LIFE SCIENCES, 2

Author: RUBIO SOMOZA IGNACIO.
Year: 2004.
University: POLITÉCNICA DE MADRID.
Place of defense: E.T.S. INGENIEROS AGRONOMOS.
Place of preparation: E.T.S.I. AGRONOMOS.

Summary: In this paper we describe the molecular cloning and characterization of two factors transcripcionales type MYBR1 the subfamily SHAQKYF barley, and their involvement in the transcriptional regulation of the expression of genes that are expressed during the development and seed germination this cereal. Both factors are present in both phases of the life cycle from seed, play an inducer of gene expression during the development of endosperm, running, however HvMCB1 as repressor and HvMYBSt1 as activating the expression of genes encoding hidrolasas in aleurona in grain germination. Finally, an analysis bioinformático the presence of members of the subfamily SHAQKYF in the genomes of Arabidopsis thaliana and Oryza sativa.

Author: PARRA FARRÉ GENÍS.
Year: 2004.
University: POMPEU FABRA.
Place of defense: DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD.
Place of preparation: DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD.

Summary: The work presented here, is studying the recognition of the signs that surround and define the genes coding for proteins, as well as their applicability to the gene prediction programs. The argument presented here, is also exploring the use of comparative genomics to improve the identification of genes in different species simultaneously. It also explains the development of two programs computational gene prediction: geneid and sgp2. The program geneid identifies genes encoded in a DNA sequence anonymously based on their intrinsic properties (mainly signals differential splicing and the use of codons). Sgp2 allows use of comparison between two genomes, which must be a certain distance evolutionary optimally, in order to improve the prediction of genes, under the assumption that the coding regions that are most conserved regions that do not encode for proteins. These two programs have been used for the annotation of different species in the context of international consortia of sequencing and annotation. Among the most important partnerships in which it has been used geneid can be highlighted: the annotation of the genome of Dyctiostelium discoideum and genome Tefraodon nigroviridis. The program sgp2, you can highlight the annotation of the genome of the mouse and chicken. This work also includes collaborations with experimental and computational groups, which has allowed thing coordinating methods bioinformatics and experimental validation of many previously unknown human genes.

Author: FONTAINE FABIEN.
Year: 2004.
University: POMPEU FABRA.
Place of defense: DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD.
Place of preparation: DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD.

Summary: In order to relate the structure and activity of a series of compounds, it is important to use relevant molecular descriptors. Descriptors GRIND and VolSurf belong to a new family of descriptors called free alignment. In other words, who do not need to align compounds in order to compare their fields of molecular interactions. This study has applied these descriptors for the selection of chemical reagents from a comprehensive database. The selection was done through a protocol that allows maximize the diversity of the sample and get some compounds very informative. It has also developed new ways of descriptors that are based on changes in curvature of the molecular surface. Our results indicate that the new descriptors will integrate very well in the original and GRIND descriptors that identify the effects of both favorable and unfavorable way. Moreover, it has developed new descriptors free alignment called "anchor-GRIND" using an atom in each molecule as a benchmark for comparison of the fields of molecular interactions. The descriptors "anchor-GRIND allows a more accurate and simpler that descriptors GRIND that makes them more relevant to the analysis of certain families of compounds.

Author: LAO GRUESO ÓSCAR.
Year: 2004.
University: POMPEU FABRA.
Place of defense: DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD.
Place of preparation: DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD.

Summary: The genetic diseases typically are classified into two major groups: mendelianas illness and disease complex. While mendelianas diseases are characterized by low frequency in the population and to be caused by mutations in a particular gene, complex diseases are the main health problem in developed countries and are produced by the interaction of genetic factors and environmental factors . In this case we can not speak of mutation in a particular gene, but that polymorphism in a small fraction increases the risk to the disease. In this thesis has studied the spatial distribution of genetic variability in mendelianas diseases (particularly cystic fibrosis, phenylketonuria and b-talasemia) as a complex disease (CHD) in European populations and the entire world. These results suggest that the geographical distribution of genetic variability of disease mendelianas depends primarily on demographic factors and the history of populations. However, this effect is not independent of factors selective. In particular phenomena of balancing selection can increase or decrease the genetic variation in a population depending on the time at which the event was selective. In the case of the disease studied complex, coronary artery disease, our results suggest that the spatial distribution of the polymorphisms of risk in European populations depends, as with other genetic markers, mainly in the history of populations, especially the settlement of European continent, the subsequent reexpansión after the last glacial period and the large population expansion of farmers during the Neolithic. Given that the incidence of coronary heart disease shows a pattern of geographic distribution north, this result implies that many polymorphisms will be correlated with the incidence of the disease, indpendientemente whether or not actually being involved in the etiology of coronary heart disease .

Author: MARTÍNEZ AMAT ANTONIO.
Year: 2004.
University: GRANADA.
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE CIENCIAS DE LA ACTIVIDAD FÍSICA Y EL DEPORTE.

Summary: The practice of physical exercise has been associated with the development of microlesiones muscle. His gravity and evolution are variable, depending on the intensity, duration and type of exercise performed. It is well established that the muscle suffered greater damage when the exercise involves eccentric contractions (active stretching muscle). Various markers have been proposed to determine the muscle damage, such as the creatin kinase activity in serum (CK), lactate deshidrogenadas (LDH), or concentration in plasma malondialdehyde (MDA). However, all parameters currently available are limited, and the release of muscle enzymes not clearly reflects the structural damage muscle, as has been estimated by analysis histólogico. In this study, our aim has been to identify the protein a-actina muscular skeletal, by western blot, in the serum of subjects with severe skeletal muscle damage, healthy subjects not athletes, elite athletes subjects and subject athletes with muscle injuries, so using it as a reliable predictive element, which is capable of detecting problems of muscle contractile dysfunction. In addition, we have carried out the identification of a number of other markers, such as mioglobina, CK total LDH troponin T and troponin I, in order to compare and evaluate, together with the -actina, which of them is more reliable for detecting skeletal muscle damage. The samples were obtained from a total of 134 subjects, which were distributed in each group commented earlier. These groups took four different studies: first compare the levels of markers among different groups of subjects, the second study was to compare the changes of the different markers in the various sporting disciplines studied: The third study was designed to analyzing markers vary as a party of high competition between two types of patterns sports (handball and rugby), and finally, we compare the changes over time of various markers, after conducting a test effort. Our results show in the various studies, the a-actina is the marker protein in skeletal muscle damage greater significance, both quantitatively and qualitatively, with respect to other proteins tested. Furthermore we find that a-actina is the protein that is released into the blood when there is a more precocious skeletal muscle damage, and therefore may be regarded as a candidate for early detection of skeletal muscle injury in athletes.

