Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of gD, gB and heterodimer gH/gL. gD serves as receptor-binding glycoprotein and trigger of fusion; its ectodomain is organized in a N-terminal domain carrying the receptor-binding sites, and a C-terminal domain carrying the profusion domain, required for fusion but not receptor-binding. gB and gH/gL execute fusion. To understand how the four glycoproteins cross-talk to each other we searched for biochemical defined complexes in infected and transfected cells, and in virions. We report that gD formed complexes with gB in absence of gH/gL, and with gH/gL in absence of gB. Complexes with similar composition were formed in infected and transfected cells. They were also present in virions prior to entry, and did not increase at virus fusion with cell. A panel of gD mutants enabled the preliminary location of part of the binding site in gD to gB to the aa 240-260 portion and downstream, with T306P307 as critical residues, and of the binding site to gH/gL at aa 260-310 portion, with P291P292 as critical residues. The results indicate that gD carries composite independent binding sites for gB and gH/gL, both of which partly located in the profusion domain. The second part of the project dealt with rational design of peptides inhibiting virus entry has been performed. Considering gB and gD, the crystal structure is known, so we designed peptides that dock in the structure or prevent the adoption of the final conformation of target molecule. Considering the other glycoproteins, of which the structure is not known, peptide libraries were analyzed. Among several peptides, some were identified as active, designed on glycoprotein B. Two of them were further analyzed. We identified peptide residues fundamental for the inhibiting activity, suggesting a possible mechanism of action. Furthermore, changing the flexibility of peptides, an increased activity was observed,with an EC50 under 10μM. New approaches will try to demonstrate the direct interaction between these peptides and the target glycoprotein B.

Aims: Ripening evaluation of two different Pecorino cheese varieties ripened according either to a traditional method in plant and in cave. Different ripening features have been analyzed in order to evaluate the cave as possible ripening environment with the aim of obtaining a peculiar product which could also establish an added value to the cultural heritage of the local place in which it has been originally manufactured. Methods and Results: Chemical-physical features of Pecorino cheese have been initially analyzed into two different ripening environments and experimentations, among which: pH, weight reduction and subsequent water activity. Furthermore, the microbial composition has been characterized in relationship with the two different ripening environments, undertaking a variety of microbial groups, such as: lactic bacteria, staphylococci, yeasts, lactococci, enterobacteria, enterococci. Besides, an additional analysis for the in-cave adaptability evaluation has been the identification of biogenic amines inside the Pecorino cheese (2-phenilethylamine, putrescine, cadaverine, hystidine, tyramine, spermine and spermidine). Further analysis were undertaken in order to track the lipid profile evolution, reporting the concentration of the cheese free fatty acids in object, in relation with ripening time, environment and production. In order to analyse the flavour compounds present in Pecorino cheese, the SPME-GC-MS technique has been widely employed. As a result, it is confirmed the trend showed by the short-chain free fatty acids, that is to say the fatty acids which are mostly involved in conveying a stronger flavor to the cheese. With the purpose of assessing the protheolytic patterns of the above-mentioned Pecorino cheese in the two different ripening environments and testing methods, the technique SDS-PAGE has been employed into the cheese insoluble fraction, whereas the SDS-PAGE technique has been carried out into the cheese soluble portion. Furthermore, different isolated belonging to various microbial groups have been genotypically characterized though the ITS-PCR technique with the aim to identify the membership species. With reference to lactic bacillus the characterized species are: Lactobacillus brevis, Lactobacillus curvatus and Lactobacillus paraplantarum. With reference to lactococci the predominant species is Lactococcus lactis, coming from the employed starter used in the cheese manufacturing. With reference to enterococcus, the predominant species are Enterococcus faecium and Enterococcus faecalis. Moreover, Streptococcus termophilus and Streptococcus macedonicus have been identified too. For staphylococci the identified species are Staphyilococcus equorum, Staphylococcus saprophyfiticus and Staphylococcus xylosus. Finally, a sensorial analysis has been undertaken through on one side a consumer test made by inexperienced consumers, and on the other side through a panel test achieved by expert consumers. From such test Pecorino cheese ripened in cave were found to be more pleasant in comparison with Pecorino cheese ripened in plant. Conclusions: The proposed approach and the undertaken analysis showed the cave as preferential ripening environment for Pecorino cheese and for the development of a more palatable product and safer for consumers’ health.

