LIFE SCIENCES (5)

Author: MARTÍNEZ DE PABLOS ROCÍO.
Year: 2004.
University: SEVILLA [www.us.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: Parkinson's disease affects 1% of people older than 50 years. Caused by the loss of the neurotransmitter dopamine its pathogenesis is not well known, although it is believed that inflammation might be involved in the development of this neurodegeneration. To explore this possibility our group developed an animal model of Parkinson's by inflammation through proinflamatorio lipopolysaccharide injection. The brain structure more sensitive to this compound proved to be the black substance, structure particularly implicated in Parkinson's. In this study we intended to find the causes of this special sensitivity, exploring the possible influence of one's own dopamine in the degenerative process induced by the injection of lipopolysaccharide: our resalados show that the action degenerative lipopolysaccharide seems mediated by endogenous dopamine through a mechanism for specific action. The discovery of this mechanism is of great importance to the development of new drugs that will allow us to combat this degenerative process and therefore against the onset of the disease. Our study also provides an explanation to the fact that one of the most treatments currently used, levodopa, can accelerate the progression of neurodegeneration.

Author: GAMANE DJAMILA.
Year: 2004.
University: SEVILLA [www.us.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: In many underdeveloped countries, bean (Phaseolus vulgaris) is regarded as a staple crop, as a fundamental source of various proteins and essential amino acids. Despite being a plant widely cultivated in the world and of great importance for human nutrition, one of its special features is its low capacity to fix nitrogen in a biological. Other species associated with this crop are symbiotically: Rhizobium leguminosarum bv.phaseoli, R.tropici, R.etil, R.giardinni bv.phaseoli and R.giardinii bv.gallicum, which have different plasmids and one of them, known as pSym, is the bearer of the genes that are directly related to biological fixation of nitrogen. The growing genomic analysis, suggests that there is direct evidence that horizontal gene transfer genetics has played a key role in the evolution of bacterial genomes. For example, the introduction of the plasmid symbiotic pRL1JI of R.leguminosarum one strain of R.leguminosarum bv.trifolii and phaseoli made that the latter acquire the ability to Nodular in pea (Dibb et al., 1984) also restores the phenotype Fix a derivative Nod when transferred into R.leguminosarum bv.viciae. The goals set for the development of these doctoral thesis have been marking and identification of pSyms and construction of hybrid strains by transferring plasmid symbiotic different genetic funds.

Author: SÁNCHEZ-PORRO ÁLVAREZ CRISTINA.
Year: 2004.
University: SEVILLA [www.us.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: Halophilic microorganisms moderates are best able to grow in media containing from 0.5 to 2.5 M NaCl. These microorganisms are capable of producing a range of extracellular enzymes large industrial interest, as proteases, lipases, DNAsas, amylases, etc.., Which are particularly stress stability saline. This thesis has been studied in detail a microorganism halófilo moderate able to produce an extracellular proteases. First performed in saline screening of southern Spain which enabled us to isolate 892 bacteria capable of producing moderate halophilic extracellular enzymes. There was selected strain CP76 as the best producer of an extracellular proteases which was characterized on the basis of phenotypic and genotypic studies and was classified as Pseudoalteromonas ruthenica CP76. That proteases called haloproteasas CP1, has been purified and characterized biochemically. This is an enzyme of 38 kDa that presents an optimal activity at 55Â ° C, pH 8.5, and is capable of acting in a wide range of concentrations of NaCl (0-4 M) with optimum between 0-1 M. By PCr reverse has amplified the gene coding for haloproteasas CP1, called cp1. The haloproteasas CP1 consists of 733 amino acids and is synthesized in the form of pre-proproteína. This is a metalopróteasa the family of termolisinas or M4. Along with the gene cp1 has found another gene, cp2, which encodes another metaloproteasas extracellular called CP2, which is composed of 614 amino acids and belongs to the family M28. Finally, flow beneath cp2 is another gene, called lipP, although it is incomplete protein that encodes presented homology with esteasas the family of a / betas hidrolasas. Finally studies have been conducted on the mechanism for secretion using Pseudoalteromonas ruthenica CP76 to secrete the haloproteasas CP1 Middle extracellular. This has been amplified by PCR reverse the gene wmpD that encodes the protein D. This protein D is part of the mechanism of type II secretion, which is the most commonly used by Gram-negative bacteria to secrete proteins on the outside. The inactivation of the gene wmpD through mutagenesis insercional interrupted secrección of haloproteasas CP1.

Author: PARADA IBÁÑEZ MARIBEL EUGENIA.
Year: 2004.
University: SEVILLA [www.us.es].
Place of defense: DEPARTAMENTO DE MICROBIOLOGÍA DE LA UNIVERSIDAD DE SEVILLA.
Place of preparation: DPTO. DE BIOQUÍMICA VEGETAL Y BIOLOGÍA MOLECULAR - UNIVERSIDAD DE SEVILLA.