Author: GARCÍA CALERO ELENA.
Year: 2004.
University: MURCIA.
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: To advance the understanding of development and regionalization of prosencéfalo vertebrate, this dissertation we have followed three approaches focused on the availability of new molecular markers. The corresponding results are discussed separately in specific chapters. First, we analyzed the expression pattern of genes prezonal and EphA7 in prosencefálo chicken in early stages of incubation (2-4). We have linked this pattern of expression with the early establishment of cross boundaries in prosencéfalo. To carry out this study we have supported in the model prosomérico revised (Puelles and Rubenstein, 1993, 2003). The gene prezonal has been cloned in our laboratory based on the previously published sequence of it. The results corroborate an early establishment of cross boundaries in prosencéfalo (stages HH16-HH17). These results are presented in the first chapter and part of chapter II of this dissertation. Secondly, we discussed the establishment of a pattern ventral in prosencéfalo secondary, in particular the development of the region mamilar and their relationship with the inductive effect from the end of the previous notocorda and plate precordal. We used as a marker early in the region mamilar gene EphA7 previously described. We have seen an inductive effect of the plate precordal on the ventral region of prosencéfalo secondary resulting in a specification which follows a temporal sequence caudorrostral, namely that the regions most rostrales specified in last place. In addition, we have observed a direct or indirect link between the basal region of telencéfalo and inductive processes arising from the plate precordal. These results appear in the second part of chapter II of this dissertation. Finally, we have analyzed the pattern of expression of the gene Enc1 in prosencéfalo chicken from three days of incubation until stages of preeclosión. This has enabled us to link the domains of speech early with the formation of structures gray in more advanced stages. Also part of this review has focused on the study of the expression of this gene in the region of telencéfalo getting information of great importance as they carry out comparative studies of telencéfalo in vertebrates. We have begun, on the other hand, analysis of the pattern of expression of this gene in telencéfalo mouse. These findings are contained in Chapter III of this dissertation.

Author: Jiménez Movilla María.
Year: 2004.
University: MURCIA.
Place of defense: Facultad de Medicina.
Place of preparation: Facultad de Medicina Universidad de Murcia.

