The reform of IMF quotas: the way towards the 2008 resolution

Policy evaluation based on the estimation of dynamic stochastic general equilibrium models with aggregate macroeconomic time series rests on the assumption that a representative agent can be identified, whose behavioural parameters are independent of the policy rules. Building on earlier work by Geweke, the main goal of this paper is to show that the representative agent is in general not structural, in the sense that its estimated behavioural parameters are not policyindependent. The paper identifies two different sources of nonstructurality. The latter is shown to be a fairly general feature of optimizing representative agent rational expectations models estimated on macroeconomic data.International Relations, International Monetary Fund, governance Classification JEL: F53, F59

Exploring ecological modelling to investigate factors governing the colonization success in nosocomial environment of Candida albicans and other pathogenic yeasts

wo hundred seventy seven strains from eleven opportunistic species of the genus Candida, isolated from two Italian hospitals, were identified and analyzed for their ability to form biofilm in laboratory conditions. The majority of Candida albicans strains formed biofilm while among the NCAC species there were different level of biofilm forming ability, in accordance with the current literature. The relation between the variables considered, i.e. the departments and the hospitals or the species and their ability to form biofilm, was tested with the assessment of the probability associated to each combination. Species and biofilm forming ability appeared to be distributed almost randomly, although some combinations suggest a potential preference of some species or of biofilm forming strains for specific wards. On the contrary, the relation between biofilm formation and species isolation frequency was highly significant (R2 around 0.98). Interestingly, the regression analyses carried out on the data of the two hospitals separately were rather different and the analysis on the data merged together gave a much lower correlation. These findings suggest that, harsh environments shape the composition of microbial species significantly and that each environment should be considered per se to avoid less significant statistical treatments

A reference map of the human binary protein interactome.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships(1,2). Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome(3), transcriptome(4) and proteome(5) data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes

NGS-baserte rDNA barkoding i identifisering og avgrensning av gjærarter : rammer, muligheter og sammenligning med fenotypisk HT FT-IR spektroskopi

