MOLECULAR BIOLOGY OF MICROORGANISMS

Author: GUISANDE ÁLVAREZ JOSÉ ANTONIO.
Year: 2001.
University: VIGO [www.uvigo.es].
Place of defense: FACULTAD DE CIENCIAS DE VIGO.
Place of preparation: FACULTAD DE CIENCIAS DE VIGO.

Summary: Spain is the largest producer of bivalve molluscs of Europe, focusing most of this activity in Galicia. Intensive farming produces an increase in the risk of outbreaks of infectious, which creates the need to track and control both on the environment and on their own growing shellfish. The objectives of the study are to identify polifásica bacteria associated with the cultivation of bivalve molluscs, the selection of phenotypic testing differentials for its characterization and design of probes olognonucleótidos specific enabling its rapid identification. A total of 488 strains isolated from water, phytoplankton, seeds, larvae and breeding of clams and oysters in Galicia and 51 strains of reference were phenotypically characterized by numerical taxonomy using 92 tests physiological, morphological and biochemical. Different species of the genus Vibrio were identified mostly from samples of clams, oysters and water cultivation, while Pseudomonas and Pesudoalteromonas were gender most frequently isolated from samples of phytoplankton. This paper proposes a set of phenotypic testing for the presumptive identification of the major groups identified bacterial. Due to the rapid identification that allows these tests and will be very useful for quality control of bivalve molluscs, treatment plants or industrial staff working in the hygienic and sanitary control. A total of 71 isolates anaerobic optional representatives of the fenones obtained by numerical taxonomy were characterized using ribotipado. The profiles ribotípicos obtained were included in 9 ribogrupos. Representatives of each were analyzed phylogenetically by sequencing of the gene coding for ARNr 16S. The identification polifásica allowed isolation for the first time V.trasmaniensis, V.kanaloae, V.pormeroyi and V.neptunius from other crops and clams, and also the design of specific oligonucleotides that allow its quick identification.

Author: RUIZ GARCÍA MONTSERRAT.
Year: 2003.
University: MIGUEL HERNÁNDEZ DE ELCHE [www.umh.es].
Place of defense: MEDICINA.
Place of preparation: UNIVERSIDAD MIGUEL HERNÁNDEZ.

Summary: The application of molecular techniques to the study of tuberculosis is producing a breakthrough in the understanding of this disease. Techniques more utilziadas are: RFLP of IS6110 (technical standard that considers a sequence insertion), and PCR-based techniques (spoligotypius, VNTR yAFLP very useful as adjuncts to RFLP of IS6110). RFLP is being used to improve the knowledge on the epidemiology of tuberculosis in populations, pear is not capable of discriminating strains with less than 6 copies of IS6110, in these cases could be useful techniques based on PCR. Our objectives were: 1-Improving understanding of the epidemiology of ABC in the area of Health Elche by applying the technique of reference IS6110-RFLP. 2-Evaluate the importance and usefulness of AFLP complementing IS6110-RFLP and applicability to the study of this disease. 3-Study matching molecular epidemiology with the classical epidemiology.

Author: NADAL MATAMALA ANNA.
Year: 2003.
University: GIRONA [www.udg.es].
Place of defense: CIENCIAS.
Place of preparation: LAB. INVESTIGACIÓN DE MEDICINA INTERNA-HEPATOLOGIA DEL HOSPITAL DE LA VALL D'HEBRON.

Summary: The hepatitis C virus (HCV) leads to a chronic hepatitis which affects more than 171 million people. It is a virus RNA positive chain is classified within the family Flaviviridae and like most of RNA virus is characterized by a high rate of mutation. One of the main biological implications of the high rate of mutation is the resistance to treatment. Wanted as other therapeutic solutions to combat viruses including the use of ribozimas dirigidad directly against the RNA genome of the virus. The ribonuclease P (Rnasa P) is a ribozina that is present in all organisms as it is the enzyme responsible for the maturation of prescursores of transfer RNA. It is a endonucleasa of very specific and differs from other natural ribozimas why recognizes elements destructurales rather than in sequence. The most interesting based on the therapy is that it has been shown that one can direct their activities towards any guide RNA using a sequence of RNA that when hybrida with RNA target, the hybrid imitate the secondary structure of the substrate narural. The ultimate objective of this work is cut, in vitro, I'RNA HCV using ribozima Rnasa P. We have studied two models of Rnasa P, Rnasa P human guided by external guide sequences (EGS) i RNA M1 of the Rnasa P E.coli linked to the sequence guided by the extreme 3 '(ribozima M1GS). Before directing ribozima, we studied the structure and variability in a region of the genome of the virus since it has been described that are factors that can limit the effectiveness of any ribozima. This paper provides data accessibility and variability of a region's internal genomo, the region E2/NS2.Los results showed that in vitro has been directing both Rnasa P human as ribozima M1GS had HCV RNA and cut in a default position as accessible. An analysis of mutations suggests that the region is variable studied. Due to the interaction with sequences guide is susceptible to variations in target these mutations could affect the efficiency of court. In front of these results and in the case of aiming for a therapeutic strategy would be to attack several targets simultaneously. On the other hand, two cuts have been characterized observed on the HCV genome when incubated with this excerpt from Rnasa P human sequences in the absence of guidance. For carecterización have applied different techniques that can be divided into direct methods (RNA fingerprinting) i indirect (inmunoprecipitación and competitive inhibition). The results show that Rnasa P human enzyme is responsible for the specific cuts that are located, one on the internal ribosome entry (IRES) and the other in the region coding structural and non structural. The Rnasa P is one of the metabolism of tRNAs. Moreover, while a virus is highly variable, these structures must be important for the virus since it is maintained in all variants natural analyzed. Whatever their role is, the presence of these structures replantea initial therapeutic strategy because its similarity to eltRNA makes them susceptible to attack Rnasa P directly, without the need for sequences guide.

