BIOCHEMISTRY (5)

Author: SANCHEZ MARTINEZ SILVIA.
Year: 2006.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE CIENCIA Y TECNOLOGÍA.
Place of preparation: FACULTAD DE CIENCIA Y TECNOLOGÍA.

Author: GOYTIA PASQUÍN M. ELISA.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS.

Summary: This Doctoral Thesis deals with the study of various aspects of the transmission mechanism of potyvirus by aphids with special attention to the search for potential systems interfere with the mechanism and facilitate new ways of protecting crops face potyvirus. It has also delved into the study of the molecular mechanisms that operate in this phenomenon and its involvement in the spread of the virus. It has analyzed the possible cause for the loss of transmission capacity by aphids in a series of variants potyvirus VTE with some changes in the region codificaste factor assistant HC-Pro. This would have used two techniques to the study of protein interactions: Far Western blot and two hybrid yeast. It has been detected in a timely modification of the protein coding region of the cápsida (COP) of this virus that affects the conserved domain DAG involved in the transmission by aphids it may be the cause for the loss of function of this collection of mutants. It has also been noted that the ability of the interaction of various factors HC-Pro both from communicable and non-communicable variants by aphid remains, but it appears to be somewhat weaker in the case of non-communicable unknown variants the biological implications that this fact may have. In the search for strategies to interfere with the transmission of potyvirus aphid has been used by virus Sharka, PPPv. It has developed a system of transient expression of the protein HC-Pro of PPV based on Agrobacterium tumefciens allowing express the protein outside the context of viral injection. Likewise have also expressed proteins HC-Pro aleradas and non-functional in transmission. The protein expression was analyzed in two different guests: N.benthamiana and N.tabacum being detected only in the first one. The levels of protein expression in this system were much higher than those produced by a systemic infection of PPV in this host. To study the activity in transmission of this protein was expressed transiently Three clones full of infectious PPV not transmissible by aphid introducing various changes in the coding region of the protein as well as CP HC.Pro factor. Thanks to these clones has been demonstrated activity in the transmission of the protein expressed outside the context of viral injection through two different approaches. In trials sequential aphids feeding protein HC-Pro of PPV expressed transiently produced values of transmission comparable generators in trials with protein HC-Pro from a viral infection. However, in trials transmission plant to plant mutant protein supplemented with active transmission, the values obtained were lower than in controls virus transmitted by aphids PPV. It has been using this system to produce protein HC-Pro of PPV not functional in transmission and has been tested in the same capacity to interfere in the process without observing a significant reduction in the values of transmission for nigua of strategies tested. It has also been proven to interfere with the transmission of the virus through infecicón mixed with active virus is not transferable without obtaining again reducicón signficativa the rate of transmission.

Author: RICO RODRÍGUEZ DANIEL.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD CC BIOLÓGICAS.
Place of preparation: FACULTAD CC BIOLÓGICAS UCM.

Summary: Genomic analysis of gene regulation Receiver X Retinoides (RXR) in macrophages has revealed that the RXR ligands caused the differential expression in a modest number of genes, which are involved in functions defensive response, chemo, complement and lipid metabolism . In addition, macrophages KO for RXR-alfa lose almost entirely responsiveness, suggesting that this isoform is limiting in this cell type. These studies found that RXR induces an increase of transcriptional quimioquinas CCL6 and CCL9 and subsequently showed that the mechanism of induction involves the union of RXR and ARNpol-ii the proximal promoters. In murine models of peritonitis, KO mice to RXR-alfa accumulate fewer granulocytes and monocytes in the outbreak of inflammation, a phenomenon correlated with a shortfall in the production of CCL6 and CCL9 in KO mice.

Author: SÁNCHEZ GARCÍA OLGA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: UNVIERSIDAD DE BARCELONA.

Summary: Stress, biomedicine, it is understood as any situation in which a particular experience produces an increase in glucocorticoids and catecholamines. This increase is a result of stimulation shaft hipotálamo-pituitaria-adrenal (HPA) system and sympathetic medulo-adrenal (SMA). In the mouse, submaxillary salivary glands (SMG) is the principal place of synthesis and storage Growth Factor Epidérmico (EGF). The cat ecolaminas, segregated during stress, stimulate a rapid secretion of EGF from SMG into the blood and saliva. Later this EGF interferes with the main actions induced metabolic catecholamines and injuries caused by these hormones in the longer term. Although the effect of this interference level metabolic not easy to understand, it is obvious that interference in the generation of tissue damage is a beneficial effect for the body. This protective action of EGF has been described in the gastrointestinal and cardiovascular system. For this reason, the main aim of this thesis has been to analyze whether the EGF exerts the same protection in another target tissue of catecholamines as in the liver. To achieve this goal we study the effect of EGF on two different models of stress in a model of social stress (paradigm intruso-residente) and a model of physical stress -inmune (administration endoteoxina bacterial or LPS). The conjutno of our results lead us to conclude that the EGF not exercise a hepatoprotective so acute, or during stress generated by the confrontation between mice, either during the inflammatory response developed by LPS. Despite this we found that the mice or SMG, sialoadenectomiados (SIALO), develop some more severe liver injury and suffer higher mortality in response to LPS. We believe that this effect is due to changes in the structure of liver, the result of a transient loss of EGF after the sialoadenectomia, affect the balance between inflammatory factors (TNF-alpha and IL-6) and anti-inflammatory (IL-10 and corticosterona).