Summary: This thesis has studied the importance of two surface polysaccharides of Sinorhizobium fredii HH103, capsular polysaccharide antigen type K (KPS) and exopolisacárido (EPS), the symbiosis that bacterium provides eta on soybeans and other legumes host. They have isolated genes S.fredii HH103 involved in the production of KPS (rkp genes) and EPS (exoA) and have generated mutants of this bacterium in rkp genes and / or gene exoA. The mutant genes rkp and exoA not produced, respectively, the KPS and EPS of the wild strain. This would have derived from HH103 defective in the production of each of these polysaccharides as a double mutant EPS-KPS. The symbiotic properties of these mutants were evaluated in various legumes host of S.fredii HH103. In these trials is studied for the first time the involvement of KPS in symbiosis with legumes forming nodules identified as soybeans and Cajanus cajan. In these interactions the KPS of HH103 plays an important role but not essential, for its part, the EPS does not appear to play a prominent role on, but functionally replaces the KPS. In legumes that are indeterminate nodules, as Glycyrhiza uralensis, the defect in EPS in KPS decrease symbiotic properties of HH103. However, a double mutant of HH103 in both polysaccharides was able to induce the formation of nodules some nitrogen-fixing legumes in this. This result is in clear contrast to that described in the symbiosis forming nodules indeterminate Sinorhizobium meliloti-alfalfa, where it is strictly required the presence of at least one of these two polysaccharides, EPS or KPS, for the formation of nodules fixators.

Author: MARTÍNEZ PITA INÉS.
Year: 2004.
University: SEVILLA [www.us.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: This research work has focused on the study of various aspects of reproduction of the species opistobranquiso nudibranquios Polycera aurantiomarginata, Polycera quadrilineata, Berghia verrucicornis and Berghia columbina in the area of El Portil, in the province of Huelva from the year 2001 until the 2003. The four species lay their eggs and fertilized in what are called put, the two species Polycera shaped tape that adhiriere the substrate by one of its edges and yellow while those of Berghia take the form of cord spiral which provides up to four laps. Each egg is on the inside of a capsule, there is a single egg capsule and the two species of Polycera are connected with each other by a gelatinous strand called capsule. The copulation is crossed, mutual and simultaneous and is not associated with the photoperiod, the bet hours later deposited with it and this is associated with the photoperiod. The productive capacity of the species vary between the two genres, Polycera deposits until 8 put per couple while Berghia between 1 and 2, however in each species varies depending on the size and condition parent, a larger size of the parent is the largest number to put. The longer put the presents P.aurantiomarginata with almost 50 mm and the shorter P.quadrilineata with just 20 mm. Eggs are spherical and its diameter varies between 65 microns in P.quadrilineata and 80 in B.verrucicornis, the capsules are oval and the largest show the two species Berghia. The four species presentna development embryonic-type plactotrófico and suduración varies from 6.8 days in P.quadrilineata and 8.4 in P.aurantiomarginata, velígeres are transparent and without any pigmentation, the shell is wrapped in a spiral and is not ornamented, belongs the type 1 Thompson. It has also analyzed the fatty acid composition of the set at the beginning and end of development, the higher the presentna polyunsaturated fatty acids and within this group dominate the series 3 and remain saturated. Over the year vary stocks, the highest abundances appear in the winter and spring with moderate temperatures of the water while in the summer, when the temperature exceeds 20Â ° C stocks dropping to almost disappear. P.aurantiomarginata reproduced throughout the year while the two species Berghia do in the spring mainly, the first is a cycle subanual while the latter are annual.

