CELL BIOLOGY (3)

Author: SANCHO BRU PAU.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAT DE MEDICINA.
Place of preparation: FACULTAT DE MEDICINA.

Author: SIMÓ OLIVAR SEGI.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: During the training period Central Nervous System (CNS) a multitude of factors involved in the development of different brain structures. These factors controlling both cell migration and the correct position of the different cells within the CNS. Reelina is a soluble protein of the extracellular matrix expressed during development and adulthood, but its main function is hipotetiza during development. This thesis covers three different roles exercised by Reelina in cell migration during development and adulthood. We have shown that Reelina can control the niel phosphorylation mode I protein MAP1B during the migration of neurons that form the crust. This phosphorylation mode I MAP1B has been associated with the control of the stability of microtubules during cell migration. The morphological study we have exercised poor animal protein MAP1B has confirmed a link between the signaling cascades triggered by Reelina and activity of the protein MAP1B. Finally, we have also described that in response to Reelina the kinase GSK3beta phosphorylated mainly MAP1B mode I, but participation, to a lesser extent, the kinase Cdk5/p35 can not be ruled out. We have found that Reelina can induce phosphorylation of mDa1 and lead to the activation of PI3K, which can fosforilar to Akt1/PKB. Based on these results, we demonstrated that the activation of PI3K in response to Reelina induces activation of MEK1 / 2 and this activation leads to the phosphorylation of proteins Erk1 and Erk2. We have also described the phosphorylated forms of Erk1 / 2 are translocated to the kernel, in cortical neurons in response to Reelina where indcuen expression of how minimal the transcription factor Egr-1. This induction in response to Reelina believe it can stimulate Expression other proteins exert important roles in development. An important point of this study is that we have connected the cascade Reelina-MAPK/ERK in the process of desadhesión of cells in the ventricular zone (SVZ) involved in the route of migration rostral (or RMS). Basánndonos in hehco that in the presence of Reelina cells emerging from explants of the SVZ not migrate homofílicamente but suffer a process of desadhesión and are individualized, we described that if genetically or pharmacologically blocking the path of activating MAPK / ERK the effect on desahesión of Reelina on cells in the SVZ is inhibited. With this result, we can conclude that in response to Reelina proteins Erk1 / 2 is fosforilan and that this phosphorylation is necessary for Reelina exert its role in desahesión of neuroblastos involved in the olfactory bulb to RMS. The third and final point in this thesis is based on migration control radial granular cells of the cerebellum by Reelina. In response Reelina the granular cells of the cerebellum inhibiting their migration and this response is induced through activation of the path of MAPK / ERK. Locking faramcológico that way inhibits way inhibits the effect of Reelina. So we can conclude that the activation of the path of MAPK / ERK is not only important in controlling migration in the brain above, but it is also important in brain and may be some post-signaling cascade that participates extensively in the roles where hipotetiza participation Reelina.

Author: IGLESIAS ARA AINHOA.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE CIENCIA Y TECNOLOGÍA.
Place of preparation: FACULTAD DE CIENCIA Y TECNOLOGÍA (UPV/EHU).

Summary: The transcription factors of the family E2F (E2F1-8) are critical elements in controlling the cell cycle, regulating the expression of many genes involved in target processes such as DNA replication, entry and progression phase So apoptosis. Studies in vivo and in vitro suggest that each member of the family E2F plays specific roles in the development and homeostasis of the body, however, is unknown mechanism of action of each member E2F, as well as a degree of functional redundancy. In this paper we have analyzed, through studies and functional genomics, the unique functions and shared E2F1 and E2F2, using mice carrying mutations inactivadoras for E2F1 and E2F2 or both (DKO), generated in our laboratory. It has been found that E2F1 and E2F2 play a critical role in maintaining homeostasis pancreatic and hematopoietic, operating through similar mechanisms in both systems. The simultaneous inactivation of E2F1 and E2F2 leads to the development of diabetes, pancreatic postanatal and atrophy and hypertrophy dysplasia of acinar cells, all complemented by an increased rate of DNA synthesis and apoptosis in vivo and a deletion of markers pancreatic differentiation. Also, the hematopoietic system of the mice DKO is characterized by reduced ability of the parents of lineage monocítico produce macrophages differentiated. The proliferative effect is also accompanied in this case a higher rate of DNA synthesis and replication of centrosomes followed by a locking cycle progression and apoptosis. The analysis of gene expression profiles in the pancreas and bone marrow indicates that the simultaneous inactivation of E2F1 and E2F2 function as repressors dela cell proliferation by regulating the expression of refusal of these genes. The significant increase in the number of cells undergoing DNA synthesis does not lead, however, to a state hiperproliferativo which could be the prelude to a tumor, after the activation of several checkpoints in these cells. The cells differentiated features of cell aging and the analysis of gene expression showed overexpression of transcripcionales targets of the path of p53, in particular, p21, suggesting that inadequate DNA replication or uncoordinated duplication of centrosomes in the cells DKO generate genomic instability that actibaría secondarily towards p53-p21 to induce the activation of cellular checkpoints or apoptosis, as a protective mechanism for homeostasis

Author: BOSQUE PARDOS ALBERTO.
Year: 2005.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: This paper has studied the pattern of expression of anti different proteins and pro-apoptóticas during the transformation process blast of human T lymphocytes from healthy donors and their relationship to susceptibilización to activation-induced death (AICD) of human T blasts. Moreover, it has studied the role of FasL and Apo2L/TRAIL in the activation-induced death (AICD) and the regulation of activation of the T blasts, both in population and in the mixed subpopulations CD4 + and CD8 +. It also has characterized the population surviving after re-estimulación blasts human T, which displays a memory cell phenotype. It has also marked the death induced by growth factor deprivation in blasts T CD4 + or CD8 +, as well as the participation of different proteins of the family of Bcl-2 in this process. In addition, it has analyzed the possible contribucción of FasL and Apo2L/TRAIL in regulating the cell cycle, especially in the run-G2 / M cell cycle. Subsequently, the defects have been analyzed and functional regulation of T blasts from patients with syndrome linfoptoliferativo autoimmune (ALPS), which has allowed us to characterize the role of FasL in inducing levels of Bim, protein implicated in the death deprivation of growth factors. These data relate for the first time the two main ways to regulate the activation of human T lymphocytes, the IACD mediated by receptors fatal and deprivation of cytokines. Finally, it has initiated the characterization of proteins in the exosomas released during the IACD of human leukemic T Jurkat and human T blasts healthy donors.