Summary: In this dissertation have studied different aspects glucídica the composition, structure, training and origin of the zona pellucida glycoproteins and that the form, with special interest in the human species. For this purpose we have developed three main lines of research, ultrastructural study of the zona pellucida and cortical granules of human oocytes and the synthesis of glycoproteins of the human and mouse ZP, biochemical and biophysical analysis of the zona pellucida glycoproteins of hamster and physiological and biochemical analysis of ZP2 and ZP3 recombinant human expressed in CHO cells. The following is a summary of the experiments and data in the three lines of inquiry. 1. There has been an ultrastructural study of the zona pellucida and cortical granules of human oocytes glucídica to determine their composition and structure. We have also investigated, ultrastructural level, the possible route of synthesis of glycoproteins that form the human ZP and structure of organelle involved in the synthesis of glycoproteins in the different stages of development of the follicles of mouse oocytes. To do this we used at cytochemical techniques of electron microscopy applied lectin-peroxidase conjugate, digoxigenina and colloidal gold in combination with enzymatic treatments. They have also been used techniques inmunocitoquímicas with antibodies directed against glycoproteins constituent of the ZP and against human protein markers organelle in the cytoplasm of mouse oocytes. Comparative studies have been performed using quantitative image analysis. The main results obtained in this study can be grouped under the following headings: A. The ZP-human MII oocytes in VG and presents lectin affinity for the following: AAA, AIA, ConA, DSA, LFA, MAA, PHA-E , PHA-L, RCA-Iy WGA. However, we found no markings in the ZP when incubamos sections ultrafine of human oocytes with the following lectin: BSA-I-B4, DBA, GNA, HPA, LTA, NAPA, SBA, SNA, STA or UEA-I. The antibody anti-ZP3 humans has a strong reactivity across the thickness of the ZP in human oocytes in VG as in MII oocytes. The human ZP was also heavily marked with antibodies anti-sialil-Lewisa and anti-sialil-lewisx in both stadiums analyzed in this study. It was not detected any reactivity with antibodies anti -Gal, anti-Lewisa, anti-Lewisb, anti-Lewisx and TEC-02 in the ZP. When ultrafine sections were treated with neuraminidase enzyme and eliminate waste acid siálico, lectin LTA, PNA and SBA presented by the ZP reactivity of human oocytes. This treatment also exposes the binding sites that recognize the antibody anti-Lewisa and anti-Lewisx. The quantitative analysis was performed with the lectin WGA and AAA, the antibodies anti-sialil-Lewisa and anti-sialil-lewisx and an antibody anti-ZP3 humans. With these results we have demonstrated that glicanos of human ZP presented as sugars terminal sequences sialil-lewisa, sialil-lewisx and Neu5Ac 2-3Gal 1.4 GlcNAc. Also, we have demonstrated the presence of sequences Neu5Ac-Gal 1.3 GalNAc and Neu5Ac-GalNAc. The data also show that the sequence polilactosamina is present in the ZP and expression of human chains biantenarias and triantenarias type N-glicanos. We found fucosa united 1.6 to GlcNAc (AAA) and fucosas contained in the sequence of the lewis ay lewis x. Lastly and unlike other spices have shown the presence in the human ZP Tn antigen (GalNAc -Ser). Through quantitative analysis of marking obtained suggest a heterogeneous distribution of carbohydrates in the human ZP. Quantitative analysis indicated that some sequences of carbohydrates as sialil-Lewisa and sialil-Lewisx was 8 encuentr 1ff8 an principally located in the region outside of the ZP. B-cortical granules were heavily marked with the lectin: AAA, AIA, DSA, LFA, MPA PHA-Ey WGA. The MAA lectina and antibody anti-sialil-Lewisx showed a weak reactivity. The marking observed in the cortical granules was not uniform. The cortical granules were not reactive to the lectin: BSAI-B4, ConA, DBA, GNA, HPA, LTA, PHA-L, NAPA, RCA-I, SBA, SNA, STA UEA-Iy other antibodies used in this study . After treatment with neuraminidase, the cortical granules showed strong reactivity to lectina NAP. In this study, we have demonstrated the presence of the following waste carbohydrates through citoquímica lectin coupled with colloidal gold particles: GlcNAc, Fuc, Gal 1.4 GlcNAc, GalNAc, Neu5Ac 2.3 Gal 1.4 GlcNAc, Neu5Ac 2.3 Gal 1, 3 GalNAc and N-glicanos complex type with chains biantenarias and / or triantenarias. We have also noticed that lectin that bind specifically to human ZP have no affinity for cortical granules confirming that the composition of the ZP different from the composition of cortical granules. Moreover also suggest that there are human oocytes in different populations of cortical granules. C. - The human oocytes in germinal vesicle (prophase I) and metaphase II were incubated with antibodies anti-ZP3 human anti-ZP total pork and anti-ZPC pork showing a strong affinity for the human ZP. In oolema of oocytes in prophase I can observe a marking moderately antibodies anti-ZP3 human anti-ZP pork and anti-ZPC pork. However, in oocytes at metaphase II, we did not see this marking. These data demonstrate that antibodies against human glycoproteins of the ZP and pig ZP recognize epitopes present in the human ZP and these glycoproteins are at the plasma membrane of oocyte suggesting that the prosecution of this glycoprotein occur at the level of oolema in oocytes in prophase I put that in oocytes at metaphase II, we found no markings. In ooplasma of oocytes in prophase I observed one marking specific antibodies in these three bodies multivesiculares and tanks of the Golgi apparatus. In the oocytes that were at metaphase II, these structures are not specifically marked. This suggests that the glycoproteins of the ZP are synthesized and endocitadas or degraded by the oocyte. D.-Sections of ultrafine mouse ovaries were inmunomarcadas with specific antibodies against organelle citoplasmáticas as the antibody anti-PDI, which recognizes the reticle endoplasmatico, and the antibody anti-GM130 recognizing the Golgi apparatus. The study was conducted in follicles unilaminares, bilaminares and multilaminares. We have seen that in follicles unilaminares the reticle endoplasmatico introduced a provision in tanks stacked to form a linear structure. In oocytes from follicles bilaminares in ooplasma found a large quantity of circular structures marked specifically with the antibody anti-PDI that were not marked with the antibody anti-GM130. In oocytes that were in follicles multilamelares not detect the presence of circular structures. However, we found, with the antibody that recognizes the protein reticulum endoplasmatico, the presence of small vesicles electrodensas marked specifically. The results, we suggest that the oocyte follicles to be found in primary bilaminares supports an important protein that affects traffic on the high presence of the organelle involved in the synthesis of proteins, as is the reticle endoplasmatico and this presents a circular shape with a diameter average 1.5 m. However, the oocyte multilaminar once they have completed their needs proteícas to cope with fertilization, reorganizes the ERs to serve as a reservoir of Ca2 + and regulate the various transits of Ca2 + that occur during fertilization may note the presence in the ooplasma the oocyte, some vesicles in average diameter of 100 nm. 2. To make the biochemical study of glycoproteins of the ZP we used as a model for testing the ZP hamster. In this study we have done, first, a study immunocytochemical ultrastructural level of ZP hamster with an antibody anti-ZP pork epitopes recognized that the region outside of the ZP and compare the pattern in different species. For this we use Immunohistochemical techniques at the level of electron microscopy and quantitative comparative studies have been carried out through image analysis. Subsequently, we conducted the purification and characterization of glycoproteins of the ZP hamster by SDS-PAGE electrophoresis techniques and western-blot with antibody anti-ZP pork. To determine which are the epitopes recognized by this antibody perform inmunoprecipitación and digestion with the enzyme N-glicosidasa F. Finally secuenciamos band that recognizes the antibody anti-ZP pork by mass spectrometry. The results are reflected below. A. The ZP of oocytes from different ovarian follicles hamster, pig, rat and mouse was specifically marked with the antibody anti-ZP pork. Note that the