GENETIC ENGINEERING

Author: FERNÁNDEZ RODRÍGUEZ MATILDE.
Year: 2003.
University: GRANADA.
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The enterocina AS-48 is a circular peptide produced by enterococcus faecalis, which shows a broad spectrum of action against many pathogenic bacteria transmitted by food and alterantes of them. This report has been completed characterization of the genetic region responsible for the character AS-48 cloned in pAM401, consisting of genes as-48ABCC1DD1EFGH, which as-48A is the structural gene. The characterization of the products encoded by the last four genes, obtenidamediante analysis of phenotypic insertion mutants with transposoón Tn5 and application software for high reliability, has revealed that genes as-48EFGH codify a conveyor tipo-ABC involved in the self versus AS-48. In this transport system has identified the role of As-48G, which are binding domains of ATP and protein As-48EH presenting four domains expansion membrane, while the protein As-48F could increasing the effectiveness of the conveyor. The analysis of transcription in the region as-48 made by Northern blotting with the results derived from studies complementation in trans-genes as-48ABC in pAM36e carried out, has led to conclude that the region as-48 is structured into two operons constitutive expression (as-48ABC and as-48C1DD1EFGH) although present promoters internal allowing regulated the expression of genes as-48BC and as-48EFGH. The region as-48 has been cloned in bacteria eigenvectors of ácidoláctico (plL253-81 and pEM76-81), but none of the new construction has gained equaled the effectiveness of pAM401-81 in the expression and resistance to AS-48. The transfer of the region as-48 strains of biotech interest has been done successfully in Lactococcus, Lactobacillus and E.faecium. The first two showed only a weak expression of resistance, while all strains of E.faecium processed express the character with similar efficacy to the wild strain,. Faecalis S-48. This result is of great interest applied to it isolates of food, lack of virulence determinants, so in the near future may be used in obtaining fermented foods, which are implemented without any difficulty.

Author: JARA RAMÍREZ MÓNICA.
Year: 2004.
University: AUTÓNOMA DE BARCELONA.
Place of defense: CIENCIAS.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: Previous studies conducted in our laboratory have analyzed the content of regulón LexA of gamma Proteobacteria (Erill et al., 2003). Along the same line, the present work seeks to extend the description of that regulón other families Domain Bacteria: Geobacter sulfurreducens, as a representative of the group Desulfuromonas, belonging to the Delta branch of the Proteobacteria: Fusobacterium nucleatum, Phylum Fusobacteria; microorganisms belonging to Alpha Class of Proteobacteria and gram-positive bacteria. First, it undertook to identify boxes union to LexA of microorganisms G.sulfurreducens and F.nucleatum, which was cloned and secuenciaron their respective genes lexA. After sobreexpresar and purify the respective proteins were to test delay electrophoretic and footprinting, which identified two reasons for binding to LexA: GGTT N2 C N4 G N3 PCA G.sulfurreducens and TGTATC N12 TACA to F.nucleatum . Next, we studied the distribution of in silico the reasons described in each genome. In both cases the single gene, a part of lexA, which proved to be under the control of the protein LexA the fen dinB. In G.sulfurreducens gene recA, besides not possessing the reason LexA and not join such a protein, presented a constitutive expression compared to the damage in the DNA. By contrast, the gene recA of F.nucleatum is inducible by DNA lesions although it is not regulated directly by lexA. Once described these two microorganisms addressed the study of microorganisms belonging to the Alfa class of Proteobacteria and gram-positive bacteria. Since the boxes LexA of these microorganisms already knew, was to seek those reasons in their genomes. Once analyzed the results biocomputaiconales, in each case was to validate these results experimentally choosing a microorganisms as representatives. In the case of Proteobacteria Alfa was chosen to Sinorhizobium meliloti, and as gram-positive bacterium Bacillus subtilis were elected. In both microorganisms experimental data have coincided wholly obtained in the in silico. The set of results has shown that there is a great variability in the content of regulón LexA in the Dominion Bacteria. This suggests the existence of an important horizontal gene transfer in the domain Bacteria in what refers to this regulón.

