CELL CULTURE

Author: BADIA MANAUT EVA.
Year: 2003.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: In developing and prgresiÃÂ charges of atherosclerosis exerts a key role environmental factors as dieto, exercise fÃÂsico, hÃÂ ¡bito tabÃÂ ¡quico and consumption alcohol.Precisamente this ÃÂ seventh last has been seen as a protective factor of apariciÃÂ ³ n and development the arteroesclerosis when consumption is moderate (up to 40 grams of alcohol at dÃÂa inthe men and 20 grams for women). At first this protective effect of alcohol is atribuyÃÂ ³ a effects on the profile lipÃÂdico (growth colesterol-hdl) and hemostasis (reducciÃÂ charges of agregabilidad platelet). However, mÃÂ ¡s recently has been shown in various jobs that alcohol may alter the inflammatory response that occurs in the arterial wall during formaciÃÂ ³ n of the plate aterme.Es precidamente this point, the theme Central the Doctoral Thesis, which has been submitted by the following published reports.

Author: REBOREDA PRIETO ANTONIO.
Year: 2003.
University: VIGO [www.uvigo.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: This paper demonstrates the intrinsic origin of the rhythmic activity and the neurons of the nuclei of the dorsal columns and characterized the mechanisms that give rise. To this end we developed a primary culture of the area and recorded the electrophysiological activity isolated from any activity synaptic using the technique of "patch-clamp." Most of the neurons fire short of action potentials groups (clusters) separated by stretches silent in which appears an oscillation subumbral (OS), either spontaneously or when they are slightly despolarizadas. The frequency of the OS (11.4 + -1.2 Hz) is strongly correlated with the frequency of action potentials in the group (12.5 + - 1.3 Hz), indicating that the OS determines the frequency of action potentials. The frequency of the OS and the frequency of action potentials also show some degree of correlation with the frequency of clusters (1.2 + -0.1 Hz) which indicates the existence of a factor that intersects the three types of frequency. The frequency of the OS and the frequency of action potentials are dependent on the voltage and the frequency of the clusters is not. The OS is generated by a continuing flow of Na + (Ina, p) sensitive to TTX and riluzole. Recording individual channels, it is established that the current is caused by bursts of openings sodium channel transient who lose their inactivation. The cells show current M also involved in the generation of the OS. TEA, Ba2 + and linopirdina significantly affect the OS or locking (TEA), or reducing their frequency (Ba2 + and linopiridina), and inhibit the flow M voltage ramps. Other blockers (Cd2 +, 4-AP, apamina and Cs +) affecting flows involved in generating rhythmic activities in other cells (flow of Ca2 +, K + Ca2 + -dependientes and Ih) did not affect significantly the OS, or the flow M. Curiously, the Cd2 + and 4-AP disrupt activity in bursts. The application of Cd2 + in fixing voltage allows us to exclude the participation and flows Ca2 + low threshold (IT) in the generation of rhythmic activity in cells in culture. These cells do not express the growing flow. The study of the activity in clusters shows that during the cluster is activating a mechanism that does not depend on the number of action potentials previous cluster and is able to maintain the unit in silicon for 1 s about and that is affected by Cd2 + and 4-AP. The OS and clusters can affect the transmission of information somatosensorial. The OS can filter entries favoring certain frequencies and synaptic activity in clusters can transform inputs into outputs rhythmic tonic to the thalamus. This mechanism may be important in the synchronization of neuronal populations in the NCD.

Author: BOSCH PITA MIGUEL.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The current research in the field of stem cells is offering new possibilities for therapy for incurable diseases of the nervous system, as is the case with Huntington's disease. This is a hereditary disease characterized by a selective degeneration of neurons GABAérgicas Kernel Estriado. In this thesis we studied the coping mechanisms of stem cells in culture and the factors involved in the differentiation toward selective phenotype GABAérgico. We have developed a protocol in vitro differentiation based on the sequential addition of retinoic acid (RA) and potassium chloride (KCI). RA promotes cell survival and induces a neuronal phenotype. Treatment with KCI selectively induces the phenotype GABAérgico. Through this procedure generated a large number of mature neurons GABAérgicas, post-mitóticas and functional, efficient and uniform manner, beginning with a line of stem cells in culture. The transplantation of these cells pre-diferenciadas in vitro in the adult rat brain and a model excitotóxico of Huntington's disease show a good degree of differentiation, survival and functional integration. The cells show a long-term survival, retain the phenotype neuronal GABérgico and elaborate process neuríticos associated with possible contacts pre - and post-sinápticos. These results support the possibility of developing cell therapies based on the differentiation of stem cells for treating diseases neurodegnerativas.