inmunomarcaje was distributed throughout the thickness of the ZP of ovarian follicles pig, rat and mouse, however, we see a different pattern in the distribution of marking obtained in the ZP hamster ovary. The ovarian follicles preantrales showed a uniform dialing throughout the ZP while the inmunomarcaje observed in the larger follicles (antral follicles multilaminares and) was distributed with greater intensity heterogéneamente marking in the area outside. The heterogeneous pattern observed in the antral follicles of hamster ovary was also seen in oocytes ovulados. However, in rat and mouse oocytes observe a marking distributed throughout the thickness of the ZP. These data demonstrate that an epitope present at the ZP pork and is also found in the mouse and rat ZP hamster, is mainly distributed in the region outside of the ZP hamster. B. By electrophoresis and western-blot of glycoproteins of the ZP hamster note that the antibody anti-ZP pork specifically recognizes the band of 56 kDa which has been described which is the ZP3 hamster. The lectina WGA recognizes two bands a lower 56 kDa and a fairly wide between 90 and 200 kDa. We inmunoprecipitación of the glycoproteins with the antibody anti-ZP pork noting that the band of 56 kDa was totally precipitous and the band remaining in the supernatant. This result would demonstrate the different structure of the ZP along its thickness because using electron microscopy note marking exclusively on the outside of the ZP, however, by testing inmunoprecipitación note that the antibody has affinity for all the band where the ZP3 hamster and this is throughout the thickness of the ZP. The presence of a supramolecular structure along the ZP consisting of the various glycoproteins can make the internal region is inaccessible to antibody while in the region are exposed external epitopes recognized that the antibody. C.-Aislamos band of 56 kDa and conducted digestion with the enzyme N-glicosidasa F of this band and the ZP total hamster. We analyze the sample obtained by electrophoresis and western-blot incubating the membrane with the antibody anti-ZP pork. With these experiments, we observed that the deglicosilación of ZP total hamster showed the presence of four bands of approximate molecular mass 67 kDa, 58 kDa, 48 kDa and 38 kDa and the band of 56 kDa digested note the presence of two bands molecular weight to 8 proximad 1df5 or 48 and 38 kDa. These data suggest to us two things; 1) that the antibody recognizes the protein part of all but these glycoproteins are inaccessible due to the presence of the molecular structure formed by the protein and chains glicosídicas. 2) The band of 56 kDa in the ZP hamster are present two glycoproteins that can be detected after treatment with N-glicosidasa F. The band of 38 kDa belong to ZP3 without chains N-unidas and the upper band of 48 kDa could correspond to a new glycoprotein that had not been described so far, therefore, the ZP hamster, like the ZP human and rat may comprise four-glycoproteins D. The band of 56 kDa detected with antibody was purified and sequenced by mass spectrometry can determine the presence of two peptides corresponding to the amino acids 334-344 (YQAHGVSQWPT) and 285-297 (VTPANQTPDELNK) belonging to the above sequence for ZP3 hamster. With these data we were able to demonstrate the presence of ZP3 hamster in the band of 56 kDa. 3. Based on three lines of CHO cells, including two other line transfected and parental control, we obtained three samples. 1) A sample containing ZP3 recombinant CHO secreted by cells that were transfected with plasmid ZP3 humans. 2) Another shows ZP2 recombinant secreted by CHO cells transfected with the plasmid of ZP2 humans. 3) A third shows the supernatant without parental CHO cell transfectar. These samples were characterized biochemically by electrophoresis and western-blot. Caracterizamos the proportion of chains N-oligosacarídicas through enzyme treatment. Once the ZP2 and ZP3 were characterized perform tests induction of acrosomal reaction with both recombinant glycoproteins. The ZP3 recombinant without chains N-glicosídicas was also tested as an inducer of the acrosomal reaction in human sperm. Data obtained the development below: A. - CHO cells transfected with the plasmid of ZP3 human were cultured and then the supernatant collected, diluimos and concentrate. This sample containing ZP3 recombinant human secreted was marked with antibodies anti-ZPC of monkey and the antibody anti-ZP pork. The antibodies specifically recognized a band of 60 kDa in the sample collection of supernatant of CHO cells transfected with the plasmid of ZP3 humans. This band had a molecular weight similar to the human ZP when the ZP and ZP3 recombinant human were detected with antibody anti-ZP pork. Do not look reactivity in the supernatant collected from CHO cells without transfectar that we used as a control. With these data we conclude that the ZP3 recombinant expressing CHO cells introduced a molecular weight of about 60 kDa suggesting that the protein was highly glycosylated since the molecular weight of protein ZP3 human secreted without glicosilar is 36.39 kDa. With these data we conclude that the ZP3 recombinant under study has a molecular weight similar to ZP3 native already expressed by the other groups. The ZP3 recombinant was digested with enzyme N-glicosidasa F. The glycoprotein without chains N-oligosacarídica presented a sharp decline in its molecular weight of 60 kDa to 38 kDa. With this result, we can conclude that with the liberation from the shackles N-unidas is the elimination of most carbohydrates that make up the molecule and therefore suggest that the ZP3 human being more heavily N-glicosilada that O-glicosilada. B. - CHO cells transfected with the plasmid of ZP2 human were cultured and then the supernatant collected, diluimos and concentrate. This sample containing ZP2 recombinant human secreted was marked with antibodies anti-ZP2 human antibody anti-ZP pork. The antibody anti-ZP2 human specifically recognized two bands whose approximate molecular weight of 110 kDa were higher and 90 kDa the bottom. The antibody did not recognize any protein in the supernatant of cultured CHO cells were transfected not. The antibody anti-ZP pork affinity filed by two bands of molecular weight of approximately 110 kDa and 90 kDa in the fraction of transfected cells. We see how this antibody affinity introduced by a band of approximately 110 kDa weight in the human ZP. These data suggest that CHO cells expressing the ZP2 recombinant human with two levels of glycosylation, a less glycosylated of 90 kDa and one of 110 kDa closer to ZP2 human native. The two classes ZP2 expressed are glicosiladas since the molecular weight of protein secreted without glicosilaciones is 67.44 kDa. C.-To determine the activity of recombinant glycoproteins obtained perform tests induction of acrosomal reaction with human sperm. The tests were performed in seven patients. The ZP3 recombinant showed the ability to induce acrosomal reaction in 72.6% of the human sperm, slightly lower than the levels shown by the induction ionóforo calcium. The ZP2 recombinant and the supernatant collected from cells without transfectar showed similar levels observed spontaneous induction in human sperm. With these data we conclude that we have obtained a ZP3 recombinant biologically active and suggest that the expression of ZP3 recombinant human biologically active in CHO cells is a very efficient system can obtain a glycoprotein recombínate with activity levels similar to native-born. Although machinery glycosylation of CHO cells is different from the human system, we suggest that CHO cells have the ability to synthesize glycoprotein ZP3 recombinant human sequence sugars needed to produce a glycoprotein capable of inducing the acrosomal reaction in human sperm. The ZP2 does not induce the acrosomal reaction in human sperm probably due to his role as a secondary receiver described above. With the glycoprotein recombinant ZP3 digested with enzyme N-glicosidasa F perform tests acrosómica induction. With this experiment we note that the proportion of sperm reaccionados with glycoprotein and treated with the glycoprotein untreated control were the same. These data suggest that the chains N-oligosacarídicas of glycoprotein recombinant ZP3 are not involved in the induction of acrosomal reaction in human sperm. These data indicate that in humans, ZP3 and probably sugars O-unidos be involved in the induction of acrosomal reaction as has been described in mice.