Author: MERCHÁN IGNACIO FRANCISCO.
Year: 2004.
University: SEVILLA [www.us.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: The leguminous plants are a family of great importance to agriculture and the environment, due in part to its ability to interact with soil bacteria of the family Rhizobiaceae to form some bodies symbiotic nitrogen-fixing atmospheric called nodules. Through this process, legumes acquire nitrogen sources that can allow a certain independence of available nitrogen in the soil (fertilizer, etc.). Colonize and facilitate certain drylands. The physiological state of both the legume as host of the bacteria determines the establishment of an effective symbiosis. There are many environmental factors that can limit the growth and activity of the nitrogen-fixing legumes. One is a high soil salinity, a problem that is increasing and that is already affecting a significant portion of farmland. Previous studies have indicated that the salinity mainly affects the plant, because the bacterial symbionts can tolerate higher levels of salinity in the soil. Despite this, different varieties of a single species of legumes that have a wide range of sensitivity compared to saline stress, and the possibilities and increase tolerance of the plants in this stress sobreexpresando certain genes, indicating that a small group gene may confer osmotelarancia a sensitive plant. In legumes however, still little known molecular markers and genes involved in regulating the response to salt stress. Based on the background commented, in this study we have concentrated our efforts on the analysis of gene expression in the roots and nodules M.truncatula, legume models for genetic studies, in response to various abiotic stress. Among the plant genes involved in response to high levels of salt in the soil, we can distinguish between two main groups, formed by the genes that encode effector elements involved in the adaptation of the plant (ion channels, metabolic enzymes ..) a second group, which includes those genes that practice in perception, signaling stress and the coordination of responses plant (kinases, transcription factors, among others) (Seki et al., 2003). In our laboratory, we tried to isolate genes regulating the symbiosis following different strategies. Thus, we have identified two genes potentially regulators, MsAPK1 (Medicago sativa Ankyrin Protein Kinase) and MsZPT2-1 (Medicago sativa Zinc finger Petunia Transcription fctor) encoding respectively, a protein kinase and transcription factor. Our work has revealed that these two genes may also be involved in the response M.truncatula to salt stress. The gene MsAPK1 part of a family of genes previously intensified both animal and plants and codifying a new type of protein kinase domain with a ankirina aminoterminal in the region. In alfalfa, its expression has been detected in the nodules and similar structures that are formed on the roots. But this gene is not tied exclusively to the nodulation since it is also expressed in other organs of plants, especially in the root during germination. The genome of A.thaliana, there are at least three gene homologues MasAPK1 to retain the same distribution intrón / exon it. For RT-PCR, we have shown two of them to a differential in the organs of the plant. In M.truncatula by contrast, the various experiments conducted have indicated that there would be no more than a counterpart of the gene MsAPK1. Thanks to the scrutiny of a bank genomic BAC M.truncatula, we have isolated and sequence the genome region where this gene. The homology between A.thaliana and M.truncatula we asylee and sequenced the genome region where this gene. The homology between A.thaliana and M.truncatula in this region genóm 8 ica s 1104 and is not limited solely to the gene MtAPK, but extends to genes next to it which indicates a high microsintenía between the two species in this region of genome. The induction by stress saline for the expression of MsAPK1 in roots M.sativa occurs within a few hours after the onset of stress, continuing for at least 48 hours, suggesting that this protein develops possibly a role in the signaling of this stress . The gene coding for transcription factor MsZPT2-1 was identified by Dr. Frugier during scrutiny differential gene M.sativa involved in the development of the nodules, showing that it was essential to the success of the symbiosis and the plants transgéncias expressing antisense gene in conformation are not nitrogen-fixing nodules. Based in addition to the transfer of this gene in yeast, conferred osmotolerancia, we studied the relationship of this gene with the response to salt stress. In the species M. Truncatula, we have identified a counterpart MsZPT2-1 and we have shown that in this plant as M.sativa, these genes are induced in response to the stress aliño roots and nodules. In the various tests conducted nodulation in the presence of salt, we were able to determine the conditions of inhibition of nodulation process in M.truncatual by this kind of stress. We have also studied the recovery process of this plant after a period of stress aliño setting their tolerance limits in vitro. Comparing these conditions limits the growth of the roots of wild plants and plant transgenic As-MtZPT2-1, we have obtained evidence that the latter is recovering more slowly after a saline stress. The results thus obtained are of MtZPT2-1 a good molecular marker of stress saline suggest their involvement in a transcriptional program designed to M.truncatula can tolerate the stress. In order to identify new molecular markers of stress saline and transcriptional program which is involved MtZPT2-1, we used two techniques of functional genomics. First, based on the tolerances of M.truncatula R108, we have built two libraries SSH (substractive supperessive hybridation) with a view to identify these molecular markers and other genes linked to the process of recovery after a stress saline. Subsequently, thanks to information provided by partial sequencing of the SSH, we have developed a macroarray to study the expression profiles of 384 genes and analyzing the differences between transcriptomas of wild plants and plant antisense As-MtZPT2-1 in response to various stress conditions saline as well as during the process of recovery of the plant. Thus, based on the results of macroarray and confirmation by quantitative PCR, we have identified at least two dozen deregulated genes in plants As-MtZPT2-1. Three of these candidates have submitted their expression profiles expected in the roots transgenic sobreexpresan MtZPT2-1, indicating that can be directly regulated by the factor MtZPT2-1. Finally, it should be noted that the analysis of the response to salt stress in plants M.truncatula that we have developed in this work may help the understanding of the mechanisms involved in the impairment of nodulation stress, and opens the doors to study in other legumes largest agro-economic usefulness.

Author: ANTUNES BARRADAS GONCALO.
Year: 2004.
University: EXTREMADURA [www.unex.es].
Place of defense: ESCUELA INGENIERÍA AGRARIAS.
Place of preparation: ESCUELA INGENIERÍAS AGRARIAS DE BADAJOZ.

Summary: With this work will help to study the growth, production and fiber quality of Gossypium hirsutum L., cultivated in ecological conditions at the perimeter of irrigation Caia (Alentejo, Portugal). With this objective install a trial with 6 varieties cycle medio-cuarto to medio-longo, planted in 3 dates of planting, in the Farm Comenda (38Â º 54'N, 7Â º 03'W) in the years 2002 and 2003 . There has been a difference between varieties in the percentage of germination and in the testes cold (18Â ° C, 7 days) and standard (30Â ° C, 4 days), recording the seeds of 'Celia' and 'Sonia' highest rates of force. The growth rate relative presented a marked downward trend, right from the early stages of plant development, while the rate of net assimilation presented an upward trend until the end of July, after declining continuously until the end of the cycle, thus the increase folia area index (LAI) and the reduction of temperature and the number of daylight hours. There had been no differences between varieties and planting dates in periods of the year when there were peak values of LAI and the rate of growth of the crop (CGR), with maximum LAI with a time lag of 10 to 31 days relatively CGR maximum. The knot insertion of the first fruiting branch (NFFB) than was found in the variety 'Celia' and the date of planting earlier. The cutout (NAWF = 5) was observed between July 20 ( 'Celia' in the early sowing) and August 19 ( 'Flora' on the late planting), reducing the delay from the date of planting, in all Variety, the number of days after seeding (DAS) until cutout. The damage caused by Earias sp. Heliothis and / Helicoverps, pests that led to a high loss of early forms fruitful, resulting in numbers DAS until NWF = 5 relatively high, characteristic plant a burden successful enough to transfer the equivalent Available vegetative growth. That fact, combined with thermal conditions unfavorable to the growth and maturation of the capsules formed later, resulting in reduced harvest rates and low retention, primarily in the top positions and fruiting in simpódiso located in low over the canópia. There have been significant differences in the production of raw cotton between varieties, with the variety 'Celia', which also showed that more characters precocity, atingir productions higher. What dela late planting date, but allow better conditions for the emergence and early growth of the plants, reduces the growing season available, the end being a negative effect on the production of algodón-bruto. Thus, the production of cotton in the region of Caia requires the use of short-cycle varieties and planting dates early, being decisive achieve high retention capsules at the top and branches sympodial so that the period of growth and maturation of capsules match environmental conditions are more favorable. In general, the fibers would be classified as Strict Low Midding presenting a fineness of normal thick, maturing very high, long fibers high degree of uniformity and low and short fibers, with a very strong and very low elongation.