Author: PUERTOLLANO VACAS MARIA ANGELES.
Year: 2004.
University: JAÉN [www.ujaen.es].
Place of defense: FACULTAD DE CIENCIAS EXPERIMENTALES.
Place of preparation: FACULTAD DE CIENCIAS EXPERIMENTALES.

Summary: Pilot studies have found an increase in the prevalence of tuberculosis in Esquimales of Greenland, while a reduction in the incidence of inflammatory diseases was also observed. At present, have discovered the reasons why these events occur, and that the consumption of diets rich in polyunsaturated fatty acids in the series n-3 (common in the diet of this population) derived from fish oil reduces resistance immune front of infectious diseases. For obvious reasons, alteration of the resistance to a bacterial infection has been tested in animal models, in which administration of a diet rich in fish oil increases the number of viable bacteria obtained from the spleen of the animals, and reduces a substantial survival of the animal during the course of an infection caused by the intracellular growth of pathogenic Listeria monocytogenes. The present research work we used mice Balb / c fed diets lipídicas. These mice were divided into four experimental groups: group I fed a diet rich in olive oil (AO), group II fed a diet rich in fish oil (PA), group III fed a diet rich in oil coconut (BC) and finally the Group IV fed a diet low in 1ipidos (control diet). Terminated the period of feeding diets lipídicas mice were infected with a virulent strain of L. Monocytogenes. Therefore root and the results established that fish oil composed mainly of polyunsaturated fatty acids of the series n-3 is the most immunosuppressive and therefore animals fed on this diet showed greater susceptibility to infection with L . Monocytogenes, while the olive composed mainly of monounsaturated fatty acids in the series n-9 exercises also a suppressive effect of the immune functions, but to a lesser degree than that promoted by fish oil. This property makes the immune resistance of animals fed with olive oil is slightly higher than that exhibited by animals fed with fish oil. The immunomodulatory properties of some 1ipidos present in the diet, especially from fish oil and olive oil reduces the survival of mice, and increase the recovery of viable bacteria from the spleen, as well as alter the bactericidal activity of peritoneal cells faced with a experimental infection induced by L. Monocytogenes. Moreover, the administration of diets containing olive oil or fish oil increases the formation of superoxide anion before and after infection with L. Monocytogenes. In our study also shows that the administration of diets containing olive oil or fish oil increases some parameters as apoptotic DNA fragmentation or activity caspasa-3. However, experimental infection of these cells with L. Monocytogenes does not produce synergy in the induction of apoptosis.

Author: TÁRREGA MOULARDE CÉLINE.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: CENTRO DE INVESTIGACIÓN PRINCIPE FELIPE.
Place of preparation: CENTRO DE INVESTIGACIÓN PRINCIPE FELIPE.

Summary: The MAP kinases are part of the large group of intracellular kinase involved in cell signaling. They kinases of serinas / treoninas involved in the generation of an adaptive response to changes arising in the cellular environment. In this work, directed by mutagenesis of the MAP kinase mammalian ERK2, we have addressed: 1) the identification of mutations hyperactive of ERK2 based on the description prior gain of function mutations of this gene in Saccharomyces cerevisiae and Drosophila melanogaster, and 2 ), the comparative study of the physical and functional interaction of ERK2 their activators (especially MEK1) and its phosphatases inactivadoras (especially PTP-SL, STEP and MKP-3), based on the prior identification of a ground anchor and similar characteristic in different types of regulators and substrates of MAP kinase. With these objectives has analyzed the kinase activity of ERK2 (wild and mutant) in vitro trials have been conducted in GST-pull down, studies of the yeast two hybrid and double immunofluorescence analysis, as well as various types of tests and kinase phosphatase. We have characterized the residue Asp319 as one of the relevant waste of ERK2 upon him and we have identified some hyperactivity in ERK2 a groove anchoring compound, at least four structural elements (the loop L16, the loop L11, the noose L5 and helix e). The groove is much more specific and sensitive to the recognition of ERK2 by their inactivadores that their activators. Our findings indicate the possibility of modulating the activity of ERK2, without directly affect its enzymatic activity by specific molecules to join the groove anchorage.