Author: CALLÉN MORÉU ELSA.
Year: 2004.
University: AUTÓNOMA DE BARCELONA.
Place of defense: ESCUELA DE POSTGRADO.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: Fanconi anemia (FA) is a genetic disorder autosomal recessive in nature and characterized by a seri of birth defects, bone marrow failure and a high propensity to acquire solid tumors and acute myeloid leukemia. The FA cells, possess a high chromosomal instability both spontaneous and induced agents inductors links Crusaders. Other features include cellular responsiveness to oxidative damage, failures in the cellular cycle, high levels of apoptosis regulation of transcription and poor telomeric dysfunction. So far, it has been described that there are 12 genes that can cause different disease, but not all of them have been cloned and characterized. Among them, NAFEC is the most frequently mutated found among the population and presents a wide spectrum of mutations, large deletions being a major. A deeper understanding of the biology of this disease will allow us to gain a more comprehensive view of the repair adaño in DNA in response to specific agents as well as a better understanding of the molecular interactions that exist between different genetic syndromes and have an indisputable diagnosis and therapeutic value. In this thesis work have been raised four main objectives and general, which are: To establish potential links between the característics hematological disease and some of the cellular characteristics such as shortening telomérico and numerical or structural chromosomal abnormalities. * Characterize genetically to people affected by Fanconi anemia Spanish. * Exploring the role of the FA route in biology and maintenance telomérico. * To study the molecular biology of the FA route in response to genomic damage. With the studies carried out, there has been a shortening telomérico accelerated in Fanconi patients accompanied by an increase in the number of fragments extrateloméricos and mergers, although this excess mergers terminals occurs independently of the presence of TRF2 in telómero. In addition, this reduction is not directly related to the severity of the disease at blood. If it relates, however with the blood disease frequency of spontaneous chromosome breakage. Moreover, it has been exhaustively studied and characterized the population Fanconi Spanish, with an emphasis on the carriers of mutations in the gene NAFEC and the gypsy population, possess more frequent carriers of mutations in NAFEC globally. One important tip drawn from these studies is the involvement of history H2AX within the Fanconi route in response to the irradicación with UV-C.

Author: MORALES GERMAN MÓNICA.
Year: 2004.
University: AUTÓNOMA DE BARCELONA.
Place of defense: ESCOLA DE POSTGRAU.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: The melon (Cucumis melo L.) is a vegetable of great economic importance in which there are many genetic improvement programs. One of the goals of these programs has been the incorporation of genetic resistance compared with pathogens that cause significant economic losses in this crop. The virus of necrotic spots of melon (MNSV) is a Carmovirus endemic to the family of Cucurbits grown under greenhouse. The only resistance described so far compared to MNSV is conferred by the recessive gene nsv melon. This resistance is effective compared to the vast majority of known isolates of the virus. Little is known about the mechanism of resistance from the interaction MNSV / nsv. Thus, the study of this resistance through the cloning of the gene nsv will allow us to understand the strategy used by the virus to infect the plant and this information may be used to control this virus. Little is known about the mechanism of resistance from the interaction MNSV / nsv. Thus, the study of this resistance through the cloning of the gene nsv will allow us to understand the strategy used by the virus to infect the plant and this information may be used for better control of this virus. The main objective of this work has been the cloning of the gene nsv through positional cloning strategy. First, the gene nsv was mapped in the group ligamiento 11 of the genetic map of melon generated in our laboratory. Then, a population of 69 LDHs was used to obtain markers AFLPs and RADPs linked to the gene nsv through the strategy of "Bullked Segregant Analysis." Two high-resolution genetic maps were constructed in two different populations, an F2 of 408 individuals and BC1 of 2727 individuals. The markers flanqueantes and that cosegregaban with the first gene population LDHs were converted into markers PCr to be used easily in these large populations. In addition, two genebanks of BACs melon, generated from a genotype resistant MNSV and another from one subject, were discussed with these markers and earned a physical map of the region of the gene nsv through chromosomal walk. The population F2 markers flanqueantes gene 1L3 and 5B3sp6 were separated by 4 recombinants, while the population BC1 markers flanqueantes were 1L3 and 10O16sp6 to 20 the number of recombinants between them. The physical distance relationship / genetic distance in this region of the genome of melon was estimated at 228 kbit / cM, optimal relationship to carry out the positional cloning of the gene. Finally, it has come to identify the BAC 1-21-10 containing physically gene nsv. The existence of microsintenia in lar region of the gene nsv with a region of the genome of Arabidopsis allowed the identification of a candidate gene for nsv for quickly. We identified the factor initiation of translation 4E (eIF4E) as the candidate for nsv, which had previously been identified as responsible for the resistance to potyvirus pepper, peas and lettuce. The development of a PCR marker from eIF4E melon confirmed that it cosegregaba with the gene nsv in populations F2 and BC1. Next, the complete sequencing of cDNA of eIF4E revealed that the only difference between allelic sequences resistant and susceptible alleles of two lay in a nucleotide, which corresponded to a change in amino acid position 228 of the protein: one in leucine genotype resist 8 entity aM 365 NSV and a histidine in the two genotypes susceptible. Probably, this mutation in the eIF4E is responsible for the resistance in PI161375.