The abundance of ribosomal DNA (rDNA) in the yeast and fungal genomes derives from their multigene nature. During the last decade of the XX century, this DNA region has become very popular for the molecular characterization of fungi. Unfortunately, the multigene nature of rDNA cannot be completely identified by the Sanger sequencing that records only the most prevalent nucleotide at each position. Conversely, Next Generation Sequencing (NGS) has unveiled the internal heterogeneity of rDNA, due to its mechanism of reporting individual reads. For these reasons, rDNA sequencing and particularly the Internal Transcribed Spacer (ITS) marker, have huge advantages in taxonomy, barcoding, ecological microbiology and diagnostics. The aim of this thesis was to achieve a closer understanding of the rDNA organization and to link molecular and phenotypical analysis in order to obtain a stable and meaningful phenetic taxonomy, which accounts for the phylogeny. The first part of the introduction of this thesis is a critical review of the literature on rDNA and its taxonomic variability. In the second part, the thesis illustrates how the application of new strategies to detect the variability within the rDNA, allows the identification and classification of species by analysing species derived from different environments that are relevant for white, green and red biotechnologies. Limitations of the significance of markers in the application of DNA-based molecular taxonomy of microorganisms are discussed. Therefore, to avoid a sterile taxonomic approach leading to a pure nomenclatural exercise, phenotypic characterization was associated to the genotyping of selected microorganisms. For this reason, as example, results obtained in studies on the ability of selected microorganisms to form biofilm in addition to their metabolomic characterization are presented. The biofilm forming ability of more than two hundred pathogenic strains belonging to Candida genus identified using ITS marker are presented. The relation between different variables was tested and results showed that species and biofilm forming ability appeared to be distributed almost randomly whereas the relation between biofilm formation and species isolation frequency was highly significant (R2 around 0.98). The identification of saprophytic filamentous fungi, which cause invasive infections, is also presented. In this case the current molecular diagnostic tools, based on the barcode marker ITS, failed in discriminating this fungi between the complex Trichoderma longibrachiatum/Hypocrea orientalis, even using different tools. The definitive identification was carried out combining molecular approach and microbiological test. A combined approach in the delimitation of ninety-six food-related strains of the complex Meyerozyma/Candida guilliermondii is presented. Results of both approaches (ITS and FT-IR spectroscopy) showed that the possibility to discriminate among strains with molecular and metabolomic analyses represents an additional tool to empower food and industrial monitoring and to gain further knowledge on the genetic variations of this species. In order to study the variability of the rDNA an NGS-like approach on a new species Ogataea uvarum sp.nov. was carried out. Results showed that the ITS marker was more variable than the LSU gene, especially in the ITS2 region. In order to test the origin of this heterogeneity the whole region was introduced in a mini library and several clones were sequenced separately. The cloning of a sample of single copy sequences showed that indeed an internal heterogeneity is present and that the process of generating a consensus using Sanger sequencing hides a large part of it. For instance, the introduction of NGS leads to a deeper knowledge of the individual sequences and of the variants between the same DNA sequences located in different tandem repeats. With this purpose, more than two hundred strains belonging to Candida genus were sequenced with NGS and a pipeline for the identification using different bioinformatics tools was carried out. The NGS also offers the possibility to evaluate this heterogeneity by analysing the Single Nucleotide Polymorphisms (SNPs) within the reads of an rDNA region amplified from a single strain DNA. Results performed on the four prevalent Candida species (C. albicans, C. glabrata, C. parapsilosis and C. tropicalis) indicated the presence of high variability among the strains and between the species, especially in the ITS2 region. Moreover, a combined approach on these four Candida species using NGS and FT-IR spectroscopy was applied in order to improve the identification of pathogenic strains. Multivariate data analysis (MVA) by Consensus Principal Component Analysis (CPCA) was carried out. Partial Least Squares Regression (PLSR) was applied to build a classification model based on most relevant IR variables. The model was then crossvalidated with the a success rate of 94.2%. Identification was also performed considering both the distance to the type strain and the central strain resulting in 97.4% correct classification. In conclusion, in this thesis an identification method for the diagnose of pathogenic yeasts was developed on the basis of NGS. The internal variability of the rDNA was exploited and the relative limitations of the current methodologies presented. The comparison of results from totally different characters (molecular vs. phenotypic) and expressed with different data types (categorical vs. continuous) is one of the challenges necessary to try a reconciliation between the molecular DNA-based taxonomy, characterized by stable and “potential” characters, and the phenotypic data