Author: LLARENA FERNÁNDEZ MARTA.
Year: 2004.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE CIENCIA Y TECNOLOGÍA.
Place of preparation: FACULTAD DE CIENCIA Y TECNOLOGÍA.

Summary: In cyanobacteria nitrate / nitrite are transported to lower cell through a conveyor "ATP-binding cassette (ABC) called NRT, composed of four protein subunits: NrtA (subunit periplásmica responsible for the binding of nitrate / nitrite), NrtB (subunit transmembranal ), NrtC and NrtD (subunits of nucleotide binding). The filamentous cyanobacterium, thermo and not fixing nitrogen, Phormidium laminosum had been identified genes nrtA, nrtB and nrtC, which encode the ABC transporter nitrate / nitrite, part of operon nir, along with the gene mirA, which consolidates nitrite reductase. This study has identified the gene nrtD, after mrtC, part of operon nir. The subunit NrtC transporter nitrate / nitrite P.laminosum has been purified from cells deficient in nitrogen and it has been shown that hydrolyzed ATP in vitro, in the absence of other components of the conveyor. Genes nrtC and nrtD were cloned into plasmid expression pQE-9 and have been expressed in E.coli, purifying as fusion proteins to queue histidinas. We have characterized subunits His6NrtC and His6NrtD for its ability to catalyze the hydrolysis of ATP, with regard to their specificity for several substrates and inhibitors sensitivity, revealing that both proteins hydrolyzed ATP in vitro. In addition, through elctroforesis in polyacrylamide gels in non-denaturing conditions has been detected formation homodímeros of NrtC, His6NrtC and His6NrtD. The formation of homodímeros of His6NrtD also has been tested by mass spectrometry MALDI-TOF.

Author: BONJOCH FORNOS XAVIER.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGIA.

Summary: The Bifidobacterium genus has been proposed as an indicator of the origin of the fecal contamination, as certain species of Bifidobacterium spp. It is found only in humans and in other warm-blooded animals. These microorganisms can be used to distinguish between human and animal fecal contamination. These are in a concentration among 10 9 and 10 10 CFU / g in faeces. In addition, anaerobic their physiology, their food requirements complex and the inability to multiply below 30Â ° C are unlikely to play in the middle extraenterico. The aim of this thesis has been the optimization of these techniques for the detection and quantification of Bifidobacterium spp.en samples of wastewater. The selective media analysis for the detection of Bifidobacterium, Beerens, HBSA and BFM, enumerations presented some very similar in wastewater, showing a high specificity for the detection of bifidobacteria. However, the medium Beerens and HBSA recovered a number significantly equal and highest bifidobacteria arable not the middle BFM. The colonial hybridization with the probe Bif allowed a confirmation practice enumerations, giving a correction to the growth fraction of giving no specific means used. The average HBSA allows calculating the ratio bibidobacterias total respect bibidobacterias fermented sorbitol / Y / T), which allowed distinguish the origin of the fecal contamination. The values of this ratio above 0.20 indicate a human origin of the pollution. The values below 0.20 indicate a contamination of animal origin. This paper has developed a PCR for multiple species B.adolescentis and B.dentium as a complementary tool to characterize the molecular origin of the fecal contamination in water. The detection limit of this technique is 10 3 CFU/100mL. To quantify these species in the environment carried out a quantitative PCR for these two species (proposed as indicator of human fecal contamination in wastewater). This technique provides a detection limit of 10 4 UCF/100mL. Finally, studies were conducted inactivation of Bifidobacterium genus in river water through all the techniques developed in the thesis. Gender Bifidobacterium is potentially a good indication of the origin of the recent fecal contamination in water. But his persistence low in the water environment is a disadvantage when fecal contamination is not new.

Author: CANTERO GONZÁLEZ SALAZAR LAURA.
Year: 2004.
University: POLITÉCNICA DE MADRID [www.upm.es].
Place of defense: E.T.S. INGENIERÓS AGRONOMOS.
Place of preparation: E.T.S. INGENIEROS AGRONOMOS.

Summary: The rhizosphere is the area of soil amended by the presence of exudates and plant roots. As a result there exists greater microbial activity in the rest of the soil, and it is in the rhizosphere where is the beginning of the interaction between the bacteria symbiotic nitrogen-fixing Rhizobium and the root of the legume. % & / Research recently revealed the existence of systems autoinduction ( "quorum sensing") in many Rhizobium, which can play an important role in the early stages of recognition and colonization of the rhizosphere of the legume host (Rodelas et al. 1999). The systems are autoinduction chemical intercellular communication systems that enable bacterial cells detect the size of the population in which they find themselves. In the best cases studied, the chemical signals are molecules acil-homoserian lactones (AHLs), which by its structure, freely cross cell membranes. These molecules are synthesized at low levels normally act as co-inductoras or co-represoras systems gene above a threshold level. Precisely because of their chemical structure that allows them to enter and leave the cells, only this threshold will be reached for a cell determined when sufficient numbers of cells is in immediate vicinity with that cell. These are therefore detection systems for the cell density, and has demonstrated its importance in processes in which the bacterial population size is important, as the pathogenicity, luminescence, and so on. The system better known in rizobios is to Rhizobium leguminosarum bv.viciae A34 in symbiosis with peas, thanks to the work carried out by Downie et al. (See review Wisniewski-Dyé Downie, 2002). The main objective of this dissertation is based on previous studies of systems autoinduction in this strain, and is to determine the nature, composition, function and regulation of such systems in autoinduction strains of Rhizobium leguminosarum bv.viciae UPM791 and 3841. Specific objectives pursued in this Doctoral Thesis: 1-Study on the production of molecules signal type acil-homoserina lactones (AHLs) strains UPM791 and 3841. This will take place techniques thin layer chromatography to separate and identify allowing individual molecules synthesized. 2-Identification and description in both strains of the different systems of autoinduction gene responsible for the production of these molecules, as well as its location in the genome. This will take place molecular biology techniques (PCRs, hybridization with probes ..) to allow the characterization of these systems. 3, - Search for genes and proteins that are regulated by systems autoinduction. This will be used for protein separation techniques using high-resolution two-dimensional gels, to allow comparison of proteomas of different strains in terms of presence and absence of acil-homoserina lactones (AHLs). Those proteins vary its expression may be identified by fingerprint peptide (mass spectroscopy by MALDI-TOF) and the comparison with the databank will provide us with the genomic sequence. 4-Determination of the role of systems autoinduction in the survival and competitiveness of R.leguminosarum in the rhizosphere and on the surface of the plant. This will take place functional studies in the presence and absence of AHLs needed to determine parameters such as adherence to the root, or effectiveness in symbiosis in early and late stages of interaction with the plant.