Author: CALERO NIETO FERNANDO JOSE.
Year: 2004.
University: CÓRDOBA.
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: F. Oxysporum infection secret for many extracellular enzymes that are responsible for degrading the plant wall of their host plants. An important group of these enzymes is the complex xilanolítico which is responsible for degradarel polímerode xilanoque apareceen fraction hemicelulósicade wall plant. It has isolated the gene foxlnR that encodes a protein containing a DNA-binding domain of the type of binúcleo zinc. This gene has a basal transcription that transiently induced in the presence of a source of carbon inducing as xilano of oat bran and who are being repressed in the presence of repressive carbon sources, such as glucose. The transcript of foxlnR not affected by the pH quantitatively environmental although his induction occurs at different times depending on the pH of growth of the fungus. The inactivaciónde foxlnR leads to the inability to produce halo hydrolysis of xilano colored solid in the middle and lower activity in terms of inducing xylanase in liquid medium. Unlike what happens in the wild strain in the mutant deficient in the gene foxlnR genes xy12, xy13, xy14, xyl5 And xyl6 not increase their transcript in terms of induction. These data indicanqueel product of gene foxlnR serving as reguladortranscripcional complex xilanolítico. Unlike what happens in species of the genus Aspergillus, overexpression of the gene foxlnR does not involve an increase in the expression of genes xy13, xyl4 And xyl5 in F. Oxysporum but on the contrary it decreases while his induction begins earlier. The product of the gene foxlnR exerts its effect by binding directly to the stream 5'GGCTAA 3 'presence in the region promotorade the geneses tructurales, comose has demostradoen this work in the case of gene xy14. It proposes a mechanism for the regulation of genes that make up the complex xilanolítico involving the performance of the product of the gene foxlnR, complejoHapy of / s protein / scapaces recognizing the secuenciacon role repressor AGAA. This paper demonstrates for the first time the involvement of genes that make up the system xilanolítico since altering levels of gene foxlnR causes a disturbance of behavior patotípico F. Oxysporumf.sp. Lycopersici.

Author: JOVER GIL SARA.
Year: 2004.
University: MIGUEL HERNÁNDEZ DE ELCHE.
Place of defense: UNIVERSIDAD MIGUEL HERNANDEZ.
Place of preparation: UNIVERSIDAD MIGUEL HERNANDEZ.

Summary: To make contributions to the development of causal analysis of the leaves of plants, we have obtained and studied various mutant strains of Arabidopsis thaliana submitting alterations of the leaf morphology, which we called incurvata (icu) and Rotunda (rum) . The leaf blades of vegetative mutants were icu recurva directly into the beam, instead of being almost level as that of the wild strains, suggesting that some process associated with the specification or the maintenance of dorsoventralidad leaf is altered. The leaves of the mutants rum are wider and rounded to the wild, which is an indication of the disruption of the division or expansion of the cells that contribute to organogenesis leaf. We have characterized genetics and molecular 9 recessive mutations belonging to groups de'complementación ICU8, ICU9 and ICU15 establishing through positional cloning that are new alleles hipomorfos or none of the genes HYPONASTIC LEA VES1 (HYL 1), ARGONAUTE1 (AGO1) and HUA Our alleles hen1 and hyl1 are allegedly invalid and caused phenotypes similar to those previously described. The alleles ag01-51 and ag01-52 are hipomorfos and causing radialización and adaxialización partial or total vegetative some leaves. Leaves not radializadas of these mutants ag01 show a transition between the stalk and limbo much more gradual than that seen in wild type. In addition, the epidermal cells of peciolos of mutants ag01-51 and ag01-52 show morphological features characteristic of the leaf blade. These results indicate the involvement of the gene AGO1 in the specification and / or the maintenance of dorsoventralidad and proximodistalidad of the blade. We molecularly characterized genetics and two alleles insercionales, recessive and probably zero (icu4-3 and icu4-4), and two off, semidominantes and gain function (icu4-1 and icu4-2) gene ICU4, which has positional analysis indicated that encodes ATHS-15, a transcription factor of the family Homeodomain / Leucine Zipper 111 (HD-Zip 111). alleles icu4-3 and icu4-4 not cause any mutant phenotype, suggesting that the gene ICU4 and some another member of her family are functionally redundant mind. The alleles icu4-1 and icu4-2 are carriers of a mutation in a sequence complementary to that of microARN miR165 and miR166, and cause an increase in the concentration of mRNA gene ICU4 which is especially evident in the leaves. The mutants icu4-1 and icu4-2 presented vegetative curvature of the leaves, delayed flowering and the transition between juvenile and adult leaves, and aberrations in the development of root and stem, as well as an insensitivity to moderate ABA yal NaCI during germination. These results indicate that ICU4 is negatively regulated by a microARN and participating in the development of root and stem vascular channels, in the role of meristemos and in establishing the polarity leaf. We conducted comprehensive analysis of gene expression in plants ag01-51/ag01-51 and icu4-1/icu4-1 through hybridization on microarrays validated by quantitative RT-PCR, we have identified genes directly or indirectly regulated by AGO1 and ICU4. It is worth noting that among the genes deregulated in mutant icu4-1 are some involved in the responses to stress and in the metabolism and signaling of certain hormones, including the ABA. We have achieved double mutant alleles of synergy between several of the genes AGO1, HEN1, HYL 1, HASTY (HST), and ICU 8 4 that co 4ab nfirman genetically that the functional relationship is assumed between the first four, which are proven components or probable machinery in the path of microARN as well as ICU4, which is a target of a microARN. We molecularly characterized genetics and two mutants ron2, which have proven to be carriers of recessive alleles new and hipomorfos gene LEUNIG (LUG), which encodes a correpresor transcriptional which was first described his involvement in the development of the flower. Our results show that RON2 (LUG) is involved in controlling the size and shape of the leaves, as it participates in the processes of cell expansion is not polar, possibly through the transcriptional regulation of genes related to growth.

