MYCOLOGY

Author: ORTONEDA PEDROLA MONTSERRAT.
Year: 2003.
University: ROVIRA I VIRGILI [www.urv.cat].
Place of defense: MEDICINA.
Place of preparation: FACULTAT DE MEDICINA, UNIVERSITAT ROVIRA I VIRGILI.

Summary: The number of serious infections caused by fungi has risen significantly in recent years, especially among caused by the so-called opportunistic fungi. The latter tend to cause infections in patients suffering from other diseases base, such as leukemia, AIDS, cancer, or who have received solid organ transplants or bone marrow. The list of species of fungi opportunistic involved in serious infections is becoming longer, and strategies to deal with these infections remain very limited. The three genres filamentous opportunists who have been studied in this thesis, Fusarium Scedosporium i Paecilomyces are within this group of opportunistic fungi. Although only causing severe in immunocompromised patients, the mortality rate of these infections is close to 100%. Amphotericin B has been for many years, and it still is today, the drug most commonly used to treat systemic infections, but has significant toxicity problems. In recent years there have developed other formulations of this drug, which present fewer problems in terms of toxicity. The main objective of this thesis was to evaluate the efficacy of one of the new formulations of amphotericin B, prepared liposómico in animal models of disseminated infection. In order to compare different treatments in the first place have had to develop animal models of infection for each of the fungi studied (using mice), which are reproducible and that can simulate the human fungal infection. Once developed animal models comparing efficacy of the two formulations of amphotericin B in the treatment of infections caused by fungi studied. This was assessed by comparing the survival time (MST) of the different groups using statistical methods. He also compared the amount of fungus present in different organs of mice, which were killed when treatment ended. A part of amphotericin B that is, as we said, the drug most commonly used to fight infections caused by opportunistic fungi, there are other antifungal agents, but have proved ineffective, at least in vitro, and clinical outcomes are very rare. This is why we wanted to evaluate the potential synergistic effect of some combinations of antifungal in vitro. In summary, the main conclusions drawn from the studies are: 1, - were established models disseminated infection in mice by the following species: Paecilomyces variotii, P.lilacinus, P.javanicus, Fusarium verticillioides. Scedosporiium prolificans, S.apiospermum in immunocompetent and immunocompromised mice. 2-We evaluated the efficacy of the two antifungal formulations of amphotericin B (desoxicolato and liposomal formulation) in different models of experimental infection developed in the first phase of the studies. In mice infected with P.variotti, the two formulations were equally effective at low doses, but the administration of liposomal amphotericin B to 10 mg / kg resulted in a significant decrease in the presence of the fungus in some organs. The low dose of liposomal amphotericin B did raise the TMS of mice infected with F.verticillioides. High doses also did significantly decrease the presence of the fungus in various organs. In mice infected S.prolificans, there was an increase of TMS, which was most evident at the highest dose of liposomal amphotericin B. The administration of factor granulocyte-colony stimulating along with the antifungal therapy did not improve the results. 3, has been evaluated in vitro activity of nine combinations of antifungal against various species of the genus Fusarium and Paecilomyces. The combinations that have behaved in a synergistic manner for a larger number of strains of Fusarium has been to ravuco 8 nazol co 299 mbinado both with amphotericin B as terbinafine, and the latter combined with voriconazole. The combinations that have behaved in a synergistic manner for a larger number of strains have been Paecilomyces all of terbinafine combined with azoles.

Author: LÓPEZ ERRASQUÍN ELENA.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FAC. BIOLOGIA.