Author: FABRA PASTOR MARIA MERCEDES.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA (UNIVERSIDAD DE BARCELONA).

Author: VACA SÁNCHEZ MARIA PILAR.
Year: 2005.
University: MIGUEL HERNÁNDEZ DE ELCHE [www.umh.es].
Place of defense: INSTITUTO DE BIOINGENIERIA.
Place of preparation: INSTITUTO DE BIOINGENIERIA.

Summary: Diabetes mellitus is a metabolic disorder caused by an autoimmune destruction or bad depending on pancreatic beta cells responsible for the production and secretion of insulin. The current therapeutic alternatives, daily injections of insulin or transplant of pancreatic islets, have multiple disadvantages. The cells trocales of embryonic origin because of their high proliferation and the ability to differentiate into all cell types of the body, are presented as a potential source of beta cells for development of cell therapies for diabetes. In this paper we demonstrate that through a system of selection of lineage and the addition of protein factors expressed during embryogenesis of the pancreas day e16.5, favoring the differentiation of stem cells of embryonic origin to cells secreting insulin. This mouse embryonic stem cells of the line D3 were transfected with constrcutro -geo regulated by the promoter of human insulin and joined the resistance gene higromicina under dela fosfoglicerato kinase promoter. After the differentiation protocol and proper selection, cells obtained decreased the rate of proliferation, had a high expression of insulin and Pdx-1 and coespresaban along with insulin, the anchor Péptido-Cy Glut2 in a high percentage. It also showed a secretion of insulin and C-peptide regulated by glucose and other secretagogues as tolbutamide. When transplanted into diabetic animals were able to normalize glucose levels for 6 weeks. With all this in this paper proposes a protocol differentiation able to get a high percentage of cells secreting insulin in a regulated and other characteristics of the cell. At the same time, a battery of techniques necessary for a proper characterization of phenotype obtained. However, the identification of the factors released during the development of the pancreas can be an essential tool for obtaining beta cells from embryonic stem cells.

Author: GALLOT ESCOBAL NATALIA.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The H2O2 is a major component of inflammatory microenvironment, generated by the interaction between tumor cells and normal stromal tissue invaded by cancer. In melanoma has been proposed that high oxidative stress microenvironmental could contribute to damaging the adjacent tissues and promote tumor progression. The present study was raised to study development metastatic of MB16 depending on the content of intracellular glutathione and determine the role of H2O2 in its machinery accession microbascular, through a pattern of colonization by liver cells of melanoma B16, which comes from the interaction molecular VLA-4/VCAM-1. Likewise, we identified cell subpopulations of MB16 with different functional activity of the integrina VLA-4 and studied its metastatic capabilities, with particular attention to their impact on the mechanisms of migration proangiogénica endothelial and the perivascular myofibroblasts in response to H2O2. Finally, the project has focused on evaluating the effect of blocking VLA-4, with a specific synthetic antagonist, and oxidative stress, with a natural antioxidant-resveratrol-, on the introduction and development of metastases cell MB16 . The results show that H2O2 stimulated adhesividad of MB16, with a low content of intracellular glutathione, the liver sinusoidal endothelial through a regulatory mechanism for activating the integrina VLA-4. Moreover, it identified a subpopulation of MB16 with cellular VLA-4 in state constitutively active, which in turn, was responsible for the development of metastases and the ability proangiogénica and estromagénica in response to H2O. "Finally demonstrated that treatment with an antagonist VLA-4 and Resveratrol reduces the introduction and development of metastasis through its inhibitory effect on the retention of microvascular circulating tumor cells. Eventually, the results concluded that the integrina VLA-4 regulates the effects prometastáticos lured by the H2O2 during the liver spread of melanoma B16. Study of synthetic molecules antagonists VLA-4 may be of a great use to try to achieve further progress in the fight against skin cancer.

Author: CANTÓN SERRANO IRENE.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE MEDICINA Y ODONTOLOGÍA.
Place of preparation: FACULTAD DE MEDICINA Y ODONTOLOGÍA.

Author: SOLAUM AGUIRRE MIREN SORNE.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: DTP. BIOLOGÍA CELULAR E HISTOLOGÍA. FACULTAD DE MEDICINA.