describing “actual” traits of the cells.Overflod av ribosomalt DNA (rDNA) i gjær og sopp genomer stammer fra deres multigen natur. I løpet av det siste tiåret av XX århundre, har denne DNA regionen blitt svært populært for molekylær karakterisering av sopp. Dessverre kan den multigene naturen av rDNA ikke fullstendig identifiseres ved Sanger-sekvensering, som registrerer bare de mest utbredte nukleotider ved hver posisjon. I motsetning har Next Generation Sequencing (NGS) avduket den interne heterogeniteten av rDNA, på grunn av sin mekanisme for rapportering av enkelte ‘reads’. Derfor har rDNA sekvensering og spesielt Internal transkribert Spacer (ITS) markører store fordeler i taksonomi, barcoding, økologisk mikrobiologi og diagnose. Målet med denne avhandlingen var å oppnå en bedre forståelse av rDNA organiseringen og å lage en forbindelse mellom molekylær og fenotypisk analyse for å oppnå en stabil og meningsfull fenetisk taksonomi, som uttrykker fylogenien. Den første delen av innledningen av denne avhandlingen er en kritisk gjennomgang av litteraturen om rDNA og dens taksonomisk variabilitet. I den andre delen, viser avhandlingen hvordan anvendelsen av nye strategier for å oppdage variasjonen innenfor rDNA, tillater identifisering og klassifisering av arter ved å analysere arter som stammer fra ulike miljøer som er relevante for hvite, grønne og røde bioteknologi. Begrensninger i betydningen av markører i anvendelsen av DNA-baserte molekylære taksonomi av mikroorganismer diskuteres. Derfor, for å unngå en steril taksonomisk tilnærming som fører til en ren taksonomi øvelse, ble fenotypisk karakterisering knyttet til genotypingen av utvalgte mikroorganismer. Derfor presenteres, for eksempel, det resultater som er oppnådd i biofilmstudier, hvor evnen av utvalgte mikroorganismer for dannelse av biofilm i tillegg til deres metabolomisk karakterisering undersøkes. Evnen til å danne biofilm ble presentert for mer enn to hundre patogene stammer tilhørende slekten Candida og som er identifisert ved hjelp av markeringen ITS. Forholdet mellom evnen til å danne biofilm og artene ble undersøkt. Resultatene viste at det ikke er noe korrelasjon mellom arten og biofilmformingsevne, mens korrelasjonen mellom biofilmdannelse og isolasjonsfrekvensen for arten. Identifiseringen av saprophytic trådformede sopp, som forårsaker invasive infeksjoner, blir også presentert. I dette tilfellet, mislykkes dagens molekylære diagnostiske verktøy basert på strekkode markør ITS i å diskriminere denne sopparten i komplekset Trichoderma longibrachiatum/Hypocrea orientalis, selv ved hjelp av ulike verktøy. Den endelige identifikasjonen ble utført ved å kombinere molekylær tilnærming og mikrobiologiske test. En kombinert tilnærming i avgrensningen av nitti-seks matrelaterte stammer av komplekset Meyerozyma/Candida guilliermondii er presentert. Resultater av begge tilnærminger (ITS og FT-IR spektroskopi) viste at muligheten til å diskriminere mellom stammer med molekylære og metabolomiske analyser representerer et tilleggsverktøy som kan styrke mikrobiell kontroll i matindustri og for å få mer kunnskap om de genetiske varianter av denne arten. For å studere variasjonen av rDNA ble en NGS-lignende metode testet for en ny art Ogataea uvarum sp.nov. Resultatene viste at ITS markøren var mer variabel enn LSU genet, spesielt i ITS2 regionen. For å teste opprinnelsen av denne heterogeniteten, ble hele regionen innført i en mini-bibliotek og flere kloner ble sekvensert separat. Kloning av et utvalg på enkelkopi sekvenser viste at faktisk en intern heterogenitet er til stede, og at prosessen med å generere en konsensus ved hjelp av Sanger-sekvensering skjuler en stor del av denne heterogeniteten. Innføringen av NGS fører til en dypere forståelse av de individuelle sekvensene og av variantene mellom de samme DNA-sekvensene som ligger i forskjellige tandemrepetisjoner. Med dette formålet, ble mer enn to hundre stammer tilhørende Candida slekten sekvensert med NGS og en rutine for identifisering ved hjelp av ulike bioinformatiske analyser ble satt opp. NGS tilbyr også muligheten for å evaluere heterogeniteten ved å analysere enkelt-nukleotider (SNPs) i lesninger av en rDNA region amplifisert fra DNAen til en enkelt stamme. Resultatene utført på de fire viktigste Candida-arter (C. albicans, C. glabrata, C. parapsilosis og C. tropicalis) indikerte tilstedeværelse av høy variabilitet blant stammene og mellom artene, spesielt i ITS2 regionen. Videre ble en studie gjennomført, hvor en kombinasjon av NGS og FT-IR-spektroskopi ble utført for de fire Candida-artene for å forbedre den identifikasjon av patogene stammer. Multivariat dataanalyse (MVA) ved Konsensus Principal Component Analyse (CPCA) ble utført. Partial Least Squares Regression (PLSR) ble brukt til å bygge en klassifiseringsmodell basert på de mest relevante IR variablene. Modellen ble deretter kryss-validert med en suksessrate på 94,2%. Identifikasjon ble også utført med tanke på både avstanden til typestammen og den sentrale artsstammen og resulterte i 97,4% korrekt identifisering. I denne avhandlingen ble en identifikasjonmetode for diagnose av patogene gjærsopper utviklet på basis av NGS. Den nye metoden utnytter den indre variasjon av rDNA. De relative begrensningene ved eksisterende metoder blir diskutert. Sammenligningen av identifikasjonsresultater som stammer fra data med helt forskjellige karakterer (molekyl vs. fenotypiske) og som er uttrykt med ulike datatyper (kategoriske vs. kontinuerlig) er nødvendig hvis man vil komme fram til en avstemming mellom en molekylær DNAbaserte taksonomi, preget av stabil og "potensielle" tegn, og en fenotypiske taksonomi som beskriver egenskapene til cellene