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Bioremediation implies the use of living organisms, primarily microorganisms, to convert environmental contaminants into less toxic forms. The impact of the consequences of hydrocarbon release in the environment maintain a high research interest in the study of microbial metabolisms associated with the biodegradation of aromatic and aliphatic hydrocarbons but also in the analysis of microbial enzymes that can convert petroleum substrates to value-added products. The studies described in this Thesis fall within the research field that directs the efforts into identifying gene/proteins involved in the catabolism of n-alkanes and into studying the regulatory mechanisms leading to their oxidation. In particular the studies were aimed at investigating the molecular aspects of the ability of Rhodococcus sp. BCP1 to grow on aliphatic hydrocarbons as sole carbon and energy sources. We studied the ability of Rhodococcus sp. BCP1 to grow on gaseous (C2-C4), liquid (C5-C16) and solid (C17-C28) n-alkanes that resulted to be biochemically correlated with the activity of one or more monooxygenases. In order to identify the alkane monooxygenase that is involved in the n-alkanes degradation pathway in Rhodococcus sp. BCP1, PCR-based methodology was applied by using degenerate primers targeting AlkB monooxygenase family members. As result, a chromosomal region, including the alkB gene cluster, was cloned from Rhodococcus sp. BCP1 genome. We characterized the products of this alkB gene cluster and the products of the orfs included in the flanking regions by comparative analysis with the homologues in the database. alkB gene expression studies were carried out by RT-PCR and by the construction of a promoter probe vector containing the lacZ gene downstream of the alkB promoter. B-galactosidase assays revealed the alkB promoter activity induced by n-alkanes and by n-alkanes metabolic products. Furthermore, the transcriptional start of alkB gene was determined by primer extension procedure. A proteomic approach was subsequently applied to compare the protein patterns expressed by BCP1 growing on n-butane, n-hexane, n-hexadecane or n-eicosane with the protein pattern expressed by BCP1 growing on succinate. The accumulation of enzymes specifically induced on n-alkanes was determined. These enzymes were identified by tandem mass spectrometry (LC/MS/MS). Finally, a prm gene, homologue to the gene family coding for soluble di-iron monooxygenases (SDIMOs), has been isolated from Rhodococcus sp. BCP1 genome. This gene product could be involved in the degradation of gaseous n-alkanes in this Rhodococcus strain. The versatility in utilizing hydrocarbons and the discovery of new remarkable metabolic activities outline the potential applications of this microorganism in environmental and industrial biotechnologies.

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The aim of this PhD thesis was to evaluate the effect of a sub-lethal HPH treatment on some probiotic properties and on cell response mechanisms of already-known functional strains, isolated from Argentinean dairy products. The results achieved showed that HPH treatments, performed at a sub-lethal level of 50 MPa, increased some important functional and technological characteristics of the considered non intestinal probiotic strains. In particular, HPH could modify cell hydrophobicity, autoaggregation and resistance to acid gastric conditions (tested in in vitro model), cell viability and cell production of positive aroma compounds, during a refrigerate storage in a simulated dairy product. In addition, HPH process was able to increase also some probiotic properties exerted in vivo and tested for two of the considered strains. In fact, HPH-treated cells were able to enhance the number of IgA+ cells more than other not treated cells, although this capacity was time dependent. On the other hand, HPH treatment was able to modify some important characteristics that are linked to the cell wall and, consequently, could alter the adhesion capacity in vivo and the interaction with the intestinal cells. These modifications, involving cell outermost structures, were highlighted also by Trasmission Electron Microscopy (TEM) analysis. In fact, the micrographs obtained showed a significant effect of the pressure treatment on the cell morphology and particularly on the cell wall. Moreover, the results achieved showed that composition of plasma membranes and their level of unsaturation are involved in response mechanisms adopted by cells exposed to the sub-lethal HPH treatment. Although the response to the treatment varied according to the characteristics of individual strains, time of storage and suspension media employed, the results of present study, could be exploited to enhance the quality of functional products and to improve their organoleptic properties.

