Catalogus Plantarum :tum exotiarum tum domesticarum, quae in hortis haud procul a Londino sitis in venditionem propagantur = A Catalogue of trees, shrubs, plants, and flowers, both exotic and domestic, which are propagated for sale, in the gardens near London :divided, accorded to their different degrees of hardiness, into particular books, or parts; in each of which the plants are ranged in an alphabetical order. by a Society of Gardeners

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Producción in vitro de Embriones Bovinos Transgénicos mediante Inyección Intracitoplasmática de Espermatozoides (ICSI) con Enzimas de Restricción. In vitro Production of Transgenic Bovine Embryos using Intracitoplasmic Sperm Injection (ICSI) and Restriction Enzymes

La ingeniería genética y la reprogramación de organismos vivos representan las nuevas fronteras biotecnológicas que permitirán generar animales con modificaciones precisas en sus genomas para un sinnúmero de aplicaciones biomédicas y agropecuarias. Las técnicas para inducir modificaciones génicas intencionales en animales, especialmente en especies mayores de interés agropecuario, se encuentran rezagadas si se compara con los avances significativos que se han producido en el área de la transgénesis de roedores de laboratorio, especialmente el ratón. Es así que, el presente proyecto persigue desarrollar y optimizar protocolos para generar embriones bovinos transgénicos para aplicaciones biotecnológicas. La estrategia propuesta, se basa en conseguir la presencia simultánea en el interior celular de una enzima de restricción (I-SceI) más un transgén (formado por casetes de expresión de una proteína fluorescente -ZsGreen1- y neomicina fosfotransferasa). Específicamente, proyectamos estudiar una vía alternativa para generar embriones bovinos transgénicos mediante la incorporación del transgén (casetes ZsGreen1 y neo) flanqueado por sitios I-SceI más la enzima I-SceI al interior del ovocito junto con el espermatozoide durante la técnica conocida como inyección intracitoplasmática de espermatozoides (ICSI). Los embriones así generados se cultivarán in vitro, inspeccionándolos diariamente para detectar la emisión de fluorescencia, indicativa de la expresión de la proteína ZsGreen1. Los embriones que alcancen el estado de blastocisto y expresen el transgén se transferirán quirúrgicamente al útero de ovejas sincronizadas y se mantendrán durante 7 días. Al cabo de este período, los embriones se recolectarán quirúrgicamente del útero ovino y se transportarán al laboratorio para determinar el número de sitios de integración y número de copias del transgén mediante el análisis de su ADN por Southern blot. Se prevé que los resultados de esta investigación permitirán sentar las bases para el desarrollo de métodos eficientes para obtener modificaciones precisas en el genoma de los animales domésticos para futuras aplicaciones biotecnológicas. Genetic engineering and reprogrammed organisms represent the new biotechnological frontiers which will make possible to generate animals with precise genetic modifications for agricultural and biomedical applications. Current methods used to generate genetically modified large animals, lay behind those used in laboratory animals, specially the mouse. Therefore, we seek to develop and optimize protocols to produce transgenic bovine embryos through the use of a non-viral vector. The strategy involves the simultaneous presence inside the cell of a restriction enzyme (I-SceI) and a transgene (carrying cassettes for a fluorescent protein -ZsGreen1- and neomycin phosphotransferase) flanked by restriction sites for the endonuclease. We plan to develop an alternative approach to generate transgenic bovine embryos by coinjecting the transgene flanked by I-SceI restriction sites plus the enzyme I-SceI along with the spermatozoon during the technique known as intracytoplasmic sperm injection (ICSI). Embryos will be cultured in vitro and inspected daily with a fluorescence microscope to characterize transgene expression. Embryos that reach the blastocyst stage and express the transgene will be surgically transfer to the uterus of a synchronized ewe. After 7 days, the embryos will be flushed out the ovine uterus and transported to the laboratory to determine the number of integration sites and transgene copies by Southern blot. We anticipate that results from this research will set the stage for the development of efficient strategies to achieve precise genetic modifications in large domestic animals for future biotechnological applications.

Development of an inspection regime for on-site domestic wastewater treatment systems

The overall purpose of this study was to develop a thorough inspection regime for onsite wastewater treatment systems, which is practical and could be implemented on all site conditions across the country. With approximately 450,000 onsite wastewater treatment systems in Ireland a risk based methodology is required for site selection. This type of approach will identify the areas with the highest potential risk to human health and the environment and these sites should be inspected first. In order to gain the required knowledge to develop an inspection regime in-depth and extensive research was earned out. The following areas of pertinent interest were examined and reviewed, history of domestic wastewater treatment, relevant wastewater legislation and guidance documents and potential detrimental impacts. Analysis of a questionnaire from a prior study, which assessed the resources available and the types of inspections currently undertaken by Local authorities was carried out. In addition to the analysis of the questionnaire results, interviews were carried out with several experts involved in the area of domestic wastewater treatment. The interview focussed on twelve key questions which were directed towards the expert’s opinions on the vital aspects of developing an inspection regime. The background research, combined with the questionnaire analysis and information from the interviews provided a solid foundation for the development of an inspection regime. Chapter 8 outlines the inspection regime which has been developed for this study. The inspection regime includes a desktop study, consultation with the homeowners, visual site inspection, non-invasive site tests, and inspection of the treatment systems. The general opinion from the interviews carried out, was that a standardised approach for the inspections was necessary. For this reason an inspection form was produced which provides a standard systematic approach for inspectors to follow. This form is displayed in Appendix 3. The development of a risk based methodology for site selection was discussed and a procedure similar in approach to the Geological Survey of Irelands Groundwater Protection Schemes was proposed. The EPA is currently developing a risk based methodology, but it is not available to the general public yet. However, the EPA provided a copy of a paper outlining the key aspects of their methodology. The methodology will use risk maps which take account of the following parameters: housing density, areas with inadequate soil conditions, risk of water pollution through surface and subsurface pathways. Sites identified with having the highest potential risk to human health and the environment shall be inspected first. Based on the research carried out a number of recommendations were made which are outlined in Chapter 10. The principle conclusion was that, if these systems fail to operate satisfactorily, home owners need to understand that these systems dispose of the effluent to the 'ground' and the effluent becomes part of the hydrological cycle; therefore, they are a potential hazard to the environment and human health. It is the owners, their families and their neighbours who will be at most immediate risk.