Author: GARCÍA MARÍN VIRGINIA.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA, UNIVERSIDAD COMPLUTENSE DE MADRID.

Summary: The work presented in this thesis is the first systematic attempt to documentation and investigation of the histological preparations by Ramón y Cajal which so far had received only sporadic attention. It has created a database of the 4529 preparation histological original Ramón y Cajal that can be found inside the museum cajal, which facilitates access to and review of the preparations by other researchers. There have been completely 415 of these histological preparations, which have been chosen 6 themes to develop in depth in the thesis. The results have been presented on the study of neurons, which cajal devoted most of its research, with an emphasis on two structures, and core neuronal neurofibrillae, giving greater importance to the body of cajal. Have also been studied preparations of some of the most significant discoveries of Cajal as growth cones or interstitial cells of cajal. It has become a study of histological preparations of neurology, an area in which introduced new methods to impregnate specific staining these components of the nervous system and ultimately highlights its work in the field of pathology, through his studies of the disease Alzheimer's. The investigation has been developed according to a dual perspective, a different historical and current. The historical perspective brings us to the context in which they conducted their investigations Cajal and allows us to observe the relevance and novelty that contributed their work, preparations have been revised from today's perspective, taking into account the latest developments neuroscience, which has permitdo review the findings of Cajal and reinterpret them. Other noteworthy implementing these unique preparations of the most innovative methods of image processing such as deconvolution, the study of three-dimensional cones growth and widespread outbreak of the procedure in order to obtain images which are all the points of focus.

Author: CELA RAMOS CAROLINA.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: PARC CIENTÍFIC DE BARCELONA.
Place of preparation: FACULTAT DE BIOLOGIA (UNIVERSITAT DE BARCELONA).

Summary: One of the main objectives of developmental biology is to define mechanisms cellular and molecular programs that establish the formation of a body. Several bodies with vital functions, such as the lungs and kidneys are comprised of tubular structures branched. The morphogenesis of such bodies, called tubulogénesis requires different cellular processes highly regulated in order to generate a final structure mature and functional. Addressing the analysis of tubulogénesis requires different cellular processes highly regulated in order to generate a final structure mature and functional. Addressing the analysis of tubulogéneis in organs of mammals is extremely complex, which is why the respiratory organ of Drosophila Melanogaste, tráqueas, is presented as an ideal model system to study the foundations of tubulogénesis both cellular, molecular and genetic . Currently known varías signaling pathways and transcription factors associated with different stages of development trachea. However, the still unknown mechanism of action and genetic programs that govern different cellular processes that occur during development tracheal such as the reorganization of epithelial cells, the formation of the lumen and maintaining the integrity of the tissue. In order to identify new elements involved in tracheal development, in our laboratory we have carried out a genetic analysis of gain function. The genetic analysis conducted by mutagenesis combines elements P and the system Gal4-UAS. As a result of our analysis, we obtained a collection lines with a P UAS integrated into the genome, which, when crossed with the line activating tracheal btlGal4 presents a particular phenotype trachea. We have identified candidate genes to generate such traqueales phenotypes. We also know the molecular nature and the pattern of expression of the proteins encoded by which many of these genes. The comprehensive study of one of these lines has shown that signaling through dela canonical way of ERK-MAPK unleashed for the activation of receptor Egfr but not by the receiver of the road FGFR Btl, plays an important role in maintaining dela epithelial integrity during the length of the branches traqueales, and such works depends on pointed. In this paper we have also shown as modulations in the path of Egfr resulting in differences in levels of DE-Cadh and cortical actin in the cell, which modulates the integrity of epithelial tissue. In addition, we have also noted that the loss of cell adhesion mediated DE-Cadh or actian produces a phenotype similar to the loss of function of the path of Egfr. So our suggests that the path of Egfr regulates the maintenance of the epithelial integrity at least in part, and a post-, cell adhesion dependent DE-Cadh.

Author: CLEMENTE BLANCO ANDRES.
Year: 2005.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: DPTO. MICROBIOLOGÍA Y FAC. CIENCIAS.

Author: AMBRONA CORDERO JESUS ENRIQUE.
Year: 2005.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The yeasts vínicas are natural strains of S.cerevisiae with a phenotype very variable that threatens the maintenance of its technological characteristics. This genetic instability alters the useful properties of industrial yeast, causing problems and biotechnological loss in the quality of products, such as bread, beer or wine. This paper quantifies the impact of this inestatiblidad gene in yeast vínicas. These trends show rapid yeast that determine their potential of biotechnology. It proposes a practical solution to the use of genetically improved clones homozygous for the elimination of deleterious alleles which are damaging their property technology. The solution is effective to industrial level. The availability of genetic marker resistance cicloheximida has enabled control inoculated in yeast fermentation vínica for over 6 years in the DO Ribera del Guadiana. We recently detected up to 10% of strains marked spontaneous fermentations of some wineries. We fear that these commercial yeasts were getting residents in the commercial wineries. To avoid this, we designed yeasts are new genetic markers for use in alternate vintages: mutants resistant sulfometurón methyl mutants that form white colonies in the midst rodamina. These markers allow us to monitor test with a simple, quick and inexpensive yeast inoculated in the wine over the fermentation and evaluate its usefulness in developing wine. The test is applicable in commercial warehouses, and is a useful tool for controlling the quality of the wine. The alternative use of distingas yeast with different genetic markers avoids become resident in cellar and disperse easily in nature. The study of sexual behavior of the yeast vínicas during the development of wine indicates that tend to be homnocigóticas in its natural environment, albeit more slowly than expected because of the physical proximity of the cells in the same tetrade, and the linkage of the gene centrómero to determine the sex of a chromosome III. Although yeast vínicas can change their sex, is a capacity not used for the production of wine. We designed yeasts vínicas, lacking the gene IME1 unable to esporular and sexually combine naturally. Using this genetic sterilization can prevent the spread of yeast vínicas transgenic in nature, although it is used heavily in the holds.

