MOLECULAR BIOLOGY (4)

Author: LAGO FEMIA EVA DE.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA (DPTO. BIOQUÍMICA Y BIOLOGIA MOLECULAR III).

Summary: One of the pockets of most interest in the study of the system cannabionoide as intercellular communication system, has been the process of completion of the biological signal mediated by this system. This has been mainly due to the high expectations that could provide therapeutic inhibitors of this process, which would serve enhancing the action of endocannabionoides on their receivers. The main objective of this thesis has been the characterization and evaluation biochemistry, pharmacology and therapeutics reuptake inhibitor of endocannabinoids UCM707. With this type of compounds is possible to minimize the undesirable effects associated with the direct activation of receptor CB1 by agonists classics cannabionoides, since levels endocannabionoides would remain within a range of values that would eliminate the psychoactive effects. Our results indicate that the UCM707 remains reuptake inhibitor more potent and selective so far, and corresponds to the high potency observed in vivo assay, to intensify the effects produced by a dose subefectiva of anandamide. It also has studied the ability of the compound to modulate the levels of endocannabionoides and neurotransmitters in the brain. We have studied the usefulness of UCM707 in various models of neurodegenerative diseases. UCM707 not exercised neuroprotection in models of Parkinson's disease and Huntington. However, it has important therapeutic benefits to improve motor dysfunctions typical of the early stages of Huntington's disease. In multiple sclerosis, UCM707 does not diminish the progression of the disease but has an important effect antispastic in modelso animals from the disease. We also analyzed as this compound is able to modulate the response nociceptiva and reduce pain response in a model of neuropathic pain. Finally, we have conducted studies to assess potential impacts strengtheners and / or toxic compound that if this were to materialize, would reduce their potential therapeutic application, but we found that UCM707 could be a pretty safe compound. From this study we can conclude that this inhibitor can be a useful drug for the treatment of diseases such as multiple sclerosis, Huntington's disease and neuropathic pain diseases for which are not yet available pharmacological good tools.

Author: WENCELAO MEZA NÉSTOR.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: Poor Piruvato Kinasa Eritrocitaria human (hRPK), an autosomal recessive disorder caused by mutations in the gene PKLR, is one of the most common causes of chronic hemolytic anemia not esferocítica. The transgenic expression of RPK in precursors eritropoyéticos poor may be an alternative therapy for those patients with severe symptoms. In this paper we built retroviral vectors that express the cDNA of hRPK and eGFP in a single messenger RNA, separated by a IRES eucriótico (SF11RPXEG). Cell Lines murine 3T3 and MEL (Cells eritroleucemia murina) and human HeLA and 293T were transducidas with infective retroviral supernatants. Analysis western-blot and FACS demonstrated the stable expression of EGFP and hRPK respectively, with stable expression of the transgene for more than 20 weeks in culture. The expression of trasngenes not significantly changed during the time the crop. Furthermore, inducing eritrodiferenciación cell MEL did not alter the expression of hRPK or eGFP. Subsequently, haematopoietic progenitors Lin-Sca-1 + were transducidos In vitro SF11RPKXEG and transplanted into recipient s normal letalmente irradiated. Three months post, a high expression of hRPK and eGFP was observed in white blood cells, platelets and mature erythrocytes. These results indicate that the retroviral vector system can be an effective way to address the phenotypic correction of the flaws eritropoyéticos associated with the deficiency through Piruvato Kinasa Eritrocitaria human gene therapy protocols.

Author: BAUTISTA AVILA MIRANDELI.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA, INSTITUTO DE BIOQUÍMICA CSIC/UCM.