Author: ARJOL SERRANO JOSÉ LUIS.
Year: 2004.
University: ZARAGOZA [www.unizar.es].
Place of defense: ESCUELA DE CIENCIAS DE LA SALUD.
Place of preparation: ESCUELA DE CIENCIAS DE LA SALUD.

Summary: Programs to improve the strength and speed of football in the literature are often difficult to carry out. The objective of this study was to compare the effects of two different training programs to improve the strength and speed of football. PROGRAM TO combined exercises with loads and jumps. PACKAGE B only used various jumps. Both programs incorporate careers submáximas and sprints, in greater numbers for the program B. He joined a group C (control), which only made the usual football workouts. The study duration was seven weeks, and an initial assessment and a final test through jump (SJ, CMJ, Ablakov, Drop, Jump from 40 cm Rebound jump 15 "and CMJ% with load 30%, 45% and 60% of body weight) and test speed (30m sprint with shots from time to 15m and 5x10m race return). characteristics of the subjects participants (n = 92 initial and n = 60 final) jurisdiction (mean +-TD ) or to the age 21.46 + -3.38, 22.04 + -3.50, 22.09 + -3.73 (years); weight 75.05 + -6.17, 75.67 + - 06.30, 75.73 + -5.68 (kg), height 177.59 + -6.13, 177.97 + -5.00, 178.15 + -5.92 (cm), for the groups A, B and C respectively. Tests hops: both groups A and B, improved in all of them, but a significant (p less 0.05), ABK, DJ (with a tendency to an increase in contact time) and CMJ% with all charges for the group A and SJ, DJ (with a tendency to reduce the contact time) and CMJ% with loads of 30 to 60% for group B. Group C did not change significantly (p greater 0.05 ), in none of them. Tests speed, both groups A and B, all of them improved, albeit significant (p less 0.05), the test 5x10m group A, while group B did in all of them (tranches 0-15m, 15-30m, 30m total and 5x10m). A training program based on various jumps, specific, it can improve the speed of the player. training programs for football, aimed at improving the explosive strength and speed, should include a high number of sprints. Tests best suited to assess the explosive strength and speed of the players would be the CMJ, ABK, DJ40, CMJ with loads not exceeding 50% PC and the two tests speed 5x10m and 30m.

Author: Galbis Martínez Liliana.
Year: 2005.
University: MURCIA [www.um.es].
Place of defense: Facultad de Biología. Universidad de Murcia.
Place of preparation: Facultad de Biología. Universidad de Murcia.

Summary: Myxococcus xanthus is a bacterium Gram-negativa that responds to light blue to produce carotenoids, pigments that protect cells from the harmful effects of radiation light. In this process they are involved a series of genes that determine both enzymes carotenogénicas as regulatory proteins. In this latter group belongs the product of the gene carF, first identified in this work, whose null mutants are unable to produce carotenoids. CarF serving thus as a positive regulator of the carotenogénesis induced by blue light. Mutations in the gene carF not affect, however, other cellular processes other than the carotenogénesis. Genetic analysis by epistasia indicates that the protein CarF exercising its regulatory function in the early stages of the response mechanism in light blue, acting either as a recipient of the light signal transducer or as the same. Somehow, CarF causes inactivation, mediated by the light of the protein CarR, a factor anti-sigma. In the darkness, CarR inactivates a protein CarQ, a factor sigma belonging to the family ECF (Extra-cytoplasmic function) that it is necessary for the expression of the structural genes of carotenogénesis. It is worth noting that in trials dual hybrid in Escherichia coli, it is observed physical interaction between CarR and CarF and between CarR and CarQ. This work also examined the interaction between molecules CarR or CarF. Our results indicate that both proteins can unite themselves, opening the possibility that CarR and CarF part of some kind of macrocomplejo in which forms homomultiméricas of both protein. It also showed that CarQ interferes, somehow observed in the interaction between proteins CarR and CarF. Through mergers transcripcionales various lacZ gene fragments carF, was protected the promoter region of the gene, their levels of expression and its regulation. These studies revealed that carF expressed moderately and regardless of the state of lighting or stage of crop growth of M. Xanthus. Using mergers also the lacZ gene was identified a fragment containing the transcription terminator gene carF. The predicted amino acid sequence for the protein CarF listed four possible areas transmembranales. That CarF is housed in the membrane is evidenced by its detection in the insoluble fraction of cell extracts and tests topology with mergers traduccionales the lacZ gene. The topological analysis indicates that the first and third domain transmembranal are oriented space periplasmático, while the second and fourth would be oriented cell cytoplasm. The protein CarF is similar to a family of proteins called "Kua" of unknown function, which appear in both prokaryotic and eukaryotic organisms, being more similar in general to protein "Kua" animals. The pattern of hydrophobicity of CarF is conserved in the other proteins "Kua", as well as several domains rich histidine residues in positions yuxtamembranales. In this work we have developed experiments complementation of a mutant of M. Xanthus in the gene carF with genes Kua of Homo sapiens, Drosophila melanogaster and two of those identified in Arabidopsis thaliana. The complementation tests also were conducted with various versions of truncated protein CarF, as well as variants with point mutations in different histidine residues, preserved or not, in the rest of the protein "Kua." Different complementation tests showed that only the C-terminal 40-amino acid fragment (of the various tested) is dispensable for the right activity CarF. The replacement of several histidinas conserved in all or some protein "Kua" by the amino acid alanine produced inactive forms of the protein, 8 while 81e s that the replacement of a histidine that is only present in CarF, did not affect their business. It has carried out the purification of the protein CarF. It used extracts of E. Coli sobreexpresaban protein and affinity columns formed by Cobalt ions. CarF binds directly to this metal, possibly through their coordination by histidine residues. The protein CarF purified was used to obtain antibodies polyclonal anti-CarF used to locate subcelularmente a protein through tests Western-blot. It also carried out fluorescence microscopy techniques to determine the subcellular localization of both CarF as CarR. The system CarF-CarR signal transduction bright, it might be mediated by a transport mail, much like as in other biological systems, where the electrons are shrinking, mainly waste tyrosine, tryptophan and cysteine. The functional significance of a C-terminal tyrosine residue of CarR in this case flow of electrons is analyzed through its replacement by amino acid alanine and phenylalanine (similar to tyrosine, but without the reactive hydroxyl group). The results showed that the waste does not mediaba transmission of the light signal from CarF to CarQ.

