Recentemente, l’ocratossina A (OTA) è stata segnalata in formaggio grattugiato confezionato: si ritiene sia stata prodotta dallo sviluppo di alcuni funghi durante il periodo di maturazione. Gli studi presentati in questa tesi hanno avuto lo scopo di migliorare la conoscenza dei funghi, Aspergilli e Penicilli, associati al formaggio di tipo grana. Si è dunque provveduto alla loro identificazione tramite approccio multifasico, eseguendo anche prove ecologiche per definire i fabbisogni delle specie presumibilmente dominanti. Obiettivo finale sarà quello di predire il rischio di contaminazione da micotossine alle condizioni ambientali di stagionatura del prodotto. Aspergillus puulaauensis e alcune specie del genere Penicillium (P.crustosum and P. solitum) sono state rilevate con maggior frequenza, mentre non sono state rilevate nei campioni di crosta le specie P. nordicum e P. verrucosum. In ogni caso, le condizioni ambientali dei locali di maturazione (15-22°C; 72-88% UR) sono risultate favorevoli allo sviluppo di queste ultime, dando adito a preoccupazione circa una possibile contaminazione da OTA; tuttavia, il ruolo di altre specie micotossigene, ad esempio P. crustosum, non va sottovalutato. Le azioni di contrasto verso questi funghi micotossigeni dovrebbero, innanzitutto, ridurre l’inoculo presente nell’aria: l’ozono sembra efficace in tal senso; inoltre, l’utilizzo della luce blu potrebbe ridurre notevolmente la crescita fungina sulla superficie dei formaggi e delle scaffalature. Le informazioni fornite in questa tesi saranno utili ai produttori e agli altri operatori della filiera di questo prodotto, aiutandoli ad attuare una miglior gestione del rischio e a garantire un prodotto sicuro e di elevata qualità ai consumatori. / Ochratoxin A (OTA) was detected a few years ago in Italian packed grated cheese, and supposed to be produced by fungal growth during cheese ripening. The works managed and presented in this thesis aimed to improve knowledge about mycotoxigenic Aspergilli and Penicillia associated to Italian grana cheese. A multiphasic approach was applied for their identification and ecological trials were organised to define fungal needs of the expected dominant species. The objective was to predict the risk of mycotoxin contamination during the ripening condition of grana cheese. Aspergillus puulaauensis and some Penicillium spp (P.crustosum and P. solitum) were the prevalent species associated to grana cheese, while neither P. nordicum, nor P. verrucosum were detected in cheese rind. However, the environmental conditions of the ripening rooms (15-22°C; 72-88% RH) are favourable to P. nordicum and P. verrucosum, reinforcing concern about possible OTA contamination, but other mycotoxigenic species such as P. crustosum must not be neglected. Actions against those mycotoxigenic fungi should mainly reduce total inoculum in the environmental air: ozone is reported to be effective in this sense; moreover, blue light may significantly reduce fungal growth on cheese and shelves surfaces. Information provided therein will be useful for producers and stakeholders to perform a better risk management and guarantee a safe, high-quality product to consumers.

The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing. Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension. The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results. Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses. Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results. <b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis

<p>The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing.</p><p>Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension.</p><p>The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results.</p><p>Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses.</p><p>Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results.</p><p><b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis</p>

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the field of bacterial diagnostics and is also used for routine analysis in smaller clinical laboratories. For identification of moulds, pre-analytical steps are more complicated and time consuming than for bacteria, and the choice of reference library has a big impact on the utility of MALDI-TOF MS. The aim of this study was to investigate if MALDI-TOF MS is applicable for identification of moulds belonging to the genus Aspergillus at the hospital laboratory in Gävle. Therefore, strains belonging to the genus Aspergillus and Penicillium, were analysed with MALDI-TOF MS after 2, 4 and 7 days of incubation. Two different extraction protocols were used and compared. Mass spectra were compared to reference spectra in two different databases: MSI-2 and RUO Compass library/BDAL (Bruker). Of the strains included, 97 % were correctly identified to species complex level with MSI-2. Only 25 % were identified to species level with RUO Compass library/BDAL (Bruker). However, totally 56 % were correctly identified to species complex level if a lower score value limit than recommended for identification, were applied. Significantly raised score values were observed with one of the protein extraction protocols used. Although, in most cases, the strains were considered identified to species complex level with either method. This pilot study conveys the feasibility of MALDI-TOF MS for identification of Aspergillus species in a clinical laboratory. While there are still issues to address, applying MALDI-TOF MS has the potential to allow for quicker and more precise identification, also in this specific clinical setting.