Author: RAMÍREZ BAJO M. JOSÉ.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The work of this thesis focuses on the genetic characterization, enzymatic, structural and metabolic enzyme from a deficit of fosfoglicerato kinase with sphincter hemolytic anemia. This secuenció entire gene of the PGK-1 found a single mutation in exon 3 (46Ile - Asn). Being a young child and due to the shortage of shows, was the cloning of the normal and mutated enzyme. Once known mutation was structural modeling, and it is concluded that only being altered stability of the enzyme. The enzymatic characterization studies focused on Km values, optimal pH, inhibitory effects, thermostability and mobility electroforética of hemolizados and clones, and it is concluded that these features are only observing a decrease in the stability of the enzyme mutated, and the remainder were normal. At the level of metabolic abnormality identified levels of intermediaries of the glucólisis and metabolic genetic nucleotide adenílicos, with only an increase in the levels of 2.3 GPGs and a decrease in ATP. As a general conclusion of the thesis, the enzyme deficiency is caused by a single mutation (46Ile-Asn), which decreases the stability of the enzyme activity levels falling and causing an increase of 2.3 GPGs and a decrease in erythrocyte ATP, which would instead of hemolysis causing anemia.

Author: MIGUEL TURU MARTA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of preparation: DEPARTAMENT NEUROLOGIA BELLVITSE.

Author: OTAEGUI GARCÍA GAIZKA.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: CENTRO DE INVESTIGACIONES BIOLÓGICAS (CSIC). FACULTAD DE CIENCIAS BIOLÓGICAS (UCM).

Summary: The control of neurogenesis in the CNS implies regulate multiple cellular processes, as will the proliferation of neural precursors and stem cells, differentiation and apoptosis. Thus, the neural stem cells of olfactory bulb (CMBO) depend on the (pro) insulin or IGF-I for suproliferación in culture. The IGF-I promotes further differentiation of CMBO to neurons and lía, both in culture and in vivo. To explore the possible involvement of the routes biochemical PI3/Akt and MAP kinases in signaling by factors of the family of insulin, CMBO were treated for 30 minutes with IGF-I. We have seen that this protein stimulates the phosphorylation of Akt markedly both at the stage of proliferation and differentiation of CMBO, while not triggering significantly ERK1/ERK2. The importance of the route biochemical PI3K/Akt found by overexpression using retroviral constructs, in these cells phosphatase PTEN, an inhibitor of this route. The increase in PTEN led to a decrease in baseline PAKT / AKT and also a reduction in the number of neurons and astrocytes derived from CMBO, but did not alter their proliferation. Finally, to test the effect of the absence of endogenous IGF-I on the activation of Akt, we have quantified this protein in BO knockout embryos for IGF-Ia different ages. Our data show a significant decrease delos levels PAKT / AKT in the OB mice null for IGF-1 at the age of E16 ,5-E18, 5.

Author: MUÑIZ PELLO ÓSCAR.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: FACULTAD DE BIOLOGÍA. UNIVERSIDAD COMPLUTENSE DE MADRID.

Summary: The experimental work conducted deals with the regulation of signaling for quimioquina CXCL12 through its receptor CXCR4. The quimioquinas proinflammatory molecules are small, in the positioning of key cells in the immune system in proper time and place. In fact, elaborates on the fact that the recipients of quimioquinas, family members of GPCr form heterodímeros with other recipients of the same family, and specifically, in this thesis is described as the recipient of quimioquinas CXCR4 is capable of heterodimerizar the opioid receptor on immune system cells, which results in a new recipient who is unable to signal in the presence of the corresponding ligands added simultaneously. This result can lock the cellular response to CXCL12 and block functions CXCR4 and opioid receptors. In addition, during the development of this first part, it was observed that opioids were able to induce the adherence of monocytes to fibronectin and proceeded to characterize the signaling path, since the effect of opioids, acting directly as the regulators immune response, it is still largely unknown. On the other hand the work that defines the physiological importance is the activation of the path JAK / STAT by quimioquinas and demonstrates how the expression of suppressor of cytokine signaling by s (SOCS) blocks the signaling pair CXCL12/CXCR4 involved in retention hematopoietic stem cells from the bone marrow, leading to the rapid mobilization of this population of cells to the peripheral circulation.

