BIOTECHNOLOGY

Author: SERRA HARTMANN XAVIER.
Year: 2004.
University: AUTÓNOMA DE BARCELONA.
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: ESCUELA DE DOCTORADO Y DE FORMACIÓN CONTINUADA.

Summary: The bovine herpesvirus type 1 (BoHV-1) is a alphaherpesvirus which is considered the most important etiologic agent of the box bovine respiratory disease responsible for high economic losses in the livestock industry. Vaccination against BoHV-1, but reduces infection and clinical signs derivatives thereof, does not prevent post-infection by BoHV-1 Pet. Consequently, these animals could become a hotbed of latent infection. Controlling BoHV-1 passes through the development of eradication programs based on serological identification and slaughter of infected animals. In implementing these programs are administered marker vaccines defectivas in one or more genes that generate an immune response different from that occurs after infection with BoHV-1 wt. Together, is being tested differential serological test that detects the presence of antibodies directed against the / s glycoprotein / absent s / s in the marker vaccine in animals infected with BoHV-1 wt. Our research group carried out the development of a live vaccine marker against BoHV-1 defective in glicoproteían E (gE) (BoHV-1 gE). Along with the construction of BoHV-1 gE, addressed the expression of recombinant Escherichia coli of gE of BoHV-1. The expression of gE was necessary both for the final characterization of the virus defectivo as for the future development of a test that pemitiera differentiation serological between vaccinated animals with the virus BoHV-1 gE and animals infected with strains BoHV-1 it was toxic to E. coli. In this thesis it has been determined that the amino acid sequence TRAPP of gE of BoHV-1 is responsible for the toxicity associated with the expression of extracellular domain of the glycoprotein in E.coli. The partial abolition of TRAPP is sufficient condition for restoring normal crop growth and accumulation of the protein expressed. It has been found also that the sequence TRAPP also is toxic when expressed integrated protein native E.coli. The toxicity of TRAPP is due to the very nature of the sequence, and no response to a use of codon alien to the E.coli nor to the activation of princpales proteases of the bacterium. Furthermore, toxicity dela sequence TRAPP correlates with the absence of such polypeptide sequence coding for E.coli themselves. Determining TRAPP has led to the identification of many other peptides also little between these proteins E.coli, and consider this fact possible relevance to the expression heterológa proteins in the bacterium. Also, through homologous recombination techniques, has obtained a strain of BoHV-1 defective for the sequence RAPPR of gE (BoHV-1 RAPPR-). Our results indicate that the sequence TRAPP is not essential to the role of gE, ie direct transmission between cells BoHV-1. Substitution dela sequence RAPPR, on the contrary, it entails a small change in the pattern of alleged virus interaction with components of the cell membrane and the extracellular matrix. Finally, in this experimental work and has also obtained a panel of monoclonal antibodies against gE of BoHV-1 for use in a test serological differential joint implementation of vaccination with BoHV-1 gE. The test design, an ELISA blocking is sensitive and specific for the detection of antibodies against 8 the gE of 333 l BoHV-1, and shows promise as an attempt to tackle the development of a definitive test.

Author: BETANCOR DUTRENIT LORENA.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: INSTITUTO DE CATÁLISIS , CSIC..

Summary: The oxidasas have a high interest of biotechnology as they are capable of catalyzing very specific oxidation of organic compounds under mild reaction conditions and using molecular oxygen as the oxidizing agent. However, these enzymes are unstable because they might suffer inactivation by dissociation of subunits, distortion of its three-dimensional structure, dissociation of cofactors or chemical alteration caused by the hydrogen peroxide produced as a byproduct of the oxidation reaction. The main objective of this thesis is optimizing biotransformations catalysed by oxidasas by preparing derivative immobilized highly stabilized. To achieve this goal we tried two complementary strategies: immobilization covalent multipuntual and multisubunidades (to avoid distortions of the enzyme molecule or dissociation of subunits) and coinmovilización of oxidas as with catalasas to eliminate in-situ hydrogen peroxide formed. The biotrasnformaciones studied were: the preparation of acid cetoadipiI7-aminocefalosporanico. From Cefalosporina C using a preparation of D-aminoácido oxidase and catalase coinmovilizadas, preparation acid glucónico using a derivative of glucose oxidase and catalase coinmovilizadas and preparing fructooligosacátidos from sucrose using a fructosiltransferasa (FST) immobilized in the the presence of glucose oxidase is required to remove glucose formed as a byproduct of the reaction, since this has a efeto inhibitor on the FST. It produced a wide range of derivatives immobilized of catalasas of different origins (bovine liver (BLC), Aspergillus niger, Micrococcus Iysodeikticus and Thermus thermophilus) using different strategies physicochemical. In all cases these enzymes stabilized by immobilization (with factors stabilization of 13, 30, 3 and 50 times more stable than the soluble enzyme. Addition, the DAAO of Trigonopsis variabilis and glucose oxidas to Aspergillus niger also immobilized and stabilized by adsorption to parent aminadas and subsequent intersecting with glutaraldehyde. immobilized preparation of DAAO was 6800 times more stable than the soluble enzyme while the stabilizing factor reached for GOX (an enzyme very robust) was 96 with respect to enzyme soluble. was also prepared a derivative FST stabilized and optimized by adsorption to parent aminadas and subsequent intersecting with glutaraldehyde. every biotransforrflaciones studied, tested the efficiency of coupling oxidase / catalase by different strategies: by disappearance of a product consisting of hydrogen peroxide (as in the case of preparation of acid cetoadipil 7-aminocefalosporanico), disappearance of inhibitory effect of glucose on the action of the FST in preparing the Fructoologosacáridos, by the ability of GOX to completely oxidize a solution of glucose 100 mM that in the absence of catalase was not possible given the inactivation by peroxide suffered by the GOX.