Author: JIMÉNEZ ORTEGA VANESA.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS, UCM.

Summary: Chronic alcohol consumption in adults has been widely studied. However, the effects of this drug in rats during the period of sexual maturation is not known at present. Therefore, this thesis project seeks to analyze the effects of chronic alcohol consumption on the activity axis hipotálamo-hipófiso-testicular (HHT) during sexual maturation. Since the pituitary hormones involved in the modulation of the shaft HHT presented a rate of secretion cicardiano, this study will be conducted from him viewpoint cronobiológico. These variations are also daily concentration reflex in the concentration of different neuromodulators studied. Given the seasonality proven secretion of prolactin this work has been carried out during the summer season, in which the secretion of prolactin presents a different pace in relation to the rest of the seasons and to some extent, this could modulate HHT axis sensitivity to alcohol. The most relevant results obtained indicate a disruption of circadian rhythm of prolactin, LH, FSH and testosterone (Alcohol 2004; 34:127-132). These changes are associated with changes in the daily variation of concentration of dopamine, dopac, serotonin, norepinephrine, GABA and taurine in adenohipófisis and median eminence; neurotransmitters involved in the modulation of the secretion of hormones adenohipofisarias (FSH, LH and prolactin) above exposed. In addition, we analyzed the effects of chronic alcohol consumption on some parameters involved in the oxidative damage of the adenohipófisis, trying to determine whether the degree of damage that is induced in this gláncula by the effect of alcohol is linked in any way with the changes the hormone secretion. The data obtained indicate that there is an increase in the synthesis of nitric oxide and carbon monoxide (through the measurement of gene expression of the enzymes that synthesize, the nitric oxide synthase and hemoxigenasa), which indicate the existence of oxidative damage in pituitary. This increase in the degree of oxidative damage to the pituitary gland may explain, at least in part, the changes in the secretion of pituitary prolactin, LH and FSH. Importantly, both the expression of genes encoding enzymes and inducible nitric oxide synthase and hemoxigenasa presented neural activity circadiana which was not described until now. The overall conclusion from the data is that chronic alcohol consumption during the sexual maturation of male rats induced hipogonadimos that can be explained by effects of the drug at the level of the hypothalamus, changing daily variations of neuromodulators studied. It also exerts effects pituitary level, modifying the degree of oxidative stress that cooperates with concentrations Amended neuromodulators arriving in the hypothalamic pituitary gland. At testicular amending pace secreting testosterone and variations encountered or are correlated with changes observed with the secretory pattern LH, the chief regulator of the synthesis of testosterone.

Author: ALAÑON RIBAS M. DEL PILAR.
Year: 2006.
University: POLITÉCNICA DE CATALUÑA [www.upc.edu].
Place of defense: SALA D'ACTES INTEXTER TERRASSA.
Place of preparation: ETSEIB, EDIFICI H PLANTA 10 Campus SUD.

Summary: Carbofuran and chlorpyrifos are two well-known pesticides widely investigated, and its effects on different organisms have been previously reported in separate studies. For this reason were considered to be good model subtances, relevant from the environmental perspective. On the other hand, we selected this kind of compounds because they are used in many tones annually in agriculture and horticulture and they are significant especially in greenhouse-based production of vegetables and fruits in southern Europe, particularly Spain. The general objective of this work was to compare the response of in vitro assays using cell lines with assays using fish in vivo in order to contribute to the development of alternative methods to the use of laboratory animals. Furthermore, we compared two types of cells, a fish cell line from an established culture and mammalian cells obtained from a primary cell culture, in order to see if there are similar responses based on common mechanisms of toxicity. A better knowledge of these mechanisms facilitates the interspecies extrapolation of the impact of environmental contaminants, which is one of the major challenges to ecotoxicologists. In order to have a general point of view of the toxicity of these pesticides, single and in mixture, acute toxicity was evaluated using a battery of ecotoxicological model systems and indicators representative of a wide range of organisms. The systems studied included: RTG-2 fish cell line, primary cell cultures from bovine granulosa cells, the marine bacteria (Vibrio fischeri), three species of freshwater microalgae (Chlorella vulgaris, Scenedesmus subspicatus and Selenastrum capricornutum) and the vertebrates zebra fish (Danio rerio). Sublethal effects were also evaluated in both types of cells using several biomarkers such as assessment of the DNA damage as genotoxic indicator, inhibition of production of progesterone as indicator of the aromatase activity and inhibiton of acetylcholinesterase activity as indicator of exposure to pesticides measured also in zebra fish. By comparing the obtained in vitro fish cell line and mammalian primary cell cultures results with currently used bacteria, algae and fish acute toxicity tests, it was possible to compare its sensitivity against these conventional toxicity tests. As conclusions of the study we can say: 1) fish cell lines can be used as an alternative to the use of fish in laboratory; 2) the simultaneous use of in vitro fish cell lines with fish species allow to assess whether cellular responses in vitro could mimic toxicity responses of individual fish, thus to directly assess the ecological relevance of the proposed in vitro cell based test; 3) cell lines are the most sensitive bioassay of the studied battery; 4) the use of a battery of multispecies tests to determine the toxicity of any product is recommended; 5) The aquatic test using bacteria or microalgae are quite interesting, but they cannot be considered as a substitute for the studies on fish, because of they assess the effect of toxicants on other trophic levels, not on fish and 6) synergistic and antagonistic toxicity effects were observed with pesticide cocktails relative to pure compound toxicities.