Author: Gómez Mateo Jorge.
Year: 2004.
University: MURCIA.
Place of defense: Facultad de biología.
Place of preparation: Facultad de Biología.

Summary: Mucor circinelloides is a filamentous fungus that presents a wide distribution. It belongs to the class Zygomycetes, characterized by a sexual reproduction by merging gametangios submit mushroom spawn generally cenocíticos and produce spores aflageladas and immobile. The light is imlpicada in the regulation of many metabolic processes and development in many different organisms. In fungi, the light is involved in regulating many processes, the major studies on the control of gene expression by light were carried out in the ascomiceto Neurospora crassa, an agency which has characterized this process more deeply at the molecular level in zigomicetos Phycomyces blakesleeanus and Mucor circinelloides. The strain of wild mushroom spawn Mucor circinelloides presents yellowish white in the dark, moving into a yellow hard to be illuminated due to the induction of synthesis of b-carotene. The complete deletion of the gene crgA in that ilk eliminates the requirement of light for the synthesis of carotenes in mushroom spawn leading to a sobreacumulación both in terms of light and darkness. The fact that the null mutant continue to respond to light indicates, however, that there must be other genes involved in inducing the expression d of genescarotenogénicos by light. The main objective of this thesis was therefore identify other genes involved in regulating the synthesis of carotenes by light. Among the candidates will be considered gene homologues crgA, addressing his counterparts characterization and genes to genes wc-1 and toilet-2 N. Crassa and its subsequent characterization. By sequence analysis has shown that there is a duplication of the region of the genome of M. Circinelloides where the gene crgA. In the copy of the duplication has been identified analyzed the gene crgB, which encrypts a protein that has a structure similar to the domains of the protein CrgA. The expression of the gene crgB is constrained by crgA and inducible by light, with a pattern of accumulation characteristic of messenger early response genes, and that includes fotoadaptación. The promoter region of the gene crgB have been identified and repeated palindromic sequences found in other genes regulated by the light of M. Circinelloides and who might be involved in regulating gene crgB by light. Experiments were conducted interruption and silencing gene crgB, which suggested that the gene crgB plays an essential role in M. Circinelloides. By crawling a genoteca genomic DNA of the strain of wild Mucor circinelloides with probes of genes wc-1 and WC-2, has cloned the gene tzf-1 that figure with features of a protein transcription factor due to the presence two domains zinc-finger and whose speech is totally dependent on the gene crgA. By amplification by PCR with primers corresponding to the conserved region of the LOV domain of fototropinas plant, also present in the protein WC-1 of Neurospora crassa has been cloned cDNA fragment of a gene mwc-1a that figure with a protein high homology to the protein encoded by the gene wc-1 N. Crassa. The expression of mwc-1a is slightly repressed by light, being the gene crgA necessary for expression in the dark. In experiments silencing of gene expression mwc-1a were isolated transformants unable to respond to light. Analysis of these transformants indicates that the gene mwc-1a is not involved in controlling the carotenogénesis by light, as well as any gene with homology to mwc-1a is responsible for such monitoring.

Author: BURILLO SANZ SERGIO.
Year: 2005.
University: ALICANTE.
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The ability of cyanobacteria to adapt its functions and metabolism to changing environmental conditions depends on cellular components that are largely unknown. The aim of this thesis has been to contribute to the knowledge of the relevant networks of interaction between elements involved in signal transduction of stress nitrogen, as well as establish interconnections between specific regulation of nitrogen shortage and global responses to stressful situations, which limiting nitrogen is an inducer effective. It took up the search for interactions between proteins of interest (NtcA, NblS, NbIR, NrtC and IIP) of Synechococcus sp PCC 7942 and were constructed and analyzed gene banks to find proteins that interaccinan with PII in the system of dual hybrid yeast. The N-acetil glutamate kinase (NAGK) and the protein was called PipX ( "PII interactin protein X") were identified by this method. The interactions between PII-NAGK and PII-PipX, both novel were subsequently validated with different experimental approaches indicated that the first of which is conserved in organisms that perform photosynthesis oxigénica and the second in cyanobacteria. PipX interacts with NtcA, which connects the two main regulators of metabolism of nitrogen in cyanobacteria. The bioinformatics analysis and co-evolution of the interactions NblS-SipA respectively bait and prey on ballots in the genebanks built in this work suggest the possible conservation dela corresponding interaction in cyanobacteria.