Summary: In this work have been isolated by DDRT-PCR (differential display) two genes coders ABC transporters (fvabc) and MFS (fvmfs), which showed an expression induced a strain of Fusarium verticillioides produces fumonisins over another non-producer. The sequence of these genes has been extended by RACE (rapid amplification of cDNA ends) for the characterization of genomic sequence and coding as well as the deduced amino acid sequence. It has also been given the presence of two genes, as well as the transporter cluster of biosynthesis fumonisins encoded by the gene fum19 in different isolates F.verticillioides producing and non-producing fumonisins and different species of the genus Fusarium, including the complex species Gibberella fujikuroi, the number of copies that were in the genome and the meadow of similarity between their sequences. The results showed that transporters fvabc and fvmfs found in the genome of all strains tested, both producing and non-producing. For the cotnrario, efum19 could only be detected in isolates producers. In all cases, were as a single copy in the genome. It has also conducted a phylogenetic analysis including sequences of other genes in the GenBank. Finally, it has been analyzed by real-time RT-PCR gene expression in different isolates F.verticillioides producers and producers of fumonisins not in a position to induce production of fmonisinas and in a position control, as well as in the presence of different toxic compounds. The results suggested an involvement of both ABC transporters in the expulsion of the toxin, and FVABC in incidence of resistance to multiple drugs.

Author: BRENA ALONSO SONIA.
Year: 2004.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE MEDICINA Y ODONTOLOGÍA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: Members of the genus Candida, particularly Candida albicans, behave as opportunistic pathogens that are often isolated from the gastrointestinal tract and reproductive healthy individuals. Under certain predisposing factors, Candida spp. May be converted into a pathogen capable of causing a variety of infections and systematic surface. Our group has developed a monoclonal antibody, AMc C7, compared with a manoproteínade C.albicans presented antifungal and antitumor activities, decreases adhisión Cell C.albicans to cell lines, hybrid composites and resins used in dental restoration, and decreases filamentation of C.albicans. The manoproteína recognized by the MAB C7 is also recognized by the secretory IgA in saliva, which is presented as an antigen especialinterés that could have relevance in the pathogenesis of C.alvicans. The identification of such antigen high molecular weight and characterization of the mechanism of action of AMc C7 through analysis microaarays have been the targets of this research. Using advanced techniques such as protein sequencing analysis by MALDI-TOF and HPLC-Trampa Iónica, was achieved identify the protein as Als3p. This protein belongs to a family of proteins C.albicans, Als family, which consists of 9 tissues of the host and different substrates, which represent a very important role in the pathogenesis of the fungus. It has recently been shown that the functionality lam olécula lies in the N-terminal portion of these proteins. In this paper we have demonstrated that the AMc C7 recognizes a fraction of the amino end of the protein Als3p, so this recognition may be related to inhibition of the activity of the accession of C.albicans presenting this AMc. It was also study the distribution of proteins similar to the Als, in different species of the genus Candida and other gender related. Moreover, the analysis of microarrays response C.albicans treatment with AMc C7 helped pinpoint 79 genes that were differentially expressed in the treated cells. The pattern of expression of genes coding for proteins involved in iron metabolism and energy metabolism allowed for a possible model for action antibody, which is engaged candidacida could be caused by an iron deficiency resulted in the treated cells.

Author: RAMOS MARTINEZ MARIA BRISA.
Year: 2004.
University: SALAMANCA [www.usal.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: MICROBIOLOGIA Y GENETICA.

Author: BÁRCENA ASENSIO MARÍA CARMEN.
Year: 2004.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: Our work focuses on ele studio of dermatophytosis in pets of the autonomous region of Aragon. We examine hair samples of 485 animals suspected of dermatophytosis (dogs, cats, cows, ovjeas, goats, horses and rabbits). The lesions were located in the region of the head, face and ears. The dermatophytes isolates have been trichophyton verrucosum (79.5%), Microsporum Canis (61%), Trichophyton Mentagrophytes (58%) Trichophyton Equinum (50%) Microsporum Gypseum (27%) and Microsporum Persicolor (2%). Trichophyton Mentagrophytes and Microsporum Canis, are the two species most commonly Microsporun Canis, both in dogs (94%) and cats (97%). The age (less than or equal to 1 year) is a statistically significant factor, which influences the appearance of dermatophytosis of lso pets. In pets, and in particular the male character is obsdrva greater prevalence of demratofitosis being admeás estadíticamente signifciatio in cat species. The species of géneor Trichophyton are responsible for the demratofitosis of animal production in rural areas. Trichophyton verrucosum inthe bovine, ovine and carpina, Trichophyton equinum inthe horses and trichophyton metagrophytes inthe rabbits. The fungus saprofítico most frequently aisaldo in all epseices in estuido belongs to the genus alternaria.