Author: ZORITA AGUIRRE IZASKUN.
Year: 2006.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE CIENCIA Y TECNOLOGÍA.
Place of preparation: UPV/EHU.

Author: ALONSO MARTÍN SONIA.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS.

Summary: The podocalicina is a glycoprotein transmembranar highly sializada. This protein, or to be more precise, its load surface is essential to maintain the structure of you podocitos of glomerular epithelial and prevent occlusion of the epithelial cracks or spaces urinals. In fact, this protein serves as a marker of podocitos kidney. These podocitos are epithelial cells that form the support delos glomerulares capillaries. They are divided into three segments: the body cell, the main projections and projections of the foot. The body cell and projections main lie in space urine and are anchored to the glomerular basement membrane by means of projections of the foot. In podocitos mature, podocalicina is the main component of the apical side of the cell where it is assumed that helps keep the space between the projection interdigitales by revulsion load. Recently, it has been found that podocalicina not exclusive to the gomérulo kidney, but also expressed in cells mesoteliales lining the cavity celómica, platelets and hematopoietic precursor cells. The podocalicina according to location could play conflicting roles, perhaps as a result of finishing postraduccinales specific to each tissue. In podocitos epithelial glomeerular, appears to have properties atniadherentes, supporting the maintenance of the structure and function of renal glomerular. However, in the vascular endothelium the podocalicina displays properties similar to cell adhesion ligands essential in controlling hemostasis, thrombosis, inflammation, and atherogenesis. Its amino acid sequence, protein structure and genomic organization suggest that podocalicina may be closely related to two other molecules such as CD34 and endoglicano. Besides localizasen similar, the CD34 and podocalicina possess a structural organization very similar: mucin domain amino-terminal type, domain disulfide bridges, region transmembranar, and finally, a domain citioplasmático. In endothelial cells of venules postcapilares, CD34 and podocalicina act as ligands accession to the L-selectina of leukocytes. However, it ignores the functional role of CD34 in hematopoietic cells in the majority of vascular cells. The endoglicano is the newest member of this gene family with a wide distribution, as it is expressed in hematopoietic progenitor cells and some mature blood cells, vascular endothelium, smooth muscle and a group of nerve cells. Its function is also unknown. In our working group, most of the projects are aimed at studying the pathophysiology platelet. For this reason, it seemed interesting that the podcalicina has been described as a molecule with features adherents in hematopoietic cells, despite being characteristics of podocitos kidney with a function antiadherente. So far, we know the role that podcalicina could play in platelet function, particularly in its interaction with endothelial cells and leukocytes. Therefore, we believe that the functional study of this glycoprotein improve our knowledge about the pathophysiology platelet. We can not speak of platelets without first mention their precursors: megaccariocitos. The latter are highly specialized cells that have the function of platelet production and release them for circulation. During the development of mammals, hematopoietic stem cells are found in the sack vitelino embryonic, fetal liver and spleen. In adults, these hematopoietic stem cells. During the process of forming platelets in the megacariocito is pr 8 oducen c 1f6d ambios molecular and structural characterized mainly by process endomitosis, where megacariocitos acquire polyploidy, assembly and translocation of organelles and specific secretory granules of platelets and by the morphogenesis of the proplaquetas. The release in platelets involves a reorganization of the cytoskeleton in megacariocitos an extensive network of pseudópodos, which are found in the platelet formation or proplaquetas thereafter, to be released in sinusoids of the bone marrow. In the bloodstream, circulating platelets next to the wall of the vessel due to its shape Disk and its small size. This pegs close to the apical side of the endothelium, providing them with the best position to detect and respond quickly to the presence of a specific vascular damage. Where are trigger factors, such as the von Willebrand factor linked to collagen and other soluble factors that are released into the blood, platelets adhere react quickly, spreading, secreting and interaciconando with each other to form a coáguulo to seal the damaged surface. The formation of the clot requires platelet activation associated with morphological changes dependent on the dynamics of polymer cytoplasmic actin: platelets stretch along the damaged surface and develop filopodios that facilitate the recruitment of additional platelets. To expand, platelet resting must reorganize its cytoskeleton and assemble new actin filaments. This change follows a temporal sequence reproducible. The first thing that is observed when they come into contact with the platelet surface, is that it loses how Disk and acquires a spheroid (Phenomenon dependent on the mobilization of intracellular calcium). Then, some extensions are issued, platelets are aplanan on the surface and extend the filopodios. During the flattening of platelets on the surface, are grouped all organelles and granules in the center of the cell acquiring a characteristic appearance. The chip extends along the growth of filopidos. This happens through the growth of many lamelipodios, which join to form a single large lamela circular, and greatly increased the surface area of the cell. The rupture of vascular endothelium and exposure subendotelio sets in motion a series of reactions that, succinctly, consist of platelet adherence to the matrix subendotelial, activation parcel by agents of diverse nature, presumably released January the site of injury and finally, the formation of cell aggregates. It forms a "cap" platelet reducing blood loss and facilitates clotting or secondary hemostasis, providing support for the assembly and activation of clotting factors that result in a retraction and strengthening of joint platelets clot and fibrinas. Under the conditions of vascular flow in vivo platelet adherence to subendotelio requires joint action receptor GPlb and integrina alfallbbeta3 to be fine respectively vWF and the collagen of the extracellular matrix. After accession to the platelet subendotelio, paleutas are activated and integrina alfallbbeta3 acquires the ability to set fibrinogen with high affinity and other adhesive proteins, as both soluble attached to the array subendotelial exposed. This leads to the extension of platelets on subendotelio, secretion grándulos dense platelet aggregation and formation of the clot retraction mixed, and eventually, seals vascular injury. OBJECTIVES The main objective of this work was to study the role of podocalicina in controlling platelet function. To do so, we set the following specific objectives: 1-Generation of transgenic mice that sobreexpresen podocalicina and phenotypic analysis of the same: functional analysis of platelets. 2, - generation mice with selective cancellation of podocalicina Platelet ( "knock out" conditional) by the system Cre-loxP. 3, Expression of podocalicina cells of Chinese hamster ovary (CHO) and functional analysis of these clones: accession, aggregation, migration and interaction with other cell types. 1-GENERATION RATONES TRASNGÉNICOS THAT SOBREEXPRESEN PODCALICINA AND ANALYSIS FENOTÍPICOS OF THE SAME: ANALYSIS OF THE FUNCTIONAL PLATELETS The first step was to generate the construction gene to obtain transgenic animals. This construction, essentially consists of three parts: a promoter necessary for the expression of the selected gene, which in our case is also specific tissue, the cDNA of this gene and a tail poli-A, which will give stability to mRNA once formed. The next step was the handling of animals and microinjection technique and referral or transfer of embryos. The strains of mice with whom they worked were: FVB / N as a donor of oocytes to microinyectar and female CD-1 as recipients of embryos microinyectados. After crossing the superovulation and the females are fertilized oocytes extracted from 0.5 dpc which was subsequently microinyectaron with DNA previously purified. This is the crux of the experiment, and that is when it will introduce the construction gene in one of two pronúcleos embryo, with the help of a invertoscopio with a phase-contrast of Nomarski two micromanipulaodres and microinyector. The embryos that survived this process was stopped growing until its recuperaicón. The next day, embirones microinyectados that had fallen into the stadium two cells were transferred to unahembra receiving. Trs childbirth is proceidó weaning dela litter and the marking of genetic individuals. He then proceeded to analyze the individuals using DNA using small biopsies of mouse tail. RESULTS For the construction of the transgene were selected by the developer of the glycoprotein GPllb, which is specific to megacariocitos and platelets. In continuaicón was clonó the cDNa of podocalicina human, and finally the tail poli-a of SV40. We obtained 2 mice positive 65 who were born alive that were analyzed by PCR and Dot-Blto.Además, both lines transmitted the transgene to their offspring. Because laimposibilidad to detect podocalicina in wwesthern was generated another construcciónsimilar but merging the cDNA of podocalicina to the green fluorescent protein (GFP). It obtuvieorn 8 mice positive 73 who were born and live in the same way as above were analyzed by PCR and Dot-Blot and it was noted that they were able to transmit the transgene to their offspring. In this case, the expression of the transgene could verify mediate a smear palquetario, where plaeutas mouse trangénico expressed GFP. He then proceeded to analyze the comportamietno funcnonal these mice sobreexpesaban podocalicina. Firstly, as was midieronparámetros biochemical Blood glucose, triglycerides, a'cido uric, cholesterol or albumin, which was seen only slight aumetno of plasma lipids, elevated uric acid and decrease inthe runlevels of amino-transferasas. However, there were no significant differences between the data from the mice sobreexpresaban podocalicina and those of the controls.