Author: Abellán Martínez María.
Year: 2005.
University: MURCIA.
Place of defense: Facultad de Biología. Universidad de Murcia.
Place of preparation: Facultad de Biología. Universidad de Murcia.

Summary: The gene carD of Myxococcus xanthus is involved in the regulation of two independent processes of this bacterium, carotenogénesis and multicellular development, and is also involved in controlling about the 1% of the genes that are expressed during vegetative growth. Among them is ddvA, whose expression, very high in the wild strain, was nullified in background mutant carD. In order to find new gene products associated with CarD, we identified a number of mutations, called mutations south, which eliminate the effect of mutations carD on the expression of ddvA, not on processes carotenogénesis and multicellular development. In this paper we have examined two of these mutations, the mutation sud-2 and the mutation sud-5 prior to the cloning of the same phenotypic by complementation. The study only strains carrying mutations sud indicated and strains carrying these mutations and the deletion of the gene carD shows that none of these exercises suppressor mutations per se any effect on the processes of carotenogénesis and development of multicellular or on the expression of ddvA, and they are able to remove the effect of the deletion of carD on the expression of ddvA with the same efficiency with which eliminate the effect of other mutations carD. The effect of the mutation sud-5 is offset by the introduction into the genome of M. Xanthus a fragment of 6984 bp DNA, recovered from a genoteca of chromosomal DNA of M. Xanthus, causing a complementation apparent. The orf that seems responsible for this complementation determines an ATPase in Class AAA +, although there appears to be no difference in the sequence of this gene and its promoter between the wild strain and the strain of carrying the mutation sud-5. To study the relationship between this mutation suppressor and orf of the ATPase were carried out experiments of expression and overexpression of the latter orf showing that the gene provides a low level of expression and that its promoter is not regulated by CarD nor sud-5, and that the observed effect is not a result of increased doses of the ATPase. Test double hybrid in yeast have not identified a physical interaction between CarD and ATPase, but the latter itself, consistent with the structure oligomérica of AAA + proteins. The deletion of the gene of ATPase does not affect either the expression of ddvA, both the absence and presence of a mutation carD of mutation sud-5 or both, neither seems to have any effect on the processes of carotenogénesis and development multicellular. The suppressive effect of the mutation sud-2, which after processing various experiments, backcrossing and cotransducción seems to be linked to the locus ddvA and a distance of 10 kb, offset by the introduction into the genome of M. Xanthus a fragment of 4344 bp located 10 kb upstream of ddvA, causing a complementation apparent, which is also observed with any subfragmento smaller included in the original piece. However, the sequence of this stretch of DNA of a strain carrying the mutation sud-2 presents no differences with the same sequence of the DNA fragment of the wild strain. Such complementarity is also apparent when the fragment of 4344 bp or any of the subfragmentos tested is integrated into a site heterologous genome of M. Xanthus. Regardless, it has conducted an analysis of locus ddvA. The location of the gene ddvA downstream and coupled traduccionalmente a gene that determines a possible factor sigma of the family SCD, along with other comments, suggested the possibility that DdvA they were a factor anti-sigma. The generation of mergers Comp 8 scripcio 572 tion lacZ gene and strains carrying deletions in the two genes cited has identified two possible located upstream promoter regions of genes identified, which are regulated by the possible positive and negative factor sigma by DdvA. Both promoters are also positively regulated by the protein CarD. It has also analyzed the dependence of CarD two new potential partners factor sigma ECF-factor anti-sigma M. Xanthus, revealing such unit in one case, so they are already three systems factor sigma ECF-factor anti-sigma on M. Xanthus regulated by the protein CarD.

Author: TÍO BARRERA LAURA.
Year: 2005.
University: BARCELONA.
Place of defense: FACULTAT BIOLOGÍA.
Place of preparation: DEPARTAMENTO DE GENÉTICA - FACULTAD DE BIOLOGÍA - UNIVERSITAT DE BARCELONA.

