by Dr M Okabe, Osaka University, Osaka, Japan) that contains an enhanced green fluorescent protein gene (egfp) under chimeric cytomegalovirus-IE-chicken β-actin enhancer-promoter control Sperm/DNA incubation Sperm/pCX-EGFP incubation was carried out in accordance with two procedures 1) In the first procedure, long sperm/DNA incubation (Long Incubation), fresh or frozen/thawed semen (pooled from eight rams) was treated as previously described Experimental design Three independent assays were carried out in order to evaluate exogenous gene-expressing ovine embryo production following the treatments described above. For LI, we used fresh sperm subjected to long or short sperm/DNA incubation to fertilize superovulated sheep. For the IVF and ICSI experiments, frozen/thawed sperm subjected to the same sperm-plasmid incubation treatments were used to fertilize oocytes collected from slaughterhouse ovaries. In order to analyze sperm/exogenous DNA interaction, we tested labeled-DNA adhesion to sperm membranes after Long and Short Incubation with fresh and frozen/thawed sperm. We used PCR and FISH analyses to determine transgene presence and localization in these embryos. Finally, in order to establish conditions for ICSI in the ovine, this study compared three different activation treatments to assist the ICSI technique. Each experiment was replicated at least three times. In vivo embryo production Superovulation treatments: Donor adult Merino sheep (n=17) were synchronized by insertion of an intravaginal sponge (60 mg of medroxyprogesterone acetate, Syntex; Lab. Syntex, Buenos Aires, Argentina) for 14 days. During days 12-14 of treatment, donor ewes were superovulated with six decreasing doses of FSH i.m. (18 mg × 2, 14 mg × 2, 8 mg × 2, NIH-FSH-P1, Folltropin ® , Bioniche, Armidale, NSW, Australia) administered twice a day starting on the morning of Day 12, 48 h before pessary removal, and finishing 12 h after progestagen withdrawal. Progestagen removal was performed on Day 14, coinciding with the fifth FSH treatment, and a single dose of eCG (200 UI Novormon 5000, Lab. Syntex) was administered. All ewes were checked twice daily for the onset of estrus 24 h after sponge withdrawal using an adult ram. Laparoscopic insemination (LI): Ewes in estrus were fasted 12 h prior to artificial insemination and randomly assigned to LI with fresh semen from the Long Incubation (n=9), Short Incubation (n= 8) or control (n=1) treatments. Merino rams (n= 8) of proven fertil- ity were utilized as sperm donors. Ejaculates were collected using an artificial vagina, pooled in a water bath at 36 C and treated as described previously. LI was performed 12 h after estrus detection. Ewes were placed on a standard cradle for laparoscopic procedures. The surgical field, cranial to the udder, was shaved and disinfected. General anesthesia was administered with xylazine (0.5 mg/10 kg of Kensol 2%, Konig, Buenos Aires, Argentina) and ketamine (25 mg/10 kg of Ketalar; Parke-Davis, Buenos Aires, Argentina) and local anesthesia was applied (lidocaine, Frankaina 2%; FatroVonFrankel, Buenos Aires, Argentina). Thereafter, an endoscope (Richard Wolf, Knittlingen, Germany) was inserted into the abdominal cavity through a trocar approximately 10 cm cranial to the udder and 5 cm to the left side of the midline for visualization of the uterine horns. A second trocar was inserted in the right side of the abdominal wall and used to deliver 200 μl of previously incubated or not (control) fresh sperm (500 × 10 6 /ewe final concentration) into the uterine horns (100 μl each) using a cannula (Aspic for pellet insemination in sheep, 23-gauge needle, IMV; L'Aigle, France). Finally, the trocar orifices were treated with a local antibiotic solution (Young Plata, Quimagro, Buenos Aires, Argentina), and oxytetracycline (1 ml/10 kg, Hostaciclina LA; Hoechst, Dublin, Ireland) was given as preventative treatment. Embryo recovery: Six days after estrus detection, embryos were collected from donors placed under general anesthesia, as described above. In brief, embryos were surgically obtained through prepubian laparotomy. Both uterine horns were flushed using a Foley catheter. The collection medium was prewarmed (38 C) commercial Complete Flush ViGro (Bioniche, Bogart, GA, USA). All the morulae and blastocysts collected from the different groups were kept separated for examination by fluorescence microscopy, as described below. In vitro embryo production Oocyte collection and in vitro maturation: Ovaries were collected from slaughterhouses and transported to the laboratory at ambient temperature. Cumulus-oocyte-complexes (COCs) were aspirated with 21-gauge needles from follicles with a diameter of 2 to 5 mm. They were collected into Dulbecco's phosphate buffered saline (DPBS, 14287-072; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, 013/07; Internegocios, Buenos Aires, Argentina) and 2% antibiotic-antimycotic (ATB, 15240-096; Gibco). Oocytes covered with at least 3 layers of granulosa cells were selected for in vitro maturation (IVM) in bicarbonate-buffered TCM-199 (31100-035; Gibco) containing 2 mM glutamine (G-8540), 10% FBS, 10 μg/ml follicle stimulating hormone (NIH-FSH-P1, Folltropin ® , Bioniche), 0.3 mM sodium pyruvate (P2256), 100 μM cysteamine (M9768) and 2% ATB. Oocytes were incubated in 500 μl of medium under mineral oil (M8410) in 4-well dishes (Nunclon ® , Nunc, Naperville, IL, USA) in an atmosphere of 6.5% CO2 in humidified air at 39 C for 24 h. In vitro fertilization (IVF): Semen was collected from two Merino rams using an artificial vagina, and the samples were frozen by standard procedures Intracytoplasmic sperm injection (ICSI): For ICSI, oocytes were vortexed (to remove cumulus cells) for 2 min [1 mg/ml hyaluronidase (H-4272) in DPBS] and washed tree times in HEPESbuffered (H4034) TCM-199. Mature oocytes were evaluated by visualizing the first polar body and immediately used for ICSI. Frozen semen were thawed in a 37 C water bath for 30 sec and subjected to either the Long or Short Incubation described above. After incubation, less than 0.05 μl of spermatozoa was transferred to a 4-μl droplet of TALP-H with 10% v/v of polyvinylpyrrolidone (PVP, 99219; Fisher Scientific, Pittsburgh, PA, USA) a used directly for ICSI, which was performed as described previously Oocyte chemical activation Injected oocytes were immediately activated in TALP-H with 5 μM ionomycin (I24222; Invitrogen, Carlsbad, CA, USA) for 4 min and placed in TCM-199 for 3 h to allow second polar body extrusion (except for Io-EtOH activation, where the 3-h window is not necessary for polar body emission). Oocytes were subsequently treated with a) TCM-199 with 1.9 mM DMAP (D2629) for 3 h (Io-DMAP Group); b) 5 μM ionomycin followed immediately by TCM-199 with 1.9 mM DMAP for 3 h (2Io-DMAP Group); or c) TALP-H with 7% (v/v) ethanol for 5 min (Io-EtOH Group). In all the cases, inhibitors were removed by washing three times in TALP-H, and culture was continued as described below. In vitro embryo culture Presumptive IVF and ICSI zygotes (15-30 per group) were cul-191 SPERM-MEDIATED GENE TRANSFER IN THE OVINE tured in 50-μl droplets of SOF medium [31] containing 2.5% FBS and incubated at 39 C in 6.5% CO2 in air. Embryos were moved to a new droplet every 48 h. Cleavage was evaluated on day 2, and the numbers of morulae and blastocysts were evaluated on day 7 postfertilization. Evaluation of EGFP fluorescence in embryos On day 7 postfertilization, all embryos recovered in vivo and produced in vitro were briefly exposed to blue light using an excitation filter at 488 nm and an emission filter at 530 nm to determine egfp gene expression. Sperm/exogenous DNA (eDNA) interaction In order to analyze sperm/eDNA interaction, pCX-EGFP plasmid was labeled with Alexa fluor (F6257) by the Nick Translation System (18160-010; Invitrogen). Label eDNA was incubated in accordance with the long and short treatments described above. Afterwards, an aliquot of each labeled-DNA/spermatozoa complex was diluted (10 μl in 90 μl of prewarmed TALP-H medium) and centrifuged 5 min at 490 × g. Then, the supernatant was removed carefully, and the pellets were resuspended in 100 μl of TALP-H (washing procedure). Immediately, 10 μl of each sample (Fresh or Frozen-Thawed and washed or non-washed spermatozoa) were mounted between coverslips to observed under blue light the presence of label eDNA in sperm cells in order to distinguish between motile and immotile ones. Autoflorescence was discarded by checking the spermatozoa before incubation treatments and after unlabelled bovine genomic DNA fragment incubation treatments. A second experiment was performed by labeling bovine genomic DNA with rhodamine by the Nick Translation System (0.1 to 1 Kb fragments) and using it for both sperm incubation treatments. All experiments were repeated at least two times using pooled semen from two different rams. Determination of sperm viability before and after exogenous DNA incubation Fresh