Summary: In this paper is submitted to study the response of the liver compared to an agent of hepatotoxicity known, as is the tioacetamida, when that liver has been pretratado with an inhibitor of the role of Kupffer cells (resident macrophages in the liver) as is the gadolinio chloride. This has enabled us to contemplate changes in the parameters of liver injury and regeneration as well as changes that occur in them as a result of interaction between the two compounds. The results presented show that the agent hepatotoxic tioacetamida enhances the activity of cells Kuppfer, which is reflected in an increase in the degree of hepatocellular necrosis and oxidative stress and an increased expression of proteins stress: metallothionein and HSP70 . It also found that the tioacetamida induces the expression and release of TNFalfa and IL-6, as well as the activity of the mieloperoxidasa serum. These cytokines and activity of the mieloperoxidasa are mainly produced by Kupffer cells and are involved in the scheduling of liver damage product of the inflammatory response. The blockade of the role of Kupffer cells by chloride gadolinio apparently interrupted a step in the sequence of events leading to hepatoxicidad. Among other things, they concluded that the modulation of the function and activity of Kupffer cells, can serve as potential therapeutic targets being useful in preventing liver damage induced by drugs.

Author: FERNÁNDEZ SAN MILLÁN ALICIA.
Year: 2005.
University: PÚBLICA DE NAVARRA [www.unavarra.es].
Place of defense: INSTITUTO DE AGROBIOTECTNOLOGÍA Y RECURSOS NATURALES.
Place of preparation: INSTITUTO DE AGROBIOTECNOLOGÍA Y RECURSOS NATURALES.

Summary: The human albumin (HSA) is the most widely used intravenous protein in the world for therapeutic purposes. The annual consumption in Spain is 10,000 kg, but its demand exceeds the 500 tons globally, representing a market value of more than 1,500 billion. Not yet developed a cost-effective system that can compete with the current method of extraction from blood, so we set out to explore the possibility of producing transgenic plants in SAH. There are precedents in which there has been HSA in several plant species by nuclear transformation, although levels of expression has always been low (less than 1% of the total soluble protein) and insufficient to be profitable. The transformation plastidial presents major advantages, which include high levels of expression of recombinant proteins. Accordingly, this project aims at optimizing dela human albumin production in transgenic chloroplasts of snuff. Several vehicles were designed to choose who optimize the expression of SAH in chloroplasts. Among several buildings designed, the best results were obtained when the HSA is expressed in the presence of promoter and the region 5 'non-translated gene psbA. The HSA production plants in the most productive line varies throughout their life cycle between 1,2-10,5% of the total protein extracted and 0,02-0,95 mg / g fresh leaf that, revealing the time of harvest can have a major impact on the performance end. It has also been observed that the HSA recombinant accumulates in the stroma of clorplastos in inclusion body, which can be solubilized towards full lead to the HSA on a monomer. Moreover, it has tackled the challenge of producing HSA with a sequence amonoacídica identical to the human being. Because the HSA is a prepro-proteína, the sequence lacks mature ATG start of translation, so that the first phase of the study, the hat-trick was added at the end of the amino HSA. Thus, the chloroplasts produce an HSA with an additional methionine amino its end. Although this albumin may be functional, certain properties residing in shaping the extreme amino could be altered. It had discussed a strategy to express SAH merged to mature peptide serves as a starting point for the translation and then processed correctly in the stroma to rid albumin mature identical to the human being. Finally, the transgenic lines have been obtained characterized at the molecular level and productive. We have studied the stability of the HSA in vivo and after different storage conditions at harvest, in order to determine the time of collection and processing system more appropriate.