Summary: This study sought to investigate the functional activity of colon carcinoma murine 51B during the interaction with sinusoidal endothelial cells in the liver, proinflamatorios factors such as LPS, as well as the effect of a antiangigénico natural is the Endostaatina in the development of metastasis pilot said carcinoma of the colon. The results obtained in this study show that the ESH encourages CC-51B increasing membership, migration and proliferation of the tumor and the action of the conditioned medium of ESH to CC-21B active on both the nuclear translocation of Nfkappa B, as the expression of TNF-alpha by cell carcinoma of the colon. All this led to the ERM ESH increase of liver metastases produced by the CC-51B. In addition, this study characterized the influence of bacterial endotoxins in the ability of CC-51B to produce metastasis. The LPS activated to CC-51B, joining directly to the tumor cell, increasing the development of liver metastases by increasing adherence to ESH, migration and proliferation, being involved as adhesion molecules VCAM-1 or VLA-4 on expressed by the action of LPS and TNF-alpha as specific antibodies inhibited the increase in membership caused by LPS. Además el LPS estimulo la producción tumoral de TNF-alfa, vía NF-kappaB, así como la expresión de su receptor, actuando como mediador del efecto prometastático del LPS, siendo tanto activador del factor de transcripción kappaB, como inductor de metástasis, puesto que when used directly found that it produced a significant increase in the same. The VEGF is another factor that contributed to the activation of CC either directly on the tumor or indirectly facilitating the action of TNF-alpha by increasing expression of your receiver. Finally, the analysis of the effect of endostatina as anti-angiogénico in this experimental model, showed that mice receiving endostatina delayed the development of metastases. The analysis historlógico showed that the volume of metastases decreased due to a smaller number of large and small metastases in mice treated, but the endostatina not acted on the emergence of very small metastases. Using flow cytometry showed that the binding of endostatina was specific to the ESH compared to other cell types of sinusoide liver and tumor cells themselves.

Author: EGUIZABAL ARGAIZ CRISTINA.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE MEDICINA Y ODONTOLOGIA.
Place of preparation: UNIVERSIDAD DEL PAIS VASCO.

Summary: The primordial germ cells (PGCs) are the precursor cells of the germline, as many factors contribute to their proliferation, both in vivo and in vitro. Some of these factors can play its role as mitogens (eg FGFb, SF, FRSK, RA and TNF-A) while others involved lengthening cell survival (eg, IL-4, IL-6, IL-11, SF and LIF). The cytokine Interleukin-2 (IL-2), is an essential element in the development of the immune response, as it is the main factor for the growth and differentiation of T and B lymphocytes Furthermore, it has been shown that IL-2 in the adult testis is able to modulate the function of Leydig cells, inhibiting the production of testosterone. However, the role that could be played on PGCs it is not clear yet. To perform its function, IL-2 binds to specific receptors (RIL-2), which are composed of at least three subunits (alpha, beta and gamma). In this paper, we have studied, on the one hand, the expression of IL-2 * System / RIL-2 in isolated PGCs in different stages of embryonic development and, secondly, the involvement of IL-2 in cell proliferation in crops in vitro PGCs. Our results show that the PGCs expresses the receptor complex function of IL-2, but do not produce IL-2. Furthermore, IL-2 is capable of increasing the proliferation of PGCs, through paracrine and autocrine not. Therefore, it seems likely that the system of IL-2 actively involved in the proliferation of PGCs in vivo during migration processes and colonization of the same towards the peak gonadales.

Author: EGILEGOR IRAGORRI EIDER.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE ODONTOLOGÍA.
Place of preparation: FACULTAD DE CIENCIAS Y TECNOLOGÍA.

Summary: The squamous cell carcinomas are tumors relapsing accelerated progression, which spread to remote organs associated with a worse prognosis. In recent years he has identified the importance of the tumor microenvironment in regulating the spread of cancer and its metastasis. In squamous cell carcinoma of murine variants PAM 212 and PAM LY-2 involvement of the tumor microenvironment on the growth and metastatic progression is mediated by a mechanism dependent on the activation of NFkB, characteristics that distinguish mainly cellular variants found constitutively active in variant GPA LY-2, giving it a character more aggressive and metastatic secrete elevated levels of proinflammatory cytokines and proangiogénicas as IL-1alfa, IL-6, KC and GM-CSF. In order to identify molecular mediators behavior proangigoénico and regulated for metastatic liver microenvironmental factors in the two variants of the cell carcinoma GPA, this project raised the following objectives: First, PAM variants were compared with respect to some molecules known tumor progression, looking at the same time its population distribution and answered proinflamatorios agents. Secondly, a model of liver metastases experimental variants PAM to identify the structure of their histologic liver metastasis and stromal components that integrate its support. Third, the responses were compared during the functional interaction between variants PAM and microenvironmental factors soluble derivative of the sinusoidal endothelial cells and liver star-forming the stromal tumor in vivo. Finally, comparing their profiles secretion through a differential proteomic analysis (2D-PAGE) and a study proteomic expression for detection and identification by mass spectrometry type MALDI-TOF molecules secreted in a specific way by each of the variants of carcinoma GPA.