Author: GARCÍA SÁNCHEZ DANIA.
Year: 2005.
University: ROVIRA I VIRGILI [www.urv.cat].
Place of defense: FACULTAT DE MEDICINA I CIÈNCIES DE LA SALUT.
Place of preparation: FACULTAT DE MEDICINA I CIÈNCIES DE LA SALUT.

Summary: Fungi ascomicetos accounted for 75% of the fungal species described so far, however stages sexual or teleomorfos have been little studied because they have been able to recover only in pure culture for 10% of the species. The main difficulty in studying these organisms is that the state is required to interrupt latency of ascosporas. To this end apply different methods of isolation and activation ascoporas. They presisamnete methods of the study that will determine our knowledge about this group of organisms. The classification of ascomicetos, until a few years ago was based solely on morphological criteria. It is currently available methods more goals, including the inference of phylogenetic relationships from ribosomal DNA sequences. These methods have enabled propose classification systems more natural. However, the classification of ascomicetes there are still many questions taxonomic. This is because many groups in the demarcations between taxa are based solely on morphological characters, and these relationships tend to have little relation to the phylogeny obtained from ribosomal DNA sequences. We have focused on the study of gender Neurospora and Gelasinospora and genres confined to the family Coniochaetaceae where problems persist several taxonomic. We raised com main objectives: on the 1st Contribute to taxonomic knowledge of the members of the class Sordariomycetes. 2 nd determine the phylogenetic relationships between the genders Gelasinospora and Neurospora. 3 ro determine the molecular phylogeny of the family Coniochaetaceae. The main results are broken down as follows: A new species of Syspastospora from tropical soils. D. Garcia, A. M. Stchigel and J. Guarro. 2002. Mycologia 94: 862-865. - It is proposed that new species Syspastospora tropicalis Soil ascomycetes from Spain. XIII. Two new species of Apiosordaria. D. Garcia, AM Stchigel and J. Guarro. 2003. Mycologia 95: 134-140. - There are proposals for new species: Apiosordaria hispanica Apiosordaria globosa A new species of Poroconiochaeta from Russian soil. D. Garcia, AM Stchigel and J. Guarro. 2003. Mycologia 95: 525-529. - It is proposed that new species Poroconiochaeta tetraspora - Proposes a new combination for gender Poroconiochaeta: Poroconiochaeta savoryi (= Thielavia savoryi) Two new species of Sphaerodes from Spanish soils. D. Garcia, A. M. Stchigel and J. Guarro. 2004. Studies in Mycology 50: 63-68. - Two new species: Sphaerodes quadrangularis Sphaerodes tenuissima-Three new combinations for gender Sphaerodes: Sphaerodes ellipsospora (= Microthecium ellipsosporium) Sphaerodes Levite (= Microthecium levitum) Sphaerodes singaporensis (= Microthecium singaporensis) Coronatomyces cubensis gene. Et. Sp. Nov., A new ascomycete from Cuban soil. D. Garcia, AM Stchigel, J. Cano, and J. Guarro. 2004. Studies in Mycology 50: 143-148. The proposed gender-new Coronatomyces order Sordariales and species new Coronatomyces cubensis A synopsis and re-circumscription of Neurospora (syn. Gelasinospora) based on ultrastuctural and 28S rDNA sequence data. D. Garcia, A. M. Stchigel, J. Cano, J. Guarro and D. L. Hawksworth. 2004. Mycological Research 108: 1119-1142. - It is proposed to reduce synonymies gender Gelasinospora with Neurospora. - Redescribe gender Neurospora are proposed and thirty-six new combinations for gender Neurospora. - Two new species of the genus Neurospora: Neurospora nigeriensis Neurospora uniporata-Not included in the gender Neurospora species Gelasinospora amorphoporcta. It suggests the new gender 8 or Pseudo 636 neurospora for reubicarla in the family Sordariaceae and the new combination Pseudoneurospora amorphoporcata. A molecular approach to the taxonomy of Coniochaetaceae. D. Garcia, A. M. Stchigel, J. Cano, M. Calduch, D. L. Hawksworth and J. Guarro. (Accepted its publication in Mycological Research) - It is proposed to reduce synonymies genres Coniochaetidium, Ephemeroascus and Poroconiochaeta with Coniochaeta - redescribe gender Coniochaeta-nine combinations are proposed for the new gender Coniochaeta - Excludes gender Coniochaeta species Coniochaeta emodensis and Coniochaeta nodulisporioides are proposed genres new Pseudorosellinia (P. emodensis) and Coniocessia (Coniocessia nodulisporioides) to relocate in order Xylariales. - Excludes gender Coniolaria order Coniochaetales, it is proposed the new genre Novaxylaria to relocate in order Xylariales.