This PhD thesis is aimed at studying the possible pathways and the mechanisms that can trigger oxylipins biosynthesis, and particularly that of short chain aldehydes and alcohols, in Lactobacillus helveticus, also in the presence of oxidative stress, using a totally labelled linoleic acid as precursor. In plants and fungi these molecules, involved in defence mechanisms against pathogens and in communication systems, derive from the oxidation of cellular unsaturated fatty acids (UFAs) and their accumulation is associated with stress exposure. Since some oxylipins are produced also by lactobacilli, it is possible to hypothesize that a metabolic pathway from UFAs to oxylipins, similar to what happens in plants and fungi, is present also in lactic acid bacteria. The results obtained pointed out that some volatile molecules are the result of UFAs catabolism, since they appear only when cells are incubated in their presence. Labelled linoleic acid is integrated in the membrane and subsequently transformed into aldehydes and alcohols, whose extent and carbon atoms number depend on stress exposure. The enzymes responsible for this metabolic pathway in plants and fungi (e.g. lipoxygenase, dioxygenase) seem to be absent in Lactobacillus helveticus and in other lactobacilli. Proteomic analyses show the over expression of many proteins, including thioredoxin reductase (part of the bacterial oxidative defence system), mainly in cells grown with linoleic acid without oxidative stress exposure, confirming that linoleic acid itself induces oxidative stress. 6 general oxidoreductases (class including dioxygenases and peroxidase) were found and therefore a deeper investigation on them could be productive in elucidating all steps involved in oxylipins biosynthesis in bacteria. Due to the multiple role of oxylipins (flavouring agents, antimicrobial compounds and interspecific signalling molecules) the identification of genes involved and regulating factors should have an important biotechnological impact, also allowing the overproduction of selected bioactive molecules.

This PhD thesis is aimed at studying the suitability of proteases realised by Yarrowia lipolytica to hydrolyse proteins of different origins available as industrial food by-products. Several strains of Y. lipolytica have been screened for the production of extracellular proteases by zymography. On the basis of the results some strains released only a protease having a MW of 37 kDa, which corresponds to the already reported acidic protease, while other produced prevalently or only a protease with a MW higher than 200 kDa. The proteases have been screened for their "cold attitude" on gelatin, gluten and skim milk. This property can be relevant from a biotechnological point of view in order to save energy consumption during industrial processes. Most of the strains used were endowed with proteolytic activity at 6 °C on all the three proteins. The proteolytic breakdown profiles of the proteins, detected at 27 °C, were different related to the specific strains of Y. lipolytica. The time course of the hydrolysis, tested on gelatin, affected the final bioactivities of the peptide mixtures produced. In particular, an increase in both the antioxidant and antimicrobial activities was detected when the protease of the strain Y. lipolytica 1IIYL4A was used. The final part of this work was focused on the improvement of the peptides bioactivities through a novel process based on the production of glycopeptides. Firstly, the main reaction parameters were optimized in a model system, secondly a more complex system, based on gluten hydrolysates, was taken into consideration to produce glycopeptides. The presence of the sugar moiety reduced the hydrophobicity of the glycopeptides, thus affecting the final antimicrobial activity which was significantly improved. The use of this procedure could be highly effective to modify peptides and can be employed to create innovative functional peptides using a mild temperature process.