Author: SANJUAN PLÁ ALEJANDRA.
Year: 2006.
University: VALENCIA [www.uv.es].
Place of defense: ESCUELA UNIVERSITARIA DE ENFERMERIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: This thesis explores the role of reactive oxygen species (ROS) of mitochondrial origin in the cellular adaptation to low oxygen concentrations. Specifically, the activation of transcription factor main regulating gene expression in response to hypoxia: the so-called 'inducible factor by hypoxia-1 (HIF-1). Using a novel antioxidant specific mitochondria (MitoQ) has shown that the mitochondrial ROS are necessary for the stabilization of the subunit, one of the two protein subunits that make up HIF-1. However, since nitric oxide (NO) might modulate the role of mitochondrial ROS in stabilizing HIF-1, this work suggests that NO derivative of a possible new nitric oxide synthase mitochondrial protein might be useful to address the connection both mediators on the road signaling HIF-1. Preliminary studies characterization of this new protein in humans show that has a ubiquitous expression and that is located in the mitochondria but its function remains unknown. Moreover, given the importance of ROS mitocodriales in the development of many diseases, in this thesis is carried out preliminary characterization of a new molecule (MitoL) with the potential to act as mitochondrial antioxidant and explores the potential contribution of ROS mitochondrial dysfunction in the respiratory chain by altering the assembly of its terminal enzyme, cytochrome c oxidase (COX). This model is used in a cell in vitro after silencing a protein implicated in the biogenesis of COX (SURF1) using RNA interference.

Author: FERRERO GUTIERREZ AMAYA.
Year: 2006.
University: OVIEDO [www.uniovi.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The contamination of food with toxic compounds from marine algal origin is a frequent phenomenon that is associated with episodes of proliferation of microalgae, but the potential risk to human health due to the presence of these toxins in shellfish have not yet clarified. As objective of the thesis, was raised studying the effects at the level of the central nervous system, the exposure of marine biotoxins food algal origin, were used for this primary cultures of neuronal and glial cells of rat cerebellum as experimental system . The first part of the work focused on the study of acid okadaico, head of the group of so-called toxins type DSP (Diarrhetic Shellfish Poisoning) for causing a characteristic syndrome diarrhea after eating contaminated shellfish, and potent inhibitor of two proteín phosphatases general mammals: proteín phosphatase 1 and proteín phosphatase 2A. Okadaico acid lowers both neuronal and glial viability through mechanisms that involve degeneration characteristic parameters of apoptosis (DNA fragmentation, condensation and fragmentation of chromatin, activation of caspases) and modifies various types of proteín kinases cell, among them includes MAP kinase (ERK 1 / 2, p38 MAP kinase, Jun kinase), proteín kinases dependent ciclina and fosfo-inositol-3 kinase. Similarly, exposure to acid okadaico increased the vulnerability of glial cells from oxidative damage to the cerebellum, characterized by an increase in the formation of free radicals and by the alteration in antioxidants catalase enzyme systems and Glutathione Peroxidasa well as in the non-enzymatic system glutathione. Experiments performed with mixed cropping neurogliales helped establish for the first time the involvement of proteín phosphatases, including proteín phosphatase 2A, in the activity of neuroprotectora glial cells of the central nervous system against the oxidative damage. Lastly, have been carried out comparative studies of the effects of acid okadaico neural and chemical substances similar in nature: the acid 19-iso-okadaico and dinofisistoxina-2. These studies have yielded for the first time data on the biological action of these toxins and support its classification in the group DSP based on their mechanism of action. The results indicate that primary cultures of neurons cerebellum are an experimental system suitable for the study of the mechanism of action of these toxins, including for the development of its power in comparison with toxic acid okadaico, head of the group.

Author: ZAFRA LÓPEZ DELIA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA E INSTITUO DE INVESTIGAICÓN BIOMÉDICA (IRB).