Author: López Serrano Daniel.
Year: 2005.
University: MURCIA [www.um.es].
Place of defense: Facultad de Biología.
Place of preparation: Facultad de Biología.

Summary: Marinomonas Mediterranean is a marine bacteria, Gram negative, which synthesizes melaninas from L-tirosina. It is the first procariota described expressing all enzymatic activities PPO characterized so far. However, the enzymatic activities typical tirosinasas like cresolasa and catechol oxidase are specifically activated in the presence of SDS in cellular extracts. Treatments bacteria with the agent mutagenic nitrosoguanidina generated mutants involved in the enzymatic activities typical tirosinasa activatable by SDS but retain the other activities (DMPO, SO, TH, OJ). These mutants, as cepoa ng56 are unable to produce melanin. Subsequent testing biochemical both purified enzymes separately. The tirosinasa presents activities cresolasa and catecolasa typical of tirosinasas. It is activated by SDS. This enzyme is found mostly in the soluble fraction of cell extracts. There is another additional PPO enzyme associated with membrane with a broad affinity for different substrates. It dominates in the laccase activity, but also possesses a degree of TH activity and OJ No activatable by SDS. In order to try to clone the gene coding for a mutant tirosinasa was obtained by transposition of this bacterium which is called T101. The transposón truncated gene that codified the enzyme multipotente membrane, which allowed the cloning and sequencing of this gene. The gene was named ppoA, and was the first case described cloning of a bacterial laccase. The fact that the mutant T101 is capable of producing melanin indicates that PpoA is not the main enzyme responsible for melanogenesis in Marinomonas Mediterranean. This thesis has obtained a second mutant amelanogénico through transposition called T105 allowed to clone the gene that encodes (ppoB1) tirosinasa soluble enzyme activated by SDS. This gene is forming an operon with another gene (ppoB2). The expression of both genes is required for the activity tirosinasa and for the synthesis of melanin in Marinomonas Mediterranean. PpoB1 is mutated in strain ng56, giving rise to an early truncation of the translation causes tirosinasa and a non-functional phenotype amelanogénico for this strain. PpoB2 is mutated in strain T105 by inserting a transposón leading to a strain also amelanogénica media not fortified with copper. The alignment of the sequence corresponding to the active center of PpoB1 with other sequences tirosinasas and catechol oxidasas allows postulate an extension in the consensus sequence for the centers connecting copper increased the number of conserved residues including the 6 histidinas that make up the two copper centers and some aromatic amino acids essential for the activity. PpoB2 encodes a protein that shows similarity to proteins of the group COG5486, predicting be hypothetical membrane protein binding to metals. The recovery of activity in the mutant T105 has shown that it is a chaperone copper responsible for transferring copper PpoB1 when the concentrations of copper in the cytoplasm are low. Thus tirosinasa can rebuild its core assets and be functional. By merging with haemagglutinin and Western Blot against it, it has been shown that PpoB2 is an integral membrane protein of Marinomonas Mediterranean belonging to the group COG5486 related to the uptake of metals. It has perfected the technique of genetic complementation in Marinomonas Mediterranean. Using transposons can complement mutations in ppoB1 or ppoB2 introducing wild copies of the genes for 8. With is 36d ta technique directed mutagenesis and through it has been shown that the N-terminal end rich histidinas, why MxxxMM and why CxxxC are essential to the functionality of PpoB2.

Author: Camps Febrer Maria Carme.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: Hospital Santa Creu i de Sant Pau.
Place of preparation: Instituto de Bioquímica Clínica.