Author: Peñalver Mellado Marcos.
Year: 2004.
University: MURCIA.
Place of defense: Facultad de Biología.
Place of preparation: Facultad de Biología.

Summary: The protein CarD of Myxococcus xanthus is a global regulator of transcription. CarD participates in the regulation of carotenogénesis and development in multicellular M. Xanthus. In addition, CarD is necessary for the expression of genes active during the vegetative growth. The protein CarD has two domains: the N-terminal domain is necessary for the function of the protein, and the C-terminal domain, which contains a source of binding to DNA previously described only in eukaryotic organisms. Proteins HMGA are necessary for the formation of different complexes multiproteicos involved in various operations on DNA. The action HMGA is mediated by binding to the DNA, causing changes in their endogenous curvature, and its interaction with various proteins. The role of CarD in regulating various groups of genes in M. Xanthus, and their structural and functional similarity with HMGA, leading to the prediction that CarD interacts with different proteins M. Xanthus involved in regulating gene or other operations on DNA. In one of the search strategies protein of M. Xanthus interacting with CarD has used the system twice hybrid yeast, to analyze the possible physical interaction between CarD and two regulatory proteins of the carotenogénesis, IfhA and CarQ, and to track a genoteca mergers traduccionales of protein prey to sections open reading obtained at random. Plasmids pGMx1 and pGMx2 identified in this search are bearing a fragment of DNA whose product interacts specifically with CarD. The orf incomplete content pGMx1 does not seem to really identify a protein in M. Xanthus. The orf truncated in pGMx2 shows a great similarity with the domain protein citoplasmático of histidine kinases, which function as sensor protein in the two-component regulatory systems. The interaction between the protein sensor and CarD occurs via the N-terminal domain of the latter. Immediately downstream of the sensor protein gene is a gene that would determine a protein regulating the answer. It is hoped that both genes constitute a two-component regulatory system, which does not appear to be related to the response to light or fasting. There has been detected physical interaction between CarD and regulatory proteins of the carotenogénesis IhfA and CarQ. Another strategy for finding protein of M. Xanthus interacting with CarD has been the deletion of the gene locus carD and the resulting phenotype analysis for the processes controlled by CarD. The protein encoded in orf4 participates, as CarD, in the regulation of carotenogénesis and development multicellular, and not proteins encrypted in orf2 and orf5. For his involvement in the carotenogénesis, orf4 renamed carG. Genes carD and carG part of the same operon. The corresponding gene products, CarD and CarG acted in a cooperative manner in the activation of several promoters analyzed. The protein CarG presented in its C-terminal end two potential binding sites for zinc. One is a cause very conserved in some zinc-dependent protease, and the other is a section that contains four cisteínas, located just below the first. The protein CarG purified zinc binds to a estequiometría CarG: Zn of 1:2. This union is likely to involve the four cisteínas one hand, and histidinas of the plea preserved on the other. CarG interact physically with CarD, through the N-terminal domain of CarD and perhaps the N-terminal domain of CarG. This interaction seems to occur with a estequometría CarD: CarG of 2:1. The protein CarG does not bind to DNA in vitro, or substantially modifies the affinity of CarD by DNA, but that leads to a supercomplejo CarD-CarG-DNA. In vivo, both CarD as CarG are located mostly in the nucleoide the cells of M. Xanthus. In the absence of CarD, CarG dissociated itself from the nucleoid 8 and, qu 3d0 and confirms its inability to unite itself to the DNA. The data from this work and other pre allow propose a model of action for the duo CarD-CarG where the function provided by the module CarG would be to interact with other proteins, and regulatory factors are specific components or machinery basal transcription.

Author: Hussein Ahmed Khedr Samiha.
Year: 2004.
University: MURCIA.
Place of defense: Facultad de Biología.
Place of preparation: CEBAS (CSIC).

Summary: The transfer of crop stalks micropropagadas Rosa multiflora var. Kordana the final stage of propagating a culture system with cooling baseline and double layer. Stimulated its elongation and inhibited sprouting axillary Secundaria a. Stems micropropagated in conditions formed earlier roots viable amid growing MS without growth regulators. The process of acclimatization conditions former vittro of seedlings mcropropagadas has a duration of 30 days, at the end of which the same are adapted to survive the conditions of the greenhouse with a survival of 90%. During the acclimatization the seedlings were sampled to 0. 2, 7, 15 and 30 days. During the final stage of the acclimatization starch content in the roots of seedlings exceeded that of sucrose as the main carbohydrate, miestras that during the first part of the process was the reverse situation, this result along with the evolution over time of enzyme activity sacarolíticas clearly reflected the transition from a metabolism initially mixotrófico until a final autotrófico. During the acclimation, the structure of the apex root evolved from a simple differentiation rich intercellular spaces to the apex of a fully developed cofia (consisting ade estatocitos differentiated), area meristématica, epidermis, and endodermis cortex that pudede adapt functionally to the ecofisilogía substrate former vitro. The acilmatación to former vitro conditions led to a reduction preogersiva the levels of ethylene and pliaminas total in the roots of seedlings micropropagadas. The evolution over time of the levels of nutrients in the roots during the acclimatization reflected the pregresiva adaptation to nutritional conditions of the new substrate. The enzymatic activities involved in the absorption of nutrients, minerals also presenteron a profile characteristic linked to the necessary movilizacióny uptake of nutrients required for the functional adaptation of the root to condicones ecofisiológicas substrate former vitro. The start of the acclimatization carries increased levels of Malondialdehyde (indicator of oxidative stress) that demonstrate the existence of this kind of stress that is shrinking as it progresses the process of acclimatization. That is why the roots takes place in the activation of antioxidant enzyme systems that allow the restoration of redox balance necessary for the proper development and function of the root system of plants acclimated.