Author: GÓMEZ-CABRERO SEGURA AZUCENA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: UNIVERSITAT DE BARCELONA.

Summary: The cells trabeculares form a spongy structure, between the cornea and iris, which regulates active filters and output the aqueous humor of the eye. Different alterations of the actin cytoskeleton, such as despolimerización of actin or inhibition of myosin, increase the development of aqueous humor, which highlights the importance of actin in the physiology of the trabecular network. Therefore, it was considered studying the role of actin in this fabric through PTD4-Pfnil as a tool to interfere with the molecular dynamics of the actin cytoskeleton. The secuéncia PTD4 (Ho et al., 2001) is a peptide transduction permeable membrane that merged with Pfn, allows access to the interior of cells. Profilina (Pfn) is a protein that binds to actin monomers, he exchanged with ATP and ADP presents actina-ATP the end of a + during the actin filament polymerization. Pfn is in domains where the intracellular actin is very dynamic, as filopodios and lamelipodios. First, it created vectors expansion for the production of fusion proteins with PTD4, they were cloned genes profilina I (Pfnl: ubiquitous) and profilina II (PfnII; neuronal), as well as a marker as *- galactosidase, and were purified proteins resulting merger. Then we checked the transduction proteins in these cells trabeculares and its dependence on time and concentration, as well as its independence from temperature. In cells trabeculares growing, it was observed that PTD4-Pfnl induced formation lamelipodios was similar to that produced for serum or growth factors, both by the presence of markers lamelipodios (Arp2 / 3 and Rac) and the absence of lamelipodios after inhibition of different components of the signaling channel (Rac and PI4K) or the contractility (myosin and MLCK). This effect of PTD4-Pfnil depended both the binding to actin binding domain as a poliprolinas (PLP) (PTD4- (GP5) 3), inhibits the formation of lamelipodios. In experiments infusion earlier segments of the eye to assess the role of trabéculo, it was observed that PTD4-Pfnl induced increased ease evacuation reversibly, probably by altering the way cell, increasing the size of intercellular spaces and loss of contractility of the cells trabeculares.

Author: CANTÓ ÁLVAREZ CARLOS.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: UNIVERSIDAD DE BARCELONA - FACULTAD DE BIOLOGÍA.