Summary: The adrenoleucodistrofia linked to the X (X-ALD) is a metabolic disorder of genetic origin that is characterized by a progressive demyelination of the central or peripheral nervous system, according to the different clinical forms, accompanied by an adrenocortical insufficiency. Patients showed an accumulation of saturated fatty acids of very long chain (AGCML), and especially C26: 0, plasma and target tissues. The accumulation of these compounds is due to a defect in its process of beta peroxisomal. The genetic cause of this disease is found in the mutations affecting gene ALD (ABCD1), leading to a loss of function of the protein ALDP. ALDP is a kind of ATP-binding cassette protein localized in the membrane peroxisomal. The biochemical phenotype of patients suggests that ALDP is implicated in the amount of AGCML the peroxisome. The membrane peroxisomal there is another protein, ALDRP, which has a high percetaje identity at amino acid with ALDP. This protein appears to be some redundancy functional ALDP because of its ability to correct levels AGCML in fibroblasts of patients when sobreexpresa. Accordingly, it opens the door to a possible therapeutic strategy for X-ALD based on the overexpression of this protein. The work done in this thesis aims to provide new data on the effects of overexpression of ALDRP in vivo, as well as the role that develop ALDP and ALDRP in the metabolism of fatty acids, using mice "knockout" and transgenic as research material . The animals used include mouse Ald ko (inactivation of the gene ABCD1), the mouse Aldr ko (inactivation of the gene ABCD2), the mouse Ald / aldr ko (inactivation of the gene ABCD1 i ABCD2) mouse Wt / tg (overexpression of the gene ABCD2 , which leads to a augmento of 5-10 times the level of laproteína ALDRP in relation to WIld-type (WT)) and mouse Ald / tg (inactivation of the gene ABCD1 + sobreespresión gene ABCD2). The determination of fatty acids in tissues delratón Ald ko can detect increases AGCML, especially C26: 0, neural tissue and adrenal gland. The mouse Ald / tg, canvio presents some normal values or even lower C26: 0 in these tissues. This result demonstrates for the first time that overexpression of ALDRP is operative in vivo, in the normalization of the levels of AGCML. The determination of fatty acids in tissue from mice Ald ko, Aldr ko and Ald / aldr ko has allowed the identification of specific substrates of ALDP and ALDRP in the series of saturated fatty acids and omega9-mono-insaturados of 20 to 26 carbons long. The study of the effects of overexpression of ALDRP through mice Wt / tg, some alterations have been detected in the levels of fatty acids in some tissues from these animals. These changes, which affect fatty acids series omega 6 i omega3-poli-insaturados suggest the involvement of changes in the process of synthesis of these compounds. Through quantitative RT-PCR studies have ruled out alterations in the expression level of messenger RNA genes encoding elongasas and desaturasas (enzymes involved in the synthesis of fatty acids) which may explain the changes detected in the levels of substrates. Finally, the work presented in this thesis includes a study based on the use of cDNA microarrays, in order to monitor gene expression in mouse embryonic fibroblasts. Samples have been used for crops or Ald ko Wt genotype to identify differences in gene expression caused by the absence of ALDP, and samples of crops treated / untreated with C26: 0 to study the role of this compound in the mechanism fisio- pathological of this disease.

Author: Garrido Ocaña Maria Elena.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: Facultat de Ciències.
Place of preparation: Autònoma de Barcelona.

Summary: The iron and zinc are essential micronutrients for the normal growth of the vast majority of microorganisms, and the mechanisms for attracting these divalent cations essential elements to the pathogenic bacteria are able to promote an infective process. In addition, receiving iron proteins are located in the outer membrane of Gram negative bacteria, which makes them candidates to be used for designing vaccines. In this context, the objective of this dissertation is to deepen the study of mechanisms for attracting divalent cations of Pasteurella multocida, a pathogenic bacteria that affects a wide variety of animals causing significant economic losses in the livestock sector. This study has identified a receiver iron 60 kDa, which has been called HbpA, introducing a mechanism for regulating different from commonly used by the bacteria, as it is system independent Fur, determined that his regulation depends on Fe2 + , Mn2 + and hemina. It has been shown that the gene encoding this protein shows a shift scheduled reading resulting in a truncated derivative of 40 kDa. It has been characterized himself the role of this protein, determined that unites both hemina as hemoglobin, and that the binding capacity is maintained in both the derivative of the truncated protein. Moreover, it has studied the antigenicity of the protein HbpA and immunogenic effect, revealing that the protein HbpA and its derivative antigenic are truncated, but is not obtained protection when administered in the whole protein experiments using a confrontation experimental animal model mouse. As regards the mechanism of incorporation of zinc P. Multocida, unknown until now, have been characterized genes catchment zinc high affinity (znuABC), determined that there is a separation of 820 kb between genes znuA and znuCB and that genes znuC and znuB form a unity transcriptional. Unlike what happens in Escherichia coli and other bacteria, in which the protein Zur regulates the expression of this system of zinc uptake in P. Multocida has not been found that a protein homologous protein Zur. However, it has been established that this pathogen is the Fur protein responsible for regulating units transcripcionales znuA and znuCB, together with ions Zn2 + and Fe2 +. Finally, through the construction of mutants znuA and znuC also has shown that these genes are essential for the virulence of P. Multocida.

Author: Janer Gual Gemma.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: Centro Investigación y Desarrollo.
Place of preparation: IIQAB-CSIC.