Author: LUQUE ALMAGRO VICTOR MANUEL.
Year: 2004.
University: CÓRDOBA [www.uco.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: In Cordoba, industry joyera generated as a result of their activity a waste containing high concentrations of cyanide free and complex cianuro-metálicos. With the goal of designing a biotechnology process to eliminate these toxic compounds have been isolated from the sludge Guadalquivir River as it passes through Cordova, a bacterium alcalófica able to degrade cyanide in alkaline conditions. The bacteria, which is classified as Pseudomonas pseudoalcaligenes CECT5344, use cyanide residue free or jewelry as a source of nitrogen for growth aerobic. The path of degradation of cyanide in this bacterium passes through cianhidrinas, while their ability to synthesize sideróforos allows you to use complex cianurados very stable, as the ferrocyanide férrico.Una approximation proteomics has revealed that P.pseudoalcaligenes CECT5344 cyanide induces a protein that could be involved in the synthesis of sideróforos (fosforribosilglicinamida formiltransferasa) proteins involved in protection against oxidative stress (alkyl hidroperóxido reductase and a DNA binding protein type ferritin), a regulatory protein normally induced in conditions of nitrogen limitation (PII- 2) and a heat shock protein. In P.pseudoalcaligenes CECT5344, metabolism cyanate could depend on a conveyor and bicarbonate are within controlled by catabolite repression. This work establishes for the first time a link between metabolism and cyanate cyanide, but in a mutant gene cynS indicates that the cyanate not esun middleman in the forced assimilation of cyanide. So far, P. Pseudoalcaligenes CECT5344 is the first bacterium that degrades described cyanide to alkaline pH and in the absence of glucose. All the features make this bacteria a perfect candidate for use in proceos biotechnology, as has been demonstrated with destoxificacióm residue of jewelry in a bioreactor and the construction of a biosensor of cyanate.

Author: MARÍN ALBERDI M. DOLORES.
Year: 2004.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: CENTRO DE BIOLOGÍA MOLECULAR SEVERO OCHOA.

Summary: This paper has obtained a strain of Saccharomyces cerevisiae industrial baker capable amilolítica for inclusion in its genome gene SWA 2 of Schwanniomyces Westerners. The new strain contains only DNA from yeast and has a high level of utilization of starch. The transformation of the yeast was carried out through a process of integration genomics led allocus ILV2. The gene SWA 2 is expressed on a constitutive promoter under the ADH 1. It has studied the growth, and capacity utilization of starch fermentation of the new strain obtained. Panaderia conducted tests revealed that there is no significant improvements when using the transformed strain of S. Cerevisiae. We raised therefore the coexpresión of -amilasa SWA 2 with other activities amilolíticas. To that end, we studied the system amilolítico of yeast Phaffia rhodozyma, as a possible donor activities amilolíticas. Yeast Phaffia rhodozyma has been widely used in the food industry because it produces the pigment astaxantina. In this paper, we studied the growth of the yeast on various substrates and analyzes your system amilolítico. It has been purified from this one -glucosidasa extracellular yeast using ion exchange chromatography. The molecular weight of the purified protein is 115 +-7% based on the evidence of molecular filtration on a non-denaturing. The SDS-PAGE gels determine that the protein is composed of two subunits of approximately 60 kDa each. The conditions of maximum activity are 45Â ° C and pH 5.5. The enzyme has a high activity on maltose, maltotriosa and oligosaccharides and not hidrolizalos substrates which are formed by links to (1.6). The new -glucosidasa presents activity transglicosidasa and can be used for the synthesis of oligosaccharides with a capacity prebiotic. We have determined the kinetic constants of the enzyme on different substrates. In order to get the gene for -glucosidasa described, it generated a genoteca of cDNA of Phaffia rhodozyma. It was also designed degenerate oligonucleotides targeting conserved regions of different a-glucosidasas, to amplify the internal sequences using PCR gene.

Author: MARTIN MARCOS MARIA DEL PILAR.
Year: 2004.
University: SALAMANCA [www.usal.es].
Place of defense: CENTRO DE INVESTIGACION DEL CANCER.
Place of preparation: INSTITUTO DE MICROBIOLOGIA BIOQUIMICA.