Author: GIMÉNEZ-CASSINA SENDÓN ALFREDO.
Year: 2005.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD DE CIENCIAS, DEPARTAMENTO DE BIOLOGÍA MOLECULAR.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The mitochondrial dysfunction is typical of most neurodegenerative diseases, and it is believed that greatly contributes to neuronal degeneration. In this paper we have tried to counter the strategies of mitochondrial dysfunction through gene transfer vectors with HSV-1, with the aim of alleviating neurodegeneration. First, we have established experimental models of nerve cells to conduct studies of neurodegeneration and neuroprotection. Yes, we have used the nerve agent rotenone to induce mitochondrial dysfunction through the inhibition of mitochondrial complex I, as a pilot model interesting to assess the influence of mitochondrial dysfunction in neurodegeneration. Secondly, we studied the effect of modulating the activity of the enzyme glycogen synthase kinase-3 (GSK-3) on the toxicity induced by rotenone, the GSK-3 is a kinase involved in the modulation of various cellular processes, including apoptosis. We demonstrate that inhibition of chronic GSK-3, either by the expression of a dominant negative mutant of GSK-3 (85R), or through the use of pharmacological inhibitors, has a neuroprotective effect in the face of rotenone cells similar to human neurons in culture and in neurons elementary bulb rachidian mouse. We evaluated several possible molecular mechanisms to explain this neuroprotective effect. We show that chronic inhibition of GSK-3 results in an increase in the consumption of glucose by means glucolítica. In addition, we have also noted that the neuroprotection induced inhibition chronic GASK-3, hexoquinasa II (HKII) is located mainly in the mitochondrial transporter while the glucose GLUT3 is translocated to the plasma membrane. Thus, inhibition chronic GASK-3 could be protected neurons against the cell death induced mitochondrial dysfunction through changes in the metabolic profile that includes an increase in the rate glucolítica which could be offsetting the deficit bioenergy induced by rotenone. Furthermore, the translocation of HKII to the mitochondria might also have a role antiapoptótico, modulating the permeability of the mitochondrial membrane. In addition, our results also show that chronic inhibition of GASK-3 could result in a significant increase in extracellular secretion in the middle of brain-derived neurotrophic factor (BDNF), the blocking of BDNF signaling through its receptor neutralized partially neuroprotective effect in front of the rotenone-induced inhibition chronic GSK-3. In addition, we have developed a different strategy for neuroprotection compared with neuronal death induced protenona expressing frataxina, a mitochondrial protein involved in the regulation of iron metabolism and oxidative phosphorylation, among other functions. We designed and generated vectors HSV-1 encoding the complete genomic locus of frataxina human and we evaluated their expression in different cell types. We also analyzed the effect of the expression of frataxina human on the toxicity induced by rotenone, demonstrating that the expression of frataxina human has a neuroprotective effect compared to cell death induced mitochondrial dysfunction. Finally, we designed and created viral vectors that can bring changes in the promoter activity of the frataxina depending on the conditions or over time. Well show that treatment with erythropoietin increases significantly to the promoter activity of the frataxina cells similar to human neurons in culture. Taken together, this study demonstrates the feasibility of carrying out gene transfer efficiently to nerve cells in order to develop strategies for neuroprotection compared to cell death induced by mitochondrial dysfunction.

Author: SÁNCHEZ DE ABAJO ANA.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: HOSPITAL CLÍNICO SAN CARLOS.

Summary: The main aim of the thesis has been the molecular characterization of families HNPCC Spanish. To that end, it has made a study of microsatellite instability and expression by immunohistochemistry of proteins hMLH1, hMSH2, hMSH6 and hPMS2 in tumors of probandos of families selected for clinical criteria. This study has been able to establish two groups of families, who had high volatility in which repairmen were studied genes involved in susceptibility to cancer and the low volatility in which explored other low-risk genes involved in cancer colorectal. This has been characterized a subgroup of HNPCC families without MMR defects in the system. In families with alterations in the genes MMR, has designed an algorithm that will allow the study to optimize the process of genetic analysis in HNPCC families.

Author: FERNÁNDEZ ALONSO JUAN MANUEL.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: This paper attempts to deepen the knowledge they currently have about breast cancer family style. Specifically, it addresses the issue from three different aspects, which in turn are integrated into the discussion. First, it seeks to develop a strategy for effective genetic analysis in Spanish families affected breast cancer, providing an algorithm optimization genetic testing paralos genes BARCA1 and BRCA2 criteria benefit / cost or benefit-time. Secondly, the development of an empirical model for predicting the risk of delivering pre-test germinal mutation in the genes BARCA1 or BRCA2. To that end, the study proposes a series of clinical parameters that are associated with susceptibility to breast cancer in families breast / ovarian Spanish. And thirdly, the study elaborates on the nature poligéncio of the disease considering four candidates for susceptibility genes in breast cancer (BARD1, CXorf53, BRE and TFGbeta-R1) and their biological relationship with BRCA1.