Author: CARRASCO CABEZAS BEGOÑA.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: CENTRO NACIONAL DE BIOTECNOLOGIA.
Place of preparation: CENTRO NACIONAL DE BIOTECNOLOGIA.

Summary: Characterization phase of the post - homologous recombination in Bacillus subtilis. The homologous recombination is the process by which the genetic material is exchanged between molecules of DNA whose sequence homology shows. The cuts in the double chain of DNA in bacteria mainly by homologous recombination repair while it plays a minor role in eukaryotes are repaired by non-union counterparts extremes. In this process it is possible to identify three phases: pre-sinapsis, where it is processed to generate the DNA substrate ADNcs on which filamenta protein RecA; synapses, where RecA produces the search for homology and exchange of chains and post-sinapsis where is the migration of chains and the formation and disposition of structures formed Holliday. In Bacillus subtilis, the genes involved in various recombination of recA were classified into groups epistáticos: (recF, recL, recO, recR, recN) (addA addB) (recP, recH) (recU ruvA, ruvB, recD) (recS, recQ, recJ) and (recG) with different susceptibility to agents that damage DNA and the gene recA in places the center connected to all groups to codify the most important protein in recombination. The genes included in the groups epistáticos and encode proteins involved in the stage pre-sináptica of recombination while those encoded by genes and groups involved in the post-. During normal growth in the mutant genes classified into groups epistáticos and problems in the segregation of chromosomes with a 3-7% of cells anucleadas, large numbers of cells with nucleoides condensates and long spaces cytoplasm free DNA . The complex RuvAB along with helicasa RecG conducting the migration of the replication fork when damage occurs to form structures Holliday and protein RecU has been characterized in this study as resolvasa cutting these structures. It is thought that in the absence of protein processing intermediaries formed during the process of recombination resolution structures Holliday is directed towards the formation recombinant molecules (dimers in circular chromosomes), or alternatively there is this resolution and are grouped chromosomes. We have isolated these suppressor mutations that are found in genes sms and subA. The protein has Sms homology with RecA and Lon proteases although it has not yet been characterized. The protein SubA has no counterpart in E. Coli and hitherto unknown function. In the absence of genes sms and subA is partially suppressed the phenotype of reparation and segregation observed in mutant of groups and. Proteins Sms and SubA could participate in the stabilization of intermediaries branched produced during recombination. In the absence of the protein RecA deleted defect segregation but not deleted defect repair of the DNA of mutant recU1 and recG. RecA performs the invasion of chains which in the absence of this protein is not formed Holliday structures which are the substrate RecU and RecG and resolution is not directed towards the formation of dímeros.En B. Subtilis there is no counterpart sequence of the protein RuvC E. Coli and unknown analog conducting the resolution of the structures of Holliday. It has been characterized protein RecU as reswolvasa majority of structures Holliday B. Subtilis. RecU binds DNA chain simple (ADNcs) and double chain (ADNcd) in a process independent of ATP-dependent and magnesium. In addition binds more effectively branched structures of three and four branches (Holliday structures) built by hybridization of oligonucleotides and efficiency of union decreases 40 times in the case of synthetic replication forks. RecU short these is 8 tructura 33d s of Holliday in a sequence determined but is not able to cut DNA linear or superenrollado this website recognition. The cut comes at the same position in chains with the same polarity.