semen (from two rams) that had been treated according to the Long Incubation protocol were stained by incubation in TCM-199 containing 1 mg/ml of Hoechst 33258 (861405) for 2 min, both before and after pCX-EGFP addition. Then, a 10-μl aliquot was placed between coverslips to visualize spermatozoa. At least 100 spermatozoa were observed under epifluorescent microscopy (UV 380) and classified as i) damaged membrane (stained sperm cells) motile and immotile or ii) undamaged membrane (unstained sperm cells) motile and immotile. Control spermatozoa were coincubated with Hoechst, but not with plasmid. PCR analysis Morulae and blastocysts from LI, IVF and ICSI Short Incubation with and without zonae pellucidae (ZPs) were washed in PBS, transferred as 1-μl aliquots into an eppendorf tube, resuspended in 9 μl of extraction buffer (1 mg/ml proteinase K in 10 mM Tris-EDTA, V302B, Promega, Madison, WI , USA) and incubated at 56 C for 1 h. The samples were then heated at 95 C for 10 min, and 5-μl aliquots were used for PCR. The primer set sequences were 5-GAAGTTCGAGGGCGACACCTG-3 and 5-TCGTCCATGC-CGAGAGTGATC-3 for amplifying a 269 bp fragment of EGFP. The PCR reaction conditions consisted of denaturation at 95 C for 2 min, followed by 35 amplification cycles of denaturation at 94 C for 15 sec; annealing at 55 C for 15 sec and extension at 72 C for 25 sec. Cycle 35 contained an additional extension at 72 C (7 min). The positive control consisted of 3.6 -11 g/ml of pCX-EGFP plasmid, and the negative control was distilled water. The ZP was removed by 5 min incubation in 1.5 mg/ml pronase (P8811) in TALP-H, followed by careful washing five times in 3 ml of TALP-H. Determination of the cell number in blastocysts or spermatozoa bound to embryos Embryos were stained (2 min) in TCM-199 containing 1 mg/ml of Hoechst 33342 (B2261) and immediately mounted between coverslips to visualize spermatozoa bound to the ZP (LI embryos) or to count total nuclei (ICSI blastocysts) using epifluorescent microscopy (UV 380). Fluorescence in situ hybridization (FISH) Ovine 8-cell embryos were incubated for 20 h with 0.1 μg/ml demecolcine (D1925). Afterwards, the embryos were treated in a hypotonic solution (1% Na citrate in distilled water for 10 min) and fixed on a poly-L-lysine-coated slide with 3:1 methanol-acetic acid solution. Indirect labeled signals of FITC anti-mouse (F6257) and anti-digoxigenin (D8156), which bind the digoxigenin-labeled pCX-EGFP (5.5 kb) probe, were labeled using the Nick Translation System (18160-010; Invitrogen). The total DNA was counterstained with DAPI. Images of each cell and their signals were recorded with an Optronics camera (CCD; Optronics, Goleta, CA, USA). Data analysis Embryo development and fluorescent expression were compared by Fisher's exact test analysis. Differences in total cell number or stained sperm were analyzed using a one-way ANOVA test. For statistical analyses, the SAS program was used Results Laparoscopic insemination, in vitro fertilization and intracytoplasmic sperm injection A pool of semen collected from eight rams and incubated (long or short treatment) with pCX-EGFP plasmid was used to inseminate seventeen ewes. After six days, LI embryos were recovered through laparotomy and uterine flushing. Fertilization rates were not statistically different betweens Long and Short Incubation treatments and the control group (90.4, 98.5 and 100% respectively). None of the 78 collected embryos (38 and 40 for the long and short procedures, respectively) were fluorescent after observation under blue light One hundred and sixty-two oocytes were fertilized by IVF with spermatozoa previously incubated with pCX-EGFP plasmid in the long or short treatments. Plasmid incubation groups were not different from the controls in terms of cleavage (long, short and control: 86.8, 87.3 and 88.4%, respectively) or embryonic development A total of 107 oocytes were injected with spermatozoa incubated with pCX-EGFP plasmid. Independent of incubation treatment, high embryo development and transgene expression rates were obtained Sperm/exogenous DNA interaction Plasmid DNA attachment to the plasma membrane was observed in most of the frozen-thawed spermatozoa subjected to the Long and Short ( Determination of sperm viability before and after exogenous DNA incubation The percentage of Fresh spermatozoa with a damaged membrane did not differ before (33/110; 33.0%) or after pCX-EGFP addition (43/110; 39.0%) for the Long Incubation treatment. However, both