Author: LUCAS ALCARAZ DANIEL.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: CIENTÍFICAS.
Place of preparation: CENTRO NACIONAL DE BIOTECNOLOGÍA.

Summary: 1. Generation of mice deficient in DNA polymerase m. DNA polymerase mu (Polm) is a DNA polymerase X belonging to the family of DNA polymerases. Because of its expression in secondary lymphoid organs, an identity with transferase terminal (42%) and mutagenic, has been suggested as one of the agents mutadores in the process of hipermutación somatic of immunoglobulin genes. To determine the specific role of Polm in this process was obtained and secuenció ellocus genomic this polymerase murine model and generate knockout. Mice deficient in Polm are viable and fertile, with no apparent abnormalities. 2. Polm is one of the components of the mechanism hipermutación somatic. The analysis process hipermutación somatic in animals deficient in Poim revealed that the B cells are capable of hipermutar and generate a response identical to that of the controls. However, the cells are hipermutando (centroblastos) accumulate in the secondary lymphoid organs of the knockout mice. The analysis process hipermutación showed a reduction in the number of clones highly mutated (with more than 10 mutations). The hipermutación occurs during the reaction of the germinal center, during which centroblasto suffers multiple rounds hipermutación until it acquires sufficient mutations to increase its affinity for antigen and escape gracious reaction. We propose that the deficiency in Polm reduces the rate of hipermutación and centroblasto requires more rounds of hipermutación to escape from the reaction of the germinal center, which explains the accumulation of centroblastos. 3. Polm involved in the DNA repair system called NonHomologous End Joining It had also proposed the involvement of Polm in the system repair breaks double stranded denomínado Non Homologous End Joining. The analysis process hipermutación somatic cell line in Ramos when sobreexpresa a dominant negative Polm, revealed the presence of deletions associated with the process, as well as chromosomal instability, demonstrating the involvement of Polm in mutagenic repair of double-stranded breaks in DNA. 4. Poor Polm alters haematopoiesis. The analysis of the deficiency in Poi mu in the knockout mice revealed a defect in both the number and capacity of proliferation of hematopoietic progenitors. Besides parents deficient in Polm are very sensitive to agents that induce double-stranded breaks. These data suggest that Polm involved in mechanisms Non Homologous End Joining which are critical to the maintenance of hematopoietic progenitor. 5. Poor Polm increases longevity. Mice deficient in DNA polymerase m live longer than control animals, this longevity is associated with a lower caloric intake and a resistance to oxidative damage, suggesting that the deficiency in Polm confers an ability to detoxify this type of injury through mechanisms not yet defined.

Author: IBORRA MARTÍN SALVADOR.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: DEPARTAMENTO DE BIOLOGÍA MOLECULAR.
Place of preparation: CENTRO DE BIOLOGÍA MOLECULAR SEVERO OCHOA. FACULTAD DE CIENCIAS..

Summary: Leishmaniasis is a set of patologias affecting 12 million people and which are caused by the intracellular protozoan parasites of genéro leishmania. There is evidence that loshospedadores this protozoan can develop a resistance and acquired immunity to reinfection, despite that there is still no effective vaccine against this disease. It has been demostardo that resistance to infection with leishmania is based on the generation of an immune response mediated by T cells of type Th1 producing mainly interferon gamma cytokine that induces the activation of nitric oxide sinUisá Tnducible in macrófago, which provokes the generation of nitric oxide and the resulting destruction of the parasite. Recently, vaccine development has been based on the techniques of recombinant DNA, which allows express antigens of the parasite in a form defined qUe even form can be obtained from industry. The recombinant products obtained in this way highlights DNA vaccines that are particularly effective in inducing Qe responses mediated by T cells of type Th1 and immune responses mediated cell CD8 +. The objective of this thesis was the generation of vaccines based on defined histone (H2A, H2B, H3 and H4) And in the acidic ribosomal protein (RB, P2a and P2b) from leishmania, two families of proteins are antigenic during infection in humans and dogs. Once generated the corresponding vaccines, discussed his inmunogenícidad in the form mouse. Generating answers Th1 address these antigens was achieved through genetic vaccination or the use of oligodeoxinucleótidos containing reason inmunoestimulador "CpG". The results show that the responses of type Th1 generated address these antigens can be associated with varying degrees of proteccíón in mice against infection with l. Majar, the principal etiological agent of cutaneous leishmaniasis in the old world. This protection consists of a decline in the pathology associated with the infection as well as the parasite burden.

Author: JIMÉNEZ GÓMEZ JOSÉ MARÍA.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: CENTRO NACIONAL DE BIOTECNOLOGÍA.
Place of preparation: CENTRO NACIONAL DE BIOTECNOLOGÍA.

