The role of the ubiquitin proteasome system in Alzheimer's disease.

Today, Alzheimer's disease (AD) is one of the most important age-related neurodegenerative diseases, but its etiology remains still unknown. Since the discovery that the hallmark structures of this disease i.e. the formation of amyloid fibers could be the product of ubiquitin-mediated protein degradation defects, it has become clear that the ubiquitin-proteasome system (UPS), usually essential for protein repair, turnover and degradation, is perturbed in this disease. Different aspects of normal and pathological aging are discussed with respect to protein repair and degradation via the UPS, as well as consequences of a deficit in the UPS in AD. Selective protein oxidation may cause protein damage, or protein mutations may induce a dysfunction of the proteasome. Such events eventually lead to activation of cell death pathways and to an aberrant aggregation or incorporation of ubiquitinated proteins into hallmark structures. Aggresome formation is also observed in other neurodegenerative diseases, suggesting that an activation of similar mechanisms must occur in neurodegeneration as a basic phenomenon. It is essential to discuss therapeutic ways to investigate the UPS dysfunction in the human brain and to identify specific targets to hold or stop cell decay.

Mechanisms of antifungal resistance in the opportunistic fungus Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus is one of the most prevalent airbone fungal pathogen and can cause severe fatal invasive aspergillosis in immunocompromised patients. Several antifungal agents are available to treat these infections but with limited success. These agents include polyenes (amphotericin B), echinocandins (caspofungin) and azoles, which constitute the most important class with itraconazole (ITC) and voriconazole as major active compounds. Azole-derived antifungal agents target the ergosterol biosynthesis pathway via the inhibition of the lanosterol 14α-demethylase (cyp51/ERG1 1), a cytochrome P450 responsible for the conversion of lanosterol to ergosterol, which is the main component of cell membrane in fungi. A. fumigatus is also found in the environment as a contaminant of rotting plant or present in composting of organic waste. Among antifungal agents used in the environment for crop protection, the class of azoles is also widely used with propiconazole or prochloraz as examples. However, other agents such as dicarboximide (iprodione), phenylamide (benalaxyl) or strobilurin (azoxystrobin) are also used. Emergence of clinical azole-resistant isolates has been described in several European countries. However the incidence of antifungal resistance has not been yet reported in details in Switzerland. In this study, the status of antifungal resistance was investigated on A. fumigatus isolates collected from Swiss hospitals and from different environmental sites and. tested for their susceptibility to several currently used antifungal agents. The data showed a low incidence of resistance for all tested agents among clinical and environmental isolates. Only two azole-resistant environmental isolates were detected and none among the clinical tested isolates. In general, A. fumigatus was susceptible to all antifungals tested in our study, except to azoxystrobin which was the less active agent against all isolates. Since mechanisms of antifungal resistance have been poorly investigated until now in A. fumigatus, this work was aimed 1) to identify A. fumigatus genes involved in antifungal resistance and 2) to test their involvement in the development of resistance in sampled isolates. Therefore, this work proposed to isolate A. fumigatus genes conferring resistance to a drug-hypersusceptible Saccharomyces cerevisiae strain due to a lack of multidrug transporter genes. Several genes were recovered including three distinct efflux transporters (atrF, atrH and mdrA) and a bZip transcription factor, yapA. The inactivation of each transporter in A. fumigatus indicated that the transporters were involved in the basal level of azole susceptibility. The inactivation of YapA led to a hypersusceptibility to H2O2, thus confirming the involvement of this gene in the oxidative stress response of A. fumigatus. The involvement of the abovementioned transporters genes and of other transporters genes identified by genome analysis in azole resistance was tested by probing their expression in some ITC-resistant isolates. Even if upregulation of some transporters genes was observed in some investigated isolates, the correlation between azole resistance and expression levels of all these transporters genes could not be clearly established for all tested isolates. Given these results, the present work addressed 1) alteration in the expression of cyp51A encoding for the azole target enzyme, and 2) mutation(s) in the cyp51A sequence as potential mechanisms of azote resistance in A. . However, overexpression of cyp51A in the investigated isolates was not linked with azote resistance. Since it was reported that mutation(s) in cyp51A were participating in azote resistance in A. fumigatus, a functional complementation of cyp51A cDNAs from ITC-resistant A. fumigatus strains in S. cerevisiae ergl 1 Δ mutant strain