Summary: Gcn4p is a transcriptional activator of at least 539 genes in Saccharomyces cerevisiae. The expression of the gene GCN4 is regulated traduccionalmente depending on the availability of amino acids, for a "resumption of the translation," which depends on four phases of reading short in the region leader mRNA-GCN4 and a series of positive effectors ( GCN) and negative (GCD). The genetic characterization of a mutant spontaneous gcd17 has identified to RPL33A as a new GCD gene whose product is required for the Suppression traduccional of GCN4 in S. Cerevisiae. L33A is a protein essential for ribosomal subunit 60S which is part of a family of highly conserved eukaryotic proteins involved in binding to tRNAs. The point mutation rpl33a-1 determines the replacement of glycine at position 76 by an arginine, in a domain of the protein L33A highly conserved evolutionarily. A model of the three-dimensional structure of L33A predicts that the G76 is part of a rigid outer loop, and that the mutation does not alter the overall folding of the protein. Mutations replace some synthetic waste within the same domain by other commodities have the same phenotypic consequences that the spontaneous mutation rpl33a-1. Both the mutation rpl33a-1 as replacing RPL33A by a null allele, causing multiple defects in the processing of the ribosomal RNAs and the assembly of pre-partículas 60S, causing a serious shortage of subunit 60S mature in the cell. The lack of subunits 60S causes serious defects in the initiation of translation of mRNAs in general yeast, but favors the translation of mRNA-GCN4 in particular. The mechanism by which this phenomenon occurs in mutants gcd17 has sought to interpret proposing several models, based on regulatory traduccional of GCN4 and integrating data obtained in this work.

Author: Busquets Martí Núria.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: Facultad de Ciencias.
Place of preparation: Universidad Autónoma de Barcelona.

Summary: Previous studies conducted in our laboratory have characterized structurally and phenotypically the bacteriófago SE1. This bacteriófago presents a high frequency transduction capable of infecting S. Enterica, and serovar Enteritidis serovar Typhimurium. Following this line of research, this work has been raised to expand the genetic description of this bacteriófago and characterize the different roles phage SE1 state lisogenia, such as the conversion lisogénica, integration and its genetic switch or regulator. The sequenciación genome bacteriófago SE1, from a genoteca fágica and walking directly on the genome has revealed that has a length of 41,941 pb, reflected in 67 orf. The comparison with the GenBank database revealed that the phage, like other fagos lambdoides, is a mosaic result of genetic recombination and transferéncias horizontal with other bacteriophages. The sequence allowed obtained show that the deficiency in the extreme carboxi terminal periplasmátic of the protein encoded by a gene conversion lisogénica (GtrC) could be the cause for qual the bacteriófago P22 would be able to infect a cell lisógena by bacteriófago SE1 . In addition, the present work has been given the sequences of operators in the region to interact with the regulatory protein cI of genetic switch or regulator. Through trial delay electrophoretic (EMSA) and footprinting has defined the consensus sequence of the reasons for joining operators protein cI: AtAN3tTN3TATT. On the other hand, análisi the composition of the unit transcriptional gene cI by RT-PCR pirmitió determine that the gene orf23 part of it. The protein Orf23 serious potential regulatory member of the superfamily of ATPasas related to the partition genomics. Mutants in this defective gene have a augmento of induction of lytic cycle in the presence of mitomycin C, which indicates that this protein may be a modulator of the protein cI or could interact with it. This is the first time that a protein characterized this superfamília of ATPasas a bacteriófago that is integrated into the genome of its host.

Author: VICENTE GARCÍA RUBÉN.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of preparation: FACULTAD DE BIOLOGÍA UNIVESIDAD DE BARCELONA.

Summary: This dissertation presents the studies carried out on Kv1.3, one of the potassium channels that exist in macrophages and its regulation to different stimuli in cell proliferation and activation. Specifically, the first contribution that is attached describes how potassium channels Kv1.3, Kv1.5 and Kir2.1 are responsible for the flow of output voltage dependent and rectification of entry to be detected in these cells . This paper shows how Kv1.3 and Kir2.1 are highly regulated by both the growth factor MCSF as factors inducing activation of macrophages as ellipopolisacárido and cytokine TNFa, there electrophysiological changes in the flow Kv. These studies show how, not only Kv1.3 is regulated in the process of proliferation and activation, but that requires their participation in these processes. In addition to regulating this (! Ion attached a job which shows that this modulation is a general mechanism that may be important in different patologias systemic in which these mediators are present as cachexia, sepsis or chronic inflammation. Furthermore, a the second paragraph in this doctoral thesis focuses on changes in the electrophysiological presenting Kv in proliferation and activation. Discusses the possibility to be due to changes in the composition of complex funcional.En this regard is a contribution which describes the the presence of macrophages in all subunits Kv ¡3 studied least Kv ¡34. proliferation induced MCSF increase the expression of all subunits assistants while various stimuli activation, LPS and TNFa, regulate gene expression di these proteins differently form. kinetic parameters of the outflow of potassium on these subunits act, as are the constant activation, inactivation and deactivación, have been analyzed in an attempt to link molecular changes with changes electrofisiológfeos in proliferating and activated macrophages. Another alignment analysis of the electrophysiological changes in activated macrophages comes as a publication under preparation in which describes how Kv1 .5 is able to associate with Kv1.3 to form a complex functional generator outflows of potassium. This paper studies show association by confocal microscopy and electronics along with pharmacology studies confirm that the formation of complex heteromérico. changes in the electrophysiological parameters of the flow, depending on the composition of the complex have been discussed at various models of heterologous expression . Lastly it is suggested that changes in gating occurring in this flow to various stimuli, such as TNFa, could be due to changes in estequiometría of the subunits that make up the complex generator of the outflow of potassium in macrophages . subcellular location and environment surrounding membrane proteins are decisive in the roles. In the third paragraph of this mémoria presented studies on the cellular biology of the complex formed by Kv1.3, both its traffic as its membrane localization. was studying the influence of different subunits present in the complex on the cell biology of this, taking into account the influence of the subunit Kv1.5 that had already been described in macrophages, and is accompanied by the latest early studies of internalization of the channel through vesículas coated clatrina.

Author: NICOLAS MOLINA FRANCISCO ESTEBAN.
Year: 2005.
University: MURCIA [www.um.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: FACULTAD DE BIOLOGIA.