Author: LOPEZ COBOLLO ROSA MARIA.
Year: 2005.
University: AUTÓNOMA DE MADRID.
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The plants respond differently to low temperatures. One such response is the process of acclimation to low temperatures, which is an adaptive process that allows enhance or develop greater tolerance to freezing temperatures after previously being subjected to low temperatures themselves. This adaptive process, called acclimation to low temperatures is very complex and involves many physiological and biochemical changes, most of them controlled by changes in gene expression. It is not yet known well as plants perceive the decline in lower temperatures and this signal is transmitted to take place the adaptive response. Hence the importance of identifying and characterizing new intermediaries in the response to the low temperatures. In our laboratory isolated a set of genes whose expression is increased by exposure to 4Â ° C at low levels in the model plant Arabidopsis thaliana. One of them, RCI1A, encodes a protein of the family of 14-3-3, which have been implicated in regulating many biological processes. Another of them, RCI5, encodes a family of containing monooxygenase FMO, which is not aware of any function related to stress tolerance in plants. In this thesis we face a deeper understanding of both genes participation in the process of acclimation to low temperatures. In our lab was described RCI1A increases their expression in response to low temperatures while maintaining remains encouraging. Encode the isoform? The family of protein 14-3-3. In this thesis, first, we carry out a merger traducional between promoter RCI1A and whistleblower GUS gene, which enabled us to demonstrate that the expression of this gene comes from very early states of development and in all international bodies, increasing only in response to cold, and that is regulated at transcriptional level. To examine the role of RCI1A in response to low temperatures aislamos a mutant zero in expressing RCI1A. This mutant, rci1a-1, which presents a small due to a smaller cell, and that flourishes early regard to genotype wild suggests that RCI1A has an important role in the control of cell size and the time of flowering of Arabidopsis. At the same time, it is more tolerant of freezing temperatures both under control as acclimatization. The molecular analysis of the mutant showed that RCI1A regulates part of gene expression that is activated in response to low temperatures, specifically negatively modulated the expression of CBF1 and CBF3. Since this gene is not regulated by the ABA or factors CBFs, is a good tool for searching intermediaries on the road signaling mediated by the low temperatures. Therefore, we have isolated and characterized 7 mutants affected in expressing RCI1A in response to 4Â ° C (drx). These results suggest that might correspond to connection points in response to various stress-related response acclimatization. En esta tesis también hemos caracterizado la expresión génica de RCI5, un nuevo gen de Arabidopsis, inducible por temperaturas bajas y estrés salino. A detailed analysis of his speech, by merging traducional promoter RCI5 with the GUS gene informer, has proved to be preferentially induces in the vascular tissue of leaves and flowers in response to both stress. RCI5 encodes a protein of the family FMO. In fish has been identified that FMOs are involved in the synthesis of TMAO, a potent osmoprotector. By the purification of the protein we found that RCI5 is containing monooxygenase function in vitro. And transgenic plants sobreexpresando gene have enabled us to demonstrate that RCI5 also is functional in vivo. The plants 35S:: RCI5 show greater tolerance to freezing temperatures, both under control as acclimatization, and in response to salt stress, implying a function of RCI5 in response to both stress. RCI5 regulates positively 8 the expr 458 esión of genes that are part of regulón CBF/DREB1 and genes involved in the removal of ROS. We have demonstrated the existence of TMAO in Arabidopsis, and that levels of this molecule accumulate in response to 4Â ° C and NaCl. The results obtained in this thesis have demonstrated that the TMAO, is due at least in part to RCI5, and that is a new signaling molecule that activates the gene expression that is induced in response to low temperatures and salinity stress.

Author: Guinea Gutiérrez Bárbara.
Year: 2005.
University: AUTÓNOMA DE MADRID.
Place of defense: Centro Nacional de Biotecnología.
Place of preparation: Centro Nacional de Biotecnología.

Summary: PKL12 is a Ser / Thr kinase that is located primarily in the Golgi apparatus. However, after drug treatments, as brefeldina Ay nocodazol, PKL12 is trasloca the nucleus. In this compartment subcellular PKL12 serving as his own self-transcriptional and co-factor gene as VEGF. It seems that its kinase activity is independent of its ability translocation to the nucleus. Also PKL12 is able to cooperate with oncogenes as v-Src and its overexpression in cell NIH/3T3 makes it capable of forming more pockets of transformation. Moreover, we have isolated a new protein, inmunorelacionada with PKL12, which we called as PKL12-big brother (PKL12-BB) and is sobreexpresada in tumor lines as CMT, as well as childhood leukemia and carciomas adult humans. Finally, we generated murine models for studying the biological function of PKL12 in vivo.