Author: ORTEGA FEBRERO NÉSTOR.
Year: 2005.
University: LEÓN [www.unileon.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS Y AMBIENTALES.
Place of preparation: FACULTAD DE CIENCIAS BIOLGÍCAS Y AMBIENTALES.

Summary: This report describes the development of a system of mutagenesis "directed" stable using the system transposicional formed by the tranposón Tan1 and the mobile element Vader Aspergillus awamori ATCC 22342. The two form a single system, in which Tan1 serving providing transposasa able to mobilize Vader. This feature, along with the proposed operation of the system described in the American patent 5,985,570, have been used in the preparation of mutagenesis system described in this report. From this operation were observed all constructions described in this report, first through a "double" transposition in which elements "Vader" and Tan1 be included in two separate plasmids. In the process, they employ resistance fleomicina first and higromicina later. The functionality of this system was demonstrated performing interruption of the gene pyrG in A. Niger and gene and A in A. Nidulans. Later, with the vector pIRFN was carried out developing a system transposicional "unique" with the use of gene-sible fleomicina resistance as the only marker selection. By this construction, were obtained mutant gene wA in A. Nidulans. The gene hph conferring resistance to higromicina B was used in the construction of the vector pHVFN with which developed plasmids pHVT2-Ry pHVT2-F. With these constructions, was the interruption of the gene tan1, thus avoiding the element Vader continue transponiéndose uncontrollably and stabilizing mutant genotype obtained as the fungal strains used in the transformations were previously found the absence of tranposones described in this report . Therefore, it has been able to obtain a system of mutagenesis functional and stable with the elements described in the genus Aspergillus, thus achieving the main objective proposed in this report. The expression of the gene tan1, while not high, if it is kept constant by following a pattern of establishing term (at least for the time interval tested) and being transcribed from their expected size. This pattern of expression was also observed when the gene tan1 is expressed from promoter gene gpdA A. Nidulans. In either case, this pattern of expression seems to be sufficient to achieve the interruption in a single gene transformation, varying the percentage of mutants obtained from 9% so counterpart (A. niger) at 3.2, 2.7% or 5.1% in a heterologous (A. nidulans). As future plans, the aim is to verify and investigate the feasibility of this system transposicional in other species of the genus Aspergillus fungal outside such as Acremonium chrysogenum, Trichoderma harzianum, Penicillum chrysogenum or Penicillum roqueforti in which it has found no native form of transposons described. This probably will require the construction of new buildings in which the gene tan1 is expressed apartir himself a promoter of the species in question although, as a preliminary step, is intended to demonstrate the viability of the system using constructs such as vector pIRFN.

Author: GÓMEZ LÓPEZ ALICIA.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: This paper describes the development and standardization of two techniques for quantifying fungal components as alternatives to traditional methods for determining the fungal infection, counting viable or determining the weight of dry mycelium, and its application to studies of sensitivity with antifungals. The molecules of chitin, a polymer N-acetil-D-glucosamina present on the wall, and ergosterol, specific component of the fungal membrane, can be quantified through espectrofotométricas or chromatographic techniques both in vitro and in tissue samples and show a positive correlation with the mass of mycelium. Therefore, techniques could be used to conduct sensitivity studies with several species of fungi. The results show a reliability for each of the techniques are superior to the reference methods used so far, however, because of its complexity, it does not seem likely to have a role frontline care, but could be used in research studies, replacing the curves death conventional or colony as a method of assessing response to therapy in animal models of infection.