Single Strain High-Depth NGS Reveals High rDNA (ITS-LSU) Variability in the Four Prevalent Pathogenic Species of the Genus Candida

Ribosomal RNA in fungi is encoded by a series of genes and spacers included in a large operon present in 100 tandem repeats, normally in a single locus. The multigene nature of this locus was somehow masked by Sanger sequencing, which produces a single sequence reporting the prevalent nucleotide of each site. The introduction of next generation sequencing led to deeper knowledge of the individual sequences (reads) and therefore of the variants between the same DNA sequences located in different tandem repeats. In this framework, NGS sequencing of the rDNA region was used to elucidate the extent of intra- and inter-genomic variation at both the strain and species level. Specifically, the use of an innovative NGS technique allowed the high-throughput high-depth sequencing of the ITS1-LSU D1/D2 amplicons of 252 strains belonging to four opportunistic yeast species of the genus Candida. Results showed the presence of a large extent of variability among strains and species. These variants were differently distributed throughout the analyzed regions with a higher concentration within the Internally Transcribed Spacer (ITS) region, suggesting that concerted evolution was not able to totally homogenize these sequences. Both the internal variability and the SNPs between strain can be used for a deep typing of the strains and to study their ecology

NGS-based rDNA barcoding in fungal species identification and delimitation : limits, opportunities and relation to phenotypic HT FT-IR spectroscopy

The abundance of ribosomal DNA (rDNA) in the yeast and fungal genomes derives from their multigene nature. During the last decade of the XX century, this DNA region has become very popular for the molecular characterization of fungi. Unfortunately, the multigene nature of rDNA cannot be completely identified by the Sanger sequencing that records only the most prevalent nucleotide at each position. Conversely, Next Generation Sequencing (NGS) has unveiled the internal heterogeneity of rDNA, due to its mechanism of reporting individual reads. For these reasons, rDNA sequencing and particularly the Internal Transcribed Spacer (ITS) marker, have huge advantages in taxonomy, barcoding, ecological microbiology and diagnostics. The aim of this thesis was to achieve a closer understanding of the rDNA organization and to link molecular and phenotypical analysis in order to obtain a stable and meaningful phenetic taxonomy, which accounts for the phylogeny. The first part of the introduction of this thesis is a critical review of the literature on rDNA and its taxonomic variability. In the second part, the thesis illustrates how the application of new strategies to detect the variability within the rDNA, allows the identification and classification of species by analysing species derived from different environments that are relevant for white, green and red biotechnologies. Limitations of the significance of markers in the application of DNA-based molecular taxonomy of microorganisms are discussed. Therefore, to avoid a sterile taxonomic approach leading to a pure nomenclatural exercise, phenotypic characterization was associated to the genotyping of selected microorganisms. For this reason, as example, results obtained in studies on the ability of selected microorganisms to form biofilm in addition to their metabolomic characterization are presented. The biofilm forming ability of more than two hundred pathogenic strains belonging to Candida genus identified using ITS marker are presented. The relation between different variables was tested and results showed that species and biofilm forming ability appeared to be distributed almost randomly whereas the relation between biofilm formation and species isolation frequency was highly significant (R2 around 0.98). The identification of saprophytic filamentous fungi, which cause invasive infections, is also presented. In this case the current molecular diagnostic tools, based on the barcode marker ITS, failed in discriminating this fungi between the complex Trichoderma longibrachiatum/Hypocrea orientalis, even using different tools. The definitive identification was carried out combining molecular approach and microbiological test. A combined approach in the delimitation of ninety-six food-related strains of the complex Meyerozyma/Candida guilliermondii is presented. Results of both approaches (ITS and FT-IR spectroscopy) showed that the possibility to discriminate among strains with molecular and metabolomic analyses represents an additional tool to empower food and industrial monitoring and to gain further knowledge on the genetic variations of this species. In order to study the variability of the rDNA an NGS-like approach on a new species Ogataea uvarum sp.nov. was carried out. Results showed that the ITS marker was more variable than the LSU gene, especially in the ITS2 region. In order to test the origin of this heterogeneity the whole region was introduced in a mini library and several clones were sequenced separately. The cloning of a sample of single copy sequences showed that indeed an internal heterogeneity is present and that the process of generating a consensus using Sanger sequencing hides a large part of it. For instance, the introduction of NGS leads to a deeper knowledge of the individual sequences and of the variants between the same DNA sequences located in different tandem repeats. With this purpose, more than two hundred strains belonging to Candida genus were sequenced with NGS and a pipeline for the identification using different bioinformatics tools was carried out. The NGS also offers the possibility to evaluate this heterogeneity by analysing the Single Nucleotide Polymorphisms (SNPs) within the reads of an rDNA region amplified from a single strain DNA. Results performed on the four prevalent Candida species (C. albicans, C. glabrata, C. parapsilosis and C. tropicalis) indicated the presence of high variability among the strains and between the species, especially in the ITS2 region. Moreover, a combined approach on these four Candida species using NGS and FT-IR spectroscopy was applied in order to improve the identification of pathogenic strains. Multivariate data analysis (MVA) by Consensus Principal Component Analysis (CPCA) was carried out. Partial Least Squares Regression (PLSR) was applied to build a classification model based on most relevant IR variables. The model was then crossvalidated with the a success rate of 94.2%. Identification was also performed considering both the distance to the type strain and the central strain resulting in 97.4% correct classification. In conclusion, in this thesis an identification method for the diagnose of pathogenic yeasts was developed on the basis of NGS. The internal variability of the rDNA was exploited and the relative limitations of the current methodologies presented. The comparison of results from totally different characters (molecular vs. phenotypic) and expressed with different data types (categorical vs. continuous) is one of the challenges necessary to try a reconciliation between the molecular DNA-based taxonomy, characterized by stable and “potential” characters, and the phenotypic data describing “actual” traits of the cells