Summary: There are numerous studies on the effects of tungstato sodium antidiabetic in different animal models of type 1 diabetes and type 2. However, before the beginning of this dissertation was not investigated the mechanism of action at the molecular level of this compound. Because the animal models studied the tungstato sodium exerts its effects on the same tissues on serving insulin, our working assumption was that the tungstato sodium might be acting on any of the components of the signaling cascade of this hormone . Through analysis of phosphorylation by Western Blot tests and kinase activity indicates that the treatment with tungstato sodium, both in cells CHOIR (an ideal model for the study of signaling cascades of insulin) and in primary cultures of hepatocytes (a system closest to the physiological), activated via Ras / Raf / MEK / ERK through a mechanism independent of phosphorylation of the insulin receptor. This activation of mitogenic path leads to phosphorylation of GSK3 independent activation of the path of PL3K-PDK1-PKB/Akt but dependent ERK and probably p90rsk. Moreover, as both the PKC-sensitive G protein toxin pertúsica are involved in the mechanism of action of tungstato, since inhibition of this protein blocks the effects of tungstato on the activation of mitogenic route. In primary cultures of hepatocytes both the activation of ERK1 / 2 as G proteins are necessary for the subsequent stimulation of glycogen synthesis observed in response to tungstato in these cells. Finally, the treatment of diabetic rats by injecting estreptozotocina with tungstato standardizes the phosphorylation of certain proteins in the signaling cascade of insulin, as well as the expression of some proteins whose levels are altered by diabetes. In conclusion, these data suggest that tungstato sodium could be a good candidate for treatment of diabetes.

Author: BEDIA GIRBÉS CARMEN.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE QUÍMICA.
Place of preparation: FACULTAD DE QUÍMICA.

Summary: The esfingolípidos are a family of lipid compounds important structural and functional in the cell. The ceramide, esfingolípido central to the metabolism of the rest of esfingolípidos, is a second messenger cell and its main function is the induction of apoptosis. The degradation of ceramide through ceramidasas and sphingosine kinase leads to another esfingolípido of great importance, esfingosina-1-fosfato, which has properties prolfierativas and mitogéncias, they remain totally opposed to the role of ceramide. The esfingosina-1-fosfato can in turn be degraded by the enzyme esfingosina-1-fosfato liasa. Therefore, the ceramidasas and esfingosina-1-fosfato liasa are of great importance and enzymes that regulate reóstato ceramida/esfingosina-1-fosfato, which can determine the survival of the cell. In order to determine the activity of these enzymes and have an effective method to find new enzyme inhibitors between libraries esfingolípidos analogues, were designed and synthesized some substrates fluorogéncios. These substrates are designed to action after suffering the enzyme, leading to a volatile aldehyde to suffer beta-eliminación release umbeliferona, which is fluorescent. Following this idea and structural requirements of these enzymes for their substrates, were designed and synthesized the two substrates fluorogénicos. From these, a methodology was developed for the mass screening of libraries of molecules synthesized in our group, as potential enzyme inhibitors. These methods, which took place on plates 96 wells, first developed in vitro, using as a source enzyme homogenados of rat liver. Subsequently, these tests were applied to cell cultures. In the case of ceramidasas, there was a specific method for determining the activity of ceramidasa acid using cells sobreexpresaban this enzyme. In the case of esfingosina-1-fosfato liasa, was a valid method to determine its activity using different cell lines. Subsequently these methods were used for mass screening of libraries of compounds, including urged good candidates. One of the molecules tested in trials in intact cells showed some toxicity was deeply studied by evidence of cell biology.