Author: ABRUSCI BERNAL CONCEPCIÓN.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: At Work for the Study of biodegradation of constituent materials of films by bacteria and fungi "have isolated microorganisms, bacteria and fungi, in samples of films from the archives of Barcelona, Madrid and Gran Canaria. These samples were provided by Spanish Film Library following a protocol established. We have isolated and identified 14 strains of bacteria of the genus Stapylococcus (5 species), and Bacillus (5 species), Sphyngomonas, Kokuria and Pasteurella and, 17 strains of filamentous fungi and levdura, Aspergillus (4 especies9. Penicillium (7 species) and Alternaria, Cladosporium, Mucor, Phoma and Trichoderma. through measures viscosimétrias, has conducted a study of biodegradation of the photographic gelatin dissolved by bacteria gelatinasa positive, setting the order of efficiency of biodegradation at 37 , 30, 20 and 4Â ° C generates only four bacteria Bacillus were efficient in the time scale of three months. however, all fungi biodegradaron gelatin, 25Â ° C in hours and 4Â ° C within weeks. also have been studied and gelatin the photographic emulsions in solid through techniques Quimioluminiscencia (QL) and spectroscopy of Laser Induced Fragmentation (LIBS), with the latter technique has been identified metals present in the stratigraphic composition of the film. Issuance of QL increases drastically samples biodegradas due to the oxidation of materials that produce reactive oxygen species (ROS) formed in metalismo microbial. Lastly, by indirect measures impedance and valuation of CO2, has been studied biodegradation of photographic emulsions and options ( triacetate cellulose, polietilén terephthalate, additives), bacteria and fungi identified in this work. has been able to establish an order of efficiency of biodegradation of microorganisms.

Author: CALERO RUEDA OLGA.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS.

Summary: This paper describes a new esteasa fungal crops produced in the liquid fungus Ophiostoma piceae. The enzyme preparations traded so far, although they are effective biological control of the pitch in conifers not yield good results in hardwood timber. This fact prompted the search for an enzyme capable of breaking down the compounds involved in the formation of deposits in these plants. Preliminary studies showed the enzymatic activity of the fungus O.piceae on esters p-nitrofenol and oleato of colesterilo. The latter was chosen as a model of sterol esters present in the removable wooden Eucalyptus globulus, mostly in Spain kind used for the production of pulp. Initially several enzymatic activities were studied, using a mean baseline containing glucose as a source of carbon and olive oil as inducer. Under these conditions, purified and characterized a single enzyme. It showed a high affinity on triglycerides and sterol esters. The degradation of complex mixtures of these compounds, present in the pulp and paper removable from different plant species, demonstrated the potential application of the esterase in the biological control of the pitch in hardwood and conifers. The determination of the N-terminal end of the protein and peptides internal, the result of hydrolysis of the same, helped obtain a specific DNA probe. Thus, it was able to carry out the gene sequencing of the esteasa of O.piceae. The comparison with the genes of other esterases showed homology of approximately 40% with codifying the lipases Candida rugosa and Geotrichum candidum. From these studies, they were able to identify the waste involved in the catalysis of the enzyme. The structural model of esterase, constructed from the structures cristalográficas of lipases CRL1 and CRL3 of C.rugosa allowed to cover the enzyme in the family of alpha / beta hidrolasas and identify some of the amino acids responsible for their substrate specificity.

Author: ALCOVER DÍAZ FCO. JAVIER.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA DE VITORIA (UPV-EHU).

Summary: The dust mite Dermatophagoides pteronyssinus and Dermatophagoides farinae, along with the species of fungus Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum and Openicillim notatum, are the main biological agents associated with the onset and / or exacerbation of allergic diseases in the domestic environment usual in genetically susceptible individuals. Because of the significant medical problem, as well as the apparent change in the quality of life of individuals atopic that these agents cause, it highlights the need for a system which, ideally with a single compound remove, in all indoors allergenic burden of such agents, and is not toxic to humans and pets. With these considerations, the objectives of this study were: to study the effect acaricide and fungicide three essential oils: Tree Tea, Lemon and Parsley. Select a single compound, or secondly a combination agent for use as domestic acaricida-fungicida.