Summary: The neuregulinas are growth factors for the family of EGF (Epidermal Growth Factor) that regulate the development and muscle function (Rimer, 2003). Data obtained previously in our laboratory (Suarez et al, 2001) showed that neurgeulinas could regulate muscle glucose transport in both the short and long term. In order to deepen the capacity of these factors regulate metabolism in skeletal muscle, we proposed: 1-To characterize and understand the regulation of the expression of the receptors neuregulinas in muscle, both during development and in adult tissue. 2, - To explore how neuregulinas are able to stimulate the transport of glucose lines miocíticas in skeletal muscle and try to integrate their effects physiologically. 3-To study the chronic effects of neuregulinas on metabolism, as well as their potential applications in situations of insulin resistance. Our results indicate that the recipients of neuregulinas is finely modulated during the formation of the muscle fibers. Thus, ErB3 show high expression in the early stages of muscle differentiation, and exchanged by ErbB4 as you go differentiation. ErB4 is the largest recipient of neuregulinas in adult muscle, and their expression can be increased by muscular contraction pattern. As for the acute metabolic action of neuregulinas our resutlados indicate that neuregulinas are able to stimulate the transport of glucose in a manner independent additive to insulin, and that, in situations of insulin resistance, the action of neuregulinas on the transport of glucose is intact. The neuregulinas stimulated glucose transport through at least the way PI3K-PDK1-PKCC. The muscular contraction is another way to increase the transport of glucose and, curiously, then try the phosphorylation of receptors ErbB. Our experiments show that the cause of this phenomenon is that the muscle contraction stimulates the release of neuregulinas through a process mediated by increases in cytosolic calcium and activation of metalloproteinases membrane. Blocking the activation of receptor ErbB4 during muscle contraction prevents proper stimulation of glucose transport without negatively affecting the activation of kinases previously implicated in this process, as CAMKII or AMPK. Furthermore, blocking the action of the neurguelina during muscle contraction promoted greater fall in the levels of glycogen, ATP and fosfocreatina into the muscle, as well as greater production of lactate. These results indicate that neuregulinas are a key mediator for proper induction of glucose transport in response wing muscle contraction, as well as to regulate the content of muscle energy metabolites. Finally, we decided to analyze the effects of incubation chronic 48 hours miocitos L6E9 with neuregulinas. Our resutlados indicate that neuregulinas increase the oxidative capacity of miocitos L6E9. This effect is due to an increase in the content and mitochondrial activity. The nodulation of mitochondrial content by neuregulinas occurs through PPAR and possibly from PGC-1alfa, whose expression and phosphorylation in serinas increase in response to neuregulinas. These effects also occur in skeletal muscle in response to aerobic exercise programs, and we have preliminary data suggest that neuregulinas could be involved in how the muscular contraction modulates the mitochondrial content. Finally, we show that the fact that neuregulinas increase the oxidative capacity also brings the awareness of miocitos to acicón of insulin. This fact prompted us to consider whether the neuregulinas could improve situations insulin resistance. In this line, our results show that neuregulinas are capable of reversing situations insulin resistance induced by incubation miocitos L6E9 with palmitate.

Author: MUSRI MELINA MARA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The adiponectina is a adipocitoquina synthesized and secreted by adipose tissue alone. She appeared in various organs and has properties anti-diabéticas and anti-aterogénicas, besides being one of the largest properties with hormone sensitive to insulin. The adiponectina exert their effects on the metabolism of glucose and lipids through its receptors, adipoR1 and adipoR2. In the first work it was shown that the expression of adipoR1 in IAAT was not altered in obesity and diabetes in humans and intraabdominal adipose tissue correlated with plasma levels of FFA, while expressing adipoR2 in IAAT was reduced by obesity and diabetes in human and intraabdominal adipose tissue was associated with metabolic components implicated in the development of cardiovascular disease in obesity. Given the importance of the expression of adiponectina and their receptors, as well as other adipocitoquinas secreted by the adipocyte mature in the pathogenesis of obesity and diabetes, understand epigenetic mechanisms of transcriptional activation in the differentiation was the goal of our second study. For this we look at the changes post-traduccionales of histone H3 in the promoters of genes adipogénicos key both early and late expression throughout the process of dipogenesis. Using the technique of inmunoprecipitación chromatin (ChiP), made in cells 3T3-L1 on different days during the process of differentiation adipocitaria, we showed that the promoters of adiponectina, glut4, gpd1 and leptin are enriched in dimetilación of H3-K4 on day 0 of differentiation (D0), when neither of these genes is still expressed. A detailed study of the locus of adiponectina revealed that dimetilación of H3K4 in these cells indiferenciadas is limited in the promoter region, which also found associated with the occupation of RNA pol II. The start of the transcript of adiponectina coincides with the hiperacetilación of H3 (D3-4) and the trimetilación of H3K4 in the promoter region of the same (D2-3). This same pattern was observed modification of histones in mouse stem cells but not in fibroblasts mouse 10T72, which is not yet committed to the line adipogénica. The partial inhibition of dimetilación in H3K4 by treatment with the inhibitor resulted in a decrease in the expression of apM1, as well as a decrease in adipogénesis, detected by a decrease in adipogénesis, detected by a decrease in incorporating lipids. In summary, the second work of this thesis shows that the dimetilación in H3K4 and recruitment of the RNA polll on the promoters of genes adiogénicos key brands are indistinguishable from cells that are determined and have undertaken towards the cell line adipogénica. Those same brands, are absent in the same regions of the promoters, pluripotenciales cells that represent a preliminary step in the process of cell differentiation from totipotenciales adipocitos mature and the promoters of genes that are silenced and not expressed in certain cells to line adipogénica (3T3-L1). In adipocitos differentiated active transcription of these genes is accompanied by hiperacetilación in H3, Say and trimetilación in H3K4, as well as the extent of these changes to the coding regions of the gene. The dimetilación in H3K4 is necessary for gene expression diponectina, as well as for the development of a normal differentiated adipocitaria. However, further studies are needed to clarify which are transcription factors and cofactors necessary to the establishment of such a pattern.

Author: CANO JAIMEZ MARÍA FELICIDAD.
Year: 2006.
University: VALENCIA [www.uv.es].
Place of defense: BIBLIOTECA DEL CAMPUS DE BURJASSOT.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS - UNIVERSITAT DE VALÈNCIA.