SVILUPPO ED ANALISI DELL’EFFICACIA PROTETTIVA DI UN PROTOTIPO VACCINALE A VIRUS-LIKE PARTICLES CONTRO LA SINDROME DELLA LEPRE BRUNA EUROPEA

La Sindrome della Lepre Bruna Europea (EBHS) appartiene al complesso delle malattie emorragiche virali dei lagomorfi. È una malattia altamente contagiosa ad elevata mortalità causata da un virus a RNA ss/+ appartenente alla famiglia Caliciviridae, genere Lagovirus. Attualmente, la profilassi vaccinale prevede la somministrazione di un vaccino inattivato a base di antigene virale. Vista l’impossibilità di poter coltivare il virus in vitro ed essendo il virus presente ad alto titolo solo nel fegato degli animali deceduti per EBHS, la disponibilità del vaccino prodotto come stabulogeno è vincolata esclusivamente alla quantità di fegati infetti conferiti presso i laboratori degli II.ZZ.SS. In questo contesto, l’obiettivo di questa tesi è stato quello di valutare l’efficacia protettiva di un prototipo vaccinale a Virus-Like Particles specifico per EBHS sulla specie di elezione sottoposta ad infezione sperimentale con EBHSV. Per le lepri del gruppo di controllo è stata osservata una mortalità del 60% in accordo con i dati epidemiologici. Le lepri immunizzate con 100 µg di VLPs sono sopravvissute al challenge. Ulteriore obiettivo della tesi è stato quello di ottimizzare il processo produttivo del prototipo a VLPs al fine di ridurre il rapporto costo/produzione e di rendere concorrenziale il prodotto finito. L’individuazione delle migliori condizioni di espressione, purificazione, auto-assemblaggio e linea cellulare ha permesso di migliorare ed ottimizzare l’iter produttivo in previsione di un suo impiego in un processo produttivo su larga scala. Ad oggi, i risultati ottenuti indicano chiaramente che tale presidio immunizzate può rappresentare una valida alternativa all’impiego del vaccino stabulogeno