Author: RODRÍGUEZ SÁNCHEZ ENRIQUE.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: FCULTAD DE CIENCIAS BIOLÓGICAS.

Summary: The white rot fungi are an interesting group with great potential biotech organisms due to their ability to degrade the polymer lignin and aromatic compounds structurally related. This potential is based principal in the system extracellular involved in the degradation of lignin, which is composed of different types of enzymes (peroxidases and oxidasas extracellular) and low molecular mass compounds. This work has been carried out the study of the ability of white rot fungi of the genus Pleurotus to transform and mineralizar aromatics 2,4-diclorofenol, organochlorine compound of natural phenolic and benzo (a) pyrene, hydrocarbon aromatic policíclico Nature not phenolic. These aromatic compounds are associated with pollution problems in terrestrial ecosystems. The results presented in this thesis show that the fungi of the genus Pleurotus are capable of trans-formar and mineralizar these compounds, confirming its potential application in bioremediation strategies. Previous studies showed some doubt on the involvement of enzymes ligninoliticas in the degradation of this kind of environmental pollutants by fungi of the genus Pleurotus. However, in vitro studies carried out in this thesis with enzymes ligninolíticas laccase peroxidase and versatile purified of the fungus Pleurotus eryngii, and the detection of these activities during the processes of transformation in culture, suggesting their involvement in the degradation 2.4 - diclorfenol and benzo (a) pyrene and confirm the results obtained with other basidiomicetos of white rot. Finally, it has conducted a molecular approach to the study of the activities ligninolíticas secreted by the fungus P.eryngii. In this regard have been identified, cloned and secuenciando two new genes in this fungus lacquers. As a complement to the caracterizaicón molecular has described the structural models of enzximas encoded by these genes. Finally, it has developed a system of heterologous expression of one of these genes, aportándose new data on the biochemical and kinetic characterization of the enzyme expressed, as well as a tool in the study of the catalytic properties of lacasas.

Author: GÓMEZ TORIBIO VÍCTOR.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS.