Author: FERNÁNDEZ MARTÍNEZ AMALIA.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: This report focuses on the study of the relationship between the expression of COX-2 and liver pathology, and there are many pathophysiological process related to the overexpression of COX-2 and are increasingly being uncovered. We have used both approaches, analyzing models of pre-existing pathology hepatectomía liver -regeneración after partial acute treatment with tioacetamida and human liver biopsies with chronic disease induced by the hepatitis C virus in addition, in vitro cellular models, and working with models overexpression of COX-2, experimental approaches both in vitro and in vivo, to study the relationship between prostaglandins produced by COX-2 and the process of apoptosis, one of the proposed mechanisms for the involvement of XOX -2 and prostaglandins in tumor progression. These models show that prostaglandins produced from the action of COX-2 play an important role in the initiation and development of a number of pathophysiological process attending with increased cell proliferation in the liver tissue.

Author: CAÑAS PENDÓN RAFAEL ANTONIO.
Year: 2005.
University: MÁLAGA [www.uma.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: UNIVERSIDAD DE MÁLAGA.

Summary: Reservations nitrogenous of pine seeds are composed of proteins with a high content of arginine. The use of nitrogen content in these stocks is special for germination and development of the plant. During germination proteins are hydrolysed releasing arginine that is transported to the plant where development is hydrolysed by arginase activity occurring ornithine and urea. So far unknown metabolic fate of ornithine but in this paper it is proposed that is used by the enzyme orinita *- aminotransferase (PsdOAT) for the synthesis of glutamate, running as the main source of glutamate during germination and stages early development plantular. Moreover, during these stages there has been a large accumulation of the amino acid asparragina. The thesis is postulated that asparragina is synthesized by a asparragina synthetase (PsAS1) from nitrogen in the catabolism of arginine. Finally, it is proposed that a gene that encodes a asparraginasa (PsASPG) employs the asparragina accumulated in the hipocótilo of plants for the development of the vascular system.

Author: CIFUENTES BUIRA DANIEL.
Year: 2005.
University: BARCELONA [www.ub.es].

Summary: At the molecular metabolic network, thousands of substrates are interconnected through biochemical reactions. Despite this high level of connection could lead to the flow of substrates in multiple directions, practice metabolic flows are channeled through specific metabolic pathways. One key to understanding dela organization and cellular homeostasis of the body is to understand how these modules are defined functional and are isolated from each other to ensure the proper functioning of the body. To answer this question we use the glycogen metabolism as a model for study, in particular the relationship that is established between the glycogen sintasas (GS) and hexoquinasas (HK1 i GK), through the glucosa-6-fosfato (G6P). With the overexpression of HK1 and GK through recombinant adenovirus show that the GK induces better than HK1 activation of GS liver mediated G6P because GK synthesizes more efficiently elevated concentrations of G6P that requires the activation of hepatic GS. By analyzing the fiogenias and the pattern of expression of isoenzymes implicated in the metabolism of glycogen, the hexoquinasas, glycogen sintasas and glucose transporters (GLUT), indicates that the emergence of tissue-specific isoforms at the origin of vertebrates through gene duplication, contributes to the creation of metabolic independent modules. The overview provided to us by evolutionary studies together with previous biochemical data confirms that the two tándems of isoenzymes, GLUT2/GK/GS liver and GLUT4/HK/GS muscle co-evolucionan to adapt to the needs metabolic characteristics of the fabric in that expresses.