Summary: The metaloproteínas are a group of proteins that are characterized mainly by the presence of metal atoms as functional. These metal ions are critical for proper structure, stability and / or function of proteins. Nearly a third of the total protein can be considered metaloproteínas and within this large group, include metaloproteínas (MT). These show some characteristics that distinguish it from other metaloproteínas: * The metal ion is always coordinated through links to the protein thiol the type of cisteínas present S in the polypeptide chain (Cys). * They are small molecular weight (about 12 kDa). * They have a very high percentage of Cys in its composition (approximately one third of the total amino acids), which gives them their high capacity heavy metal chelator. In mammals have been isolated for up to four genes coding for metalotioneínas (isoforms NMTI, MTII, MTIII, MTIV). These genes are all in the same chromosomal region, forming the "cluster metalotioneínas" which is interpreted as the result of evolutionary processes of gene duplication. In the human species, some family (MTI) still has a higher degree of duplicity, double subformas (MTIa, MTIb, etc.), which globally is unknown functional significance. In other mammalian species, such as the mouse, has been described only the existence of one member of each family (MTI to MTIV). There is a great similarity between the different TM isoforms of the same species: mouse differences aminoacídicas ranging between 15 aa (MTI-MTII) and 27 aa (MTI-MTIV). This similarity is accentuated between the same protein from different species (between MTIV mouse and the human there are only 4 substitutions of aa). In contrast, there are major differences in the scope of the patterns of expression of these isoforms. While MTI and MTII summarizes ubiquitously, although mainly in the liver and kidney after metal poisoning, MTIII and MTIV have a specific expression, sintetizándose only in the nervous system and in the stratified squamous epithelium, respectively. All MT are expressed simultaneously in decidual tissue. In the working group had previously studied the capabilities coordinativas of ITNs and MTII, so given the differences in sequence and pattern of expression presented, it was considered appropriate to also consider the MT4 and compare their performance with their predecessors. Since the metalotioneínas in birds insertion amonoacídica in the same place as MTIV, and bearing in mind that studies of evolution put MTIV closer to them not MTI is the proposed capacity study coordinativa of ckMT (metallothionein chicken ) and its domains, in order to ascertain whether more resembled the behavior of MTV or MTI. The MT system of Drosophila melanogaste comprises 4 isoforms (MTO, MTN, ERM, BAT) which is known characteristics coordinativas of the first two. Given the total ignorance about possible interactions of MT's was considered timely study Two-Hybrid with the MTO and a library D.melanogaster adult. Summing up the most important data of the work are: * MT4 has preferred to coordinate monovalent metals. * CkMT behaves in a manner similar to the MT12 in the presence of divalent metal and MT4 in the presence of monovalent metal. * Cl-ions and S2- appear to have an important role in stabilizing some MTs and depending on the metal are coordinating. In the two-hybrid experiments have obtendio 17 clones that are positive: * Tioredoxina peroxidase: interaction has been found by a trial GST pull-down. * Hmólog of guanine nt binding protein. * Vhac39 then was that the sequence was cloned in a position that did not match their pattern of reading which generated a protein of aa does not fall into any hitherto described. This result is considered negative.

Author: LOPEZ PEREZ ANTONIO JOSE.
Year: 2005.
University: MURCIA.
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: UNIVERSIDAD DE MURCIA.

Summary: The report presented research addresses two main objectives: 1 - Develop a protocol for regeneration of plants vine by embriogenesis somatic. 2-To establish the basic parameters for the transformation of embryogenic callus cells through cocultivo with agrobacteium tumefaciens. The results have allowed the first goal to establish a protocol for regeneration of plants vine by somatic embryogenesis in three varieties of table grapes (crimson seedless, sugraone and gift Marian). The type of explanto (anthers or ovaries), the addendum to the culture medium of activated charcoal and morphology of somatic embryos differentiated from embryogenic callus are the three factors that have affected the efficiency of the protocol for regeneration. Once embryogenic callus and established the protocol for regeneration, addressed the transformation of plant cells using agrobacterium in crimson seedless varieties and sugraone. The establishment of a procedure with a high conversion efficiency was divided into three phases: 1-Establishment of the appropriate concentration of the antibiotic kanamycin. The experiments showed that tripe embriogénicos of crimson seedless and sugraone showed different sensitivity to kanamycin. While in the crimson seedless variety is inhibited the differentiation of embryos with 20 mg / l kanamycin, in the range sugraone took 50 mg / l. 2, - Influence of strain agrobacterium and the density of inoculum. Studies transformation dealt with two different strains of agrobacterium (EHA 105 and C58 (pMP90)). Likewise the two strains were used at different inoculum densities (DO600 = 0.06 and 0.2). In all cases, the strain EHA 105 showed greater efficiency and showed that the density of inoculum more efficient was different depending on the variety (0.06 in crimson seedles and 0.2 in sugraone). 3-Effect of culture method. Experiments conducted at this stage concluded that the cultivation of embryogenic callus after infection and cocultivo with agrobacterium membrane on enhancing the efficiency of the transformation process. The growing callus embrogénico on membrane allowed to recover more of transgenic embryos and regenerate finally Transgenic Plants crimson seedless and sugraone.