Author: RODRÍGUEZ RODRÍGUEZ M. DEL MAR.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CC. QUÍMICAS.
Place of preparation: UNIVERSIDAD COMPLUTENSE DE MADRID.

Summary: The Hepatitis C virus (HCV) is the leading cause of acute hepatitis and chronic liver disease, including cirrhosis and liver cancer. This virus has been classified as belonging to a new genus Hepacivirus belonging to the Flaviviridae family. Given the importance of health hepatitis C, it is necessary to search for new therapies as well as an effective vaccine for the prevention of disease. The current data pertaining to the controversial form of entry of the virus into the cell is disturbing, being necessary to delve into the processes which mediate viral union, as well as the entry and merging of the viral and cell membranes, in order to contribute to knowledge of the molecular virology of hepatitis C. Low levels of viral particles in the serum of infected patients and the lack of a system for in vitro replication of the virus had prevented direct analysis of the envelope protein of HCV, and in particular, the protein E2. Therefore, in the first place have been conducted various tests of expression in order to find an expression system for protein E2, allowing obtain sufficient quantities of soluble protein after purification to carry out further structural characterization by spectroscopic techniques . Moreover, the protein in a native conformation has been made characterization antigéncia well as studies designed to ascertain the status of oligomerización and pattern of glycosylation. Then, have been identified properties fusogénicas E2 posing as various tests aggregation of vesicles, the mixture of lipids and lost l content intravesicular, with different species fosfolipídicas, as well as in various conditions of pH and the presence / absence of cholesterol. These interactions induce changes in phospholipids that have been studied by polarization measurements of fluoresencia. In order to know how you can affect the elimination of the amino terminal region of E2 to the properties fusogénicas E2 has expressed protein E2 430 (amino acids 430-661) to subsequently carry out its structural characterization and study its properties fusogénicas. Finally, considering that the region that exhibits the highest degree of genetic heterogeneity is in the N-terminal segment of E2 region hipervariable 1 (HVR1), and several authors have proposed as a factor in the union viral have been obtained four mutants HVR1 in order to characterize its properties fusogénicas and destabilization membranes. In addition, these studies are of great importance in the design of a vaccine, since it has been shown that some of the mechanisms of immune evasion reside in this region. In conclusion, the results obtained in this dissertation provide data and tools to advance the design of new recombinant molecules that could be used for preventive purposes in the infection for the virus that causes hepatitis C.

Author: CORROCHANO SÁNCHEZ SILVIA.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: CENTRO DE INVESTIGACIONES BIOLÓGICAS (CIB-CSIC), FACULTAD DE CIENCIAS BIOLÓGICAS DE LA UCM.

Summary: Diseases heredo-degenerativas of the retina pose a major cause of blindness in the world. There is a scenario which assumes that the mechanisms that operate during development could be reactivated and effective in terms of traumatic injury or degenerative tissue. The type of cell death that occurs during the development of the nervous system, especially the queen neural, physiological happening in a way and in our lab has been the role anti-apoptotico exercising proinsulina on natural death in development. In this Doctoral Thesis has found expression proinsulina during development dela mouse neural retina, as well as in the retina and the normal adult who suffers from a degenerative process. The main objective of this thesis was to test the Doctoral neuroprotective effect of the proinsulina on diseases heredo-degenerativas of the retina. For this study has been selected models of mice with retinitis pigementaria rd1 and rd10, where the poles of the retina degenerate through a process of programmed cell death. The contribution of proinsulina exogenous to the suffering retinal degeneration was performed in cultured explants of these, also with subcutaneous injections daily along the degeneration with injections and punctual intravítreas. With none of these approaches was achieved see a neuroprotective effect of the proinsulina on the degeneration of sticks. When input from proinsulina was conducted in a manner constituting endogenous and, through the generation of mice rd10 and producers proinsulina human striated muscle, we were able to observe an effect of proinsulina on attenuator the degenerative process of the retina.

Author: GONZÁLEZ HORTA AZUCENA DEL CARMEN.
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 pulmonary surfactant is a complex mix lipoproteica developed two vital roles. First the surfactant reduces the surface tension in the alveoli decreasing labor and preventing respiratory alveolar collapse due to the interaction between the phospholipids, neutral lipids and proteins both hydrophobic SP-By SP-C. These proteins promote the formation of a functional film increased adsorption of surfactant and outreach at the interface aire-líquido respiratory surface-active molecules. Second, the surfactant develops important functions associated with the innate immune system. Most of these activities have been allocated to the protein hidrofilicas SP-Ay SP-D, which interact directly with pathogens and alerfenos and stimulates immune system cells. A direct interaction between SP-Cy lipopolysaccharide bacterial has suggested the involvement of this protein in lung defense mechanisms. The research developed in this thesis has sought to characterize at the molecular level the role of the N-terminal segment of the SP-C plays in these two functions using this synthetic peptides that could be potentially useful as an additive in new surfactant preparations clinical.