Author: GUIJARRO LARRAZ PATRICIA.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: The factors are proteins essential guide in the process and guide neurons axones that occurs during the development of the nervous system. Netrinas and Semaforinas and compose two of the five families of factors existing guide. In this work, we studied the effect quimiotáctico of Netrina1 and Semaforinas secretables Sema3A and SEma3F in the guide and its interneuronas axones during development of the cerebellum and hippocampus, as well as the participation of Sema3A in the regeneration process of axones axotomizados in a model in vitro. The first goal of our study was to study the role of Netrina1 in the postnatal development of the cortex interneuronas cerebelar, and we found that: A-Netrina1 is a factor quimiorepulsivo in vitro for the crust interneuronas cerebelar postnatal. B-Netrina1 and their receptors Dcc, Unc5h2 and Unc5h3 expressed in the cortex cerebelar during postnatal development. C-interneuronas the crust postnatal cerebelar express NEtrina1 and their receptors Dcc, Unc5h2 and Unc5h3. D-Dcc does not seem to participate in the signal quimiorepulsiva of Netrina1 before interneuronas cerebelar of the crust. E-interneuronas the crust cerebelar migrate both in vitro and in vivo to associated with glial fibers. Our second objective was to study the effect of Sema3A and Sema3F on axones GABAérgicos of training hipocampal developing. We have noted that: A-Sema3A and Sema3F are agents quimiorepulsivos in vitro axones hipocampales embironarios and postnatal, while only Sema3F repels axones entorrinales embryonic. B-Sema3A, Sema3F and their respective receptors Neuropilina1 and Neuropilina2 expressed in the hippocampus of embryonic and postnatal ages. C-interneuronas GABAérgicas of the pyramidal layer and stratum oriens express Neuropilina1 and 2, while those in strata radiated and lacunosum-moleculares not express these receptors. Our third and final goal was to study the possible inhibitory effect of Sema3A on axones adults axotomizados using cultivation organotípico entorrino-hipocampla as a model for in vitro injury. We demonstrated that: 1-After axotomía to 15 days in vitro (DIV) m when axones entorrionales not regenerate, it increases the expression of Sema3A both in the hippocampus and in bark entorrinal, and also increases the expression of your receiver Neuropilina1 in crust entorrinal. 2, In-crop organotípicos entorirno-hipocampales obtained from homozygous mice deficient in Sema3A, is part of the regeneration axones entorrinales axotomizados to 15DIV.

Author: BORRAZ ORDÁS CARMEN.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA/H.U.BELLVITGE.

Summary: Staphylococcus aureus resistant to methicillin (SARM) is a pathogen of great importance to public health both hospital and in the community. Presents a rapid spread throughout the world and creates resistance to different antibiotics with great ease. In Spain was first described by the end of 1970 and to this day has often followed up a course in which there has been remarkable changes and molecular epidemiology. To know the current status of SARM in Spain, a multicenter study was conducted during the period June 2003, in which 64 Spanish hospitals across 14 regions. It found the proportion of methicillin resistance in strains isolated during this period, we studied the sensitivity to other non-beta-lactámicos antibiotics, as well as the properties of genotypic strains SARM, detection of resistance genes to macrolides and finally to the study the production of leucocidina of Panton-Valentine those strains acquisition community and in selected strains of nosocomial origin. The methodology used was: biochemical characterization of microorganisms, conventional microbiological techniques, molecular methods of nucleic acids such as PCR, pulsed field Electrophoresis (CCA), multilocus sequence tuyping (MLST), staphylococcal cassette chromosome mec (SCCmec). Detection of virulence factors. The results showed the following conclusions: * The proportion of methicillin resistance was 20% (similar to that obtained in other studies conducted in our country and in Europe). * There were detected strains resistant to glycopeptide, although a 1.4% of the strains presented heterorresistencia vancomycin. * 60% of the strains resistant eirtromicina submitted a phenotype MLSB encoded by genes ermA and ermC and 40% restant submitted a phenotype MSB encoded by the gene msrA. * We identified a complex clonal dominant, 73% of the strains (ST, 125:: Q / Q: mecIV) whose genetic ancestor might be related to the international epidemic clone named Clone Pediatric (ST5: mecIV). I was present in all hospitals participating in the study. * In less frequently detected other clones epidemic, EMRSA-16 (ST36: mecll), 8% isolated preferably in the Galician community, EMRSA-15 (ST22: mecIV), 2%, isolated in a hospital in Palma de Mallorca , and one isolation Clone Iberian (ST247: mecl) isolated in a hospital in Toledo. * The prevalence of SARM isolates of Community origin, producers leucocidina of Panton-Valentine was low (3%). The three strains were isolated in two hospitals in Barcelona and belonged to the same clone (ST8: BG: mecIV).

