TISSUE CULTURE

Author: CAÑADILLAS LÓPEZ M. SAGRARIO.
Year: 2004.
University: CÓRDOBA [www.uco.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: UNIDAD DE INVESTIGACIÓN HOSPITAL REINA SOFÍA , CÓRDOBA.

Summary: The parathyroid glands constribuyen the proper functioning of many organs and cell types through which they exercised control over the maintenance of the concentration of calcium (Ca2 +) within a physiological range. The cell paratiroídea is able to detect small changes in the concentration of Ca2 + extracellular, which responds by modifying the secretion of parathyroid hormone (PTH). The ability of the parathyroid cell to sense changes in the concentration of Ca2 + is due to the expression in its membrane calcium receptor (CaR). The CaR belongs to a family of G protein coupled receptors (GPCRS): activation by binding to the ligand (Ca2 +) generates intracellular signals that result in the reverse regulation of PTH secretion and cell proliferation, as well as in regulating the expression of some genes. Therefore, the calcium acts as a regulator of parathyroid function, both of hormone secretion, and at the level of gene expression, however intracellular mechanisms involved in the cellular response to the calico are not known accurately. The CaR stimulation, possibly through an increase in intracellular calcium (Cai), the active phospholipase A2 (PLA2), which synthesizes acid Araquidónico (AA). We know why the AA acting as a mediator in the inhibition of secretion of parathyroid hormone (PTH) for calcium. Preliminary data from our laboratory showed that the extracellular calcium upward regulates gene expression of Vitamin D receptor (VDR) in parathyroid cells. Since calcium activated via PLA2-AA is possible that the AA regulate gene expression vdr since this metabolite in other cells is related to expersión of some genes. Moreover, it has recently been demonstrated in different cell types, the ability of CaR to trigger a cascade of MAPKS (protein kinase activated by mitogenic). This paper has shown the involvement extracellular, leading to inhibition of PTH secretion and the positive regulation of the expression ARNmvdr.

Author: FLAMES BONILLA NURIA.
Year: 2004.
University: MIGUEL HERNÁNDEZ DE ELCHE [www.umh.es].
Place of defense: INSTITUTO DE NEUROCIENCIAS.
Place of preparation: INSTITUTO DE NEUROCIENCIAS.

Summary: The cerebral cortex is the region most complex brain of mamíferon. We can group neurons into two main classes: the projection neurons, also called pyramidal neurons and the neurons interneuronas and local loop. The projection neurons are excitadoral (used as a neurotransmitter glutamate), on the contrary, are interneuronas inhibitory (GABA used). For a smooth functioning of the cerebral cortex there is a need for a delicate coordination between the activity of neurons and interneuronas projection. The interneuronas cortical originate outside of the cerebral cortex, the subpallium, and arrive at their destination through a long migration tangential furante development. This doctoral thesis describes mecanísmos molecular involved in guiding this migration. Most of the interneuronas born in a region called Eminence Ganglionar Medial and migrate dorsalemente the crust. We discovered that, repulsive signals from the ventral part of telencéfalo cooperating with attractive cortical signals to guide interneuronas the crust. The analysis of the double mutant mice to proteins Slit1 and Slit2 showed that its function is not necessary for the repulsion of the ventral interneuronas. Multiple experiments in experimental embryology, and the analysis of mutant mice allowed us to assert that the protein Neuregulina-1 with signaling through its receptor ErbB4, is responsible, at least in part of the cortical activity attractive.

Author: NAVAL MARINO MARIA DEL MAR.
Year: 2005.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: UNIVERSIDAD POLITÉCNICA DE VALENCIA DEP. BIOTECNOLOGÍA.
Place of preparation: UNIVERSIDAD POLITÉCNICA DE VALENCIA.