Summary: The white rot fungi are an interesting group of organisms with great ability to degrade lignin. This aromatic polymer is very heterogeneous and, along with cellulose and hemicellulose, the cell walls of wood. During the last two decades have been identified numerous enzymes involved in this degradation, both oxidasas and fenoloxidaas extracellular and intracellular reductasas. However, because of the compact structure of lignin, are low molecular weight compounds which begin ligninolisis. Among them, the free radicals of oxygen are a group of highly oxidizing compounds capable of reacting with lignin and / or its products despolimerización. Pleurotus eryngii is capable of producing such sweeping through cycles redoz of aromatic aldehydes and quinones. This research work is studied in detail the formation, by P.eryngii of reactive oxygen species through redox cycles of quinones derived from lignin, paying particular attention to the enzymes involved in the process, the synergies that occur between these elements can promote this mechanism and the possibility that this process can be generated in other white rot fungi. First, it detected the production of reactive oxygen species through oxidation metoxihidroquinonas by enzymes ligninolíticas of P.eryngii, laccase or peroxidase versatile. This mechanism consists of monovalent oxidation of hidroquionas by the enzyme, or Mn3 +, followed by autooxidación of semiquinonas arising with the consequent production of radical O2, and dismutación them, H2O2. It has been shown that there is a direct relationship between the number of groups metoxilo replacing the hidroquinonas, effectiveness thereof by oxidation enzymes and autooxidación of semiquinonas. It has also been demonstrated for the first time the production of OH radicals in reactions of oxidation metoxihidroquinonas by these enzymes, conducted in the presence of complex Fe3 + -EDTA. The generation of OH radical is done in a reaction Fenton led mostly by radical semiquinona and to a lesser extent, by radical O2. Trials with P.eryngii have shown that the fungus produces extracelularmente, continuous and linear radical O2, H2O2 and OH radicals through the "redox cycling" three benzoquinonas derived from lignin and naphtoquinone. The three basic steps of this mechanism, leading primarily to the production of radicals O2, are as follows: 1-Reduction of intracellular quinones to hidroquinonas through a quinone reductase. 2, extracellular-Oxidation of hidroquinonas to semiquinonas by enzymes ligninolíticas. 3-Autooxidación of semiquinonas. It has been shown that, then the radicals O2 generated in the latter reaction are transformed into H2O2 by three different ways: dismutación, reduced by some hidroquinonas as the derivative of menadione, and reduction by Mn2 +, with the consequent production of agent ligninolítico Mn3 +. Finally, the production of OH radicals requires action FE3 + as a catalyst. It has been found that the reduction of this ion is not only involved radicals semiquinona and O2, but also an enzyme system associated with the mycelium of the fungus. This mechanism cyclic activation of oxygen is characterized by sustained operating for several hours, presenting as a whole low substrate specificity and be more efficient in terms of production of H2O2, the "redox cycling of aromatic aldehydes. The production of reactive oxygen species by P.eryngii in cycles of redox quinones can be enhanced by promoting the oxidation of hidroquinonas, and that this reaction is the limiting cycles under incubation conditions studied. We have achieved higher levels by increasing the amount of laccase incubation of the mixture by adding Mn2 +, which catalyzes the oxidation of hidroquinonas by superoxide and replace EDTA, used as a chelating artificial Fe3 + 8, ác 631 Ido oxalic, which oxidizes complex directly to hidroquinonas. The production of oxalic acid by P.eryngii has also been demonstrated for the first time. Similarly, stimulation of the production of H2O2 in P.eryngii way "redox cycling of aromatic aldehydes, it increases the generation of OH radicals during redox cycles of quinones. The simultaneous action of the cycles of the two types of compounds shows that all the enzymes involved (laccase, peroxidase versatile, aril-alcohol oxidase, aldehyde reductase and quinone reductase) may cooperate in the generation of extracellular reactive oxygen species. Finally, the demonstration of the existence of the "redox cycling of quinones in other white rot fungi, in addition to P.eryngii, pointing to its consideration as a general mechanism of extracellular production of reactive oxygen species in these microorganisms.

Author: JURADO GARCÍA-POSADA MIGUEL.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLÓGICAS.

Summary: The contamination of cereals with fungi producing toxins is a problem that produces losses in the agriculture, livestock, to affect animal health, and is a public health problem, as the cereal part of the basic human diet. The diagnosis of the species fungícas involved is a basic requirement for the control of the problem. This thesis has developed a method of identifying specific, through the implementation of the PCR technique, the main species of Fusarium producing toxins in cereals: F.graminearu, F.culmorum, F.poae, F.sporotrichioides F . equiseti and F.proliferatum. It has also developed a protocol that allows integrated diagnosis using DNA extracted from plant material directly, without the need of prior isolation of the fungus. The primers were designed from copying multiple genomic sequences, which increases the sensitivity of the test with respect to the single copy sequences, as has been demonstrated. This thesis has conducted an extensive epidemiological study in wheat, barley and maize from various origins in Spain, as well as samples of imported for the distribution and frequency of the main species of Fusarium in these cereals. It has also been given the presence of other fungal species that produce mycotoxins. The species of Fusarium more important in wheat Spanish were F.graminearum F.culmorum F.sambucinum and F.equiseti, and in the case of maize F.verticillioides and F.proliferatum. Other species so important in barley and wheat Spanish were P.purpurogenum, Altemaria altermata and A.niger var.niger. We have conducted studies of genetic variability in fungal isolates obtained through the analysis of the gene elongation factor, which have shown greater variability in F.equiseti, followed by F.avenaceum, F.graminearum and F.sambucinum. In the case of F.verticillioides and F.proliferatum has built a phylogeny including isolates from various hosts and global geographic origins. The results of these tests allow us to conclude that F.proiferatum is a more variable F.verticilioides, and probably presents a degree of adaptation to different hosts. In addition, it has developed and evaluated a method for the determination by real-time RT-PCR gene expression fum1 in F.proliferatum related to the biosynthesis of fumonisins, a group of mycotoxins with carcinogenic activity, which has allowed an assessment in shorter time to the traditionally used, the production of this toxin in different isolates, indicating a large intraspecific variability. The results obtained in this thesis provides a useful information for the implementation of control measures encaminadosas to reduce the presence of mycotoxins in cereals.