Author: CARRILLO VICO ANTONIO.
Year: 2005.
University: SEVILLA [www.us.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: Melatonin is a compound that was first described in 1958 as a hormone synthesized by the pineal gland. Today, it can be considered as a compound pleiotrópico with important properties cronobióticos, antioxidants, oncostáticas and inmunomodulares. To exercise its functions through various mechanisms of action, including through the union to membrane receptors and nuclear weapons. In recent years, it has revealed the presence of this compound in a large number of organs, tissues and cells, which has highlighted the possibility of sources extrapineales of melatonin, helping to redefine the classic line of thought about melatonin as a pineal hormone alone. Based on this background, the goals set were: 1-Locate and identify melatonin binding sites for cells in the thymus and spleen of mice. 2-detect gene expression of the receptors for melatonin MT1, MT2 and RORalfa, and the expression of proteins MT1 and RORalfa1 in mouse thymus and spleen. 3-To examine the effect of melatonin through their membrane receptors on the inhibition of the production of IL-2 induced by PGE2, in primary cultures of PBMCs human. 4-Identify active melatonin synthesis in primary cultures of PBMCs human. 5-examine the effects and mechanisms of action of endogenous melatonin on the system IL-2/IL-2R. The conclusions that the study after evaluating the results were: 1 - There are binding sites for melatonin in plasma membrane and nucleus of cells of mouse thymus and spleen. 2 - The genes encoding the receptor melatonin MT1 and RORalfa expressed in the thymus and spleen of mice, whereas the expression of MT2 only detected in thymus. 3-Both the thymus and spleen of mice presentna proteins MT1 and RORalfa1. 4-Melatonin is reversing the inhibitory effects of PGE2 on the production of IL-2 in primary cultures of hPBMCs through the union to its membrane receptors. 5-neutralized action of melatonin on the inhibition of the production of IL-2 mediated by PGE2 takes change by a decline in the levels of cAMP stimulated by PGE2. 6 - The hPBMCs express the gene receptor MT1, which shares half of melatonin on the effects of PGE2. 7 - The hPBMCs express the genes encoding enzymes AA-NAT and HIOMT and presents the enzymatic activities of these proteins. 8 - The hPBMCs cultured in vitro have the capacity to synthesize and release melatonin. 9-Melatonin endógenamente synthesized for the hPBMCs acting through their union membrane receptors and nuclear weapons as a factor intra, auto and / or para-crino regulating system IL-2/IL-2R.

Author: ALONSO SARASQUETE Ma. EUGENIA.
Year: 2005.
University: SALAMANCA [www.usal.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: HOSPITAL UNIVERSITARIO DE SALAMANCA.

Summary: The Multiple myeloma (MM) is an incurable disease in which the majority of patients lies at the persistence of tumor cells that regenerate the disease, a concept known as minimal residual disease (ERM). The analysis of the ERM has proven its usefulness in the early detection of relapses in other haematological malignancies, why try to standardize a technique for follow-up. Our first finding was based on the use of a new target, incomplete DJ rearrangement of immunoglobulin genes for amplification by PCR for the quantitative study of MRA. This target has as its main advantage to be present in 60% of patients with MM and also is not mutated, allowing the proper primers and probe hybridization to complementary sequence. The standard techniques for tracking MRA are Cytometry Flow (CMF) and quantitative real-time PCR (RQ-PCR). His simultaneous application in the analysis of ERM in a series of patients with MM in complete remission (n = 24) demonstrated that, although the RQ-PCR is slightly more sensitive and detects MRA in more cases (17/24 ), Cytometry is the simplest and applicable to more cases (90% vs. MFC. 75% RQ-PCR) becoming the technique of choice for monitoring ERM at GM. Both techniques agree that a reduction in tumor burden below 0.01% confer a better prognosis. Analysis of gene expression in the tumor cell itself is a marker and is one of the main determinants of the biology of cells. Therefore we analyze the levels of expression of a number of tumor suppressor genes (p14, p15 and p16) in patients with symptomatic MM (N = 52) and quiescent Myeloma (N = 7) and find greater expression in the GM quiescentes respect the symptomatic. Considering only the latter, we find a statistically significant correlation between high levels of gene expression and factors clínico-biológicos good prognosis, the lower proliferative activity, better response to treatment and greater overall survival. These data suggest a correlation between high levels of gene expression and less aggressive forms of the disease, similar to GM quiescent.