Summary: Parkinson's disease (PD) is a neurodegenerative disease characterized by disorder engines because of the effect on the central nervous system. In this disease are lost on the one hand neurons of the substantia nigra produce, as a result, a shortage of dopamine neurons projected that these nuclei, and secondly, the remaining neurons may present some cytoplasmic aggregates characteristic called Lewy bodies. Besides earmarking central peripheral autonomic nervous system is also affected in these patients. Among the symptoms resulting from sympathetic dysfunction include orthostatic hypotension, problems of sweating, decreased in salivation, urinary problems and / or constipation and impotence. So far, known both potential environmental reasons as genetic cause of the outbreak. Among the genes known that, when mutated, cause PD is the alfa-sinucleína, whose protein product is the largest component of Lewy bodies. To try to understand the disease models are used toxic and / or genetic. In this thesis has studied the peripheral involvement in various models of PD through different approaches. First, we have characterized the pattern of expression of alfa-sinucleína and their identification cell in the peripheral nervous system during embryonic development and in the postnatal mouse. Secondly, we have studied the development of the lymph SCG, sympathetic mode, or nodosum of sensory modality, in mice deficient in alfa-sinucleína and survival after deprivation neurotrófica, genotoxic damage, or inhibition of several intracellular signaling pathways . Thirdly, we have studied the interaction between alfa-sinucleína and toxic parkinsonian MPTP / MPP + in primary cultures, and cell lines and in the sympathetic nervous system in vivo in mice null mutants for alfa-sinucleína and transgenic alfa-sinucleína human . This work is key to understanding the implication of the alfa-sinucleína in the peripheral nervous system and its relation to the JV and failures arising sympathetic.

Author: MARTIN PEÑA JOSÉ ALFONSO.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: INSTITUTO CAJAL (C.S.I.C.).

Summary: Drosophila is an appropriate body for the study of human disease and its nervous system is often used in the investigation of neurological after lathes. Here, we used the 40 neurons that make up the type R ellipsoid body of adult brain as experimental system. During metamorphosis, these cells are differentiated by issuing its extensions towards the median line according ending its proliferation to its size and arborización end in the form of ring. The complete set of neurons in the body includes ellipsoid system nuerotransmisores cholinergic and GABAérgicos besides peptidérgicos. We have used this cell system to perform two types of studies: A-characterize a new ccb Drosophila gene, and its role in axonal guidance. B-examine the effect sinaptogénico of fosfatidil-inositol 3 kinase (PI3K) and other factors cellular signaling. The choice of the cellular system is justified by the peculiar morphology of their projections, being in a ring must cross the line of the brain, and for the role it plays in the brain center that controls locomotion, which enables functional testing in the individual completely. The protein corresponding to the unit transcription CG 14579, is key for the correct guidance of axones on its way to the passage of the median line. The mechanism that allows this function is based on the regulation of intracellular trafficking of proteins and its translocation to the plasma membrane. Furthermore, we use these cells to study the signaling of PI3K in sinaptogénesis. The data show that PI3K has a strong power sinaptogénico through the action of Akt. These sinápsis ectopic result from a functional standpoint behavioral and electrophysiological. Also, we wonder how long it could keep this capacity during adulthood. PI3K retains its power sinaptogéncio throughout the life cycle of Drosophila and in all types neural analyzed. In the same way, ectopic synapses promoted by PI3K retains its power sinaptogénico throughout the life cycle of Drosophila and in all types neural analyzed. In the same way, sinápsis ectopic promoted by PI3K require the sustained activity of this enzyme. Finally, the activity of PI3K seems preserved in humans, at least in line SHSY5Y of neuroblastoma, and, therefore, opens up new prospects for combating cognitive symptoms associated with neurological diseases.

Author: SERRANO GÓMEZ DIEGO.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA - UCM.

Summary: DC-SIGN is a type C lectina that recognizes major clinical pathogens (including HIV, HCV Mycobacterium), operating as the recipient of accession, providing a bridge between the innate and adaptive immunity. This thesis seeks a comprehensive study of this lectina, and this has investigated four fundamental aspects of their biology. The first part discusses how it regulates the expression of DC-SIGN. It proposes to leukemic cells THP-1, as a cellular system useful for characterizing the functional DC-SIGN and to dissect the molecular mechanisms that control their expression is regulated and specific myeloid dendritic cells and macrophages. The second part, through the structural and functional analysis of splicing variants and mutants DC-SIGN, seeks to determine the molecular basis of the partnership between the polymorphic variants of this lectina and altered susceptibility to viral infections (HIV, Dengue) or bacterial (Mycobaterium tuberculosis). The third part focuses on the ability of DC-SIGN to recognize pathogens. It shows that DC-SIGN half the union and capture Aspergiullus fumigatus by dendritic cells and macrophages describes the requirements for such interactions and discusses their potential involvement in the establishment and persistence of pulmonary fungal infections. The fourth part discusses the interaction between DC-SIGN and Inmunoferon ®, which modulates drug regulator and effector functions of the immune system. Inmunoferon ® inhibits binding and capturing pathogenic viral, fungal and parasitic by dendritic cells. Our resutlados indicate that DC-SIGN is the recipient of this immunomodulator, and suggest that the actions immunomodulator and adjunct Inmunoferon ® are due, at least in part, to the disruption of functional activities of this lectina.

Author: BELLO COLLANTES RAQUEL.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE BIOLÓGICAS.
Place of preparation: CENTRO DE INVESTIGACIONES BIOLÓGICAS.