Author: PORTA PELAYO JAVIER.
Year: 2005.
University: MÁLAGA.
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS, UNIVERSIDAD DE MÁLAGA.

Summary: S.senegalensis is a species with high economic interest which is in the initial stages of their crop. Therefore, there is need for molecular tools to study the genetic characteristics of their breeding stock, as well as their evolution in the course of successive generations of cultivation. The objectives of this work are: 1-Obtaining demarcadores microsatellites for the species S.senegalensis. 2-Valuing these tools for the characterization of genetic breeding stock of a company in the sector. 3-Studying the genetic variability of different players with different stocks reproductive behavior. 4-player Redeployment stock of a company in the sector in five tanks estimators put through kinship, thereby optimizing resources stock. 5, - Development of a PCR multiple automatic tool for application to the study of pedigree in this species. The findings and conclusions are presented below. 1, - have been isolated 15 loci microsatellite species Solea senegalensis and other 3 adapted Solea solea to study s.senegalensis, which represent a useful tool for the study of genetic and natural populations cultivated. 2 - The complete for the 5 loci: AF441385, AF441387, AF441389, AF41390 and AYA426693 has shown characteristics appropriate to conduct genetic analysis of pdigrí under culture conditions. 3-pedigree studies conducted over a period of putting in a company, show that one female and two males have been responsible for more than a million descendants, which gives an idea of the high reproductive capacity this species. 4 - The group formed by the 8 loci: AF441385, AF441387, AF441388, AF441389, AF441390, AY426692, AY426693 and AF173849, have proven useful in the characterization of genetic groups both home grown and wild. It has also proved its worth in the estimation of the values of kinship in the absence of data pedigrees. 5 - The genetic analysis of several groups of breeders captured in the Bay of Cadiz show to be representative samples of a wild population homogeneous. The genetic characterization of this population through 8 loci revealed high values of genetic variability, with an average number of alleles per locus of 15.1 and a hetrocigosidad expected 0.84. These values are in the range of those described in various marine species. 6-Genetic analysis of groups of people born in captivity and used as players in several facilities devoted to the cultivation of S.senegalensis show values of genetic variability much lower than those exhibited by individuals of the natural environment. This drastic reduction in the levels of variability is due to the incorporation of breeding stock to a high number of individuals with close ties of kinship. 7 - The widespread use of individuals born in captivity, the strong effects of selection and genetic drift caused by artificial conditions of cultivation, ignorance of the kinship relations between individuals, the high reproductive capacity and trafficking of animals between facilities , resulted in a significant deterioration of the genetic variation in much of the stock players employed in the operation in the investigation of this kind. 8 - The significant loss of genetic variation of these home grown stock can be adversely affecting the adaptive capacity of these groups. In addition, the inclusion in stock breeding of a large number of individuals from severe bottlenecks can cause a substantial increase dela inbreeding in the next generations causing 8 Ndo efec 59b cough adverse derived from homocigosis and therefore unsuitable for laying groundwork the future of the crop of this species. 9 - The use of these genetic tools to solve many doubts cultivation of this species, and in their reproductive behavior. Such knowledge would enable us to carry out a thorough control of reproduction and consequently make a proper selection of individuals who are used as breeding, so as to ensure the conservation of existing genetic resources. 10, has been developed an automated tool for the analysis of 5 loci microsatellite through multiple PCR methodology. This method reduces the time and cost of the art, minimizing handling, and hence the risk of error handling. To perform such analysis is required only a small amount of DNA obtained by non-invasive methods. Furthermore, the standardization of loci allows employees to compare results from different laboratories.

Author: RELAT PARDO JOANA.
Year: 2006.
University: BARCELONA.
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE BIOLOGÍA - UNIVERSITAT DE BARCELONA.