Author: BLANCO BENAVENTE SANDRA.
Year: 2005.
University: SALAMANCA [www.usal.es].
Place of defense: CENTRO DE INVESTIGACION DEL CANCER.
Place of preparation: CENTRO DE INVESTIGACION DEL CANCER.

Summary: VRK is a new family of serina-treonina kinases with unknown function. VRK2 endogenous is located in the nucleus and in the cytosol, as a result of the expression of two isoforms generated through alternative processing of messenger of VRK2, which gives rise to proteins of 508 and 397 amino acids. VRK2A contains a hydrophobic region that anchor the protein to the membrane of the endoplasmic reticulum and mitochondria and is expressed in most cell types studied, while VRK2B is located in the nucleus and free in the cytosol and is expressed in cell lines in which VRK1 is cytosolic. Both isoforms share a catalytic domain identical and fosforilan p53 in vitro only in threonine 18. Only overexpression of the isoform nuclear VRK2B induces stabilization of p53 through a mechanism postraduccional, mainly because of the phosphorylation on threonine 18, which induces the decrease in ubiquitinación by Mdm2 and promotes acetylation by p300. In this context, VRK2B could replace functionally to the kinase nuclear VRK1 as part of signaling pathways involved in proliferative processes. The protein anchoring JIP1 assembles signaling complex composed of MAPK kinase. The latter activity is modulated by interactions with other proteins. VRK2 co-localiza with JIP1 in the perinuclear region, and interact on a regular basis with the C-terminal domain of JIP1. But this association does not interfere with the interaction between JIP1 and TAK1, MKK7? 1 or JNK. However, VRK2 decreases the activation of JNK detected as a reduction in its phosphorylation and decreased activation of the trasncripción dependent AP1. Thus VRK2 modulates the cellular response induced by interleukin-1? And DFO-hipoxia, mediated by the activation of TAK1, suggesting that VRK2 can alter the balance of signals generated by these stimuli in the cell.

Author: CABALLERO VELAZQUEZ ROSALIA.
Year: 2005.
University: SALAMANCA [www.usal.es].
Place of defense: DEPARTAMENTO DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Author: DE LOS RIOS ALFONSO LAURA.
Year: 2005.
University: SALAMANCA [www.usal.es].
Place of defense: INSTITUTO DE MICROBIOLOGIA-BIOQUIMICA.
Place of preparation: DEPARTAMENTO MICROBIOLOGIA Y GENETICA.

Summary: The DNA methylation is essential for the development of mammals and is a key element in the control of genetic information, this process takes place at position 5 'of the cytosine, which stands at dinucleótido CpG. The CpG islands are groupings of these dinucleótidos, approximately 1 kb, which is located in the promoter region of 60% of genes in humans. They remain free methylation under physiological conditions, except those in the inactive X chromosome and the inactive allele of genes regulated by imprinting. Genes subject to imprinting is characterized by an expression monoalélica depending on the parental origin. These genes are associated with CpG islands and have important roles in the development and growth of fetal mammals. Its mechanisms of inactivation monoalélica are mediated by changes epigenéticas, such as DNA methylation and changes histónicas. Studies to identify changes in gene expression in murine embryonic fibroblasts (MEFs), when cells are under stress situation, revealed that the accumulation of cell divisions in cultured MEFs leads to the transcriptional inactivation of genes undergoing imprinting genetic. This stress also causes the de novo methylation of CpG islands that are active in the alleles of these genes. The aberrant methylation affects exclusively or preferentially subjected to imprinting genes, since it was not detected in CpG islands associated with genes located on chromosomes autosómicos nor in the genes associated with the active X chromosome. This phenomenon occurs not only in terms of cultivation, but also affects the same genes in tumor development in mice in vivo. The sensitivity of these genes to the cell stress opens the possibility of its use as an early tumor markers. The de novo methylation of the promoter gene regulated by imprinting -Cdkn1c- not the case when the metilado allele is not present, or in hybrid mice when it is inhibited the process of recombination in the region that contains the gene. This suggests that the pattern of methylation could be transferred from active to inactive allele through a process of mitotic recombination, it would generate an intermediary hemimetilado acting as a substrate for DNA metiltransferasa maintenance.