Summary: Mucor circinelloides is a filamentous fungus that has a wide distribution, found in the soil, manure and other organic substrates decomposing. It belongs to the class Zygomycota characterized by having a mating Fusion gametangios, presenting a mycelium generally cenocítico (in some species may show some septa), and produce spores aflageladas and immobile. The RNA-mediated gene silencing is a complex mechanism of gene regulation, preserved in the world eucariota (a notable exception is S. cerevisie), which leads to suppression of gene expression mediated by small RNA molecules that induce the destruction of mRNA , inhibit translation or inhibit transcription. Initially described as a molecular mechanism of defense against viruses and transposons, in recent years it has been shown that this mechanism is also involved in regulating complex processes like the formation of heterocromatina, development and physiology of the organisms. In addition to reveal a world of hitherto unknown gene regulation, RNA-mediated gene silencing has become a fundamental tool in the study of the role of genes, even allowing for the development of new disciplines such as functional genomics The overall objective of this work has been the molecular characterization of the mechanism of gene silencing in the post - fungus M. Circinelloides: M. Circinelloides presents a mechanism transgene-induced gene silencing, which is reflected by introducing copies of exogenous gene buck carB. The silenced phenotype is unstable and reversible, and is a direct result of a sharp reduction in levels of mature mRNA gene carB. This reduction is not due to a lower rate of transcription of this gene in individuals silenced but to the degradation of mRNA mature, indicating that the observed gene silencing is post-transcripcional.El silencing gene in M. Circinelloides is not associated with methylation of transgenic sequences. However, it is related to the presence of a high number of copies of the transgene, which remain as episomas structures multiméricas. In M. Circinelloides, the transgene-induced gene silencing is associated with the presence of molecules siRNAs antisense and sense. There are two distinct classes of siRNAs antisense, 21 and 25 nt, which is differentially accumulated during the vegetative growth of the fungus. The silence on M. Circinelloides is associated with an amplification process that occurs in the molecules siRNAs side of the two size classes, corresponding to sequences located downstream of the molecule inducing. Both kinds of siRNAs are generated primarily from the region 3 'mRNA target. It has cloned the gene dicer1 M. Circinelloides that figure with a protein domains structural characteristic of the enzyme Dicer. The expression of this gene occurs throughout the vegetative growth and leads to several transcribed, which differ on the site of polyadenylation and / or prosecution of any of its alternative introns. It is not possible to know the functionality of alternative transcripts. The functional analysis of null mutants for the gene dicer1 indicates that the gene is not essential in the mechanism of silencing and suggests the existence of at least one gene dicer further. It is not excluded, however, that the gene dicer1 to be a redundant role in the mechanism of gene silencing. The phenotype of null mutants for dicer1 indicates that this gene is involved in the mycelial growth and morphology of the hyphae, suggesting the involvement of dicer1 in endogenous regulatory processes mediated by the miRNAs

Author: CATANESE GAETANO.
Year: 2005.
University: CÓRDOBA [www.uco.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The melva (Auxis thazard and Auxis Rochei) is a species of tuna with utmost importance at the Andalusian fishing industry, and more specifically in the canning industry. Despite its economic importance, so far has not carried out any detailed study to determine the degree of genetic variability in natural populations, nor has developed no specific system for authentication of the products processed melva. In this regard, the lack of data on these molecular species has prevented undoubtedly tackling these problems. Therefore, in this thesis has been isolated and characterized molecular markers mitochondrial and nuclear weapons, have investigated the genetic characteristics of natural populations of A.rochei and has developed a system of authentication of canned goods. They have analyzed copies of A.rochei coming from the western Mediterranean, the Atlantic Ocean and the eastern Pacific Ocean east. Of the species Auxis thazard were obtained copies from the western Indian Ocean. As mitochondrial markers, it has sequenced the mitogenomas of A.rochei (Atlantic and Pacific) and A.thazard as well as tuna T.thynnus, T.alalunga, Elalletteratus and K.pelamis. The total sizes ranged between 16,501 and 16,503 pb for A.rochei and 16,506 pb for A.thazard. Comparing the sequences were found between 761 and 773 positions polymorphic. The content and organization followed the general pattern of vertebrates. The phylogenetic analysis revealed the existence of two mitochondrial lineages (mitotipo I and II) at A.roche. In addition, there was a monophyletic origin of A.rochei and A.thazard with regard to other species of tuna. For the study population A.rochei analyzed mitochondrial markers (cytochrome control by region) and nuclear (spacers ribosomal ITS-1 and Its-2 and microsatellites). Both markers revealed the lack of genetic differentiation among populations Atlantic and Mediterranean. By contrast, the Pacific population showed a clear separation respect to 2 above and for the three markers (cytb Fst between 0025-0031, ITS-1 and ITS-2 Fst between 0083-0087, microsatellites Fste between 0015-0027). Biological data and isolation by distance explain these results. The differentiation of samples Pacific support the data described by other authors based on morphological characteristics and merísticas, on the existence of subspecies A.rochei eudorax in this Pacific region. Finally, it has developed and optimized a system of multiple MS-PCR products for authentication preserved melva. That system was based on the simultaneous amplification of three different regions mitochondrial: cytochrome b, specific A.rochei, ATPase 6, specific A.thazard, ARNr 12S, as a positive control amplification. The tests conducted on canned products on the market have demonstrated the specificity and reliability of the system for authentication of melva.

Author: SANTIAGO MORA RAQUEL MARIA.
Year: 2005.
University: CÓRDOBA [www.uco.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The widespread use of herbicides in agriculture has led to the current study on the effects of these products on the environment, as well as the study of the processes of degradation acquire great importance. Simazine is a herbicide triazínico which was widely used in a variety of crops, including in the olive grove. In this work has been quantified by PLC, simazine residual soil of olive groves in the province of Cordoba, only one of the samples was detected a concentration of simazine exceeding 0.1 ppm. It has been determined the kinetics of mineralization of simazine in two floors of olive grove. We have managed to isolate microorganisms capable of growing with simazine as the sole source of carbon and nitrogen. Among the microorganisms isolated, are those that can degrade the herbicide and isolated microorganisms, bacteria, which need to form a consortium to degrade simazine. These micro-organisms, bacteria and fungi have been identified or at least been able to determine the taxonomic group to which they belong by molecular techniques. Among the microorganisms are identified Xanthomonas, sp Variovarax sp Pseudothantomonas Mexican Acidovarax sp and Methylopila capsulata. Three routes have been described degradation of simazine in various microorganisms were detected and isolated the genes coding for enzymes involved in such degradation.