"2003". / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Department of Biological Sciences, 2004. / Bibliography: leaves 136-160. / Introduction: The Antarctic environment; Antarctic inhabitants; Microfungi; Identification of microfungi; Physiological factors affecting Antactic microfungi; Flow cytometry and microfungi; Hydrolytic enzymes of industrial interest; Isolation of genes from microfungi; Aims of this study -- Materials and methods: Fungal strains and cultivation conditions; Molecular identification of fungal isolates; Fungal physiology; Hydrolase activity of secreted proteins; Gene cloning and expression -- Results and discussion: Microfungal identification; Physiological factors affecting Antarctic microfungi; Activity in microfungi when grown on solid media; Characterisation of hemicellulases from selected Antarctic microfungi; Cloning of an Antarctic Penicillium allii lipase gene and its expression in Trichoderma reesei -- Conclusions and future prospects. / The Antarctic occupies that region of the planet that falls below the 60th parallel of South latitude. Although it has been frequented by adventurers, journeyman scientists and tourists for the past 100 years, the Continent has remained virtually unoccupied. The intense cold, the absence of human occupation and the limited range of local higher animal species have combined to create the impression that the Continent is virtually devoid of life. -- Although the microbiota of the Antarctic has attracted some small level of attention in the past, the examination of filamentous microfungi has been largely overlooked and fallen to a small group of dedicated investigators. In this study it will be shown that far from being an insignificant component of the Antarctic network, microfungi represent a potentially large and so far untapped bioresource. -- From just 11 bryophyte samples collected at four sites in the Ross Sea/Dry Valleys region of Southern Antarctica, some 30 microfungal isolates were recovered. Using molecular techniques, the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) was sequenced to reveal no less than nine unique microfungal species. For only two of these species did the ITS sequence data produce a 100% match with records held on the public databases. This investigation also highlighted the problems inherent in the traditional morphological identification system which are now being perpetuated in the molecular database records. -- A set of seven notionally identified isolates obtained from ornithogenic soil samples gathered in the Windmill Islands in Eastern Antarctica (offshore from the Australian Antarctic Division's Casey Station) were also subjected to molecular identification based on ITS sequence data. Each of the seven isolates was identified as a unique species; six were cosmopolitan in nature and the one remaining bore very little resemblance at the molecular level to any of the recorded species although it was provided with an epithet commonly used in the identification of Antarctic microfungal species. -- To evaluate their potential as a bioresource, samples of Antarctic microfungi were examined to determine if the same physiological factors common to mesophilic species also applied to their Antarctic analogues. It is known that when placed under stress, trehalose can act as a protectant against cold (cryoprotection) and dehydration in mesophilic yeasts and fungi. The level of trehalose produced by the Antarctic isolates and their mesophilic analogues when subjected to stress was compared. A similar comparison was made for the production of glycerol which is well established as a compatible solute providing protection to mesophilic species against osmotic stress. Only in the case of trehalose production by an Antarctic Embellisia was there any indication that either of these two compounds could play a significant role in providing protection to the Antarctic fungi against the rigours of their environment, which leaves open to question what in fact does. -- In the course of investigating the means by which Antarctic microfungi guard against the damage which can ensue when subjected to oxidative stress, flow cytometry was introduced as an investigatory tool. It was established that there is a window of opportunity during which flow cytometry can be used to undertake a detailed analysis of the early stages of fungal growth from germination through hyphal development. -- Of major significance in determining the potential of Antarctic microfungi as a resource is their ability to produce new and novel enzymes and proteins. The microfungal isolates were screened for hydrolytic activity on solid media containing indicative substrates and proved to be a fruitful source of enzymes active over a range of temperatures. A detailed characterisation of two hemicellulases, β-mannanase and xylanase, secreted into a liquid medium by a subset of the Antarctic fungi and a high producing mesophilic reference strain permitted direct comparisons to be made. It was shown that the maximum hemicellulase activity of the Antarctic strains occurred at least 10°C and as much as 30°C lower than that of the reference strain and that mannanase activity for two of the Antarctic isolates exceeded 40% of their maximum at 0°C. These assay results highlight the potential of Antarctic microfungi to yield novel cold-active enzymes. -- As a final measure of the capacity of the Antarctic to yield novel enzymes from its microfungal stock, a lipase gene was selected as a target for isolation and expression in a heterologous fungal host. Using PCR techniques, the gene of interest was isolated from an Antarctic isolate of Penicillium allii, transformed into the mesophilic production host Trichoderma reesei and the active protein successfully produced in the growth medium. The recombinant lipase was assayed and found to exhibit novel characteristics consistent with a cold-adapted enzyme. / Mode of access: World Wide Web. / 186 leaves ill