Summary: Ninety-five percent of all animal species are invertebrates, and like vertebrates, they are susceptible to endocrine disruption. Nevertheless, there are important gaps on the knowledge of the endocrine system of invertebrates that hinder the understanding of the endocrine disruption phenomena in those species. Steroid synthesis and metabolism is one of several possible targets of endocrine disruptors. This thesis aimed to characterize sex steroid metabolism in different invertebrate species, to test the in vitro effect of model endocrine disruptors in those species, and to test the in vivo effect of model endocrine disruptors on steroid levels and metabolism in species from a selected phyla (Mollusca). The species investigated (Mollusca: Marisa cornuarietis, Mytilus sp., and/or Crassostrea virginica; Crustacea: Hyalella azteca; and Echinoderma: Paracentrotus lividus) were able to form a series of phase I metabolites of testosterone that are also formed by vertebrates, with the exception of 4-androstene-3[beta],17[beta]-diol, a metabolite formed by P. lividus and H. azteca. One of the major differences with vertebrate species was the lower contribution of hydroxylases in the metabolism of testosterone, and the metabolic fate of androstenedione in molluscs (5[alpha]-reduction instead of 17[beta]-reduction). Regarding phase II metabolism of testosterone, sulfotransferases were found at high levels in P. lividus, but low or undetectable levels were present in H. azteca and the molluscan species investigated. In contrast, the conjugation of steroids with fatty acid moieties was present in all invertebrates investigated, showing that acyl-CoA:steroid acyltransferases are well-conserved through evolution. Steroid levels and the enzymatic activities for some metabolic pathways showed differences between males and females, among tissues, and between seasons in some invertebrate species, which adds further evidence for a physiological role of sex steroids in invertebrates. Some xenobiotics modulated steroid metabolic pathways in invertebrates in vitro. Among the pathways investigated, 5[alpha]-reductase and sulfotransferase showed the highest sensitivity to inhibition/activation by tributyltin (TBT), triphenyltin (TPT) and fenarimol. Finally, the effects of the exposure to estradiol, crude oil, and the mixture of crude oil and alkylphenolic compounds in Mytilus sp. and the exposure to methyltestosterone, TBT, TPT and fenarimol in the gastropod M. cornuarietis were investigated. The type of effects induced by estradiol depended greatly on the concentration. Some of the responses observed in mussels exposed to crude oil and the mixture of crude oil and alkylphenols were similar to those observed in mussels exposed to the highest concentrations of estradiol. Those experiments also showed that esterification seems to play a key role in the regulation of free steroid levels in bivalve molluscs. The exposure to the organotin compounds (TBT and TPT) caused imposex and decreased esterified steroid levels in females of M. cornuarietis, but not in males. Methyltestosterone and fenarimol induced imposex in females, but did not lead to alterations in esterified steroid levels. Therefore, the connection between a decrease of esterified steroids and the phenomena of imposex could not be proved. The exposure to TPT inhibited 5[alpha]-reductase in female M. cornuarietis. However, neither TPT nor TBT altered the sexual dimorphism in androstenedione metabolism that exists in the digestive gland/gonad complex of this snail species. Overall, this thesis contributes to the knowledge of androgen metabolism in invertebrate species and identifies possible targets of endocrine disruption.

Author: FERNANDEZ ISERN GUERAU.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: HOSPITAL UNIV. GERMANS TRIAS PUJOL.
Place of preparation: AUTONOMA DE BARCELONA.

Summary: This Doctoral Thesis has had as its main objective the study of the genetic and phenotypic variability of the human immunodeficiency virus type 1 (HIV-1). The high genetic variability is the main feature that distinguishes the evolution of the virus's RNA other agencies possessing DNA as the genetic material. This high genetic variability of HIV-1 originated the virus is constantly subjected to a process of change, competition and selection of those genomes adapted to a particular environment. The answer adaptatiba an environmental change is not driven by a single type of viral genome, but by a series of related but not identical genomes called cuasiespecie. The exhaustive study of the variability in different populations in HIV-1 is essential for understanding the evolution of disease and to develop strategies to combat it. In this paper we present two studies, which seek to genotype of a given region (env or pol) with their phenotype. The study focuses env gene in the region V3-V5 of glycoprotein gp120 as a result of their involvement in the fundamental cell tropism. In the process of merger, the glycoprotein gp120 interacts with the receptor CD4 and with a host of chemokines, which acts as a coreceptor virus which mainly are CCR5 and CXCR4. The viral variants that use the coreceptor CCR5 are called variants R5 while those using the coreceptor CXCR4 are called variants X4. Samples of patients for this study were selected by the low number of T cells CD4 + (less than 200 cel. / Â µ l) because at this stage of the disease the selective pressures from the host immune system has decreased sharply and was unaware of the implications real in the evolution of viral variants R5 and X4 at this stage. Likewise, through genetic screening based on the use of bacteriófago lambda, identified the catalytic activities of the HIV protease-1 three viral populations that had not been previously treated with protease inhibitors. The study of these cuasiespecies becomes important to the extent that this protein is one of the main targets of treatment antiretrovírico and essential in the production of mature viral particles. The HIV protease-1 is a good model for study because with this system of screening, we can identify a large number of catalytic efficiencies of a simple and reproducible and therefore can relate to a wide range of genotypes with their phenotypes .

Author: VIÑOLY ABREU RAQUEL.
Year: 2005.
University: LA LAGUNA [www.ull.es].
Place of defense: INSTITUTO DE BIORGANICA ANTONIO GONZALEZ.
Place of preparation: FACULTAD DE BIOLOGIA.

Summary: We have studied the localization and characterization of glial cells from the visual system (retina, optic nerve, chiasm, optic tract and conus paillaris) reptile Gallotia galloti during ontogeny. We Immunohistochemical techniques using anti-GS and coexpresión with anti-GFAP as markers of cells macrogliales and histochemical techniques using lectina tomato (Lycopersicon esculentum) as a marker of microglial cells. We noticed that the expression of GS is confined in retinal cells muller as in the rest of vertebrates, conferring such a role of protector cells and neuronal that coexpresión with GFAP is limited to the feet of these cells. On the other hand, the nerve, optic chiasm and tract is the maximum expression in the adult, this expression may be necessary to maintain the levels of glutamate in a position not toxic to nerve oligodendrocytes, as has been described in mammals. In addition, we have identified three distinct cell subpopulations located in different regions, suggesting a wide variety glial. As for the existing population microglial during ontogeny, this presents different morphologies, the same as those observed in the brain of the same species and similar to those observed in the CNS of different vertebrates. In addition to discussing possible avenues of entry for the microglía to the retina and optic nerve are similar to those described in mammals and birds and propose a home mesodérmico.