Author: Punzón Gau Ma. Isabel.
Year: 2005.
University: AUTÓNOMA DE MADRID.
Place of defense: Centro Nacional de Biotecnología.
Place of preparation: Centro Nacional de Biotecnología.

Summary: Design of a method of transgenesis subzonal embryo using vectors lentivirales in pure strain of mouse, C57BL / 6. Characterization of transgenic mice as well as segregation lentiviral. Using the technique for obtaining transgenic mice immunodeficient NOD / scid as a model of human xenotransplantation / morino, and expressing biologically active human protein.

Author: Mateos Gil Álvaro.
Year: 2005.
University: AUTÓNOMA DE MADRID.
Place of defense: Facultad de Ciencias.
Place of preparation: Príncipe Felipe.

Summary: In this thesis entitled "Survey and supervised application of methods for analysis of gene expression patterns" has conducted a study on the implementation of supervised learning methods for data analysis of gene expression level genomics. The patterns of gene expression are obtained by the technique of microarrays and consist of expression of the values of most or all genes in a genome at the level of transcription. The learning methods are overseen a series of techniques in the field of statistical learning and artificial intelligence. Through such methods can be classified multivariate data sets. The defining characteristic of these methods is defined using information independently to the data themselves, thus the name "supervised". Typically, such information may be belonging to various classes of the different elements present in the data set. Through a process of learning from examples whose class is known, this type of decision-making functions in accordance with methods that can be used for classification of elements of class unknown. In this thesis have studied the methods of learning and supervised perceptrón perceptrón multilayer (two known types of neural network) and support vector machines (SVM). Where required the combined perceptrón and SVM with a method called unsupervised clustering SOTA, to reduce the dimensionality of the original data. Applying methods supervised listed datasets of gene expression, has conducted a study of gene function prediction of the yeast Saccharomyces cerevisiae. There have also been applied to the prediction of phenotypic class of tumor samples and healthy. Lastly, he has used a method of bi-clustering supervised, known as "signature algorithm" for the study of the modular structure of the transcript in different cancers.

Author: ESTRUCH LORENDEAU MARIA ILONA.
Year: 2005.
University: AUTÓNOMA DE MADRID.
Place of defense: INST. DE CERAM.Y VIDRIO, CSIC..
Place of preparation: INST. DE CATAL. Y PETROL. CSIC,.

Summary: Penicillin G acylase (PGA) catalyses the cleavage of the amide bond in the benzylpenicillin (penicillin G) side-chain to produce phenylacetic acid and 6-aminopenicillanic acid, the starting material for the synthesis of several semi-synthetic antibiotics. This enzyme can also be used to catalyze the inverse reaction of semi-synthesis of Ã-lactam antibiotics. PGA from Escherichia coli is the most widely used hydrolytic and synthetic enzyme. This acylase is used as immobilized derivative on solid supports in order to ensure good activity-stability properties under operational conditions. However, it has been reported that different immobilized derivatives of PGA may exhibit different catalytic properties. The main objective of this Ph.D. Thesis is the preparation of immobilized derivatives of native and recombinant PGA with good synthetic properties for the kinetically controlled synthesis of a precursor of Cephamandole. This main objective has been focussed via three main sub-objectives: a.- Evaluation of synthetic properties of different derivatives of native PGA. Different derivatives has been prepared: a.- with different orientation of the enzyme on the support, b.- by using supports with different physical properties, c.- by blocking the support (after immobilization of the enzyme) with different small ligands. These derivatives have been evaluated regarding to a critical parameter in enzymatic synthesis: the ratio between synthetic and hydrolytic activity of the immobilized enzyme. b.- By using a recombinant PGA (with a number of Lys groups on the opposite face to the active centre) we have been able to immobilized the enzyme (through this region) on glyoxyl-agarose. The synthetic properties of these new derivatives were also evaluated. c.- A new mutant of PGA was designed in order to achieve the best immobilization and the best synthetic properties. The pac gene from E.coli ATCC 11105 was mutagenized by PCR at its 3' end, corresponding to the end of the à chain. The overproduction of the recombinant enzyme was obtained by cloning the mutagenized pac gene into the pET101/D-TOPO expression vector. After purification, the mutant was immobilized and the catalytic properties of the free and immobilized enzyme were compared with those of the native enzyme, in the kinetically controlled synthesis of an intermediate of Cefamandol. The introduction of this new tag improves the immobilization efficiency as well as the catalytic properties of the immobilized enzyme. In particular, the immobilized mutated enzyme shown much better synthetic properties than the native enzyme and maintained its catalytic efficiency also after immobilization.