results differed significantly with respect to the control group (26/120, 21.6%; P<0.05). All stained sperm cells (membrane damaged) were immotile. PCR analyses PCR analysis showed and egfp amplification product for LI whole embryos (with a ZP). Nevertheless, when LI embryos were released from their ZPs by pronase treatment, no egfp PCR product was visible in any of the embryos analyzed. For IVF, no embryos (with or without a ZP) showed PCR amplification products. On the other hand, all ICSI embryos showed amplification of the transgene In vitro development of transgenic-ICSI ovine embryos assisted by different chemical activation treatments In total, 304 ovine oocytes were injected with spermatozoa incubated with pCX-EGFP plasmid. All ICSI activation protocols were capable of producing blastocysts FISH analyses FISH analysis displayed various possible integration loci in eight of twelve (66.6%) ovine 8-cell stage embryos Discussion After both the long and short sperm/exogenous DNA (eDNA) incubation treatments, LI embryos did not express egfp transgene Several aspects of spermatozoa/DNA interaction were studied. Both fresh and frozen/thawed spermatozoa bound eDNA, as has been found in mice and some domestic species [35-37; for a review, see 38]. Labeled DNA signals were detected on the surfaces of all fresh and frozen-thawed sperm after both treatments. However, in agreement with observations in the bovine We saw that the fresh spermatozoa that maintained exogenous attached DNA were nonmotile. Unexpectedly, embryos produced by LI (with fresh sperm) showed amplification of the transgene by PCR analysis. Nevertheless, when the PCR analysis was performed on ZP-free embryos, we did not find a specific transgene band. In order to clarify these results, we stained LI embryos with Hoechst. We observed numerous spermatozoa adhered to the ZP in 100% of the stained recovered embryos, verifying that PCR amplification was derived from spermatozoa attached to the ZP and not from the embryos. This raised the question of how spermatozoa carrying the transgene arrived at the ZP. We propose two hypotheses. 1) The first is that a small proportion of motile spermatozoa carrying the transgene were able to reach the oocytes, but none of them were able to fertilize it, and 2) the second is that nonmotile spermatozoa carrying the transgene remained in the uterine horns and could adhere to the ZP during embryo migration to the uterus. The first hypothesis has been reported in pigs, where a low percentage of motile spermatozoa (1-3%) carried eDNA, although non transgene-expressing-embryos were detected SPERM-MEDIATED GENE TRANSFER IN THE OVINE the results were independent of ZP presence. The three different activation treatments evaluated induced blastocyst development after ICSI. In order to replace the activating effect produced by piezoelectric microinjection Simple PCR amplification or RT-PCR can be used to detect the presence of a transgene, but they cannot determine its localization. An alternative technique, fluorescent in situ hybridization (FISH), can be used for this purpose. For ICSI embryos, FISH analysis at interphase found possible signals inside the nucleus compatible with at least two or four integration loci In conclusion, under the conditions studied, ICSI was the only method of fertilization to successfully produce exogenous geneexpressing ovine embryos, and the Short Incubation treatment enhanced transgene expression percentages. In addition, we found it necessary to carefully remove the ZP from embryos in order to avoid false positives by PCR. To our knowledge, this is the first study to explain possible failures involved in SMGT by LI or IVF in the ovine. The results obtained in this work demonstrated that sperm-mediated gene transfer by ICSI could be useful for inducing exogenous gene expression in ovine embryos

«Donotforgetme». Promotion of mental health welfare conditions for the elderly

El artículo da cuenta de las producciones realizadas en el proyecto interdisciplinario «Nomeolvides», que tuvo como objetivo la promoción de las condiciones de bienestar de la salud mental en una comunidad de adultos mayores en la ciudad de Córdoba (Córdoba, Argentina).The article gives an account of the productions made in the interdisciplinary project «Forget-me-nots», which aimed at promoting mental health welfare conditions in a community of older adults in the city of Córdoba (Córdoba, Argentina).Facultad de Periodismo y Comunicación Socia