was attempted. Expression in S. cerevisiae allowed the testing of these cDNAs with regards to their functionality and involvement in resistance to specific azote compounds. We could demonstrate that Cyp51A protein with a G54E or M220K mutations conferred resistance to specific azoles in S. cerevisiae, therefore suggesting that these mutations were important for the development of azote resistance in A. fumigatus. In conclusion, this work showed a correlation between ITC resistance and mechanisms involving overexpression of transporters and cyp51A mutations in A. fumigatus isolates. However, azole resistance of some isolates has not been solved and thus it will be necessary to approach the study of resistance mechanisms in this fungal species using alternative methodologies. RESUME Aspergillus fumigatus est un champignon opportuniste répandu et est la cause d'aspergilloses invasives le plus souvent fatales chez des patients immunodéprimés. Plusieurs antifongiques sont disponibles afin de traiter ces infections, cependant avec un succès limité. Ces agents incluent les polyènes (amphotericin B), les échinocandines (caspofungin) et les azoles, qui représentent la plus importante classe d'antifongiques avec l'itraconazole (ITC) et le voriconazole comme principaux agents actifs. Les dérivés azolés ciblent la voie de biosynthèse de l'ergostérol via l'inhibition de la lanostérol 14α-demethylase (cyp51/ERG11), un cytochrome P450 impliqué dans la conversion du lanostérol en ergostérol, qui est un composant important de la membrane chez les champignons. A. fumigatus est également répandu dans l'environnement. Parmi les antifongiques employés en agriculture afin de protéger les cultures, les azoles sont aussi largement utilisés. Cependant, d'autres agents tels que les dicarboximides (iprodione), les phenylamides (benalaxyl) et les strobilurines (azoxystrobin) peuvent être également utilisés. L'émergence de souches cliniques résistantes aux azoles a été décrite dans différents pays européens. Cependant, l'incidence d'une telle résistance aux azoles n'a pas encore été reportée en détails en Suisse. Dans ce travail, l'émergence de la résistance aux antifongiques a été étudiée par analyse de souches d'A. fumigatus provenant de milieux hospitaliers en Suisse et de différents sites et leur susceptibilité testée envers plusieurs antifongiques couramment utilisés. Les données obtenues ont montré une faible incidence de la résistance parmi les souches cliniques et environnementales pour les agents testés. Seulement deux souches environnementales résistantes aux azoles ont été détectées et aucune parmi les souches cliniques. Les mécanismes de résistance aux antifongiques ayant été très peu étudiés jusqu'à présent chez A. fumigatus , ce travail a eu aussi pour but 1) d'identifier les gènes d' A. fumigatus impliqués dans la résistance aux antifongiques et 2) de tester leur implication dans la résistance de certaines souches. Ainsi, il a été proposé d'isoler les gènes d' A. fumigatus pouvant conférer une résistance aux antifongiques à une souche de Saccharomyces cerevisiae hypersensible aux antifongiques. Trois transporteurs à efflux (atrF, atrH et mdrA) et un facteur de transcription appartenant à la famille des bZip (YapA) ont ainsi été isolés. L'inactivation, dans une souche d'A. fumigatus, de chacun des ces transporteurs a permis de mettre en évidence leur implication dans la susceptibilité d'A. fumigatus aux antifongiques. L'inactivation de YapA a engendré une hypersusceptibilité à l' H2O2, confirmant ainsi le rôle de ce gène dans la réponse au stress oxydatif chez A . fumigatus. La participation dans la résistance aux antifongiques des gènes codant pour des transporteurs ainsi que d'autres gènes identifiés par analyse du génome a été déterminée en testant leur niveau d'expression dans des souches résistantes à l'ITC. Bien qu'une surexpression de transporteurs ait été observée dans certaines souches, une corrélation entre la résistance à l'ITC et les niveaux d'expression de ces transporteurs n'a pu être clairement établie. Ce présent travail s'est donc porté sur l'étude de 2 autres mécanismes potentiellement impliqués dans la résistance aux azoles : 1) la surexpression de cyp51A codant pour l'enzyme cible et 2) des mutations dans cyp51A. Cependant, la surexpression de cyp51A dans les souches étudiées n'a pas été constatée. L'effet des mutations de cyp51A dans la résistance aux azoles a été testée par complémentation fonctionnelle d'une souche S. cerevisiae déletée dans son gène ERG11. L'expression de ces gènes chez S. cerevisiae a permis de démontrer que les protéines Cyp51Ap contenant une mutation G54E ou M220K pouvaient conférer une résistance spécifique à certains azoles, ainsi suggérant que ces mutations pourraient être importantes dans le développement d'une résistance aux azoles chez A. fumigatus. En conclusion, ce travail a permis de mettre en évidence, dans des souches d'A. fumigatus , une corrélation entre leur résistance à l' ITC et les mécanismes impliquant une surexpression de transporteurs et des mutations dans cyp51A. Cependant, ces mécanismes n'ont pu expliquer la résistance aux azoles de certaines souches et c'est pourquoi de nouvelles approches doivent être envisagées afin d'étudier ces mécanismes.