Author: VILLAESCUSA RAMIREZ JUAN CARLOS.
Year: 2006.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: INSTITUTO DE BIOTECNOLOGIA Y DE BIOMEDICINA.
Place of preparation: DIBIT (Milan), CEINGE (Napoles), IFOM (Milan).

Summary: This thesis is composed of two different parts. The aim of the first part was to study the role of Cup protein in the control of translation initiation during Drosophila oogenesis, and to identify a possible mouse homolog. We focused our work on the role of Cup during ovary development through its interaction with eIF4E. The resulting data shows that Cup colocalizes with eIF4E to the posterior cytoplasm of developing oocytes and is required for the correct accumulation and localization of eIF4E within the oocyte. We also demonstrate that Cup and eIF4E interact genetically to control ovary development and growth. Our results demonstrate that the interaction between eIF4E and Cup is essential to achieve the correct level of translational activity required for the normal development of Drosophila ovary. A search of the protein database for a mouse homologue of the Drosophila Cup protein identified the product of the Clast4 gene. We performed a yeast two-hybrid screen in order to identify specific interactors of Clast4, and we isolated the product of eIF4E gene. We also show that Clast4 mRNA and protein are highly expressed within the cytoplasm of growing oocytes. The Clast4 protein is stable during this developmental window and post-translationally modified by phosphorylation upon oocyte meiotic maturation. Moreover, we demonstrate that Clast4 and eIF4E directly interact by a canonical and functional eIF4E-binding motif. In summary, our results suggest that Cup functionally interacts with eIF4E, pointing to a crucial role for Cup in the control of translation initiation during oogenesis in Drosophila. Clast4, similar to Drosophila Cup, may act at the translational level during murine female germ-line development. The aim of the second part was to study the role of Prep1 protein in vivo during embryo development. Due to the early embryonic death of the Prep1 null mutant mice, we have analyzed a hypomorphic mutant mouse (Prep1i/i), which produces between 3 to 10% of the Prep1 protein. Prep1 belong to the MEIS class of the TALE superfamily of homeodomain containing proteins. MEIS proteins interact with Pbx proteins, and are able to form ternary complexes with Hox proteins, in order to increase the especifity of the target DNA sequence. In zebrafish, down-regulation with morpholino antisense oligonucleotides of the prep1.1 gene causes an embryonic lethal phenotype with extensive brain apoptosis, loss of hindbrain rhombomeric segmentation, lack of cartilage differentiation of neural crest cells, pericardial edema, and lack of fins. In this thesis, we demonstrate that an insertion of a retroviral vector in the first intron of the Prep1 gene (Prep1i/i) results in a hypomorphic mutation that exhibits variable penetrance and expressivity. Prep1i/i embryos die at embryonic day 17.5 to birth with an overall organ hypoplasia, severe anemia, and eye anomalies, particularly in the lens and retina. The anemia correlates with delayed differentiation of erythroid progenitors and may be, at least in part, responsible for intrauterine death. The levels of cMyb and Pax6 in fetal liver and retina, respectively, are significantly decreased in Prep1i/i embryos, consistent with the hematopoietic and eye phenotypes. The Prep1i/i embryonic phenotype recapitulates, at least in part, the Meis1 and Pbx1 phenotypes. We also show that in wt embryos, Prep1 is expressed in the AGM region (including SAPs, aortic floor, HIACs and aortic blood) and in a subset of cells of the fetal liver displaying surface markers of long term repopulating hematopoietic stem cells. Prep1i/i hypomorphic fetal livers show reduced protective activity after irradiation. Lethally-irradiated mice that are protected by high doses of Prep1i/i cells show major anomalies in all lineages both 8 in bone 52c marrow and in peripheral organs. Finally, competitive repopulation assays show that the Prep1i/i mutation induces a deficient RU activity for all hematopoietic lineages. We conclude that Prep1 is essential at multiple steps of embryonic as well as adult hematopoiesis, from the activity of LTR-HSC to the differentiation of different lineages. In summary, in this thesis we show that Prep1 is essential for hematopoiesis, eye development, and it is expressed in early embryonic hematopoietic progenitor/stem cells, due to the overall organ hypoplasia, severe anemia, eye anomalies and general hematopoietic deficiency present in Prep1i/i hypomorphic embryos.