Author: GIRAL ARNAL HÉCTOR.
Year: 2006.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: MAP17 (Membrane associated protein 17 Kda) is a small membrane protein was identified sobreexpresada in several epithelial tissues carcinomas (colon, lung and breast). The main physiological expression of MAP17 focuses on the kidney proximal tubule contouring. It has not yet been possible to determine the physiological function of this protein, although recent studies have implicated MAP17 in the regulation of renal phosphate transporter, NaPi-IIa. This involvement in controlling the activity of the conveyor is linked to the formation of macromolecular complexes via interaction with proteins ADS, family members NHERF (NHERF1-4). These four proteins interact with both MAP17 with NaPi-IIa, through a matter of binding domains ADS located in the carboxyl end of the two proteins. The coexpresión of MAP17 with protein NHERF3 (or NHERF4) in the cell line OK, induced a process of internalization of phosphate transporter from the apical membrane to the Golgi apparatus, causing a decrease in the activity of phosphate transport. This effect did not occur with protein NHERF1 and 2. The work done on this dissertation intended to be a continuation of studies that have linked to protein ADS and MAP17 with regulating phosphate transporter, NaPi-IIa, in the renal tubule. For this reason, we study possible interactions between MAP17 and ADS other proteins that are expressed in the proximal tubule: PIST / CAL and Shank2E. We determined that both proteins ADS interaccionaban with MAP17 through in vitro biochemical techniques. Furthermore, we note that the co-expresión of Shank2E with MAP17 induced a change in the pattern of expression of the protein ADS, which now appeared in the Golgi apparatus. The protein ADS PIST, on the other hand, seemed to interact also with the conveyor NaPi-IIa. Since the co-expresión of both proteins, PIST and NaPi-IIa in OK cells, also resulted in the expression of the transporter in the Golgi apparatus. It is most likely that the overexpression of the protein PIST induced the "kidnapping" transporter expressed de novo, similar to what happens with the CFTR chloride channel. To try to minimize these effects of overexpression of the protein in the cell line OK, we decided to identify endogenous gene sequences in this cell line. The cloned genes we got were associated with the conveyor NaPi-IIa: MAP17, NHERF1, NHERF3, NHERF4, PIST and AKAP10. It also obtained partial sequences of other genes endogenous cell OK including: Shank2E, ezrina, SGLT1, SGLT2 and NHE2. We get an antibody capable of identifying a protein endogenous MAP17 cells OK. With the help of this antibody was determined that the expression of MAP17 in cell line WOK (Warnock OK) was affected by conditions such as the degree of confluence of the cells and the concentration of fetal bovine serum in the culture medium. It was found that the phosphate transporter endogenous NaPi4, also was affected in a similar way by these factors. Finally significant differences were found when it studied cell lines that we called WOK and NOK. Both lines derived from the line isolated by the group Koyama in 1978, but the cells WOK correspond to the cells that have been studied as a model of the proximal tubule, whereas the NOK cells were obtained from tissue bank of ATCC (American type cell culture). These two cell lines showed marked differences both morphological, genetic and functional. The differences were most important: The absence of mRNA expression of genes NHERF4 and PTH1R in cell line NOK, as well as a significantly reduced expression of transporters NaPi4 and NHE3; An activity of transporting phosphate reduced in cells compared with NOK cells WOK, which was related to differences in the expression of 8 transpo 4b7 rtador NaPi4 between the two cell lines. The inability of the cells to produce a NOK inhibitory effect on the transport of phosphate in response to treatment with parathyroid hormone (PTH), which correlates with the lack of expression of the receptor PTH1R in cells NOK. All these evidences indicated that the cell line WOK appears to be an acceptable model for the study of the functional relationship between proteins NHERF, MAP17 and conveyor NaPi4, while NOK cells are not a cell line suitable for this purpose.

Author: MARTÍNEZ OCAÑA VERÓNICA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAT DE FARMÀCIA - UNIVERSITAT DE BARCELONA.