Activation mechanisms of the protease MALT1 and cleavage of one of its targets - the ribonuclease Regnase-1- in lymphocytes and lymphoma cell lines

Persistent infection induces an adaptive immune response that is mediated by T and B lymphocytes. Upon triggering with an antigen, these cells become activated and turn into fast expanding cells able to efficiently defend the host. Lymphocyte activation is controlled by a complex composed of CARMA1, BCL10 and MALT1 which regulates the NF-KB signaling pathway upon antigen triggering. Abnormally high expression or activity of either one of these three proteins can favor the development of lymphomas, while genetic defects in the pathway are associated with immunodeficiency. MALT1 was identified as a paracaspase sharing homology with other cysteine proteases, namely caspases and metacaspases. In order to be active, caspases need to dimerize. Based on their sequence similarity with MALT1, we hypothesized that dimerization might also be a mechanism of activation employed by MALT1. To address this assumption, we performed a bioinformatics modelling based on the crystal structures of several caspases. Our model suggested that the MALT1 caspase-like domain can indeed form dimers. This finding was later confirmed by several published crystal structures of MALT1. In the dimer interface of our model, we noticed the presence of charged amino acids that could potentially form salt bridges and thereby hold both monomers together. Mutation of one of these residues, E549, into alanine completely blocked the catalytic activity of MALT1. Additionally, we provided evidence for a role of E549 in promoting the MALTl-dependent growth of cells derived from diffuse large B cell lymphoma (DLBCL) of the aggressive B cell-like type (ABC). To our initial surprise, the E549A mutation showed only a partial defect in dimerization, indicating that additional residues are essential to form a stable dimer. The MALT1 crystal structures revealed a key function for E549 in stabilizing the catalytic site of the protease via its interaction with an arginine which is located next to the catalytic active cysteine. In an additional study, we discovered that MALT1 monoubiquitination is required for the catalytic activity of the protease. Interestingly, we found that the MALT1 dimer interface mutant E549A could not be monoubiquitinated. Based on these findings, we suggest that correct formation of the dimer interface is a prerequisite for monoubiquitination. In a second project, we discovered a novel target of the protease MALT1, the ribonuclease Regnase¬la It was described that the RNase activity of Regnase-1 negatively regulates immune responses. We could show that in ABC DLBCL cell lines, Regnase-1 is not only cleaved by MALT1 but also phosphorylated, at least in part, by the inhibitor of KB kinase (IKK). Both regulations appear to restrain the RNase function of Regnase-1 and thereby allow the production of pro-survival proteins. In conclusion, our studies further highlight and explain the importance of the catalytic activity of MALT1 for the activation of lymphocytes and provide additional knowledge for the development of specific drugs targeting the catalytic activity of MALT1 for immunomodulation and treatment of lymphomas.  SUMMARY IN FRENCH PhD Thesis Katrin Cabalzar 2 SUMMARY IN FRENCH Une infection persistante induit une réponse immunitaire adaptative par l'intermédiaire des lymphocytes T et B. Quand elles reconnaissent l'antigène, ces cellules sont activées et se multiplient très rapidement pour défendre efficacement l'hôte. L'activation des lymphocytes est transmise par un complexe composé de trois protéines, CARMA1, BCL10 et MALT1, qui régule la voie de signalisation NF-KB lorsque l'antigène est reconnu. L'expression ou l'activité anormalement élevée de l'une de ces trois protéines peut favoriser le développement de lymphomes, tandis que des défauts génétiques de cette voie