Summary: Current data receptor T lymphocyte antigen (complex TCR/CD3) have been obtained using various types of cells or cell lines and meditate different experimental approaches, and it would be reasonable to conclude that the complex TCR/CD3 is a constant structure whose components, except variable regions of TCR. They are equal in all T lymphocytes On the other hand, we had previously identified differences in the sequence amino-terminal of CD3 epsilon and interactions TCR-CD3 weaker. In this thesis show that the complex TCR/CD3 the surface of T lymphocytes from mice and various cell lines expressing different chains CD3 epsilon with different isoelectric point, which correspond to points isoeléctricos theorists chains who have lost one or two negatively charged residues of extremoamino-terminal of this protein. In addition, different cell lines presented proportion of different isoforms of CD3 epsilon, correlacionándose greed of the union antibody YCD3.1 with plenty of strings isoelectric point high. Moreover, we have observed the existence of complex TCR/CD3 monkey and multivalent in these cell lines, with a higher proportion of complete chains in the complex monovalent. Our data also indicate that the waste acids end amino-terminal of CD3 epsilon are involved in the interaction with the TCR, and that there are different proteases involved in the degradation of this protein. So using an inhibitor metaloproteasas we have seen a decrease in the degradation of CD3 epsilon and on the contrary, using an inhibitor aminopeptidasas we have seen an increase in degradation. The existence of various chains CD3 epsilon gives rise to different functional consequences. On the one hand there are differences in the structure of the complex TRC/D3, identified through biotinilación of membrane proteins in cell lines with different proportions of strings complete and degraded CD3 epsilon. On the other hand, the activation of lymphocyte affected, as demonstrated in the different phosphorylation of ZAP-70 or the ERK MAP kinase after treatment with various protease inhibitors, or production of IFN-gamma intracellular in without mutants negative charges at the end amino-terminal of CD3 epsilon. Therefore, we propose that these negative charges involved in the interaction TCr-CD3, so that chains CD3 epsilon degraded favor the flexibility of the complex, allowing happen more easily change conformacional after antigen recognition by the domains variables TCR, and a decrease in the threshold of lymphocyte activation.

Author: LÓPEZ TÉLLEZ JUAN FÉLIX.
Year: 2006.
University: MÁLAGA [www.uma.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS - UNIVERSIDAD DE MÁLAGA.

Summary: The hippocampus is a region of vital encefálica limbic system, which is involved in many processes of learning and memory, and provides a structure for exchanging information between different types of sensory crusts and associative, and subcorticales nuclei. The study of normal development, and in particular the changes during development in the expression of markers of neurotransmission systems, allows lay the groundwork for later comparison with certain pathological changes associated with the development, such as schizophrenia and some forms epilepsy. Applying Immunohistochemical techniques for optical and electronic microscopy techniques immuno woe, we have studied the postnatal maturation of three systems neurotransmission of great importance to the role hipocampal, such as GABAérgico, glutamatérgico and dopaminérigco. The pre - and post-synaptic markers were analyzed: neurotransmission GABAérgica, subunits alfa1 and alfa5 of GABA A receptor proteins ligadoras along with calcium claretinina and parvalbúmina: glutamatergic neurotransmission, the enzyme glutaminasas type kidney KGA, vesicular transporters glutamate (VGlu T1-3) and receivers NMDAR1 and mGluR5 in the case of dopaminergic neurotransmission analyze the subtype receptor D4, along with the enzyme tyrosine hydroxylase TH and the dopamine transporter membrane DAT. The main findings and conclusions are: 1 - The subunit alfa1 of GABA A receptor is expressed in cells main glutamatergic in nature interneuronas GABérgicas inhibition perisomática expressing protein ligadora calcium parvalbúmina, with a pattern different from that of other subunits type alpha suggesting the existence of specific control of the temporary expression of GABA receptor subunits A. The expression of alfa1 in interneuronas, precedes the time of expressing parvalbúmina, reflecting the temporary delay in the development of inhibition perisomática. 2 - The GABA receptor subunit alfa5 of A has a characteristic pattern of expression in the cells of the hippocampus main glutamatérgicas, with a change of expression somático-dendrítico between the first and second week postnatal which coincides with the change in the role of the neurotransmitter GABA the exciter to inhibitor, which suggests a possible implication of this subunit in effect despolarizante of GABA during the early stages of postnatal development. 3 - The enzyme KGA, which was responsible for the synthesis of glutamate, is expressed in neurons from the main day of birth, prior to the maturation of glutamatergic neurotransmission, support the important role as a signal trophic glutamate in the development of the nervous system hippocampus in particular. This enzyme is also expressed in different subpopulations of interneuronas GABAérgicas since early stages of postnatal development, indicating the role of glutamate as a possible precursor to the neurotransmitter GABA or as an energy. This enzyme also presents a distinctive marking on the periphery of some blood vessels from the second week postnatal, identified ultrastructural level in axones perivascular and dendrites, suggesting an important regulation mediated neuronal glutamate on the vascular system hipocampal. 4 - The vesicular glutamate transporters, VGluT1, VGluT2 and VGluT3, show differential expression during postnatal development, suggesting the existence of functional differences and / or cell types expressing these transporters. VGluT1 is most abundant in the hippocampus from the day of birth until adulthood, introducing a distribution basically complementary to VGluT2. However, unlike the previous ones, VGluT3 has been located in interneuronas, especially in the subpopulation expressing colecistoquinina, suggesting a dual phenotype GABAérg 8 ico-glut 531 amatérgico this neuronal population. 5-glutamate receptors (NMDAR1 and mGluR5) are expressed abundantly since early stages of postnatal development, both in pyramidal cells in interneuronas highlighting significant participation by both receivers in the maturation of connections glutamatérgicas and GABAérgica. 6 - The receiver D4 dopamine begins to expresares beginning of the second week postnatal, although there is an obvious innervation dopaminérigca from the day of birth. Therefore, the action of dopamine in the first fortnight postnatal and prenatal certainly in the stadiums, it is mediated by other subtypes of dopamine receptors. The area CA1 presents the greatest expression of D4, demonstrating an important modulating dopaminergic through this type of receptor on the main activity of the cells of this region.