Author: Vidal Conde Luis.
Year: 2005.
University: AUTÓNOMA DE BARCELONA.
Place of defense: Escuela Técnica Superior de Ingeniería (ETSE).
Place of preparation: Ingeniería Química.

Summary: Using aldolasas as biocatalysts for the formation of links with estereoquímica defined CC essentially depends on the discovery of new aldolaas and the ability of disponerlas in the market for power desmostrar power in the synthesis of sintones chiral This paper is a contribution to the field the asymmetric synthesis based on the use of enzymes through cloning new aldolasas natural, and the development of processes for obtaining aldolasas recombinant dependent DHAP, glycine and acetaldehyde for use in the synthesis of compounds with complex two new chiral centers of estereoquímica defined and complementary. Enzymes objective of this work are the four aldolasas dependent DHAP (ramnulosa 1-fosfato aldolasa, fuculosa 1-fosfato aldolasa, fructose 1.6 bifosfato aldolasa, tagatosa 1.6 bifosfato aldolasa), two aldolasas dependent glycine (threonine aldolasas) and aldolasas dependent acetaldehyde (desoxiribosa 1-fosfato aldolasa). Specifically, this work is summarized in five basic points: First, we have cloned seven aldolasas prokaryotes microbial from various sources, primarily E. Coli, in a homogeneous platform, which enables simple and versatile overexpression of soluble intracellular enzymes such as protein fusion to a queue histidinas. Second, it has developed an overall strategy for purification giving the product functional in a minimum number of steps with high performance and stability. Thirdly, we have to point methodologies for the determination of activities aldolásicas specific tests based on enzymatic espectrofotométricos based on the reaction with the natural substrate of each. Fourth, the system has been selected to produce ramnulosa 1-fosfato aldolasa (RhuA) as a model for defining a reproducible process to production scale, optimizing core conditioning overexpression of recombinant proteins in E. Coli. Fifthly, it has worked on developing a strategy for growing semi suitable for obtaining high cell density crops, optimizing the criteria for induction of the expression of the recombinant protein to maximize production and productivity RhuA. Finally, using techniques of molecular biology, has worked on improving the platform of expression to dispense with the use of antibiotics as selection markers, always thinking about its application to industrial scale. Specifically, it has desarrolado a plasmid complementation of a auxotrofía for glycine allowing the growth of an E. Coli mutant media identified without the use of antibiotics. This new system will enable future design strategies for growing more economic for the production of recombinant proteins with a lower environmental impact

Author: ESNAOLA CAMPOS URKO.
Year: 2005.
University: PAÍS VASCO.
Place of defense: FACULTAD DE INFORMÁTICA.
Place of preparation: FACULTAD DE INFORMÁTICA.

Summary: This thesis presents a new way of interaction máquina-persona, máquina-máquina based on a musical language, which has been called 'Language Musical MiReLa'. Such language can be generated by the majority of musical instruments, mobile phones and PDAs. It can be silvado. It has made a special effort to design a language to its musical phrases are easy to be silvadas. This thesis also presents a review of a distributed architecture that allows easily integrate different aspects to be considered in the operation of a mobile robot. He has been named 'Association of Agents'. It proposes a way to create networks of agents. Each agent adds new capabilities to the robot. This uses the capabilities of the players already added before implemented in the network. Communication between players is simple.

Author: Mir Llorente Mònica.
Year: 2006.
University: ROVIRA I VIRGILI.
Place of defense: Escuela Técnica Superior de Ingeniería Química.
Place of preparation: Escuela Técnica Superior de Ingeniería Química.

Summary: The work done in this thesis describes the development of new platforms electrochemical biosensors to get systems that allow for easy detection of analyte, so we needed a pre-marking of this either have to add reagents for detection. This platform biosensórica also allow the detection of a wide range of analytes on the same team at a low cost. To avoid marking DNA samples are developing a system of movement. This method of self-branding is based on the displacement of oligonucleotide mutated molecules and marking, which although containing mutations is capable of hibridar with the probe reconnaissance immobilized in the biosensor, i therefore when this is in the presence of analyte due that probe has a higher affinity for the analyte that the mutated molecule, the analyte displaces oligonucleotide mutated and marking diminuyendo well biosensor signal, which is proportional to the concentration of the analyte. It also carried out various strategies to develop an electrochemical biosensor based on oligonucleotides (aptámeros) for the detection of protein without a prior marking this analyte. Getting a generic system based on oligonucleotides, which can detect both nucleic acids and proteins. It showed five configurations of electrochemical biosensors based aptámeros to detect thrombin not checked.