de signalisation sont associés à l'immunodéficience. MALT1 a été identifiée comme étant une paracaspase qui partage des séquences homologues avec d'autres protéases à cystéine, comme les caspases et les métacaspases. Pour être actives, les caspases ont besoin de dimériser. Etant donné leur similarité de séquence avec MALT1, nous avons supposé que la dimérisation pouvait aussi être un mécanisme d'activation utilisé par MALT1. Pour vérifier cette hypothèse, nous avons conçu un modèle bioinformatique à partir des structures cristallographiques de plusieurs caspases. Et notre modèle a suggéré que le domaine catalytique de MALT1 était effectivement capable de former des dimères. Cette découverte a été confirmée plus tard par des publications qui montrent des structures cristallographiques dimériques de MALT1. Dans l'interface du dimère de notre modèle, nous avons remarqué la présence d'acides aminés chargés qui pouvaient former des liaisons ioniques et ainsi réunir les deux monomères. La mutation de l'un de ces résidus, E549, pour une alanine, a complètement inhibé l'activité catalytique de MALT1. De plus, nous avons mis en évidence un rôle d'E549 dans la croissance dépendante de MALT1, des cellules dérivées de lymphomes B diffus à grandes cellules (DLBCL) de sous-type cellules B actives (ABC). Dans un premier temps nous avons été surpris de constater que cette mutation révélait seulement un défaut partiel de dimérisation, ce qui indique que des acides aminés supplémentaires sont indispensables pour former un dimère stable. Les structures cristallographiques de MALT1 ont révélé un rôle primordial d'E549 dans la stabilisation du site catalytique de la protéase via son interaction avec une arginine qui se trouve à côté de la cystéine du site actif. Dans une autre étude, nous avons découvert que la monoubiquitination de MALT1 est requise pour l'activité catalytique de la protéase. A remarquer que nous avons trouvé que le mutant E549A de l'interface dimère de MALT1 n'a pas pu être monoubiquitiné. Sur la base de ces résultats, nous suggérons que la formation correcte de l'interface du dimère est une condition préalable pour la monoubiquitination. Dans un second projet, nous avons découvert une nouvelle cible de la protéase MALT1, la ribonucléase Regnase-1. Il a été décrit que l'activité RNase de Regnase-1 régulait négativement les réponses immunitaires. Nous avons pu montrer que dans les lignées cellulaires ABC DLBCL, la Regnase-1 n'était pas seulement clivée par MALT1 mais également phosphorylée, au moins en partie, par la kinase de l'inhibiteur de KB (IKK). Les deux régulations semblent supprimer la fonction RNase de Regnase-1 et permettre ainsi la stabilisation de certains ARN messagers et la production de protéines favorisant la survie. En conclusion, nos études mettent en évidence le rôle-clé de la dimérisation de MALT1 et expliquent l'importance de l'activité catalytique de MALT1 pour l'activation des lymphocytes. Ainsi, nos résultats apportent des connaissances supplémentaires pour le développement de médicaments spécifiques ciblant l'activité catalytique de MALT1, qui pourraient être utiles pour modifier les réponses immunitaires et traiter des lymphomes.

Activation of Srk1 by the Mitogen-activated Protein Kinase Sty1/Spc1 Precedes Its Dissociation from the Kinase and Signals Its Degradation

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. The Schizosaccharomyces pombe SAPK Sty1/Spc1 orchestrates general changes in gene expression in response to diverse forms of cytotoxic stress. Here we show that Sty1/Spc1 is bound to its target, the Srk1 kinase, when the signaling pathway is inactive. In response to stress, Sty1/Spc1 phosphorylates Srk1 at threonine 463 of the regulatory domain, inducing both activation of Srk1 kinase, which negatively regulates cell cycle progression by inhibiting Cdc25, and dissociation of Srk1 from the SAPK, which leads to Srk1 degradation by the proteasome.