Author: PAZ GONZALEZ BEGOÑA.
Year: 2005.
University: LEÓN [www.unileon.es].
Place of defense: FACULTAD DE CIENCIAS BIOLOGICAS Y AMBIENTALES.
Place of preparation: UNIVERSIDAD DE LOEN.

Summary: OBJECTIVES OF THIS OFFERED TO HOME WORK OF INVESTIGATION WERE THE FOLLOWING: 1. DEVELOPMENT OF A PROGRAM OF CLASSICAL SELECTION BASED ON CHANCE TO CHANGE. 2. CLONACIÓN OF GENES OF B. TRISPORA INVOLVED IN THE BIOSÍNTESIS OF -CAROTENO, AND / OR PROVIDED AN EXPRESSION CONSTITUENT. 3. DEVELOPMENT OF A PROTOCOL TO TRANSFORM B. TRISPORA. 4. EXPRESSION OF GENES BIOSINTÉTICOS OF -CAROTENO IN M. CIRCINELLOIDES AND B. TRISPORA. 5. EFFECT OF LIGHT ON THE PRODUCTION IN CAROTENOIDES M. CIRCINELLOIDES AND B. TRISPORA. BE IDENTIFICADO, CLONADO AND THE GENES CARACTERIZADO RECORD, CARB AND CARRA. ORF GEN OF THE RECORD (1,561 PB) POSEE 4 INTRONES, CODIFICA A G-ACTINA, AND THEIR REGION IS PROMOTING IN FUNCTIONAL E. COLI AND A. CHRYSOGENUM. GENES AND THE CARB CARRA IS LIGADOS IN A REGION OF 4.5 KB OF THE GENOME B. TRISPORA, AND IS TRANSCRIBEN IN SENSES OPUESTOS UNDER THE CONTROL OF A BI-DIRECTIONAL PROMOTER OF 611 PB. GEN OF THE ORF CARB (1,955 PB) POSEE 2 INTRONES And CODIFICA A PROTEÍNA WITH ACTIVITY FITOENO DEHYDROGENASE. GEN OF THE ORF CARRA (1,894 PB) POSEE 1 INTRÓN And CODIFICA A PROTEÍNA WITH ACTIVITY FITOENO SINTASA / LICOPENO CICLASA. GEN OF THE CARB ECA SUPERPRODUCTORA OF? -CAROTENO B. TRISPORA F-744 OWN THE MUTATION A1791C, THAT LEADS TO THE REPLACEMENT OF AMINOÁCIDO BE IN THIS B. TRISPORA NRRL 2457 IN THE POSITION 528 BY THIS IN ARG F-744 (S528R). GEN OF THE CARRA ECA F-744 OWN THE MUTATION T497C THAT LEADS TO THE REPLACEMENT OF AMINOÁCIDO PRO THIS IN NRRL 2457 IN THE POSITION 143 BY BEING IN THIS F-744 (P143S)). THE GENES OF CARB AND CARRA B. TRISPORA ARE IN FUNCTIONAL M. CIRCINELLOIDES. GEN OF THE CARB B. TRISPORA supplementing the DEFICIENCY IN ACTIVITY FITOENO DEHYDROGENASE OF THE ECA M. CIRCINELLOIDES MS23 AND GEN CARRA supplementing the DEFICIENCY IN THE ACTIVITIES FITOENO SINTASA And LICOPENO CICLASA OF THE ECA M. CIRCINELLOIDES MS8. THE EXPRESSION OF GEN OF CARRA B. TRISPORA IN M. CIRCINELLOIDES MS12 INCREASES PRODUCTION G-CAROTENO AND OF INTERMEDIARIES BIOSINTÉTICOS G-CAROTENO, LICOPENO And NEUROSPORENO. THE PRESENCE OF LIGHT INCREASES PRODUCTION IN CAROTENOIDES M. CIRCINELLOIDES, WHILE THAT THIS PRODUCTION DECREASES IN BOTH CROPS OF INDIVIDUAL AS MIXED B. TRISPORA. PRODUCTION OF CROPS IN MIXED ON CAROTENOIDES B. TRISPORA IS SUPERIOR TO NETAMENTE OBTAINED AT THE SAME CROPS OF INDIVIDUAL strains. THE TRANSCRIPTION OF GENES OF CARB AND CARRA B. TRISPORA ARE INDUCED IN TERMS OF DARKNESS, THE CONTRARY OF WHAT HAPPENS IN M. CIRCINELLOIDES.

Author: Blanco Calvo Moises.
Year: 2005.
University: A CORUÑA [www.udc.es].
Place of defense: Facultad de Ciencias.
Place of preparation: Facultad de Ciencias.