Author: GONZALEZ HERRERO INES.
Year: 2005.
University: SALAMANCA [www.usal.es].
Place of defense: INST.DE BIO. MOLE. Y CELU.DEL CANCER.
Place of preparation: INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DEL CANCER.

Summary: SLUG (SNAI2) is a transcription factor with type zinc finger domains related SNAIL essential for the normal development of cells derived from the neural crest. Mutations SLUG loss of function contributes to piebaldismo and Waardenburg syndrome type 2 in a dose-dependent. However, little is known about the overexpression of SLUG in pathological state and embryonic development. We studied a duplication of SLUG in a child with a single duplication de novo 8q11.2? Q13.3 associated with tetralogy of Fallot, closing submucoso palate, kidney abnormalities, hypotonia and developmental delay. To test the effects of overexpression of Slug developing and cancer, we generated mice that have a transgen Slug punishable by tetracycline. These mice were morphologically normal at birth, and developed tumors mesenquimatosos (leukemia and sarcomas) in the majority of cases reviewed. The removal of transgen Slug not rescued the malignant phenotype. Also, the BCR - ABL oncogene, which induces the expression of Slug in leukemic cells, did not cause leukemia in mice deficient in Slug, involving Slug in leucemogenesis BCR - ABL in vivo. Moreover, in adult mice, there was a 20% incidence of sudden death, cardiomegaly and heart failure, associated with an incipient tumorogénesis mesenchymal. The data indicate that while overexpression of Slug is not enough to produce defects morfogenéticos evident in mice, can contribute directly to cardiac phenotype and development of cancer conducting a specific and essential role in the pathogenesis of tumors mesenquimatosos.

Author: GONZALEZ NUÑEZ VERONICA.
Year: 2005.
University: SALAMANCA [www.usal.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: In this work we have studied some of the biochemical mechanisms responsible for the activity of opioid agents, using zebrafish as a model pilot. First we cloned and characterized the pronociceptina and prodinorfina of zebrafish, finding new streams in these precursors for opioid peptides. Then we have characterized pharmacologically the peptide Met-encefalina-Gly-Tyr, demonstrating that binds to opioid receptors in zebrafish and mammals, and may act as an endogenous ligand-receptor ZFOR1 and ZFOR4. Also, the dinorfina A zebrafish joins all opioid receptors in zebrafish. The study of the internalization receptor ZFOR4 has shown that this receptor is internalized by etorphine and endogenous peptides, introducing differences in the average effective dose, but not in kinetics. The receiver ZFOR3 only assimilated by dinorfina or by dinorfina 1-13, with a median effective dose and kinetics similar to those encountered in ZFOR4. Studies of the activation of MAP kinases have shown that recipients ZFOR1 and ZFOR4 activated temporarily ERK1 / 2 after the union of etorphine and endogenous peptides, with the existence of an activity constituting these two receivers. The receiver ZFOR3 active MAP kinases after the union of agonists and antagonists such as naloxone, being this activation longer in time. Finally, we note that despite differences structural, functional and pharmacological between the opioid system of the zebrafish and mammalian, processes union and activation of the opioid receptors, as well as signal transduction, are similar between these two groups agencies. Therefore, we can establish that the opioid system of the zebrafish is a good model for studying the mechanisms governing the activities of agents opioids.

Author: REYES MARTIN DAVID.
Year: 2005.
University: SALAMANCA [www.usal.es].
Place of defense: EDIFICIO HISTORICO.
Place of preparation: FACULTAD DE BIOLOGIA.