Author: ALVAREZ LOPEZ CARMEN MARIA.
Year: 2005.
University: OVIEDO [www.uniovi.es].
Place of defense: DEP. BIOQUMICA Y BIOLOGA MOLECULAR.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The living organisms show rhythms that are controlled by clocks

Author: COSTALES MARCOS LORENA.
Year: 2005.
University: OVIEDO [www.uniovi.es].
Place of defense: FACULTAD DE BIOLOGIA DE OVIEDO.
Place of preparation: FACULTAD DE MEDICINA DE OVIEDO.

Summary: The ossification endocondral involves the formation of ossification centers replacing the cartilage by bone tissue. The development of secondary ossification center presents similarities with the primary ossification of the diaphysis but there are substantial differences between the two processes. The formation of secondary ossification center begins with the invasive growth of cartilage channels through non-calcified hyaline cartilage of the epiphyses. These channels provide an avenue for vessels and the precursor cells osteogénicas. The centripetal growth of the channels is linked to the expression of MMP-9, MMP-13 and MMP-14, and with the presence of cells from the line of osteoclasts, but does not depend on VEGF. The clodronate is a known inhibitor of osteoclasts. Animals treated with the bisphosphonate show no changes in the patterns of secondary ossification. The most noticeable effects, a delay in the start of the invasion and an accumulation of trabeculae in the medullary cavity. Inhibition of reabsorption does not mean the death of osteoclasts or loss of expression of MMPs.

Author: ESCOBEDO LUCEA Ma. CARMEN.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: INSTITUTO VALENCIANO DE INFERTILIDAD - I.V.I..
Place of preparation: FACULTAD DE MEDICINA E INSTITUTO UNIVERSITARIO IVI.

Author: Sàbat Bofill Maria.
Year: 2005.
University: GIRONA [www.udg.es].
Place of defense: Facultad de Ciencias, Univesidad de Girona.
Place of preparation: Universidad de Girona.

Summary: This thesis describes the structure gonadal gametogenesis and annual cycle of Scorpaena porcus, S. Scrofa and S. Elongata, to deepen the understanding of the varying degrees of specialization within the oviparismo in fish. The aim is to highlight those unusual characteristics which differ from those that have been described so far by most species ovíparas of teleósteos, and to relate the reproductive strategies of the species that are being presented. The sampling was conducted in intensively between 2000 and 2002, but continued collecting samples until August 2004, more or less often depending on the need. In total we have obtained 258 copies of S. Porcus, 119 S. Scrofa and 46 S. Elongata. S. Porcus, S. Scrofa and S. Elongata are species ovíparas ovulíparas specialist, as its structure ovarian presents a whole series of very specific traits to the rest i rare fish ovíparos studied so far: central position Rachis músculo-conjuntivo; presence stems in oócitos in growth phase secondary shortage and small size of the alveoli cortical; area radiata very thin i secretion of a viscous mass that includes eggs at the time of entry. All these features have also been observed in Scorpaena notata, suggesting that the specialization of oviparismo ovulíparo occurs throughout the gender Scorpaena. The structure testicular i spermatogenesis of these species is also very special. First testicles are of lobular unrestricted, but with some features such as the management part of cistos according to the stage of maturing germ cells that contain, and the absence of central lumen, which currently has only been observed in the testes lobular restricted type of species in the series Aterinomorpha. And secondly, spermatogenesis is the type semicística, ie cistos open before the end of the whole process i germ cells have recently developed in the light of the lobe. The placing of these species is multiple and consists of a mass of gelatinous pelagic eggs, which in the case of S. Porcus are released between the months of June and August, and S. Scrofa, from the month of July. The presence of this mass has been associated with a macho-hembra mating, so that the male sperm release directly on the mass of eggs, thus ensuring fertilization total of the same.

Author: ROCA PÉREZ LUIS.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE MATEMÁTICAS - UNIVESITAT DE VALÈNCIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: The biosynthesis of secondary metabolites in plants is regulated by factors inherent in the plant (genotype and stage of development) and from the environment (biotic and abiotic). Of all the luminous intensity abiotic factors, climatic conditions and availability of nutrients are those that primarily affect the synthesis of these natural products. The greater or lesser availability of nutrients in the soil is reflected in the nutritional status of the plant, being content in mineral nutrients in the different organs plant which directly affect the metabolism of the plant. Many of these natural compounds, including the cardiac glycosides, which are produced by plants have great interest because of its use for the development of drugs. Therefore, it is necessary to know the factors that influence the synthesis of these in order to improve their production and earn higher yields. In this Doctoral Thesis has studied the influence of biomass and soil characteristics on the nutrient content and the production of cardenólidos in sheets Digitalis obscura, so it has been analyzed and interpoblacional seasonal variation in the nutrient content , cardenólidos and level of expression of the gene Dop5, 8r in natural populations of D. Obscura. The results show the influence of genotype, the state of development of the plant and environmental conditions in the biosynthesis of these natural products.