Summary: The hemo is a molecule of interest from the point of view of energy metabolism, since in addition to intervene in the capture and transport of oxygen (indispensable to the process of obtaining energy via respiration) also forms part as a functional proteins the transport chain mail and mitochondrial oxidative phosphorylation. Moreover, in the case of yeast, acts as an oxygen sensor at the cell and is involved in regulating gene dependent on the availability of oxygen. Therefore, the hemo a molecule is essential that, given its importance, it could help unravel why the metabolic differences between yeast Kluyveomyces lactis and Saccharomyces cerevisiae and we provide tools to improve their farm biotechnology. The gene HEM13 S. Hypoxic cerevisiae is a gene that encodes the enzyme coproporfirinógeno oxidase (CPO), which catalyzes the sixth step of the route biosynthesis group hemo. Both the gene and the protein it generates are key points in the regulation of synthesis of hemo in S. Cerevisiae. On the one hand, the PCO is the first enzyme of the route needed oxygen as a substrate in their reaction, so that the face of a fall in oxygen can become a bottleneck for the generation of hemo (hence there is a direct relationship among hemo levels and oxygen) On the other hand, ScHEM13 is a gene hypoxic, ie increases sharply expression in hypoxic conditions. In regulation ScHEM13 intervenes further hemo as the initiator of a regulatory cascade leading to the suppression aerobic or desrepresión hypoxic gene. This thesis has isolated the gene KlHEM13 from a genoteca K. Lactis and has conducted a trial complementation of a mutant Schem13, noting that KlHEM13 encodes a PCO so similar to the S. Cerevisiae that both are functionally interchangeable. At the root of the foregoing has developed a prediction of tertiary structure for the PCO K. Lactis drawing on the recent crystallization and structural elucidation of the PCO S. Cerevisaie, with a close resemblance between the two. It also has studied the regulation of KlHEM13 in response to oxygen through measures of activity? -galactosidasa And northern. KlHEM13 is regulated in a similar way to how it does ScHEM13, speaking so prevalent in hypoxia. However, KlHEM13 seems to follow a pattern of regulation other than that described for ScHEM13, because while the latter is suppressed by a factor Rox1 aerobically, KlHEM13 not affected by KlRox1. It reveals the weak aerobic expression of KlHEM13 in a mutant strain in the gene KlROX1 coding repressor of the same name. Likewise, it seems that KlHEM13 is not regulated by factors in S. Acting on loa cerevisiae gene expression hipóxicos. We have identified points of initiation of transcription for KlHEM13 and two of the most intense met 3Â'del first ATG (ATG1) pautra of the ORF and 5Â'de one second internal ATG (ATG2). They also found other points weaker 5Â'del ATG1 among them one present only able hipóxicas. The analysis of the transcribed by RT-PCR revealed that there are some who have both ATGs and mergers to lacZ using both ATGs indicate that is likely to generate two proteins (although it is not known whether this occurs in a natural way) also has obtained a null mutant of KlHEM13 not feasible in the absence of precursors hemo, has been verified and measured their activity PCO as well as their strain isogénica wild from crops and aerobic cell in the soluble fraction (free mitochondria membranes and ) null mutant of KlHEM13 as expected, no activity PCO. But the strain if presented with what, at least in the testing conditions, the PCO K. Lactis is cytosolic as its counterpart S. Cerevisiae. Finally has been studied by arrays, the transcriptional regulation in response to the oxygen availability of a number of genes K. Lactis counterparts in the qu 8 and S. 4 e1 cerevisiae encode transcription factors involved in gene regulation dependent on oxygen. The genes studied were: HAP1, ROX1, MOT3 And MGA2. In K. Lactis none of these genes show after six hours in hypoxia, show changes in expression with respect to aerobic. Within this study also analyzed the behavior of genes in the biosynthetic route of hemo K. Lactis yet studied: KlHEM2, KlHEM3, KlHEM4, KlHEM14, KlHEM15. KlHEM14 show a slight induction after six hours in hypoxia, nothing comparable to the induction of almost three times that shows ScHEM14.

Author: CORDON PRECIADO VIOLETA.
Year: 2005.
University: SALAMANCA [www.usal.es].
Place of defense: CENTRO DE INVESTIGACION DEL CANCER.
Place of preparation: CENTRO DE INVESTIGACION DEL CANCER.

Summary: To ensure the integrity of the genome, eukaryotic cells have developed coping mechanisms, known as "checkpoints", which co-ordinate the repair of DNA damage in the progression of the cell cycle. In the yeast Saccharomyces cerevisiae the kinase Rad53 is a central regulator of these mechanisms. In turn, is a protein essential for the normal growth of cells. By labeling end carboxi-terminal of protein Rad53 with epítopo HAS we have created a mutant strain characterized by a reduction in levels of this kinase. The yeast cells carrying the allele rad53HA are apparently normal, as they are viable and have no obvious defects in cell growth. However, when exposed to treatment with genotoxic agents show a sensitivity uneven respect to wild type, which indicates they are defectivas in the activation of some checkpoints. When exposing these cells to treatment with hydroxyurea, a drug that inhibits the synthesis of DNA, lose viability due to a collapse of the replication forks in progress, as shown by two-dimensional electrophoresis of DNA. In contrast, when exposed to sublethal doses of alkylating agent metil-metano-sulfonato, cells rad53HA are more tolerant than the wild type of drug, introducing higher growth in his presence that could be explained by in a premature deactivation of the checkpoint. The fusion protein is functional, as demonstrated by tests autofosforilación spot. In addition, overexpression reverses its sensitivities to genotoxic agents observed in the strain labeled, indicating that the defects that should be introduced to the low basal levels of the kinase. This work shows that the modulation of the activation of Rad53 is critical to the proper functioning of the checkpoints and a reduced activation of the same cause, as opposed to certain genotoxic agents, increased cell survival in the presence of DNA damage.

Author: GODIO FERNÁNDEZ RAMIRO PEDRO.
Year: 2006.
University: LEÓN [www.unileon.es].
Place of defense: FACU.DE CIEN. BIOL. Y AMBIENTALES.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS Y AMBIENTALES.