Summary: SUMMARY: Among the diseases that affect melon, Cucumis melo L., caused by viruses causing heavy loss of] production. The fight preventive methods are ineffective in many cases non-existent and phytosanitary available] farmer. Therefore, the development of new cultivars with genetic resistance is the best control strategy. In this context, genetic engineering has become a valuable tool in improvement programs aimed at the introduction of disease resistance by allowing the use of sources of variation extraespecífica and addressing strategies based on the use of resistance derived from pathogen. Within viral etiology of the disease, screening melon, caused by the virus of necrotic spots of melon (MNSY), causing serious losses. In order to open a new path leading to melon cultivars with resistance to the disease, the central objective of this PhD thesis has been to obtain transgenic plants melon cv. Arizo (type Gaul) with sequences of the genes related to the rep] icasa, protein and protein cover movement of this virus. Although the genetic transformation has become a standard technique in some laboratories, their application to specific genotypes (eg those that are relevant from the viewpoint of the improvement) is far from routine, since there is a strong dependence of the response morfogénica In vitro respect to genotype. Therefore, in the first phase of this project have been carried out various studies to adapt the method via Agrobacterium transformation (developed in our lab in different types of melon)] selected genotype (cv. Arizo type Gaul). Specifically, it has studied the changing pattern polisomático in the bodies used as a source of explant (ie cotyledon, leaf, hypocotyl), regeneration from primary explants (cotyledon and leaf) in culture media with varying hormonal balances, and the response after infection and co-cultivo with A. Tumefaciens. In these prior testing was achieved informer GFP gene transfer, which allows tracking the course of the transformation process in a direct way, and the gene asnA from E. Coli, which encodes a asparagina synthetase and could confer a certain greater tolerance to salinity. Once adapted the method of processing the geno type selected, have been carried out transformations genetic sequences of the genes of replicasa, protein and protein deck turnover MNSY. Following the molecular characterization, transgenic plants have been evaluated using a bioassay which involves mechanical inoculation of the virus and the subsequent analysis of the infection. It has been found that a high percentage of transgenic plants remained healthy, or have fewer injuries in local cotyledons inoculated, falling, too, the percentage of plants with systemic infection.

Author: CASTELBLANQUE SORIANO LOURDES.
Year: 2005.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: UNIVERSIDAD POLITÉCNICA DE VALENCIA BIOTECNOLOGÍA.
Place of preparation: UNIVERSIDAD POLITÉCNICA DE VALENCIA.

Summary: SUMMARY: The ornamentals are consumer products for which there is a continuous increase in demand yen which apreéian mostly novelty and attractiveness. The breeding of ornamental plants focuses on both characters led to the ultimate consumer (eg changes in the architecture of the plant, adapting to new formats, delay senescence, modification of the architecture, color, ftagancia and shape of the flower, etc..) or characters irigidos producer (resistance to pests and diseases, tolerance to various types of abiotic stress, adapting to new production systems, etc..). The biotechnology plant puts at our disposal a number of tools to the improvement of these plants by channels faster than conventional. Our group is carrying out a program aimed at improving ornamental plants using biotechnological approaches and in this context falls this Doctoral Thesis, whose main objective has been to the application of various techniques for the production and development of new varieties of ornamental plants . As working materials have been elected three ornamental species: Codiaeum variegatum, an indoor plant attractive by its leaves in shades of green, yellow and red; Begonia rex, which is used as an indoor plant and whose main attraction lies in its spectacular leaves and Kalanchoe blossfeldiana, a pot plant is very popular for its abundant flowering. It has developed a protocol for micropropagation Codiaeum variegatum that enables the large-scale production of various cultivars. In addition, it has developed a method for the isolation and cultivation of protoplasts of this kind to address improvement programs based on somatic hybridization. We have developed efficient and reproducible methods for obtaining Transgenic Plants Kalanchoe blossfeldiana and Begonia rex through cocultivo of explant.

Author: ALVAREZ FERNANDEZ RUBEN.
Year: 2005.
University: OVIEDO [www.uniovi.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: FACULTAD DE BIOLOGIA.