Ionic Conductivity as a tool to study biocidal activity of sulfobetaine micelles against Saccharomyces cerevisiae model cells

Zwitterionic sulfobetaine surfactants are used in pharmaceutical or biomedical applications for the solubilization and delivery of hydrophobic molecules in aqueous medium or in biological environments. In a screening on the biocidal activity of synthetic surfactants on microbial cells, remarkable results have emerged with sulfobetaine amphiphiles. The interaction between eight zwitterionic sulfobetaine amphiphiles and Saccharomyces cerevisiae model cells was therefore analyzed. S. cerevisiae yeast cells were chosen, as they are a widely used unicellular eukaryotic model organism in cell biology. Conductivity measurements were used to investigate the interaction between surfactant solution and cells. Viable counts measurements were performed, and the mortality data correlated with the conductivity profiles very well, in terms of the inflection points (IPs) observed in the curves and in terms of supramolecular properties of the aggregates. A Fourier transform infrared (FTIR)-based bioassay was then performed to determine the metabolomic stress-response of the cells subjected to the action of zwitterionic surfactants. The surfactants showed nodal concentration (IPs) with all the techniques in their activities, corresponding to the critical micellar concentrations of the amphiphiles. This is due to the pseudocationic behavior of sulfobetaine micelles, because of their charge distribution and charge densities. This behavior permits the interaction of the micellar aggregates with the cells, and the structure of the surfactant monomers has impact on the mortality and the metabolomic response data observed. On the other hand, the concentrations that are necessary to provoke a biocidal activity do not promote these amphiphiles as potential antimicrobial agents. In fact, they are much higher than the ones of cationic surfactants

A novel, rapid and automated conductometric method to evaluate surfactant-cells interactions by means of critical micellar concentration analysis

Conductometry is widely used to determine critical micellar concentration and micellar aggregates surface properties of amphiphiles. Current conductivity experiments of surfactant solutions are typically carried out by manual pipetting, yielding some tens reading points within a couple of hours. In order to study the properties of surfactant–cells interactions, each amphiphile must be tested in different conditions against several types of cells. This calls for complex experimental designs making the application of current methods seriously time consuming, especially because long experiments risk to determine alterations of cells, independently of the surfactant action. In this paper we present a novel, accurate and rapid automated procedure to obtain conductometric curves with several hundreds reading points within tens of minutes. The method was validated with surfactant solutions alone and in combi- nation with Saccharomyces cerevisiae cells. An easy-to use R script, calculates conductometric parameters and their statistical significance with a graphic interface to visualize data and results. The validations showed that indeed the procedure works in the same manner with surfactant alone or in combination with cells, yielding around 1000 reading points within 20 min and with high accuracy, as determined by the regression analysi

Phenotypic and molecular diversity of Meyerozyma guilliermondii strains isolated from food and other environmental niches, hints for an incipient speciation

Meyerozyma guilliermondii is a yeast species widely isolated from several natural environments and from fruit; in medical microbiology it is known as the teleomorph of the opportunistic pathogen Candida guilliermondii, which causes about 2% of the human blood infections. This yeast is also promising in a variety of biotechnological applications as vitamins production and post-harvest control. The question if isolates from different sources are physiologically and genetically similar, or if the various environments induced significant differences, is crucial for the understanding of this species structure and to select strains appropriate for each application. This question was addressed using LSU and ITS sequencing for taxonomic assignment, i-SSR (GACA4) for the molecular characterization and FTIR for the metabolomic fingerprint. All data showed that fruit and environmental isolates cluster separately with a general good agreement between metabolomics and molecular analysis. An additional RAPD analysis was able to discriminate strains according to the isolation position within the pineapple fruit. Although all strains are members of the M. guilliermondii species according to the current standards, the distribution of large variability detected suggests that some specialization occurred in the niches inhabited by this yeast and that food related strains can be differentiated from the medical isolates