Summary: The transition flower is one of the most important processes in the development of the plants. The timing of the transition to flowering is regulated to occur when the environmental conditions are more favorable for the production of seeds and fruits, and to coordinate the time of flowering species cross-pollinated. With regard to the requirements of length of the day (photoperiod) to flourish, plants can be classified into plants long day, short-day plants and day-neutral plants. Plants Long Day are those that bloom when the day length is greater; short day plants bloom when the day length is less, and the day-neutral flourish regardless of photoperiod. The molecular mechanisms that control the time of flowering plants have been studied in the short day as rice and plants Long Day as Arabidopsis, we found that many of the genes responsible for controlling flowering time are common to both models. On the other hand, they have not been extensively studied the molecular mechanisms that control the time of flowering plants in the day-neutral, so we decided to use the tomato for this purpose. This analyzed 21 varieties belonging to 8 of the 9 species that make up the genus Lycopersicon and analyze the number of leaves until flowering (LN leaf by number), a reflection of the time of flowering. This study demonstrated that there is variation among LN species of the genus Lycopersicon analyzed, and also this variation is also among the different varieties of each species. To analyze the genetic determinants of the time of flowering in tomatoes were selected 2 varieties. Among those analyzed presenting enough variation to this character. These varieties were Lycopersicon esculentum L777 (tomato grown, it is also the kind of reference for genetic studies) and Lycopersícon chmielewskii CH6047 (a wild species that blooms later than L. esculentum). From these two varieties have generated a population F2 segregating for the time of flowering on which measured both the variable LN, as the number of days from seed to flowering (DTF for days to flowering). On the other hand, we used molecular markers of type CAPS, SCAR, microsatellites and AFLPs obtained from other laboratories or designed by us for this work to build a genetic map of the crossbreeding of L. X L. esculentum ChmielewskiL. Using time measurements flowering of the population F2 and the linkage map, we performed an analysis of QTLs allowing locate 6 QTLs affecting variable LN and explaining a joint 66.7% of the variance found in the population F2. These QTLs are found on chromosomes 1, 3, 4, 5, 7, and 8, with the chromosomes 5 and 7 which had greater effect in LN. Plants carrying alleles of L. Esculentum for QTLs of chromosomes 4, 5, 7, and 8 had lower number of leaves until flowering compared with the carrying ale L. ChmielewskiL However, contrary to expectations plants carrying alleles of L. Esculentum for QTLs of chromosomes 1 and 3 showed a higher number of leaves, therefore flourished later, the plants with alleles of L. ChmielewskiL The QTLs on chromosome 1 and 8 of the ale of L. Esculentum were dominant on the L. ChmielewskiL As for the variable DTF, analysis detected QTLs two loci on chromosomes 5 and 12 behind an 56.3% of the variance found in the population F2 to this character. The plants carrying the ale of L. Esculentum for both loci accelerated its flowering plants with respect to the alleles of L. ChmielewskiL Moreover, in both cases, the ale of L. Esculentum presented dominance on the part of L. ChmielewskiL In regions where were detected QTLs affecting flowering time is mapearon genes that could be responsible for the effects detetctados by these QTLs. As a candidate gene for QTLs of chromosomes 3 and 5 are proposed loci PHYB2, FA AND LeFLC belonging to families who have submitted gene natural variation in Arabidopsis and further control and 8 l time 1b8 flowering in Arabidopsis and tomato.

Author: PRIETO ALCEDO MANUEL.
Year: 2004.
University: SANTIAGO DE COMPOSTELA.
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: The plant cell wall is responsible dela consistency of genetic tissue in plants. The cell wall is composed of various molecules. This comprised a series of cellulose microfibrils embedded in a matrix formed for various polysaccharides (hemicelulosas and pectins) and glycoproteins. The pectins are a group of complex polysaccharides, were found in the intercellular spaces, as part of the film media, and therefore are primarily responsible for the consistency of the tissue of plants. One of the enzymes act on the pectins are poligalacturonasas, hidrolizanla molecules homogalacturonano, a component of pectins. The poligalacturonasas have important industrial applications, and which are used for the processing of fruits for maceration and liquefaction prior use in industrial processes. They are also used for obtaining essential oils and carotenoids from the skins of citrus. Although his or clarification is the most widespread, the addition of these enzymes allows reducing viscosity and get drunk much juice a less filling of the filters. The wine industry also employ, adding at the beginning of fermentation. This thesis has studied a poligalacturonasa alfalfa involved in the process of symbiosis between the plant and Rhizobium, the gene MsPG3. It clonó this gene for expression in bacteria and yeast Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris. In every agency employees detected the production of certain amount of enzyme, but the amount is so small that the use of these cell lines transformed to obtain the enzyme in large quantities is not viable. Another of the goals scored in the thesis has been the effect of the study dela overexpression of the gene in plants. Arabidopsis thaliana was used because it is the plant model for studying genetic plants. As a result of gene expression were observed in the early stages of development leaves of the plants seriously damaged, confirming the correct expression of the gene. However, in later stages of development transgenic plants had a normal, due to a mechanism of the plant to cope with the overexpression of the gene. When making cuts histological of transgenic plant leaf damage was observed with a clear disruption d tissues of the leaf, which supports the expression of lapoligalacturonasa is correct, and that by acting on the cell wall would cause damage to the plant tissue . Through real-time quantitative PCR was determined the levels of gene expression in transgenic plants, confirming that the gene is expressed correctly, with a high level of expression. Measures of activity detected lower levels in the transgenic plants compared with control plants, resulting contrary to expectations. The reason was probably due to the action of molecules small interfering RNA (siRNAs). Ultimately the results in plants confirmed the correxta expression of the gene MsPG3 and showed that it is a poligalacturonasa.

Author: MARTIN CASTRO FRANCISCO ANTONIO.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: DEPARTAMENTO DE BIOLOGÍA MOLECULAR.
Place of preparation: CENTRO DE BIOLOGÍA MOLECULAR SO.

Summary: Discs imaginales of Drosophila originate as groups of 20-40 cells in the embryonic ectoderm (primordios imaginales). During the larval development growing rapidly and the number of cells is increased a thousand times. During the metamorphosis give rise to structures cuticulares adult (wings, legs, eyes.). There is evidence that the activity of the via Dpp is necessary for the growth of disk wing. This thesis has analyzed the role of the route signaling Dpp in the growth of disc-wing generating different genotypes with different levels of signaling Dpp and examining their effects on the size of the wing. The level of activation via correlates with the final size. So Dpp promotes growth concentration-dependent manner. By contrast, the gene brinker (brk) behaves as a repressor of growth, and also No inhibitory effect "is dependent on concentration. Given that the activity of Dp" p suppresses the expression of brk, the effect of Dpp growing is mediated repression of brk. We have studied some aspects of the mode of action of brk apoptosis and there is a place where there is a discontinuity in its expression, but his main role with respect to growth is to reduce the rate of division. Brk probably suppresses the expression of genes that control cell proliferation, as a bantam. In addition, there is an ectopic expression of miRNA in bantam that correlates with the loss of expression of brk and higher rate of division.

Author: ARJONA MAYOR M. DOLORES.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: FACULTAD DE BIOLÓGICAS.
Place of preparation: FACULTAD CIENCIAS UAM..