Summary: It presents a system of Agrobacterium tumefaciens-mediated transformation in embryos of cork (Quercus suber L.) mature selected. We have studied the parameters that influence the efficiency of transformation, which hallándose: kanamycin at a concentration of 100 mg L-1 is a selective agent adequate, while the glufosinate ammonium (as commercial Finale ®) is not ; despite obtained chimeras, the cultivation of them for at least 3 months in the presence of 100 mg L-1 of kanamycin allows embryos to obtain homogeneous. Of the three strains of A. Tumefaciens tested (AGL1, LBA4404 and EHA105) AGL1 is the most efficient. Of the combinations of inoculum density (A600nm = 0.25, 0.5 or 1) and time cocultivo (2 or 3 days) tested, the best is A600nm = 1 and 2 days cocultivo. In relation to the type of explanto inoculated, aggregates embriogénicos provide more efficient processing embryos isolated; addition, explantos inoculated after 20 days of subculture provide the highest performance. The light during cocultivo affects the efficiency of transformation, which is higher when explantos is cocultivan with light or dark photoperiod that on the contrary, the temperature during cocultivo not influenced significantly between 22-26  ° C. Finally, the use of mutant virGN54D not significantly increases the efficiency of AGL1. There have been lines transgenic embryogenic after inoculation of different clones with AGL1 pBINUbiGUSint, pBinUbiBar and pBIN19-p35SsGFP. The two factors that play important roles in the efficiency of transformation are the choice of Agrobacterium strain (AGL1) and the time of collection of explantos for inoculation (20 days after the last subculture). Once implemented the protocol processing is optimized has obtained a conversion efficiency (ie transgenic lines obtained for total explantos inoculated) of up to 42%. It has also crioconservado in liquid nitrogen ten transgenic lines for the bar gene, and all have continued expressing once recovered.

Author: MUHIALDIN JANGI SH..
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE MEDICINA Y ODONTOLOGÍA.
Place of preparation: FACULTAD DE MEDICINA - UPV.

Summary: It has been described that histamine is an amine biogénica involved in the proliferation of different tumor and normal cells. However, in some human cancers, as in T Cell Acute Leukemia (ALL) and the Cutaneous Melanoma, its effect has not yet been clarified. In the present work we studied the effect of histamine and its antagonists in ALL cell lines, known as EMF and Jurkat, and in cell lines Melanoma called A375, HS294T, HT144 and MJOI. We noticed that in vitro treatment with histamine is not exercising any effect on the cells leucímicas, but increases the proliferation of human melanoma cells. In addition, the endogenous histamine acts as a survival factor and stimulator of cell proliferation, both in leukemic cells in the melanoma since all receptor antagonists H1 histamine used dihphenidrmina, astemizole, terfenadine and inhibited cell proliferation and induced cell death by apoptosis significantly. However, the viability of cells after treatment as normal lymphocytes from peripheral blood, melanocytes and embryonic fibroblasts, remained unchanged. To characterize the time of cell death induced by antihistamines in tumor cells, we analyzed the morphological and biochemical changes induced in the cells in response to these compounds. The morphological studies, conducted by light microscopy, transmission electron and fluorescence, revealed in the treated cells all cellular processes of apoptosis, as condensation cellular condensation of the chromatin and nuclear fragmentation. We have also detected the biochemical changes of apoptosis, as the externalization of the fosfatidlserina in the plasma membrane, DNA fragmentation, the collapse of the mitochondrial membrane, release of Citrogromo C to cytosol and the activation of caspases. Apoptosis induced antihistamines is dependent caspases and caspase-2 is the factor regulating the initiation and progression of the process apoptótico. The caspase-8 and recipients of cell death does not appear to be involved in this process. The route mitochondrial process apoptótico seems to play an important role, as we have seen dissipation of mitochondrial membrane potential, activation of caspase-9 and release of Cytochrome C to cytosol. In addition, the endoplasmic reticulum also appears to be implicated, because we have shown that treatment of cells, malnoma with terfenadine induces an increase in the concentration of intracellular Ca2 + as a result of the depletion of its deposits in the endoplasmic reticulum. This finding has been confirmed by treating the cells with neomycin, an inhibitor of fosofolipasa C (PLC), the results showed that treatment with neomycin prevented the release of Ca2 + to the cytosol and apoptosis induced by terfenadine. With these results we conclude that histamine is a factor in the survival and proliferation of cell lines studied human melanoma and leukemia. The receptor antagonists H1 histamine trigger route intrinsic process apoptótico in tumor cells but not in normal cells as you lymphocytes, melanocytes and fibroblasts. At the start of the process apoptótico induced by treatment with terfenadine, calcium plays a very important role, and the damage in the DAN is the first structural change occurred. The mechanism apoptótico is dependent on caspase-2 and fosflipasa Cy both mitochondrial route as the endoplasmic reticulum appear to be involved. Taken together, our results indicate that the antagonists H1 histamine could have a potential therapeutic application in certain neoplastic diseases.