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

Hexokinase 2, Glycogen Synthase and Phosphorylase Play a Key Role in Muscle Glycogen Supercompensation

Background: Glycogen-depleting exercise can lead to supercompensation of muscle glycogen stores, but the biochemical mechanisms of this phenomenon are still not completely understood. Methods: Using chronic low-frequency stimulation (CLFS) as an exercise model, the tibialis anterior muscle of rabbits was stimulated for either 1 or 24 hours, inducing a reduction in glycogen of 90% and 50% respectively. Glycogen recovery was subsequently monitored during 24 hours of rest. Results: In muscles stimulated for 1 hour, glycogen recovered basal levels during the rest period. However, in those stimulated for 24 hours, glycogen was supercompensated and its levels remained 50% higher than basal levels after 6 hours of rest, although the newly synthesized glycogen had fewer branches. This increase in glycogen correlated with an increase in hexokinase-2 expression and activity, a reduction in the glycogen phosphorylase activity ratio and an increase in the glycogen synthase activity ratio, due to dephosphorylation of site 3a, even in the presence of elevated glycogen stores. During supercompensation there was also an increase in 59-AMP-activated protein kinase phosphorylation, correlating with a stable reduction in ATP and total purine nucleotide levels. Conclusions: Glycogen supercompensation requires a coordinated chain of events at two levels in the context of decreased cell energy balance: First, an increase in the glucose phosphorylation capacity of the muscle and secondly, control of the enzymes directly involved in the synthesis and degradation of the glycogen molecule. However, supercompensated glycogen has fewer branches.

Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors

Plant growth and development are particularly sensitive to changes in the light environment and especially to vegetational shading. The shade-avoidance response is mainly controlled by the phytochrome photoreceptors. In Arabidopsis, recent studies have identified several related bHLH class transcription factors (PIF, for phytochrome-interacting factors) as important components in phytochrome signaling. In addition to a related bHLH domain, most of the PIFs contain an active phytochrome binding (APB) domain that mediates their interaction with light-activated phytochrome B (phyB). Here we show that PIF4 and PIF5 act early in the phytochrome signaling pathways to promote the shade-avoidance response. PIF4 and PIF5 accumulate to high levels in the dark, are selectively degraded in response to red light, and remain at high levels under shade-mimicking conditions. Degradation of these transcription factors is preceded by phosphorylation, requires the APB domain and is sensitive to inhibitors of the proteasome, suggesting that PIF4 and PIF5 are degraded upon interaction with light-activated phyB. Our data suggest that, in dense vegetation, which is rich in far-red light, shade avoidance is triggered, at least partially, as a consequence of reduced phytochrome-mediated degradation of transcription factors such as PIF4 and PIF5. Consistent with this idea, the constitutive shade-avoidance phenotype of phyB mutants partially reverts in the absence of PIF4 and PIF5

Mechanisms of apoptosis in retinitis pigmentosa.

Mutations in humans are associated with several forms of inherited retinal dystrophies, such as Retinitis Pigmentosa which lead to retinal cell death and irreversible loss of vision. Genes involved in affected patients mainly encode proteins related to vision physiology including visual cycle and light-dependent phototransduction cascade. As reported in spontaneous and genetically engineered mouse models, apoptosis is a common fate in retinal degeneration, although the triggered signals to retinal apoptosis remain largely unraveled. Several studies highlighted that many of the molecular pathways involved in ocular diseases rely on caspase-dependent or -independent apoptotic mitochondrial pathway involving the Bcl-2 family of proteins. Anti- and pro-apoptotic Bcl-2 members are present in retinal tissues and are thought to play a role in the pathogenesis of several retinal disorders. Since almost no efficient treatments are available so far, it remains a great challenge to decipher the molecular pathways involved in retinal dystrophies and to develop alternative therapies to prevent or inhibit eye defect. Toward this goal, mutation-independent strategies such as molecular therapy provides promising and exciting approaches to deliver anti-apoptotic molecules targeting the Bcl-2 pathway through the use of cell permeable transport peptides. Modulation of common apoptotic signaling pathways may be of outstanding potential to target multiple retinal dystrophies regardless of the primary genetic defect.