Summary: Tumors astrociticos form, despite advances in las1écnicas surgical, an unresolved problem. The forms of higher degree of malignancy still does not have a higher survival of 9-12 months. The molecular understanding of 21teraciones that these tumors appear in a sequential manner is essential to start chemotherapy or radiotherapy treatment, since they have found varying degrees of response as these changes. In our series we have included 86 tumors astrocíticos of distint0s grade malignancy, proceeding to search them lost heterocigosidad, hypermethylation and mutations, as well as amplification and overexpression géníca, regions and genes involved in malignant transformation of this tumor type. The findings of our study are as follows: 1. The losses heterocigosidad of arms chromosomal 9p, 1Op and 10q-se relate significantly with a high degree of malignancy of the tumor astrocíticos, especially in the development path of glioblastomas "de novo". Losses in 19q, however, would be related to the path of gradual evolution tumora!. Less significant losses appear to be in 1p in the development of tumors astrociticos. 2. Mutations in the PTEN, and the EGFR gene amplification events are related LOH of chromosome 10, appearing later these deletions associated with an increase in the degree of tumor malignancy. 3. The mutations of TP53, the amplification PDGFR-ay of genes located in the region 12q 13 are implicated in the emergence of forms tumor astrocíticas grade 11. 4. The gene MDM2 seems to be the target amplification process, at early stage, the region 12q13 in a subgroup of tumors that no amplification MDM4 or mutations in TP53. However, the amplification of CDK4, also located in that region, would be associated with an increased degree of malignidap of tumors via the progressive IIA to AA. 5. The results of the analysis of EGFR gene confirm a decisive role in the development of gliomas astrocíticos. The EGFR amplification appears related LOH in the region chromosome 7p11, where the gene is located, as well as overexpression of mutant transcripts. Have been found, in addition, six alterations in the sequence not previously described. 6. The hypermethylation of CpG islands in the promoter regions of genes p16ink4a, p14arf, MGMT and RB1 plays an important role in the development and progression of tumors astrocíticos. 7. The hypermethylation of the MGMT gene p16ink4a and are associated with an increase in the degree of malignancy of tumors via progressive. 8. The promoter hypermethylation of the gene RB1 is significantly higher in GBM2arios that GBM1 Aryans. 9. The route RB1 was deregulated predominantly front of the path of TP53 in our series. In addition, both disorders seem to happen at different times of the progression of tumors astrocíticos it would play a different role in the process, TP53 in the genesis of forms of low grade and RB1 in the progression toward aggressive form of high malignancy.

Author: CUESTA ROJO ISABEL.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: INSTITUTO DE INVESTIGACIÓN BIOMEDICAS.
Place of preparation: INSTITUTO DE INVESTIGACIONES BIOMEDICAS CSIC-UAM.

Summary: The organization of the genetic material in the nucleus is called chromatin and is a dynamic structure that regulates the establishment and maintenance of gene expression. The members of the family of transcription factors Forkhead regulate the expression of tissue-specific genes, which are essential for the differentiation and development. The factor Forkhead FoxA1 part of the family of functional factors pioneers, who are able to join compact chromatin structure during development and remodelarlas. We used the model of the gene of Tiroperoxidasa (TPO) for the study of the role of factors Forkhead during processes of differentiation. FoxE1 is essential for the activation of the expression of OPTs in response to hormones and this is because of its ability to join the promoter inactive and generate an open structure boosting the following events leading up to his speech. Using the model for the regulation of gene albumin by FoxA1 we have studied the molecular mechanisms of regulation of chromatin, identifying regions of interaction of the gene albumin by FoxA1 we have studied the molecular mechanisms of regulation of chromatin, identidicando regions interaction with the nucleosoma. This feature is conferred by a domain binding to the DNA forkhead and a domain able to interact with the surface of nucleosoma inhibiting the compaction of chromatin fibers. These results establish a novel mechanism of activation of genes by transcription factors, which takes place thanks to a functional domain of the protein identified in this study we call "Hélice Opening." Thus, we identified and characterized the molecular mechanism of the overall role of factors Forkhead as factors Pioneers, who are able to start opening phenomena in chromatin silenced, required for the activation of genes during development and differentiation.

Author: OCHANDO SÁNCHEZ ISABEL.
Year: 2004.
University: MIGUEL HERNÁNDEZ DE ELCHE.
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: INSTITUTO DE BIOINGENIERIA UNIVERSIDAD MIGUEL HERÁNDEZ.

Summary: The bodies side of the plants show polarity and asymmetry axis próximo-distal and abaxial-adaxial. Surgical Experiments have suggested that the apical meristemo produces a signal required for the cells to adopt the correct destination on the shaft abaxial-adaxial, and recent genetic studies have led to the identificaicón several genes that are expressed in the body side and are probably involved in interpreting the signal emitted by the meristemo apical. Two mutant gene INCURVATA4 (ICU4) presented the phenotype incurvata characteristic (leaves vegetative curved towards the beam). These traits can be interpreted as a transformation paracial structures abaxiales in adaxiales. The synergistic effect between mutations icu4-1 and hst-1 (a mutation adaxializante) includes, among other phenotypic traits, a strong adaxialización of leaves and transforming the replum abaxial in adaxial placenta. These obervaciones support the hypothesis that the product ICU4 has role adaxializante. We located an identical mutation in both alleles (icu4-1 and icu4-2) in the gene Athb-15 which encodes a transcription factor that belongs to the family of genes HD-ZIP II. In this family belong other genes involved in the establishment of the axes adaxial-abaxial of lateral organs and central-periférico stem.

Author: VÁZQUEZ LÓPEZ M. ARACELI.
Year: 2004.
University: SANTIAGO DE COMPOSTELA.
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Author: RUIBAL VILLASEÑOR CONTASTINO.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: FACULTAD DE CIENCIAS DEPARTAMENTO DE BIOLOGÍA MOLECULAR.
Place of preparation: MERCK, SHARP Y DOHME DE ESPAÑA S.A..

Author: DIAZ VALDEZ FARRAY NANCY.
Year: 2004.
University: OVIEDO.
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE MEDICINA.