Summary: The increase in triglycerides and acil-CoAs can occur in cases of obesity or insulin hypersecretion and their accumulation in peripheral tissues such as muscle or pancreatic may affect the uptake of glucose and functionality of beta cells, respectively. Controlling levels of fatty acids in early stages of obesity and diabetes type2 would, as this will reduce hyperinsulinemia and insulin resistance and associated avoid lipotoxicidad caused by the accumulation of fat are metabolized is beta mitochondrial . This is a finely regulated process that presents a critical point at the entrance of the fatty acids to the mitochondrial matrix. Due to their hydrophobicity, long chain fatty acids are unable to pass through the mitochondrial membrane and require a shuttle system, the system carnitine palmitoiitransferasa. This system consists of three enzymes that catalyze the translocation of long chain fatty acids through a mechanism dependent carnitine. It is a system regulated mainly by the enzyme carnitine palmitoiitransferasa1 (CPT1), which catalyzes the formation of tioésteres of acils-CoA of long chain fatty acids carnitine, generating acilcarnitinas in the mitochondrial membrane outside. The CPT1 is a protein controlled transcriptional level and level alostérico through its physiological inhibitor, malonil-CoA. To study the mechanisms of transcriptional regulation of the CPT1B were used 380 bp region of the promoter of CPT1B human where they had already been marked an element of response to receptor peroxisome proliferators-activated (PPAR), a response to factors miogénicos of family MyoD. The results of transient transfection experiments describe a synergistic and cooperative mechanism between PPARalfa and MEF-2c in the expression of CPT1D human, as well as an effect ransactivador differential between PPARalfa and PPARdelta on promoter studied. Parallel has described the involvement of a G / C response to factors of the family Sp in the basal promoter activity studied. Parallel has described the involvement of a G / Cde response to factors dela Sp family in the basal activity of the promoter and the gene transactivation by PPARalfa, through the union of the transcription factor Sp1 to the DNA. Besides delas described functional interactions have been described in vitro physical interactions between PPARalfa and MEF-2C and between PPARalfa and Sp1. The study of the regulation of enzyme activity of CPT1 has been cloned and characterized enzyme CPT1B pork and have generated protein quiméricas between CPT1A rat and CPT1A pork, with the objective of locating regions of the protein important in determining the kinetic constants of these enzymes, especially those who can influence the sensitivity malonil-CoA of the protein. From the results it appears that the CPT1B pork features kinetic coming to a isotipo A, despite having a high amino acid identity with isotipos By that chimeras between CPT1A rat and CPT1A pork confirm the existence of a dominant positive and a dominant negative in the N-terminal region of isotipo A of CPT1. It also concludes that the C-terminal region of CPT1 behaves as a single domain functional. Finally, the paper describes a mechanism independent malonil-CoA in the activation of the system by the CPT synthetic compound C75.

Author: TÉLLEZ BESOLI NOÈLIA.
Year: 2006.
University: BARCELONA.
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The transplant of pancreatic islets is an emerging therapy for the treatment of type 1 diabetes mellitus. Previous studies in our group have shown that during the first days after transplantation of islets, where there is a loss of over 60% of the mass initially transplanted, there is an increase in expression of IL-1. The receptor antagonist of interleukin 1 (IL-1Ra) is a natural antagonist that competes with the IL-1 to interact with the receptor type 1 but does not initiate the signal transduction. The assumptions on which it has based this work is that proinflamatoria cytokine, IL-1, is involved in the transplant failure. Appointing the overexpression of IL-1Ra as a strategy to improve the prognosis of transplant singénico of pancreatic islets. Thus, the overall objective of the study is to determine if overexpression of IL-1Ra in the islets pancréaticos protects beta cells from the deleterious effects of IL-1 on the islets in culture and improves prognosis transplant. For the entire study period were used islets from Lewis rats that were infected with 6.26 x 106 pfu of adenovirus that codified by the green fluorescent protein gene dela (Ad-GFP), the beta-galactosidasa (Ad-LacZ) or by the atagonista receptor IL-1 (Ad-IL-1Ra). 48 h after infection, 100% of the blocks were infected and 30% of the cell sinsulars express the transgene. The islets were exposed to 50U/ml of IL-1beta during 48h, after this time were included in paraffin and processed for immunohistochemistry. Exposure of the islets proinflamatoria cytokine IL-1 led to an increase in apoptosis inhibition of the replication of beta cells which were reversed by the overexpression of IL-1Ra. Later, groups of 500 islets uninfected or infected with Adl-lL-1Ra were transplanted under the capsule rental Lewis diabetic rats by injection simple estreptozotocina. The islets grafts were recovered after 3, 10 or 28 days of transplantation and were processed for immunohistochemistry. The grafts islets sobreexpresaban 1Ra showed high levels of apoptosis (determined for the TUNEL technique) of beta cells, lower than grafts control at all times studied. The replication of beta cells (Determined by incorporation of BrdU) was only stimulated grafts day 10, though the islets were under conditions of hyperglycemia where it is expected stimulation delas beta cell replication. Instead, the grafts islets sobreexpresaban 1Ra showed high levels of beta-cell proliferation stimulated respect which are in a normal pancreas. Finally, overexpression of IL-1Ra led to the recovery of the body of beta cells (determined by morphometry) lost in the first days after transplantation of islets. The positive effects of the overexpression of IL-1Ra on the viability and proliferation of mass beta cells resulted in a better prognosis transplantation of islets, and that 100% of animals transplanted with 800 islets sobreexpresaban 1Ra recovered the normoglicemia to 14 days after transplantation, however only 40% of the transplanted islets control reverted to diabetes. From these resutlados concluded that IL-1 is involved in the transplant failure and must play a central role, because only block his acicón clearly improve the prognosis of transplantation.