The mechanisms of agglomeration: Evidence from the effect of inter-industry relations on the location of new firms

The objective of this paper is to explore the relative importance of each of Marshall's agglomeration mechanisms by examining the location of new manufacturing firms in Spain. In particular, we estimate the count of new firms by industry and location as a function of (pre-determined) local employment levels in industries that: 1) use similar workers (labor market pooling); 2) have a customer- supplier relationship (input sharing); and 3) use similar technologies (knowledge spillovers). We examine the variation in the creation of new firms across cities and across municipalities within large cities to shed light on the geographical scope of each of the three agglomeration mechanisms. We find evidence of all three agglomeration mechanisms, although their incidence differs depending on the geographical scale of the analysis.

Conduction mechanisms and charge storage in Si-nanocrystals metal-oxide-semiconductor memory devices studied with conducting atomic force microscopy

In this work, we demonstrate that conductive atomic force microscopy (C-AFM) is a very powerful tool to investigate, at the nanoscale, metal-oxide-semiconductor structures with silicon nanocrystals (Si-nc) embedded in the gate oxide as memory devices. The high lateral resolution of this technique allows us to study extremely small areas ( ~ 300nm2) and, therefore, the electrical properties of a reduced number of Si-nc. C-AFM experiments have demonstrated that Si-nc enhance the gate oxide electrical conduction due to trap-assisted tunneling. On the other hand, Si-nc can act as trapping centers. The amount of charge stored in Si-nc has been estimated through the change induced in the barrier height measured from the I-V characteristics. The results show that only ~ 20% of the Si-nc are charged, demonstrating that the electrical behavior at the nanoscale is consistent with the macroscopic characterization.

Pitfalls in cefepime titration from human plasma: plasma- and temperature-related drug degradation in vitro.

While developing a high-pressure liquid chromatography assay for cefepime in plasma, we observed significant drug degradation at 20 and 37 degrees C but not at 4 degrees C. This plasma-related degradation persisted after protein removal. This warrants caution regarding cefepime assays for pharmacokinetic and pharmacodynamic studies of cefepime in vitro and in vivo.

The mechanisms of agglomeration: Evidence from the effect of inter-industry relations on the location of new firms

The objective of this paper is to explore the relative importance of each of Marshall's agglomeration mechanisms by examining the location of new manufacturing firms in Spain. In particular, we estimate the count of new firms by industry and location as a function of (pre-determined) local employment levels in industries that: 1) use similar workers (labor market pooling); 2) have a customer- supplier relationship (input sharing); and 3) use similar technologies (knowledge spillovers). We examine the variation in the creation of new firms across cities and across municipalities within large cities to shed light on the geographical scope of each of the three agglomeration mechanisms. We find evidence of all three agglomeration mechanisms, although their incidence differs depending on the geographical scale of the analysis.

Molecular analysis of the bacterial diversity in a specialized consortium for diesel oil degradation

Diesel oil is a compound derived from petroleum, consisting primarily of hydrocarbons. Poor conditions in transportation and storage of this product can contribute significantly to accidental spills causing serious ecological problems in soil and water and affecting the diversity of the microbial environment. The cloning and sequencing of the 16S rRNA gene is one of the molecular techniques that allows estimation and comparison of the microbial diversity in different environmental samples. The aim of this work was to estimate the diversity of microorganisms from the Bacteria domain in a consortium specialized in diesel oil degradation through partial sequencing of the 16S rRNA gene. After the extraction of DNA metagenomics, the material was amplified by PCR reaction using specific oligonucleotide primers for the 16S rRNA gene. The PCR products were cloned into a pGEM-T-Easy vector (Promega), and Escherichia coli was used as the host cell for recombinant DNAs. The partial clone sequencing was obtained using universal oligonucleotide primers from the vector. The genetic library obtained generated 431 clones. All the sequenced clones presented similarity to phylum Proteobacteria, with Gammaproteobacteria the most present group (49.8 % of the clones), followed by Alphaproteobacteira (44.8 %) and Betaproteobacteria (5.4 %). The Pseudomonas genus was the most abundant in the metagenomic library, followed by the Parvibaculum and the Sphingobium genus, respectively. After partial sequencing of the 16S rRNA, the diversity of the bacterial consortium was estimated using DOTUR software. When comparing these sequences to the database from the National Center for Biotechnology Information (NCBI), a strong correlation was found between the data generated by the software used and the data deposited in NCBI.