Summary: It has been developed and optimized an effective strategy for conversion to H. Sublateritium, mediated by A. Tumefaciens. It has shown the importance of using sequences belonging promoters, at least at the same Branch (Basidiomycotina) to transform efficiently species of the genus Hypholoma. There has been a strong correlation between the pH of the culture medium and acid production clavárico in different culture media tested. There has also been the influence of various factors on acid production clavárico, including the addition of copper, agitation cultivation or supplementation with oil. Have been identified in the genome of the strain HS898 H. Sublateritium two genes encoding enzymes important in the biosynthesis of sterols and acid clavárico, erg1 and osc1; these genes were cloned and sequenced. The gene erg1 of 1659 bp, encodes for a protein of 461 amino acids with a molecular mass of 49079 kDa deducted. The protein encoded by this gene, shows the regions of conserved FAD binding characteristics of some monooxigenasas. The overexpression of the gene erg1, carries increased acid production clavárico commensurate with the increase in gene dosage but also increases the growth of mycelium. The attenuation of gene erg1 generates a phenotype typical dependent ergosterol (transformants only grow if the culture medium is added ergosterol), suggesting that the gene erg1 possibly be involved in the metabolism of primary H. Sublateritium, and also in the secondary metabolism, as it reduces the production of acid clavárico. The gene osc1 of 2840 bp, encodes for a protein of 721 amino acids with a molecular mass of 82388 kDa deducted. The protein encoded by this gene, introduced conserved regions "QW" characteristic of the majority of terpenociclasas. The overexpression of the gene osc1, carries increased acid production clavárico commensurate with the increase in gene dosage, without increasing the growth of mycelium. The disruption of the gene osc1 causes interruptions in the synthesis of acid clavárico. Moreover, the growth of mutant gene osc1 interrupted with is similar to the parental strain, and do not require any supplementation with ergosterol for their normal development, and this suggests that the gene osc1 only is involved in the metabolism of secondary H. Sublateritium, particularly on the road biosintética acid clavárico. The attenuation of gene osc1, either through the strategy of antisense RNA or the strategy of RNA interference confirms the results obtained when the gene osc1. Requirements were H. Sublateritium mutant superproductores as subproductores acid clavárico. The analysis of the mutant revealed that some of the genes that are sobreexpresados in mutants superproductores, are directly related to the biosynthesis of sterols.

Author: GONZALO ASENSIO JESUS ANGUEL.
Year: 2006.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: DEPARTAMENTO DE MICROBIOLOGIA.

Summary: Tuberculosis is now a leading cause of death due to infectious diseases, more than two million people die each year and the third of the population is infected with Mycobacterium tuberculosis. Although tuberculosis is a disease treatable with antibiotics and can be prevented through vaccination, the emergence of strains resistant to anti-tuberculosis drugs and the variable protective efficacy of the BCG vaccine against pulmonary manifestations of the disease left the victory over tuberculosis out of reach. The search for an effective vaccine continues, joint initiatives of European and American laboratories have made possible the construction of effective vaccine candidates. In fact, the starting point for this work has been the gene phoP, whose role in the virulence of M. Tuberculosis was previously studied in our laboratory in collaboration with the Institut Pasteur in Paris. A mutant phoP built in strain clinic MT103 gives better protection against tuberculosis that the current BCG vaccine in several animal models, these results suggest that mutant as a promising candidate to live vaccine. However, although the virulent phenotype of mutant phoP has been well studied, the molecular mechanisms that lead to the attenuation are not characterized. Therefore, this thesis has been focused on understanding the function of the gene phoP and decipher its role in the virulence of tuberculosis. PhoP is a transcriptional regulator that is part of the two-component system (2CS) PhoPR M. Tuberculosis. The 2CSs median transcripcionales changes in response to certain stimuli and are involved in the regulation of virulence in pathogenic bacteria. In order to characterize the role of genetically system PhoPR, we have built by delección mutant in the gene phoP or both genes phoPR in three different strains of M. Tuberculosis. The biochemical analysis of these mutants show that PhoP regulates coordinated and positively synthesis of lipids involved in the virulence of M. Tuberculosis. These results in addition to being a good explanation for the attenuated phenotype of mutant phoP represent one of the first experimental evidence of the transcriptional regulation of lipid metabolism in tuberculosis. This work has also focused on understanding the mechanism of action of the system PhoPR. The fact that some of these 2CSs autorregulan his own words led us to study the interaction between PhoP with its own promoter and transcription of the gene phoP. Our results show that the mRNA of phoP is synthesized from three sites of initiation of transcription (tsp's) suggesting a complex regulation of its expression. We have also shown that PhoP joins his own promoter. The region union of PhoP includes tsp's of this gene. These findings suggest that PhoPR is a self-regulating system and makes us assume that the expression of genes regulated by PhoP depends largely on their own expression. One of the most ambitious objectives of this study was to identify genes regulated by PhoP in an attempt to understand the transcriptional attenuation level of the mutant strain. To identify the regulón of PhoP have conducted two studies: the identification transcripcionales profiling using DNA microarrays to study patterns of speech by two-dimensional electrophoresis of proteins. Both experiments show a good correlation, which gives strength to our study. The results indicate that PhoP could be implicated in the regulation of controlled transcripcionales three routes: the remodeling of the cell envelope; metabolic adaptation to the shortage of oxygen and the response to heat shock. These responses transcripcionales are related to lifestyle intracellular M. Tuberculosis, and with so 8 to s 4c5 or virulence, therefore alteration in mutant phoP could cause attenuation. Although attenuated phenotype of the mutant strain should be mainly due to the mutation in the gene phoP, own polymorphisms strain parental MT103 might have contributed to the phenotypic characteristics and properties of the vaccine mutant phoP, therefore, we set out to identify polymorphisms the strain MT103. The results obtained using DNA microarrays demonstrate the loss of some genes in the strain MT103 when compared with the strain of laboratory H37Rv.

Author: REVERTER BRANCHAT GEMMA.
Year: 2006.
University: LLEIDA [www.udl.es].
Place of defense: FACULTAD DE MEDICINA UDL.
Place of preparation: FACULTAD DE CIENCIAS DE LA SALUT UDL.