Summary: This work is part of a research project focused on the search and synthesis of new surfactants derived from amino acids. Surfactants are chemical compounds due to its physicochemical properties can cause skin and eye irritation. Therefore, it is interesting to develop new compounds which are more biocompatible surfactants business that they are currently using. Moreover, the testing of Draize, based on the use of animals, are the only accepted methods and officially required to assess the dermal and ocular irritation prior to the commercialization of new compounds. However, because of social, ethical and scientific there is a need to have methods that allow replace animal experimentation in those toxicological tests. For that reason, has studied the potential effect of dermal and eye irritant different surfactants derived from amino acids using various alternative methods to animal testing models based on cellular and organotípicos. To evaluate the ocular irritation has been used, first, hemolysis of the test based on a cellular model. The existing test surfactants derived from amino acids are menso irritant eye that commercial surfactants used as a reference. Moreover, it appears that the burden of ionic surfactant determines its biological tissues, so that the anionic has a greater effect on proteins and on cationic membranes. It also has been evaluated by eye irritation test HET-CAM and CAM-TBS which are based on a model organotípico, membrane corioalantoidea egg hen. For purposes of comparison was also made to the test Draice eye with some of the compounds. The results of the in vivo test have shown that the membrane corioalantoidea egg hen is a model too sensitive to surfactants and it is discarded its use to evaluate this type of compounds. To assess the dermal irritation has been used in the cultivation of single cell lines (fibroblast 3T3, fibroblast 3T6, keratinocytes NCTC2544, keratinocyte HaCaT) and have analyzed various markers irritation. First, it valued the cytotoxicity induced by the test compounds catchment neutral red and reducing the tetrazolium salt and calculated concentrations of surfactant that inhibited by about 50% cell viability (Cl50, inhibitory concentration 50 ). According to the test results, has proposed a prediction model in the line of fibroblast 3T3, so that the compounds with a Cl50 less than 50 ug / ml of potential irritant high, compounds with a Cl50 between 50 and 100 ug / ml are moderate and irritating compounds with a Cl50 more than 100 ug / ml presents a potential irritant low. He also has studied the production and release of interleuceina 1 alpha, a mediator proinflamatorio, in line with keratinocytes NCTC 2544. According to the results surfactants derived from amino acids have a lower capacity to induce neosíntesis of cytokine that surfactants business, therefore, have less ability to trigger an inflammatory response. However, the implication of this marker in the intensity of inflammatory reactions is not clear what should utilizares along with other markers. Finally, it has analyzed the accumulation of intracellular lipid neutral as a possible marker of irritation. Most mammalian cells packed neutral lipid droplets in the form of spherical intracelul 8 ares. Pa. 732 pray that these be neutral lipids play a very important role in the repair of the membrane when it suffers some sort of aggression, therefore, is proposed as a potential marker. It has studied the accumulation of neutral lipids in the cell line keratinocytes NCTC 2544 following treatment with different surfactants. For the detection of neutral lipids was used red color of nilo that is specific and neutral lipid analysis was performed using citofluorometría and fluorescence microscopy. Surfactants induced fluorescence increases with a profile concentration dependent. In addition, increases in fluorescence was detected before seen increases in lactate dehydrogenase activity in the middle of the crop, a marker of damage to the membrane. This fact implies that the neutral lipid accumulation is a marker that indicates damage to the membrane prior to cell death. Finally, for comparison purposes has also been conducted testing Draize dermal with some of the compounds. The existing test surfactants derived from amino acids were classified as slightly irritating. In addition, the results showed that in vitro tests are more sensitive and can detect small differences in the potential irritant compounds, especially when it comes to compounds with a potential irritant light and moderate.

Author: DURAN PRADO MARIO.
Year: 2006.
University: CÓRDOBA [www.uco.es].
Place of defense: CAMPUS RABANALES.
Place of preparation: CAMPUS DE RABANALES.

Summary: In this Doctoral Thesis deals with the study of receptor somatostatinta pigs (sst) and new truncated isoforms of sst5 human and pig, trying to establish their possible link with normal and pathological cellular processes. First, it was cloned pigs receptors expressed in pituitary, psst1, psst2, psst3 and psst5A, the most important involved in controlling the secretion of growth hormone (GH). Subsequently transfectaron in heterologous cellular models consisting their ability to regulate both the level of AMP as the cytosolic free calcium in response to their ligands, somatostatin (SRIF) and cortistatina (TSA). Moreover, studying physical interaction through FRET, it was observed that psst exist in the form dimérica and oligomérica may also heterodimerizar of selectively. One of these receptors, psst2 showed a pharmacological profile and functional typical sst2, but some unique features in terms homodimerización / dissociation and internalization in response to SRIF. During the characterization of psst, it was cloned truncated Two isoforms of psst5 called psst5B and psst5C, 6 and 3 transmembrane domains (DTMs), both functional and that respond selectively to SRIF and TSA respectively. These receptors are located primarily in intracellular accumulations and colocalizan with isoforms not dashed, altering the typical location of the latter in the plasma membrane when coexpresan in various cellular models. Two isoforms have been cloned truncated the sst5 human similar to the pig, called hsst5B and hsst5C, 5 and 4 DTMs respectively. They behave in a similar manner similar to their pig in functional terms, and subcellular localization of physical interaction with hsst not truncated and also are present in Pituitary tumors and tumors of the breast, where coexpresan with other hsst. His contransfección with isoforms not truncated hsst2 and hsst5 resulted in the disruption of their functional ability, reducing the ability to mediate increases in the level of free cytosolic calcium in response to SRIF and CST.