Author: MURCIA FLORES LAURA.
Year: 2006.
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 several answers to the light blue, including the induction of biosynthesis of carotenes. The gene crgA, a gene induced by light, acts as a negative regulator of the carotenogénesis in this fungus, as the complete deletion of the gene in the wild strain leads to a sobreacumulación carotenes in mushroom spawn in terms of light and darkness. The effect repressor of crgA on the biosynthesis of carotenes is performed by controlling the expression of genes carotenogénicos structural. The comparison of the amino acid sequence of CrgA with sequences stored in databases suggests that the translation of CrgA could begin in a codon GUG located 201 bp upstream of AUG codon initially proposed as a starting point for the translation of crgA. To determine whether this codon GUG is the originator of CrgA have been caused by directed mutagenesis, replacement AUG codons GUG and located in the extreme 5 'crgA. These mutant alleles were used to transform the race null to crgA, and the effect of these alleles on carotenogénesis compared with caused after the introduction of the wild allele. The results have confirmed that the codon GUG is the start codon of translating crgA. The deduced amino acid sequence of the gene crgA shows significant structural different regions: a domain RING-finger a nuclear localization signal, stretches and a rich glutaminas domain isoprenilación. The domain RING-finger and the first tranche of poliglutaminas are essential for the regulation of synthesis of carotenes by CrgA on M. Circinelloides. However, the demonstration that the translation of the protein CrgA begins at codon GUG confirmed the presence of a domain RING-finger in addition, has recently identified a domain LON. In order to characterize the role in carotenogénesis of new domains identified by directed mutagenesis were obtained mutant alleles of crgA altered residues conserved in these pleas and these alleles have been used to transform the null mutant for crgA. The results have shown that both domains are essential for the proper function of CrgA as regulator of carotenogénesis. It has also carried out the study of protein CrgA, through the production of polyclonal antibodies specifically directed against the protein CrgA. Our results indicate that this protein accumulates mainly in the light, despite exercising its regulatory function of the carotenogénesis in the dark. Likewise, it has been shown that CrgA undergoes changes post-traduccionales, being present in the mycelium as N-glicosilada. Moreover, the levels of the protein CrgA on M. Circinelloides are extremely low. Low levels of CrgA are not due solely to the use of an initiation codon presumably ineffective, as the replacement of codon GUG by the AUG codon does not produce a significant increase in the amount of CrgA. The analysis of the stability of complete and truncated versions of CrgA in yeast suggests that a protein is unstable and this instability is mediated by the presence of at least the first domain RING-finger. Also, this analysis suggests that the instability is associated with the degradation in the proteasome yeast. The structural characteristics of the protein CrgA suggest that belongs to the family of ligasas of ubiquitina E3, and that could regulate carotenogénesis by binding to proteins involved in the response to light, upon his ubiquitinación and subsequent degradation in the proteasome. In order to identify these proteins, we used the yeast two-hybrid, isolated several genes that encode proteins of M. Circinell 8 oides qu 57b and interact with CrgA. One of the proteins identified is a counterpart of nucleoside diphosphate kinase involved in the response to light in N. Crassa. The isolated gene was named ndkA. To confirm the interaction of proteins CrgA and NdkA tests were carried out trawling protein in vitro. Our results indicate that CrgA interacts with NdkA, suggesting that it could regulate the function of this protein involved in the response to light in N. Crassa. In addition, CrgA serving as a promoter for the expression of NdkA, as the absence of CrgA leads to a decline in levels of messenger and protein ndkA. This result shows that CrgA may act as some gene activator and repressor others like.